The results of this study show that the Fmask 4 algorithm consistently performs better than the alternatives for Sentinel-2 images of the Amazon rain forest.

Fmask 4 had an overall accuracy of 90%, followed by Sen2Cor (79%), MAJA (69%) and s2cloudless (52%).

Our results are different from those of Baetens et al. [ 26] who concluded that MAJA and Fmask 4 perform similarly with an overall accuracy around 90%, while Sen2Cor had an overall accuracy of 84%.

We now consider some hypothesis that could account for such significant differences.


As noted by Baetens et al. [ 26], for satellites without thermal bands cloud detection methods use thresholds.

Different thresholds are set for the visible bands, the 1.38 µm band, and the Normalized Difference Snow Index.

These approximations address important challenges for cloud detection methods: distinguishing clouds from snow, mountain tops, bright deserts, and large built-up objects.

Since each cloud detection method relies on different ad hoc hypotheses, its usefulness varies from scene to scene.

For this reason, no single study can provide definitive guidance.

Studies that target specific regions, such as the current paper, provide valuable advice even though its results cannot be generalized to non-forest areas.


The comparison done by Baetens et al. [ 26] uses 10 different sites, including equatorial forests, deserts and semi-deserts, agricultural areas, mountains, and snowy areas.

Their results provide a balance between different targets that could be confused with clouds.

By contrast, our study deals only with forest and agriculture areas; the images tested have no deserts, mountains or snow.

By focusing on the Amazon biome, our results are intended as guidance for experts interested in measuring land change in the region.

Given its focus, these results cannot be generalized to non-forest regions.


A further consideration that could explain part of the differences between our work and that of Baetens et al. [ 26] is the choice of training data sets.


While we use random sampling, those authors preferred to rely on active learning.


An active learning model uses a few good quality samples instead of a large ensemble of random points.


These good quality samples are used to train a machine learning model (random forest) whose output provides labels to a large set point for classification.


In theory, this method has the advantage of being able to provide a larger number of points to test the algorithm.


Machine learning models have a tendency to overfit their training data, which could cause wrong predictions [52].


The alternative is to use random samples, which rely on standard statistical assumptions.


However, random samples can miss some cloud properties.


Clouds come in different shapes, sizes, and transparencies; it is often hard to distinguish overlapping clouds at different heights from images.


Random samples can also misrepresent minority labels such as cloud shadows.


Therefore, both random sampling and active learning have their advantages and shortcomings for evaluating cloud detection algorithms.


Further testing and comparison are required to evaluate these approaches.


Another source of divergence between our result and that of Baetens et al. [ 26] is due to class relabeling.

Cloud detection algorithms codify their results using different levels of detail.

To enable comparisons, we had to recode them to the same set of labels.

This process implies a loss of information, in particular for MAJA, which provides the most detailed data about its detection process.

Thus, our recoding process could have had a negative impact on our evaluation of MAJA.


Despite the differences discussed above, there are points of convergence between our work and earlier papers such as Baetens et al. [ 26] and Qiu et al. [ 25] related to Fmask 4 performance.

The overall user's and producer's accuracy values for Fmask 4 are broadly consistent in the three studies.

Qiu et al. [ 25] report producer's accuracies for clouds, shadows and clear pixels to be 93%, 70%, and 97%, while our results are 96%, 75%, and 90%.

Thus, we consider that Fmask 4 to be a reliable method that we recommend to be used for cloud detection in Sentinel-2 images of the Amazon rain forest.


In this work, we compared four cloud detection algorithms on Sentinel-2A images of the Amazon tropical forest, and we found that Fmask 4 performs the best.

We tested four cloud detection algorithms-FMask 4, Sen2Cor, MAJA, and S2cloudless-on 20 images with a different amount of cloud coverage, spread over five regions of Amazonia.

We validated the results of the cloud detection algorithms using the criteria of experts on remote sensing who classified approximately 400 random points on each image.

To determine the best algorithm, we computed the F1 score and the overall, user, and producer accuracies.

We found that FMask 4 has an overall accuracy of 90% to detect clouds, while Sen2Cor's OA is 79%, MAJA's OA is 69%, and S2cloudless's OA is 52%.

Based on these results, we recommend the use of Fmask 4 for cloud detection of Sentinel-2 images of the Amazon region.


The choice of method depends on the intended use.

Therefore, users should consider the benefits of each method before making their choices.

Since MAJA reduces the number of false positives by design, users that aim to improve the producer's accuracy should consider its use.

These characteristics could make MAJA suitable, for example, to build cloud-free monthly mosaics.

Despite the poor performance of S2cloudless in our study, we consider that the use of machine learning methods for cloud detection is a promising way forward.

As more good quality samples become available, its performance will improve.

Finally, Sen2Cor is an efficient method to detect clouds in Sentinel-2 images.

Despite not having the best performance, its ease of use may appeal to those that need fast processing of large data sets.


We expect our work to impact on the building of data cubes of analysis-ready data from satellite imagery, like those currently under construction by the Brazil Data Cube project. ( Brazil Data Cube project http://brazildatacube.org/) Another application is for improving the time series analysis of Land Use and Land Cover change of deforested areas, which is particularly hard because of cloud coverage.

Given the performance of FMask 4, space agencies and committees such as CEOS should consider the value of working together to develop a standardized best quality cloud detection methods that could be shared and used for remote sensing optical imagery.


The R and Python scripts used to compare the performance of cloud detection algorithms are available on GitHub: https://github.com/brazil-data-cube/compare-cloud-masks.


In their recent study, Sanchez et al. compared various cloud detection methods applied to Sentinel-2, specifically on images acquired over the Amazonian region, known for its frequent cloud cover.

Comparison of cloud screening methods for optical satellite images is a complex task, which must take several parameters into account, such as the definition of a cloud, which can differ according to the methods, the different coding of the cloud and shadow masks, the possible dilation of masks, and also the way the method must be used to perform in nominal conditions.

We found that the otherwise serious and useful comparison of cloud masks by Sanchez et al. is not fair to the real performances of MAJA cloud detection, for two reasons: (i) two thirds of the images used in the comparison were acquired before the launch of Sentinel-2B satellite, when the revisit of the Sentinel-2 mission was 20 days instead of five days for the nominal conditions of the mission, and (ii) there is an error in the understanding of how MAJA cloud masks are coded which also probably artificially degraded the results of MAJA as compared to the other methods.


In their study over Amazonia, Sanchez et al. [ 1] found that the performances of MAJA cloud screening software are not as good as those obtained in a similar study by Baetens et al [2].

A large part of the discussion in the article was dedicated to explaining the differences.

It concluded that they are probably related to the specificities of cloud cover and surface reflectances in Amazonia.

The authors also noticed that MAJA (MACCS-ATCOR Joint Algorithm) was surprisingly not able to detect cloud shadows in Amazonia.

While most cloud detection methods process successive images independently, our method for cloud detection and atmospheric correction, MAJA, uses the generally slow variations of surface reflectances as a function of time to better detect the clouds and their shadows [3].

Our cloud detection method uses time series of images as input, processes the images in chronological order, and the quality of its results improves when the interval between successive observations of the land surface is reduced.


In their study, Sanchez et al. used twenty manually classified images taken over five tiles scattered in the Amazonia (Table 2 of their paper).

Thirteen of these images were acquired by Sentinel-2A, before Sentinel-2B was put in operation in July 2017.

Moreover, until November 2017, the revisit of each Sentinel-2 satellites was 10 days in Europe and Africa, and 20 days on the other continents.

As an example, Table 1 shows the number of images acquired by Sentinel-2A or Sentinel-2B over an Amazonian tile used in the Sanchez et al. study, for the same three-month period from 2016 to 2020.

MAJA has been designed to perform optimally in the nominal conditions of Sentinel-2, with a revisit of five days from the same path.

For most images in the time series used by Sanchez et al., the revisit was 20 days.

Given the frequent cloud cover in Amazonia, MAJA does not always have sufficient cloud free observations of the surface, and works with a fallback mono-temporal method to detect the clouds and shadows.

Sanchez et al. do not provide validation statistics per image, but Table 5 of Sanchez et al. provides results per tiles, accumulating the four available dates for each tile.

The two tiles which have only dates in 2016 and 2017 (T20NPH and T22NCG) exhibit the worst performances for MAJA, which shows that MAJA’s performance should be much better for the nominal configuration of Sentinel-2 with a revisit of five days.


Moreover, we found that the authors wrongly interpreted MAJA’s cloud mask.

For each pixel, MAJA not only provides a binary valid/invalid mask, but the mask is expressed as bit mask which tells which of MAJA’s tests declared a pixel invalid.

Such a representation is a little complex but has the advantage to provide a complete information on a small volume (one byte per pixel).

Table 3 of the original paper provides a conversion table for MAJA cloud mask which is partly false, for instance, only the value 0 in MAJA cloud masks correspond to a cloud free/shadow free pixel while Table 3 lists 0 and 1.

Two figures of the Sanchez et al. paper (the graphical abstract and Figure 3) show that the shadows detected by FMASK are also classified as invalid in MAJA, but the authors wrongly indicated that MAJA classifies them as clouds instead of shadows.

For the sake of comparison, we provide in Figure 1 the MAJA cloud mask for the same tile and date as the Figure 3 in Sanchez et al. with the correct representation of the cloud bit-mask.

We understood that, in Sanchez et al., the classification performances have been computed for three classes (clear, cloud, shadow), and, in that case, the wrong interpretation of MAJA mask values must also have a large impact on the performances.

In addition, we are not sure it is optimal to separate cloud and cloud shadows in distinct classes for validation purposes, as shadows on other clouds and mask dilation process lead to mixed classes.

End-users usually expect a mask to discriminate between valid and invalid pixels with the highest possible accuracy, whether invalid ones are clouds or cloud shadows should remain a second order performance criterion.


The study of Sanchez et al. is a very interesting initiative, since the Amazonian regions pose specific challenges to cloud masking, as the cloud cover is usually high and the surface reflectances are low.

In such regions, the multi-temporal method, which is a corner stone of MAJA, partly loses its advantages as the surface is seldom visible and reflectances can significantly evolve from one cloud free observation to the next.

Even if Amazonia is not a use case where MAJA will obtain its best results, we think that the two shortcomings detailed above have resulted in performances for MAJA that are not the nominal ones for Sentinel-2, even over Amazonia.


In their comments about our paper, the authors remark on two issues regarding our results relating to the MACCS-ATCOR Joint Algorithm (MAJA).

The first relates to the sub-optimal performance of this algorithm under the conditions of our tests, while the second corresponds to an error in our interpretation of MAJA’s bit mask.

To answer the first issue, we acknowledge MAJA’s capacity to improve its performance as the number of images increases with time.

However, in our paper, we used the images we had available at the time we wrote our paper.

Regarding the second issue, we misread the MAJA’s bit mask and mistakenly labelled shadows as clouds.

We regret our error and here we present the updated tables and images.

We corrected our estimation and, consequently, there is an increment in MAJA’s accuracy in the detection of clouds and cloud shadows.

However, these increments are not enough to change the conclusion of our original paper.


In our paper [1], we compare four cloud detection algorithms for the Amazon forest using Sentinel images from October 2016 to November 2018.

Three of the algorithms (Fmask4, s2cloudless, and Sen2Cor) identify clouds using only spacial features.

On the other hand, MACCS-ATCOR Joint Algorithm (MAJA) uses time series of images for this purpose.

We compared the results of the four algorithms using points collected and classified by experts on remote sensing.


In their comments, Hagolle and Colin [2] pointed to two issues in the method reported in our paper [1], both related to MAJA.

The first issue is related to the sub-optimal performance due to the interval between successive observations.

The second issue indicates a misinterpretation of MAJA’s bit mask.


Concerning the first issue, it is noteworthy that in the experimental design, we delimited the evaluation period between October 2016 and November 2018.

This period was the same used in the comparison of the evaluated cloud detection algorithms.

We recognize that this temporal delimitation may have impacted the accuracy of MAJA, since its detection improves with the inclusion of more images observed over time, as demonstrated in the results presented in [2].


Regarding the second issue, we misread the MAJA’s bit mask.

We mistakenly took shadows for clouds.

We apologise for our error.

To fix this error, the MAJA column in Table 3 of our paper must be ignored and instead one should use Table 1.

To correctly interpret MAJA’s cloud mask, please use Table 2.

This is because of the bit structure used by MAJA.


Because of the misinterpretation of MAJA’s bit mask, MAJA’s accuracy in identifying clouds and cloud shadows needs to be updated, while the accuracy of clear observation was not affected.

We updated the accuracy reported on Tables 4 and 5 of our paper and now they are presented here in Tables 3 and 4, respectively.


However, these updates do not change the conclusion in our original paper.


Xenopus egg extracts represent a complete system for regulated cyclin degradation (Murray and Kirschner 1989; Murray 1991).

Interphase extracts supplemented with a nondegradable form of sea urchin cyclin B (⌬90) enter and remain in a mitotic state (Glotzer et al. 1991), in which they degrade mitotic APC substrates, such as securin/Pds1 (Zou et al. 1999) and cyclin B (Glotzer et al. 1991).

Cdh1 is absent in the egg and does not appear during embryonic development until the mid-blastula transition (Lorca et al. 1998), so Xenopus egg extracts do not show degradation of known Cdh1 substrates.


How- ever, by supplementing interphase extracts with baculo- virus-purified hCdh1 protein, we were able to reconsti- tute Cdh1-dependent degradation (Fig. 1).


hCdc20 and the amino-terminal fragment of Xenopus cyclin B are both stable in interphase extracts in which APC, presumably, is inactive because of the absence of a bound activator.

Both proteins become unstable upon the addition of Cdh1 to the interphase extracts (Fig. 1A).

When ⌬90 cyclin B is added to an interphase extract, the extract enters mitosis, in which the active form of APC is the form associated with Cdc20.

hCdc20 remains stable, but amino-terminal Xenopus cyclin B degrades rapidly.

This demonstrates a clear difference in specific- ity between the Cdc20- and Cdh1-activated forms of APC in the complete extract system.


The direct recognition of hCdc20 by Cdh1-APC can be studied by generating ubiquitin conjugates in a purified system.

In this system, APC is immunopurifed from in- terphase extracts with anti-Cdc27 antibody coupled to beads and activated with baculovirus-expressed hCdh1 or hCdc20 protein.

The subsequent addition of purified E1, Ubcx, ubiquitin, and an energy-regenerating system results in ubiquitination of [ 35 S]-methionine-labeled in vitro translation products of APC substrates (Fang et al. 1998).

As substrates become conjugated to ubiquitin, the signal corresponding to the unmodified substrate disap- pears and higher molecular weight species are produced.


In the purified system there is always some very low level of ubiquitin conjugation of APC substrates.

Hence, it is the quantitative loss of the unmodified protein and its conversion of the input peptide into high molecular weight conjugates that we consider evidence for signifi- cant activity on a given substrate.


Few conjugates of ubiquitin to hCdc20 or the amino terminus of xcyclin B occur with control beads (beads incubated with extract, but lacking Cdc27 antibodies), or with unactivated interphase APC on beads (Fig. 1B).

In the presence of Cdc20-APC, the pattern of conjugation to hCdc20 and to xcyclin B is strikingly different.

Except for a few monoubiquitinated species, there is no evi- dence of conjugation of Cdc20 to ubiquitin.

In contrast, the cyclin B signal is converted completely to conjugated species.

With Cdh1-activated APC, there is conjugation of ubiquitin to both substrates.

The difference in speci- ficity between Cdc20-APC and Cdh1-APC for degrada- tion, therefore, is preserved in the purified system for ubiquitination and does not require additional elements from the extract.

In the presence of unactivated inter- phase APC, there is a small background of monoubiqui- tination, but little quantitative conversion of the un- modified substrate.


The budding yeast homolog of Cdc20 contains two de- struction boxes (shown schematically in Fig. 2A), but the vertebrate homologs lack any motif similar to the R-L-N of the D box.

Furthermore, previous work (Fang et al. 1998; Gieffers et al. 1999) reports the ability of Cdh1- APC in the purified system to recognize the amino ter- minus of Xenopus cyclin B with the D box deleted.


To determine how Cdh1-APC recognizes substrates lacking a D box, whereas apparently both WD-repeat-containing proteins confer on APC the capacity to recognize D box- containing substrates, we first tested the less interesting possibility that Cdh1-APC simply shows little substrate selectivity.

Previous work (Fang et al. 1998) had indi- cated that Cdh1 was not completely permissive in the purified system.

In the Cdh1-APC ubiquitination assay on a random sampling of in vitro-translated individual proteins as well as in vitro-translation products from cDNA pools, they showed that Cdh1did not recognize any of the proteins chosen.


We performed a similar ran- dom sampling of 10 cDNA pools from a Xenopus-dorsal- ized gastrula library containing ∼30 clones each.

We tested the in-vitro translation products of each pool in the purified system Cdh1-APC ubiquitination assay as well as the degradation assay of Cdh1-supplemented in- terphase extracts.

Of the 300 clones assayed in both sys- tems, we found only 2 positives.

The positives were iso- lated from the pools, sequenced, and identified to be xcy- clin B1 and xcyclin B3.

Because the only two clones to respond positively in these assays are well-documented


Given the absence of an obvious D box in vertebrate Cdc20, we systematically deleted sequences from the amino and carboxyl termini of human Cdc20 (repre- sented schematically in Fig. 2A) and assayed ubiquitina- tion using Cdh1-activated APC (results summarized to the right of the schematic).

Deletion of the amino-ter- minal 96 amino acids did not alter the ubiquitination profile in terms of amount of full-length protein con- verted to polyubiquitinated species, or the average num- ber of ubiquitins added per molecule, but deletion of the amino-terminal 100 amino acids blocked ubiquitination almost completely.

A few conjugates formed, but most of the original protein remained unconjugated.

Those conjugates that did form were probably monoubiquiti- nated on, at most three sites.

The amino-terminal 120 amino acids showed a wild-type ubiquitination profile, and the amino-terminal 100 and 105 amino acids were each sufficient for a ubiquitin laddering of up to nine ubiquitins added per molecule, even though we did not observe the smearing associated with branched polyu- biquitinated chains.

The amino-terminal 90 amino acids remained largely unconjugated or formed only mono- ubiquitinated species.


Despite the lower stoichiometry of ubiquitination, the 100 and 105 amino acid amino-terminal fragments formed conjugates with almost complete modification of the initial input peptide.

The reduced level of conjuga- tion as compared with that of the amino-terminal 120 amino acid fragment might have been due to the pres- ence of additional lysines and not specific sequence ele- ments involved in recognition.

To test this idea, we tagged the amino-terminal 105 mutant with four addi- tional lysines at its carboxyl terminus.

This restored the branched polyubiquitin smearing seen with the 120 amino acid mutant, confirming that the smearing profile resulted from availability of lysines, not from specific sequence recognition elements.


Given the identification of the region between amino acids 97-105 as required for ubiquitination, we created a series of alanine substitutions in this region within the ⌬96 construct.

Substitution of four amino acids pro- duced a marked drop in ubiquitination (Fig. 2B) com- pared with the ⌬96 construct.

The K97, E98, N99, and N103 alanine substitutions showed a collapse from the polyubiquitin smearing to a monoubiquitin laddering.


Because we had seen significant ubiquitination of an amino-terminal 100 amino acid fragment, we were puzzled by the effect of alanine substitution at position N103, outside of the amino-terminal domain.

To study the role of the N103 position, we created a ⌬96 mutant containing an internal deletion of amino acids 101-105.

This mutant formed polyubiquitinated chains of signifi- cantly greater average length than the ⌬96 mutant (Fig. 2B).

N103, in the context of the amino-terminal frag- ment of Cdc20, therefore, is not required for recognition, but it might contribute to recognition, partially in the context of surrounding residues.


PEST sequences are sequences enriched for the amino acids proline, glutamic acid, serine, and threonine and have been shown previously to contribute to protein in- stability (Rogers et al. 1986).

Amino acids 97-108 of Cdc20 qualify as a PEST sequence (http://www.at.emb- net.org/embnet/tools/bio/PESTfind/).

Changing S101, E102, S104, T106, or P107 to alanine should reduce ubiq- uitination of Cdc20 if recognition depends on this se- quence serving as a PEST sequence.

However, these point mutants within the ⌬96 context did not reduce recognition.

The average length of the polyubiquitin chains formed actually increased following substitution of alanine for the PEST residues S101, E102, and S104.


The human, rat, and Xenopus homologs of Cdc20 in this region (Fig. 2C) are conserved for the required K-E-N residues.

The fourth position, shown to contribute to recognition, although not being required, is an aspara- gine in the human sequence, and an aspartic acid in the other vertebrate homologs shown.

Replacement of N103 in the ⌬96 context with aspartic acid has no effect on the ubiquitination profile (data not shown), confirming that a similar structure that carries a negative charge is ac- ceptable at this position.


To confirm the importance of the K-E-N-X-X-X-N se- quence for ubiquitination and subsequent degradation of Cdc20, we constructed a mutant in which all four resi- dues shown to be important by alanine scanning (K97, E98, N99, and N103) were changed to alanine within the full-length protein.

This mutant (hCdc20KENN-A) showed decreased ubiquitination compared with wild- type Cdc20, both in terms of the extent and rate of con- jugation (Fig. 3A).

Furthermore, hCdc20KENN-A was much more stable than wild-type protein in Cdh1- supplemented extracts (Fig. 3B).

To rule out gross mis- folding of the mutant protein, we compared the ability of hCdc20KENN-A with wild-type hCdc20 to activate APC.

Mutant protein showed similar activity to wild- type protein in stimulating cyclin ubiquitination in the purified system (Fig. 3C).


Previous work showed that Cdh1-APC can promote the ubiquitination of an amino-terminal fragment of Xeno- pus cyclin B lacking the D box at position 36-44 (Fang et al. 1998; Gieffers et al. 1999).

Although there were no KEN-box sequences in this fragment, we did find another R-L-N motif at the extreme amino terminus, positions 7-15 that had been overlooked previously (Fig. 4A).

This Pfleger and Kirschner 658 GENES & DEVELOPMENT Cold Spring Harbor Laboratory Press on September 23, 2025 - Published by Downloaded from potential D box is present only in Xenopus cyclin B, and is not found in other species.

Because deletion of the conserved 36-44 D box blocks degradation in mitotic extracts (King et al. 1996), the 7-15 D box of cyclin B cannot in itself promote mitotic degradation.

To test whether this additional R-L-N motif was responsible for the recognition of cyclin ⌬D box by Cdh1 in the purified system, we constructed mutants of the amino-terminal fragment that lacked this second D box or double mu- tants lacking both D boxes.


Ubiquitination assays re- vealed that deletion of the 7-15 D box alone resulted in a slight drop in ubiquitination by Cdh1 as compared with wild type.

However, deletion of both D boxes elimi- nates ubiquitination completely (summarized quantita- Both in degradation assays and in ubiquitination as- says, we have found quantitative differences in target sequence recognition.

For example, in Figure 4b, Cdh1- APC recognizes the standard D-box deletion (⌬36-44), by promoting nearly 30% of the original protein into poly- ubiquitinated species; wild type show nearly 100% con- version.


Even though there is some ubiquitination of this deletion in the purified system, that particular deletion is completely inactive for degradation in the Cdh1- supplemented extracts (Fig. 4C).

In contrast, both the wild-type and ⌬7-15 constructs were rapidly degraded under similar conditions.

The difference between ubiq- uitination with purified components and degradation in extracts may be due to the optimized conditions for ubiquitination in the purified system, in which there is no isopeptidase activity.

Alternatively, certain crucial components may be limiting or be subject to competi- tion in the cell and in extracts, so that recognition occurs only for optimal substrates.


To restore artificially some of the selectivity to the purified system, we reduced the concentration of ubiq- uitin.

At a ubiquitin concentration of 20% of the original amount, selectivity at the level of ubiquitination was much more apparent (Fig. 4D).

Conjugation of the ⌬36- 44 D-box mutant was reduced to monoubiquitination with no higher molecular weight conjugates, whereas the ⌬7-15 mutant was ultimately fully conjugated to ubiquitin, although there was a significant decrease in the rate and extent of conjugation compared with wild type.

Thus, under less optimal conditions, substrate se- lectivity was enhanced and different D boxes were dif- ferentially active.


The amino-terminal peptides of Xenopus cyclin B (Mc- Garry and Kirschner 1998; Zou et al. 1999) and sea ur- chin cyclin B (Holloway et al. 1993) containing the con- served D box, have been well characterized as competi- tive substrates for Cdc20-APC degradation in mitotic extracts.

Because Cdh1-APC recognizes D-box sub- strates, we asked whether the amino-terminal peptide of cyclin B can compete for Cdh1-dependent degradation.

We find that the amino-terminal cyclin B peptide com- peted for hCdc20 degradation in Cdh1-supplemented, whereas the corresponding deletion mutant of the D box did not compete (Fig. 5).

Because the amino-terminal 120 amino acids of hCdc20 is an excellent Cdh1 substrate, we tested the ability of this peptide also to inhibit Cdh1- dependent degradation.

As shown in Figure 5, wild-type amino-terminal Cdc20 blocked degradation of full- length hCdc20 5 µM. The KEN-box mutant amino-ter- minal peptide shows partial inhibition at 10 µM and none at 5 µM. At 5 µM, the mutant caused no increase in in- hibition over a buffer control, whereas 5 µM of wild-type KEN-box peptide stabilized initial substrate protein to >75%.


A key property of the cyclin B D box is that the degra- dation signal is transposable.

Fusion of a small region containing the cyclin B D box with the immunoglobulin binding region of protein A (a protein stable in mitotic extracts, therefore not recognized by Cdc20-APC) re- sulted in rapid degradation of the fusion protein in mi- totic extracts (Glotzer et al. 1991; King et al. 1996).

The immunoglobulin-binding region of protein A is not rec- ognized by Cdh1-APC in ubiquitination or degradation assays.


We constructed a series of fusions of the region containing 97-103 to protein A and assayed these fusion proteins for recognition by Cdh1-APC using both the ubiquitination and degradation assays.

Fusion of the amino-terminal 103 amino acids, or even the amino-ter- minal 100 amino acids (containing KENQ, but lacking the second asparagine residue shown to contribute to recognition in the point mutant analysis) to protein A resulted in a ubiquitination profile similar to full-length wild-type Cdc20 protein (not shown).

Smaller fusions re- duced the ubiquitination profile but still yielded visible ubiquitin conjugates, whereas protein A alone was not recognized at all.


These fusion proteins were stable in interphase extracts supplemented with buffer, but de- graded in the presence of Cdh1 (Fig. 6).

Fusion of the KEN box alone did not result in ubiquitination or degra- dation (not shown), but fusion of the 28 amino acid re- gion from 75-103 was sufficient to allow ubiquitination and degradation, although the degradation occurred somewhat more slowly than full-length hCdc20 or the larger fusion proteins.


Because the KEN box directs the ubiquitination of Cdc20 by Cdh1 and can be transferred to another protein, we investigated to see whether this sequence serves a gen- eral targeting signal or is specific to Cdc20.

Database searching for other KEN-box-containing proteins re- vealed that the serine-threonine kinase Nek2, a NimA related kinase, contains a region extremely similar to the recognition signal in Cdc20 (Fig. 7A).

NimA has been identified recently as an APC substrate in Aspergillis nidulans (for review, see Zachariae and Nasmyth 1999) making Nek2 a likely Cdh1-APC candidate.

Further- more, Nek2 has been reported to vary with the cell cycle, peaking in protein expression level and activity in S and G 2 and disappearing during mitosis and G 1 (Schultz et al. 1994).

The sequence of B99, a p53-inducible gene that peaks in expression during G 2 (Utera et al. 1998), also contains a putative KEN box (Fig. 7A).


In the purified system, both Nek2 and B99 show sig- nificant ubiquitin conjugation by Cdh1-APC (not shown) but not with antibody control beads.

Nek2, but not B99, shows some degree of recognition by unacti- vated interphase APC, but conjugation is still not as great as that achieved by Cdh1-APC.

Nek2, in particu- lar, forms significant branched chains of varying lengths that extend over a much broader range than for other substrates.

Because the conjugates are more spread out as they run on a gel, we do not visualize the dark smear commonly seen for hCdc20.


Nek2 is degraded in interphase extracts with a half-life of 50-60 min (not shown).

Addition of Cdh1 to these extracts results in complete degradation of Nek2 by 10 min (Fig. 7B), a 12-fold increase in rate.

Alanine substi- tution of the KEN box in Nek2 significantly stabilizes the protein against both ubiquitination in the purified system (not shown) as well as degradation in extracts (Fig. 7B).

B99 is completely stable in interphase extracts but is degraded with a half-life of 25-30 min when the extracts are supplemented with Cdh1.

Alanine substitu- tion of the B99 KEN box completely stabilizes the pro- tein to degradation (Fig. 7C).


Genetic and biochemical studies suggest that the WD repeat proteins, Cdc20 and Cdh1, sequentially activate the ubiquitin ligase APC during mitosis to achieve a temporal order of substrate degradation.

Cdc20 itself is a substrate for APC when APC is activated by Cdh1, and therefore, Cdc20 is no longer present at the end of mito- sis; Cdh1 is a stable protein present throughout the cell cycle, whose interaction with APC is most likely regu- lated by phosphorylation (Kotani et al. 1999).

Although it has been suggested that Cdc20 and Cdh1 might confer different substrate specificities on APC, the nature of substrate recognition is still unclear.


In the SCF com- plex, the corresponding WD proteins such as HOS or ␤TrCP (Margottin et al. 1998; Yaron et al. 1998; Fuchs et al. 1999) bind directly to the substrate, whereas the af- finity of Cdc20 and Cdh1 with potential substrates has not been shown, possibly because it is too low to mea- sure.

Mutagenesis experiments in many species defines a target sequence recognized by APC that has been acti- vated by Cdc20.

This sequence, which includes the de- struction box plus some nonconserved lysine containing sequences, has been identified on all species of cyclin B plus several other proteins degraded at metaphase or soon after, such as Geminin, Pds1 (securin), Ase1, and others.


The targeting sequence to APC activated by Cdh1 has, up to now, been undetermined.

Its character- ization, the subject of this work, would allow a better understanding of specificity and temporal order of pro- tein degradation during mitosis and in G 1 .


We have used Cdc20 itself as a substrate for Cdh1- APC to define recognition sequences within Cdh1-APC substrates.

Cdc20 has been shown to be degraded late in mitosis (Weinstein 1997) in an APC-dependent manner (Prinz et al. 1998; Shirayama et al. 1998) and to be a


GENES & DEVELOPMENT 661 Cold Spring Harbor Laboratory Press on September 23, 2025 - Published by Downloaded from substrate of Cdh1-APC in vitro.

Using ubiquitination assays in a completely defined system and degradation assays in complete extracts, we have examined mutants of Cdc20 to learn the requirements for ubiquitination and degradation by Cdh1-APC.

Deletion mutagenesis narrowed down the recognition region to amino acids 97-105, and point mutants in this region identified four residues that decreased recognition when changed to ala- nine, K-E-N-X-X-X-N, (in which aspartic acid in the final position supported similar polyubiquitination as the as- paragine).

However, within a truncated fragment of Cdc20, deletion of residues 101-105, containing the sec- ond crucial asparagine, showed a greater than wild-type response.

This indicates to us that the second asparagine is not required for recognition overall, but that it may be required within the context of the surrounding residues, perhaps modulating a negative regulatory region.


Sequence alignment between the vertebrate Cdc20 se- quences and the Cdc20 sequence of S. cerevisiae show no KEN-similar motif in the same region of the protein.

However, there is a potential KEN box at position 586 near the carboxyl terminus of S. cerevisiae Cdc20 (pro- tein YGL116w), K-E-N-X-X-X-N (pointed out to us by E. Hildebrant and A. Hoyt, pers.

comm.).

The carboxy-ter- minal KEN boxes of hNek2 and mB99 support ubiquiti- nation and degradation, suggesting the possibility that the KEN box in any part of the protein could be func- tional.

We do not know if recognition of the KEN box extends to the yeast system or is limited to vertebrate systems, or whether there are undiscovered Cdh1-APC recognition signals in other proteins.


Previous reports already suggested that Cdh1-APC dif- fered from Cdc20-APC in that it could mediate the ubiq- uitination of substrates like hCdc20 that lack a D box and substrates like amino-terminal xcyclin B from which the conserved D box had been deleted (deletion of residues 36-44).

A possible interpretation of these results is that Cdh1-APC is promiscuous in substrate recogni- tion, particularly since there were no known conserved sequence elements in the amino-terminus of cyclin B other than the D box that could serve as a targeting se- quence.

Yet, countering this conclusion is the lack of activity of Cdh1-APC against 300 random substrates.

By mutagenesis of Cdc20 and Xenopus cyclin B, we could demonstrate that Cdh1-APC shows specificity for two classes of sequences, those that contain the R-L-N motif of the D box and those that show a new targeting se- quence, the KEN box.

The paradoxical observation that Cdh1-APC recognizes a cyclin fragment lacking both a D box and a KEN box was explained when an additional D box sequence in Xenopus cyclin B was found upstream of the conserved D box.

Deletion of this second, noncon- served motif blocks polyubiquitination completely.


There are notable quantitative differences between ubiquitination in the purified system and degradation in extracts.

For example, there is significant polyubiquiti- nation of cyclin B from which the conserved D box has been deleted (⌬36-44 N), whereas the protein is very stable in Cdh1-supplemented extracts.

The degree and rate of ubiquitination must be a very complex function of many factors in extracts and in living cells.

It may depend on competition among substrates, the activity of isopeptidases, and other factors that could modify any of the several steps in the process.

In the purified in vitro system, ubiquitination, is optimized and inhibitory fac- tors are absent.

Dilution of ubiquitin reduced the effi- ciency of ubiquitination, but most importantly, it also elicited a difference between the behavior of the two D boxes in Xenopus cyclin B in the purified system, a dif- ference reflected in the aggregate rates of degradation in extracts.


Like the D box, the KEN-box domain was transposable to other proteins.

A fusion of only 28 amino acids around the KEN-box sequence (residues 75-103 of hCdc20) to protein A resulted in ubiquitination in the purified sys- tem and degradation in the extract system.

Degradation was slower than larger fusion constructs and wild-type protein.

Fusion of the KEN box alone did not confer deg- radation, suggesting that there may be additional struc- tural elements required for recognition.

Alternatively, there could be separate signals mediating recognition and degradation, as suggested by work with cyclin pro- tein A fusions (Klotzbü cher et al. 1996).

There also may be a minimum size for proper degradation.


In the initial mutagenesis studies of KEN box, we found unexpectedly that alanine substitution of the two serines and one of the glutamic acids in a region carboxy- terminal to the KEN sequence noticeably increased polyubiquitination.

A similar increase in the average chain length of the polyubiquitin chain was seen upon deletion of five amino acids including the previously mentioned serines and glutamate.

This increased activ- ity in the mutants suggested that negative charge in that region could inhibit ubiquitination and that phosphory- lation might be used physiologically to regulate the pro- cess.

It may be significant that there is a conserved Cdc2 site just carboxy-terminal to the KEN box, making it a likely target for cell cycle regulated phosphorylation.

Al- though phosphorylation has been postulated to control the ubiquitination machinery in APC-dependent pro- cesses, it has not yet been implicated in controlling sub- strate selectivity.

For the SCF complex the situation is reversed, phosphorylation regulates substrate selectivity positively, although there is no known regulation of the ubiquitination machinery.

These mutagenesis results suggest that it would be useful to examine APC sub- strates for phosphorylation regulation, which might fur- ther specify the timing of protein degradation at the end of mitosis.


The explanation for the partially overlapping specifici- ties of Cdc20-APC and Cdh1-APC for substrates will ultimately require studies at an atomic level resolution.

The amino-terminal peptide of cyclin B containing the D box shows no regular structure in solution by NMR (H. Yu and M. Kirschner, unpubl.), so there is little hope that the structure of the peptide independent of APC would be informative.

One way of thinking of the structural basis for the overlap in specificity mediated by Cdc20 and Cdh1 is a bump and hole model (assuming binding of both sequences to the same site).

In this model, the KEN Pfleger and Kirschner 662 GENES & DEVELOPMENT Cold Spring Harbor Laboratory Press on September 23, 2025 - Published by Downloaded from box peptide contains a bump that is accommodated by a hole in Cdh1 when bound to APC.

It would not be ac- commodated by Cdc20; hence, KEN peptides would not be able to bind to Cdc20-APC.

The D-box sequence would not have this feature and would bind to both Cdc20-APC and Cdh1-APC.


Knowledge of the respective roles of the D box and KEN box has provided us with a new understanding of how APC manages to achieve temporal, spatial, and sub- strate specificity throughout the cell cycle.

In addition, it has given us new predictive tools to identify Cdh1 sub- strates.

Given the low stringency of the KEN sequence, each candidate substrate needs to be validated by other means.

However, using this information, we were able to identify two new substrates, Nek2 and B99, which show a pattern of abundance in vivo expected for a Cdh1 sub- strate.

Both have functional KEN boxes.

In addition, the Aurora kinase family all have the conserved KEN resi- dues (for review, see Bischoff and Plowman 1999), mak- ing them likely candidates to test for KEN-box-depen- dent degradation.


Recently, we carried out an unbiased screen by in vitro expression cloning for Cdh1-APC sub- strates and identified two novel proteins, which on se- quencing, have KEN sequences which, by mutagenesis, are essential for degradation (S. Rankin, N. Ayad, and C. Pfleger, unpubl.).

Because Cdh1-APC activity is found to be high in nondividing cell populations, such as brain (Gieffers et al. 1999), we anticipate finding substrates that are involved in cellular processes beyond cell cycle events.


Extracts were prepared as described previously (Murray 1991; Fang et al. 1998).

In brief, eggs from HCG-injected PMSG- primed frogs were collected and washed in 1XMMR.

Eggs were dejellied with 2% cysteine and activated by calcium ionophore, and then washed into XB and XB containing protease inhibitors.

A 1-min packing spin at 1000 rpm was performed, followed by a crushing spin at 10,000 rpm.

The cytoplasmic layer was col- lected and subjected to a clarifying spin also at 10,000 rpm.

The clarified cytoplasmic layer was collected.

After addition of an energy-regenerating system, protease inhibitors, cytochalasin B, and glycerol to 4%, extracts were frozen in liquid nitrogen, and stored at −80°C.


Mutants were generated by PCR with primers designed for the desired mutations.

Construction of deletion mutants was achieved by designing primers with a restriction site, a start ATG (for amino-terminal deletions) or stop codon (for carboxy- terminal deletions) and sequence for at least five amino acids at the position of the desired deletion.

Point mutations within the 96 amino acid deletion mutant were achieved using an amino- terminal primer encoding the altered codon for the desired amino acid change.

Insertion of an alanine cassette in place of the KEN box in the full-length Cdc20 was achieved by designing both up and down primers introducing a NotI restriction site and sequence for amino acids on either side of the deletion.

Two separate fragments were PCRed, digested with NotI, and ligated together and into a pCS2 + based vector, in position for in vitro translation with the Sp6 primer.

For more information on primer sequences used for construction of mutants of Cdc20 and other proteins used in this study, please contact C. Pfleger or M. Kirschner.


Insertion of a StuI site at the beginning of the IgG-binding region of protein A allowed ligation to small regions of Cdc20 PCRed with a carboxy-terminal primer lacking a stop site and contain- ing a StuI site in the same frame.

Coding sequence for three additional lysines was added just amino-terminal to the StuI site to ensure the close proximity of lysines to the KEN box in the fusion protein.

Inserts were cloned into a pCS2 + vector for in vitro translation with the Sp6 primer.


His-tagged hCdc20 and hCdh1 were expressed in insect cells by the baculovirus system (virus obtained from G. Fang, Stanford University, Palo Alto, CA).

Sf9 cells were infected with the appropriate virus, and harvested 2 days later.

Protein was puri- fied over a nickel column and eluted with high imidazole.

The resulting eluate was run over a sizing column with PBS and containing 1 mM DTT.

The appropriate fractions were collected and reconcentrated.

Glycerol was added to 10%, then protein was frozen in liquid nitrogen and stored at −80°C.


Amino-terminal Cdc20 peptides corresponding to the first 120 amino acids of either human wild-type sequence or KENN-A mutant were cloned into a pET 28 bacterial expression vector tagging them with an amino-terminal His tag.

Protein was ex- pressed in bacteria and purified on nickel beads.

Previously de- scribed Xenopus cyclin B amino-terminal peptides (amino acids 1-102) containing or lacking the ⌬36-44 D box (Fang et al. 1998) were expressed in bacteria, purified on nickel columns, and eluted with high imidazole.

Peptides were dialyzed into XB- sucrose containing 1 mM DTT, reconcentrated, and glycerol was added to 10%.

Peptides were frozen in liquid nitrogen and stored at −80°C.


Ubiquitination assays were described previously (Fang et al. 1998).

In brief, interphase APC (iAPC) was immunopurified from interphase extract with anti-Cdc27 antibodies covalently coupled to protein A beads and washed 5 times with XB (minus sucrose) containing 500 mM NaCl and 0.5% NP-40 detergent and two times with XB (minus sucrose).

iAPC beads were incu- bated for 1 hr with baculovirus-expressed and purified Cdc20 or Cdh1, unless otherwise stated.

Activator-bound APC on beads was then washed three times with XB (minus sucrose) followed by addition of 50 µg/ml Ubcx, 1.25 mg/ml ubiquitin, 200 µg/ml recombinant E1, 0.1 mg/ml cycloheximide, 1-5 µM ubiquitin aldehyde, and one-fifth the volume of 35 S-labeled in vitro-trans- lated substrate (typically, 2 µl of beads would be added to a 5-µl reaction).

Ubiquitination assays were scored either visually, by observing the decrease in original signal with the appearance of polyubiquitinated (greater than three ubiquitins added per mol- ecule) conjugates, or quantitated with NIH Image 1.61.


In vitro degradation assays were performed in the presence of increasing amounts of bacterially expressed his-tagged peptides of amino-terminal Cdc20 wild-type or KEN-box mutant or amino-terminal cyclin B wild type or ⌬D box.


Nek2 was cloned by PCR using the marathon cDNA library and the advantage polymerase from Clontech.

B99 was obtained from L. Collavin in Claudio Schneider's laboratory (Trieste) and cloned into a pCS2 + vector.

Mutants of Nek2 were made by PCR by introducing an alanine cassette containing a NotI site in the manner described for the Cdc20 KENN-A mutant.

The B99 mu- tant was also made by PCR, using a carboxy-terminal primer encoding the alanine substitution.


The precise order of molecular events during cell cycle progression depends upon ubiquitin-mediated proteolysis of cell cycle regulators.

We demonstrated previously that Hsl1p, a protein kinase that inhibits the Swe1p protein kinase in a bud morphogenesis checkpoint, is targeted for ubiquitin-mediated turnover by the anaphase-promoting complex (APC) [1].

Here, we investigate regions of Hsl1p that are critical both for binding to the APC machinery and for APC-mediated degradation.

We demonstrate that Hsl1p contains both a destruction box (D box) and a KEN box motif that are necessary for Hsl1p turnover with either APCCdc20 or APCCdh1.

In coimmunoprecipitation studies, the D box of full-length Hsl1p was critical for association with Cdc20p, whereas the KEN box was important for association with Cdh1p.

Fusion of a 206-amino-acid fragment of Hsl1p containing these motifs to a heterologous protein resulted in APC-dependent degradation of the fusion protein that required intact D box and KEN box motifs.

Finally, this bacterially expressed Hsl1p fusion protein interacted with Cdc20p and Cdh1p either translated in vitro or expressed in and purified from insect cells.

Binding to Cdc20p and Cdh1p was disrupted completely by a D box/KEN box double mutant.

These results indicate that D box and KEN box motifs are important for direct binding to the APC machinery, leading to ubiquitination and subsequent protein degradation.


Ubiquitin-mediated protein degradation is necessary for the linear progression of molecular events during the cell cycle.

Protein ubiquitination and subsequent degradation by the 26S proteasome is required for the initiation of DNA replication, the onset of anaphase, and for mitotic exit [2][3][4].

The ubiquitin pathway is a multi-step process in which the 76-amino-acid ubiquitin molecule is activated by an E1 enzyme, transferred to an E2 enzyme, and then attached covalently to the protein substrate either directly or in conjunction with an E3 enzyme (ubiquitin ligase) [5][6].

Proteins destined to be degraded are subject to multiple rounds of ubiquitin attachment (polyubiquitination) and are then proteolyzed by the 26S proteasome.

The E3 or ubiquitin ligase is involved in protein substrate recognition and thereby confers specificity to the ubiquitination reaction [5][6].


The SCF (Skp1p–cullin–F-box) ubiquitin ligases are a family of multi-subunit E3s that are responsible for ubiquitination of cell cycle regulators at the G1–S phase transition [2][7][8].

SCF complexes have common Skp1 and cullin subunits, but distinct F-box protein subunits that contain either a WD40 domain or a leucine-rich repeat domain that binds directly to phosphorylated SCF substrates [8][9][10].

In Saccharomyces cerevisiae, the G1-cyclins, Cln1p and Cln2p, bind SCFGrr1 [11][12], whereas the Cdc28p kinase inhibitor, Sic1p, has been shown to bind SCFCdc4 [13][14][15], where Grr1p and Cdc4p are the F-box components of these SCFs.


The anaphase-promoting complex (APC), or cyclosome, another multi-subunit E3, is responsible for the ubiquitination of cell regulators at the metaphase–anaphase and mitosis–G1 transitions [4].

The APC is comprised of a core complex of proteins and two WD40-containing proteins, Cdh1 (called Cdh1p or Hct1p in S. cerevisiae) and Cdc20, that bind to, and have been proposed to activate, the APC [16][17][18][19][20][21][22].

Cdc20 binds the APC (APCCdc20) during mitosis.

APCCdc20 is required for the degradation of the anaphase inhibitors known as the securins (Pds1p in S. cerevisiae and Cut2p in Schizosaccharomyces pombe), thereby triggering sister chromatid separation [23][24][25][26][27][28][29].

Cdh1 binds to the APC (APCCdh1) in late mitosis and G1 and is responsible for ubiquitination of the mitotic cyclins (Clb1–4p in S. cerevisiae) during these cell cycle phases [16][17][19][22][30].

APCCdh1 is also responsible for the degradation of Cdc20, thereby restricting APCCdc20 activity to a narrow window from G2 when Cdc20 is synthesized to late mitosis when Cdc20 is degraded by APCCdh1 [18][31][32].

In contrast, Cdh1 levels remain relatively constant during the cell cycle, but phosphorylation of Cdh1 by the mitotic kinase Cdc2 (Cdc28p in S. cerevisiae) blocks Cdh1 binding to the APC, thereby preventing APCCdh1 activity during S, G2, and M phases when Cdc2/Cdc28p activity is high [22][30][33][34][35].


Two degradation motifs have been identified in APC substrates.

The destruction box (D box), with the consensus sequence R-x-x-L-x-x-x-x-N/D/E, is important for the degradation of most APC substrates [36][4].

Recently, human Cdc20 (hCDC20), which lacks a D box, was found to contain a new degradation signal called a ‘KEN box’ with the consensus sequence K-E-N-x-x-x-D/N that was critical for its degradation by APCCdh1 [37].

Subsequently, a few other APC substrates containing both KEN box and D box motifs have been identified.

These APC substrates are human CDC6, a protein required for DNA replication, human securin, and the mitotic cyclin A in Drosophila melanogaster [38][39][29].

Functional KEN boxes in yeast proteins have not yet been reported.


Because direct interactions between Cdc20 or Cdh1 with APC substrates have not been demonstrated, generally these proteins are believed to activate the APC, possibly by inducing allosteric changes in core APC subunits leading to specific substrate binding and ubiquitination.

However, indirect evidence for substrate binding has led others to suggest, by analogy to the F-box proteins of the SCFs, that Cdc20 and Cdh1 may bind and target protein substrates to the APC [16][17][32][1][40][41].

We found previously that Hsl1p, a 170-kD budding yeast protein kinase that negatively regulates the Swe1p protein kinase in a bud morphogenesis checkpoint pathway [42][43][44][45], is degraded via the APC [1].

Like most APC-substrates, Hsl1p contains a D box that is important for its degradation.

Hsl1p associates with both Cdc20p and Cdh1p based on both two-hybrid assays and coimmunoprecipitation from yeast extracts, indicating that it might interact directly with these proteins [1].

Given these findings, we believed that Hsl1p might serve as a useful model substrate for understanding how proteins destined for degradation are recognized by the APC.


In this study we set out to understand which domains of Hsl1p are important for both recognition and turnover by the APC machinery.

We noticed that Hsl1p contains a potential KEN box and were interested in addressing the following questions using Hsl1p as a model APC substrate: 1) Do KEN boxes serve as degradation signals in yeast? 2) Can Hsl1p bind directly to Cdc20p and/or Cdh1p? 3) What is the role of the D box and/or KEN box in APC recognition?

We discuss a model for substrate recognition by the APC machinery.


The specificity of ubiquitin-mediated protein degradation with regards to the selection of substrates to be polyubiquitinated has only been determined rather recently.

Substrate targeting by the N-end rule and HECT (homology to E6AP carboxyl terminus) domain ubiquitin ligases occurs through substrate-specific binding domains.

In contrast, the SCF complex recruits substrates through a substrate adaptor protein, the F-box subunit.

Despite evidence showing that Cdc20 and Cdh1 bind and activate the anaphase-promoting complex (APC) in a substrate-specific manner, there is no evidence that the activating protein and substrate interact directly; hence, no clear model exists for the mechanism of APC activation or recruitment of substrates.

We show here that the activators Cdc20 and Cdh1 can associate with substrates via their N termini.

In the absence of APC, Cdc20 and Cdh1 bind substrates reflecting Cdc20–APC and Cdh1–APC specificity.

The N termini of Cdc20 and Cdh1 provide specificity functionally, as demonstrated by the generation of active chimeras that display the specificity corresponding to their N termini.

Thus, Cdc20 and Cdh1 act as both substrate recognition and activating modules for APC.


Ubiquitin-mediated protein degradation regulates important cellular processes from cell cycle progression to Wnt signaling [1][2][3].

Attachment of a polyubiquitin chain to a protein targets that protein for degradation by the 26S proteasome [4][5].

Ubiquitin becomes covalently attached to a substrate by interaction with a cascade of enzymes.

The last in the series, a ubiquitin protein ligase (E3), transfers the ubiquitin to a lysine on the substrate protein [6].

In the N-end rule pathway, extending from bacteria to humans, a destabilizing residue at the N terminus directs proteins for ubiquitination via the E3 N-recognin/Ubr1p (Saccharomyces cerevisiae) or E3α (in mammals).

Direct binding of substrates to N-recognin/E3α is observed [7][8][9].

Similarly, HECT domain E3s are highly conserved from yeast to vertebrates.

They share homology in their C termini to E6AP and contain a conserved C-terminal cysteine residue that forms a thioester with ubiquitin; they bind substrates via their N termini [10].

E6AP targets the src kinases [11], HHR23A and HHR23B [12].

The HECT domain RSP5 in yeast targets Gap1 and Fur4 by interaction with its N-terminal WW domains [13][14][15].

The fission yeast pub1 targets Cdc25 [16].

Xsmurf1 binds and targets Smad1 for degradation [17].

Two ubiquitin protein ligases play a role in the cell cycle: the SCF (Skp1/Cullin/F-box) complex and the anaphase-promoting complex (APC) [1][2][3].

Both complexes contain a protein with cullin homology (Cul1/Cdc53 in the SCF complex, APC2 in the APC) [18][19][20] and a RING-H2 finger protein, (Rbx1 in the SCF complex and APC11 in the APC) [19][21][22][23].

Recruitment of substrates to the SCF complex occurs via the F-box subunit.

F-box proteins interact with the Skp1 subunit of the SCF complex via the F-box motif [24].

F-box proteins fall into three categories, those with WD repeats (Fbw), leucine-rich repeats (Fbl), or other domains (Fbx) [3].

Binding to substrates requires substrate phosphorylation and is thought to occur in the non-F-box domains [3].

Substrate binding has been shown to occur for Cdc4 via its WD domain [25], for Grr1 via its leucine rich repeats [26][27], and for Skp2 via its other domain [28].

In some cases, however, the F-box protein may not be sufficient for substrate interaction; homo- or heterodimerization of the F-box or another factor may be required [3].

The APC contains several additional subunits without a defined role; substrate interaction could occur through one of these proteins.

APC activation requires its binding to the WD repeat containing proteins Cdc20 or Cdh1.

The Cdc20- and Cdh1-bound forms of the APC demonstrate different substrate specificities [29][30].

Cdc20–APC recognizes destruction box (D box) containing proteins [31][32][29], and Cdh1–APC recognizes proteins containing either a D box or a KEN box [30].

No direct binding of activating protein to substrate has been demonstrated.

In one case, the APC substrate Hsl1p, a Swe1p inhibitor, was identified through a two-hybrid interaction screen with Cdc20, but without addressing direct interaction [33].

In another case, the putative substrate cyclin D was shown to bind to other APC subunits by αCdc27 immuno-precipitation analysis [34].

More recently, the interaction between cyclin A–Cdk2 with the WD repeats of Cdc20 [35] or Cdh1 [36] has been reported for the recognition of Cdc20 and Cdh1 as substrates of cyclin A–Cdk2.

Recruitment of substrate to the APC might occur through activating protein, through binding to one of the other subunits of APC, or through a combination of both.

In this report we present evidence that Cdc20 and Cdh1, the substrate-specific adaptors of the APC, interact with substrates in an APC-independent manner.

We show that this interaction occurs through their N termini and does not require the WD repeats.

We show that this binding does not require APC and can occur directly between purified proteins.

Specificity is retained by the N terminus, as demonstrated by active chimeras between Cdc20 and Cdh1 when their N termini and WD repeats are swapped.

N-terminal fragments of the activators are potent APC inhibitors both in vitro and in vivo, and we introduce a binding assay that could be used to identify additional APC targets.


Brown dwarfs, gravitationally condensed objects whose masses are too low for equilibrium hydrogen burning, occupy the mass range between the lowest mass stars (∼ 0.07 M ⊙ ; Burrows et al. 1997) and the giant extrasolar planets (∼ 0.01 M ⊙ ; Marcy & Butler 1998).

The lowest mass stars and brown dwarfs only slightly cooler than M dwarfs are classified as spectral type "L" (Kirkpatrick et al. 1999, Martín et al. 1997) and have T ef f in the range ∼1500-2000 K. Deter- mining if L-type objects are brown dwarfs is difficult, because the luminosities and effective temperatures of brown dwarfs are a function of both age and mass (e.g. Burrows et al. 1997).


The first unambiguous brown dwarf, Gl 229B, was discovered as a compan- ion to a nearby M dwarf by Nakajima et al. ( 1995).

This object is cooler than any L dwarf and has CH 4 in its atmosphere implying T eff <1300 K (e.g. Fegley & Lodders 1996) and a sub-stellar nature.

The re- cent discoveries of methane dwarfs (tentatively given a "T" spectral clasification) in the field by Strauss et al. ( 1999), Burgasser et al. ( 1999, 2000), Cuby et al. ( 1999) and Tsvetanov et al. ( 2000) demonstrate that objects like Gl 229B can form singly.


A striking characteristic of known T dwarfs is the similarity of their spectra, which resemble those of the gaseous solar system planets and are very differ- ent from even the coolest L dwarfs.

The L dwarf spec- tra are characterized by the disappearance of the TiO and VO bands (which are strong in M dwarf spectra), the presence of atomic alkali lines and CO bands, and increasing depth of the H 2 O bands as T ef f decreases.

The T dwarfs are characterized by very deep H 2 O and CH 4 bands in the 1-3 µm region.

Alkali metal fea- tures are present between the bands, but the 2.3µm CO bands are absent, due to reduced CO abundance and the overwhelming strength of the CH 4 absorp- tion.


A large remaining gap in the stellar to gas giant planet spectral sequence lies between the previously observed types L and T. In this paper, we report pho- tometry and 0.8-2.5 µm spectroscopy of three very red, faint objects identified in the imaging data of the Sloan Digital Sky Survey (SDSS) and observed at the United Kingdom Infrared Telescope (UKIRT) and the Hobby-Eberly Telescope (HET).

These ob- jects have spectra intermediate between those of the previously known L and T dwarfs, showing the on- set of CH 4 absorption at 1.6 and 2.2 µm, while still retaining observable CO absorption at 2.3 µm.

We propose that they represent the warm end of the "T" spectral sequence, yet to be defined in detail.


The three objects described here were selected from SDSS photometric data.


SDSS photometry is ob- tained with a CCD camera at Apache Point Obser- vatory (APO) which images the sky almost simulta- neously in five filters: u ′ , g ′ , r ′ , i ′ , and z ′ (the data presented here use a preliminary calibration; while we denote the bands by u ′ etc, the magnitudes are de- noted by u * etc.).


The details of the data acquisition and the photometric and astrometric calibration are described by Gunn et al. ( 1998), York et al. ( 2000) and Lupton et al. ( 2000).


SDSS photometry is in the AB ν system (Fukugita et al. 1996) and the magni- tude scale is modified to deal with low signal-to-noise ratios (Lupton et al. 1999).


The L and T dwarfs identified in the SDSS (e.g. Strauss et al. 1999, Fan et al. 2000) are undetected in the u ′ and g ′ bands.

The L dwarfs have i * − z * > 1.6 while the T dwarfs have i * − z * > 3.

We prepared a candidate L and T dwarf list for follow-up obser- vations by searching the SDSS data for point sources with i * − z * > 1.6.


Because of their extremely red colors, some very red, faint objects are detected only in the z ′ band and a search for them can be contam- inated by defects.

Therefore, additional constraints were imposed: (1) all objects blended with neighbors, or affected by data defects, were removed; (2) an ob- ject was required to be detected at ≥3σ twice: in both i ′ and z ′ , in two observations of that region of sky, or detected in the 2MASS database; (3) a z ′ -band- only single detection was required to have z ′ <19.0.


The three objects discussed in this paper were selected from a total sky area of 225 deg 2 , giving an approxi- mate and very preliminary surface density similar to the 1 per 75 deg 2 for SDSS T dwarfs found by Tsve- tanov et al. ( 2000).

Table 1 gives, for the three new objects, the J2000 position (accurate to 0.

′′ 2) and AB magnitudes at i ′ and z ′ .

Table 1 also lists photometry obtained for the L dwarf SDSS 0539 (Fan et al. 2000).

Here- after we identify the objects by the first four digits of their Right Ascension.


SDSS 0837 was found in two SDSS runs, 1999 March 21 and 2000 February 8; the z * values agree to 0.1 mag and the positions to 0.

′′ 3.

SDSS 1021 is a z ′ -only detection in data from 2000 February 12 and is also in the 2MASS database.

The brightest object, SDSS 1254, has z * =18.0 and was found in data taken on 2000 February 2.

Finding charts for the new objects are shown in Figure 1.


J, H and K photometry was obtained using UKIRT's near-infrared camera, UFTI, for SDSS 0837 and SDSS 1254 on 2000 March 2 and for SDSS 0539 and SDSS 1021 on March 14.

Both nights were photometric with seeing ∼0.

′′ 8.

The results are given in Table 1 in the UKIRT (not AB) system - we give JHK on the UFTI system as well as colors on the established IRCAM system for comparison to earlier work.


Fig- ure 2 shows a composite color-color plot containing the data from Table 1 and, for comparison, a sample of late M and L dwarfs as well as the SDSS T dwarfs SDSS 1624 and SDSS 1346 (Strauss et al. 1999; Tsve- tanov et al. 2000), whose spectra closely resemble that of Gl 229B.

Lower limits are shown for i * − z * for the z ′ -only detections.

The late M and L dwarfs show a steady progression towards redder J−K and i * − z * colors with later spectral type, however the T dwarfs have redder i * −z * colors and bluer J−K colors due to the strong absorption by CH 4 in the H and K bands.

The J−K colors of the new objects lie between those of the previously known L and T dwarfs.


A red spectrum of SDSS 1254 was obtained on 2000 February 29 with the Low Resolution Spectrograph (LRS; Hill et al. 1998a,b, Schneider et al. 2000) at the prime focus of the of the HET (Ramsey et al. 1998).

The spectrum covers the wavelength range 5100-9800Å at a resolution of ∼ 20Å, but no flux is detected below ∼6500Å.


Spectra of the objects in Table 1 were obtained in the J, H and K bands at UKIRT on 2000 February 28 - March 1 and March 13-15 using the facility spectrograph CGS4 (Mountain et al. 1990) at R∼400.

Integration times were 30-60 minutes per band.

Tel- luric absorption was removed and the broad-band spectral shapes corrected by comparison with spectra of bright F stars (with photospheric lines removed) taken immediately before and/or after each observa- tion.

The spectra in each band were scaled to match the UKIRT magnitudes using the UFTI filter profiles.

UKIRT z-band spectra were also obtained for SDSS 1254 and SDSS 0539.

For SDSS 1254 the HET spec- trum is used for λ < 0.84µm, and for SDSS 0539 the red APO spectrum (Fan et al. 2000) is used for λ < 0.89µm.


The final flux-calibrated spectra are shown in Fig- ures 3 and 4.

Figure 3 shows the spectrum of SDSS 1254 split into four panels, with the main absorption features marked.

Figure 4 compares the spectra of the new objects with those of the L5 dwarf SDSS 0539 (Fan et al. 2000) and the T dwarf SDSS 1624 (Strauss et al. 1999).


The spectra show: increasingly red col- ors at λ < 1 µm, probably due to pressure broadened Na I and K I absorption (Burrows et al. 2000) com- bined with decreasing T ef f ; increasing absorption in the H 2 O bands at 1.15 µm, 1.4 µm and 1.9 µm; in- creasing absorption at 1.6-1.7 µm and longward of 2.2 µm by CH 4 combination and overtone bands; de- creasing absorption in the CO 2.3µm band.

In par- ticular, the absorption maxima of the CH 4 2ν 2 +ν 3 , 2ν 2 , and ν 2 +ν 3 bands at 1.63, 1.67, and 2.20 µm be- come progressively deeper.

The CO 2-0 bandhead at 2.294 µm is seen in the new objects, but the 3-1 band- head at 2.323 µm is absent; it is considerably weaker at these temperatures and also is overwhelmed by the strong CH 4 ν 3 + ν 4 absorption at 2.315 µm.


The spectra in Figure 4 reveal a clear spectral sequence.

Thus SDSS 0837, SDSS 1254 and SDSS 1021 are examples of the sought after "L/T transi- tion" objects.

However, as suggested by Figure 4, they might more properly be regarded as examples of early T dwarfs, where the "T" spectral type is de- fined by the presence of CH 4 absorption in the H and K bands.

The strongest short wavelength infrared band of CH 4 is the ν 2 band, centered at 3.3 µm, and objects in which this band is present but the shorter wavelength bands reported here are absent must ex- ist over a narrow temperature range.


However, mea- surements at 3.3 µm are considerably more difficult from the ground than are H and K band measure- ments, and objects which show only the 3.3 µm CH 4 band perhaps should be classified as L rather than T (Kirkpatrick et al. ( 1999) suggest that the T class be defined by the presence of CH 4 absorption in the K band).

As the three objects presented here are at the CO/CH 4 transition temperature they are likely to have T ef f ≈ 1300 K (Fegley & Lodders 1996) and therefore masses of 20-70 M Jupiter for ages in the range 0.3-5 Gyr (Burrows et al. 1997).

Figure 2 shows that SDSS easily identifies T dwarfs by their extremely red i * − z * color, including the early T dwarfs identified in the present paper.


On the other hand, the J−K colors are similar to those of the common M dwarfs and these early T objects are thus very difficult to select on the basis of the near- infrared colors alone (although the later T dwarfs are blue and can be selected this way).

Many more L and T dwarfs will be found in the SDSS imaging data.


The SDSS is a joint project of the University of Chicago, Fermilab, the Institute for Advanced Study, the Japan Par- ticipation Group, Johns Hopkins University, Max-Planck- Institute for Astronomy, Princeton University, United States Naval Observatory, and the University of Washington.

The site, Apache Point Observatory, is operated by the Astrophys- ical Research Consortium.

Funding has been provided by the Alfred P. Sloan Foundation, the member institutions, NASA, NSF, the U.S. DoE, and Monbusho Japan.

The SDSS Web site is at http://www.sdss.org/.


UKIRT is operated by the Joint Astronomy Centre on behalf of the UK Particle Physics and Astronomy Research Council.

We thank Tim Carroll for his expert and cheerful help with the UKIRT observations, and M.-C. Liang for assistance during the 28 Feb - March 1 run.


The Hobby-Eberly Telescope is a joint project of the Univer- sity of Texas at Austin, Pennsylvania State University, Stan- ford University, Ludwig-Maximillians-Universität München, and Georg-August-Universität Göttingen.

The HET princi- pal benefactors are William P. Hobby and Robert E. Eberly.

We thank Phillip MacQueen, Grant Hill, Matthew Sheltrone, and Marsha Wolf for help with the HET data.


XF and MAS ac- knowledge support from the Research Corporation, NSF grant AST96-16901, the Princeton University Research Board, and a Porter O. Jacobus Fellowship.

GRK is grateful for support from Princeton University and from NASA via grant NAG- 6734.

DPS thanks the NSF for support via grant AST99-00703.


We present near-infrared spectra for a sample of T dwarfs, including 11 new discoveries made using the 2 Micron All Sky Survey.

These objects are distinguished from warmer (L-type) brown dwarfs by the presence of methane absorption bands in the 1–2.5 km spectral region.

A first attempt at a near-infrared classification scheme for T dwarfs is made, based on the strengths of bands and the shapes of the 1.25, 1.6, and 2.1 km flux peaks.

Subtypes T1 V–T8 V are defined, and spectral indices useful for classification are presented.


The subclasses appear to follow a decreasing scale, based on bands and the properties of L and T dwarfs with known distances.

However, we speculate that this scale is not linear with spectral type for cool dwarfs, due to the settling of dust layers below the photosphere and subsequent rapid evolution of spectral morphology around T K. Similarities in near-infrared colors and continuity of spectral features suggest that the gap between the latest L dwarfs and earliest T dwarfs has been nearly bridged.

This argument is strengthened by the possible role of as a minor absorber, shaping the K-band spectra of the latest L dwarfs.


Finally, we discuss one peculiar T dwarf, 2MASS 0937–2931, which has very blue near-infrared colors due to suppression of the 2.1 km peak.

The feature is likely caused by enhanced collision-induced absorption in a high-pressure or low-metallicity photosphere.


We present 0.6–2.5 µm, R >∼ 400 spectra of twenty-seven cool, low luminosity stars and substellar objects.

Based on these and previously published spectra we develop a preliminary spectral classification system for L and T dwarfs.

For late L and T types the classification system is based entirely on four spectral indices in the 1-2.5 µm interval.

Two of these indices are derived from water absorption bands at 1.15 µm and 1.4 µm, the latter of which shows a smooth increase in depth through the L and T sequences and can be used to classify both spectral types.

The other two indices make use of methane absorption features in the H and K bands, with the K band index also applicable to mid to late L dwarfs.

Continuum indices shortward of 1 µm used by previous authors to classify L dwarfs are found to be useful only through mid L subclasses.

We employ the 1.5 µm water index and the 2.2 µm methane index to complete the L classification through L9.5 and to link the new system with a modified version of the 2MASS “Color-d” index.

By correlating the depths of the methane and water absorption features, we establish a T spectral sequence from types T0 to T8, based on all four indices, which is a smooth continuation of the L sequence.

We reclassify two 2MASS L8 dwarfs as L9 and L9.5 and identify one SDSS object as L9.

In the proposed system methane absorption appears in the K band approximately at L8, two subclasses earlier than its appearance in the H band.

The L and T spectral classes are distinguished by the absence and presence, respectively, of H band methane absorption.


In the last six years, brown dwarfs, objects of mass too low to sustain equilibrium hydrogen burning, have gone from being a theoretical concept with no observed examples to being observationally ubiquitous [1].

Young brown dwarfs have been discovered in large numbers in star-forming regions [2][3][4][5].

Objects with masses ranging from just substellar to those of the Solar System’s giant planets have been found directly or indirectly as companions to nearby stars [6][7][8][9][10][11][12].

In addition, large numbers of isolated brown dwarfs have been found in the solar neighborhood [13][14][15].

These observations have led to controversy regarding the distinction between brown dwarfs and planets, with some suggesting that the lower mass limit for brown dwarfs be defined by the ability to burn deuterium (mass greater than ∼13 MJ, [16]), and others preferring the definition that a brown dwarf is any substellar object that forms via the same process as a star (i.e. not from a protoplanetary disk).

Brown dwarfs are of interest in their own right. They have compact structures, low temperatures, and atmospheric constituents that are markedly different from low mass stars.

The lowest mass brown dwarfs more closely resemble the gas giant planets than stars, and as such provide insight into the physical and chemical properties of extrasolar giant planets that are at present inaccessible for direct study.

After an initial period of deuterium-burning and, for the more massive brown dwarfs, hydrogen-burning, there is no internal energy source and a brown dwarf evolves without reaching thermal equilibrium.

This leads to age- and mass-dependent spectral types, in contrast to main sequence stars, and indeed eventually to effective temperatures lower than that of any stellar photosphere.

It is generally agreed that the very coolest stars and the warmer brown dwarfs (1300 K <∼ Tef f <∼ 2200 K) require a new spectral class, commonly known as ‘L’ [17][18] and currently known cooler brown dwarfs (Tef f <∼ 1300 K), require the addition of an additional class, ‘T’ [18].

The L dwarf sequence is characterized by the disappearance of the red TiO and VO bands from the optical (0.6–1.0 µm) spectrum, the increasing dominance at those wavelengths by broad absorption resonance lines of Na I and K I, and strong H2O absorption bands and persistent CO overtone bands in the 1-2.5 µm region [19][18][20][21].

In T dwarfs the CO bands are replaced by stronger and more extensive absorptions of CH4 in the H and K bands, and there is further strengthening of the water bands [8][22][23][24][25][26].


The range of L spectral types has been characterized using optical spectroscopy of objects from the Deep Near-Infrared Survey, DENIS [27] and the Two Micron All Sky Survey, 2MASS [28], while first attempts to describe the range of the ‘T’ spectral sequence are given by [29][30][31], whose sample is drawn from the imaging data of the Sloan Digital Sky Survey, SDSS [32].

The SDSS imaging data allow the discovery of T dwarfs covering a fairly wide range of spectral characteristics.

In pure infrared surveys such as 2MASS, the degeneracy of the colors of T dwarfs with those of stars (see [31]) permits only the latest T dwarfs to be selected readily (without the use of optical photometry).

This paper presents optical (0.6–1.0 µm) and infrared (1.0–2.5 µm) R >∼ 400 spectroscopy of new L and T dwarfs selected from the SDSS together with new observations of some previously known M, L, and T dwarfs.

Following the discovery of transition objects with properties between those of the then known L and T classes [31], our goal has been to obtain sufficient spectra of L and T types over the entire 0.6-2.5 µm range and at sufficiently high resolution to allow the continuous characterization of the L and T dwarf spectral sequences.

Until now the L sequence has been described only on the basis of optical spectra, and hence it has not been not clear if its full extent has been observed.

In addition there are significant differences in the assignments of L subclasses for the latest objects [18][19].

Using new and previously published spectra, we have devised a set of infrared flux indices from which we have defined a complete L spectral sequence, a T spectral sequence extending to the coolest known brown dwarfs, and a distinct boundary between the L and T sequences.

One of the new infrared indices provides a firm link between our L sequence and those proposed by [19] and [18] on the basis of optical spectral indices.

The L and T sequences we have created are purely empirical and the ranges of spectral indices describing them are defined so that they progress smoothly through the known L and T dwarfs.

We have made no attempt to model the spectra or to derive physical quantities such as temperature or mass.

Some observational gaps remain in the T sequence, and it is quite likely that the sequence will eventually extend to one or more later subclasses as lower temperature objects are found.


The next two sections describe the selection of objects and the observations.

In Section 4 we discuss various indices for spectral typing and present the classification system.

Section 5 discusses the results, with emphasis on the definition of the L//T boundary and the link between infrared and optical classification of L dwarfs.

The conclusions are given in Section 6.

In an adjoining paper, [30] present an independently derived spectral sequence for T dwarfs, which bears many similarities to the one proposed herein.

In a companion paper by [21] we provide ZJHKL′M′ photometry of many of objects presented herein and we discuss the behavior of broad-band colors as a function of spectral class.


Nine morbidly obese (BMI=46.2± 7.7 kg/m 2 [mean±SD] and fat-free mass [FFM]=84.6± 16.6 kg) type 2 diabetic patients and six sex- and age- matched healthy volunteers (BMI=24.4±1.1 kg/m 2 ) were studied.

The patients were all characterised as having type 2 diabetes according to the criteria established by the American Diabetes Association (ADA) [24].

HbA 1c was 6.0-9.5%.


All individuals were randomly assigned to the 24 h metabolic study or to the OGTT either at baseline and 7-9 days after bariatric surgery.

The two tests were performed at an interval of 24-48 h.


At the time of the baseline study, all individuals were on a diet with the following average composition: 60% carbo- hydrate, 30% fat and 10% protein (≥1 g/kg body weight).

This dietary regimen was maintained for 1 week before the study.

All patients received the same parenteral nutrition regimen (about 7,100 kJ/day) during the first 6 days after surgery, then they were on free diet.

For the basal study, each individual spent 24 h (starting at 08:00 hours) on the metabolic ward.

Four meals were administered in the calorimetric chamber for a total energy intake of 104.5 kJ (25 kcal)/kg FFM: 16.4% at breakfast at 09:00 hours, 36% at lunch between 12:00 and 13:00 hours, 13.4% at an afternoon snack at 16:00 hours and 34.2% at dinner at 20:00 hours.

Diet composition was 16.7% protein, 9.8% fat and 73.5% carbohydrate at breakfast; 18.8% protein, 51.2% fat and 30% carbohydrate at lunch; 13.4% protein, 27.7% fat and 58.9% carbohydrate at snack time; and 18.9% protein, 49.8% fat and 31.3% carbohydrate at dinner.

The average diet composition was 16.9% protein, 34.6% fat and 48.5% carbohydrate.

Hourly blood samples were drawn from a central venous catheter for the measurement of glucose, C-peptide and NEFA concentrations.


OGTT A standard 75 g OGTT was performed in each patient at baseline and after surgery as well as in each volunteer, with blood sampling at 0, 30, 60, 90, 120, 150 and 180 min.

Insulin sensitivity was computed by the OGTT minimal model [25].


Body composition was evalu- ated, on a separate day, by the determination of total body water (TBW) using 0.19 Bq 3 H 2 O in 5 ml of saline administered as an intravenous bolus injection [26].

Blood samples were drawn before and 3 h after the injection.

Radioactivity was determined in duplicate on 0.5 ml of plasma in a beta scintillation counter (Model 1600TR; Canberra-Packard, Meriden, CT, USA).

Corrections were Diabetologia (2008) 51:1276-1284 1277 made for non-aqueous hydrogen exchange [27].

Water density at body temperature was assumed to be 0.99371 kg/l.

TBW (kg) was computed as 3 H 2 O dilution space (litres)× 0.95×0.99371.

FFM was obtained by dividing TBW by 0.732 [28].

Fat mass was obtained as the difference between body weight and FFM.


All procedures and measurements described for the basal study were repeated in the obese individuals at the follow-up.


This malabsorptive surgical procedure [29] consists of an approximately 60% distal gastric resection with stapled closure of the duodenal stump.

The residual volume of the stomach is about 300 ml.

The small bowel is transected at 2.5 m from the ileo-caecal valve, and its distal end is anastomosed to the remaining stomach.

The proximal end of the ileum, comprising the remaining small bowel (involved in carrying bilio-pancreatic juice but excluded from food transit), is anastomosed in an end-to- side fashion to the bowel, 50 cm proximal to the ileo- caecal valve.

Consequently, the total length of absorbing bowel is reduced to 250 cm, the final 50 cm of which, the so-called common channel, represents the site where ingested food and bilio-pancreatic juices mix.


The study was approved by the Institutional Ethics Committee of the Catholic University of Rome.

The nature and purpose of the study were carefully explained to all indi- viduals before they provided their written consent to participate.


Plasma glucose was measured by the glucose oxidase technique on a Beckman Glucose Analyzer (Beckman, Fullerton, CA, USA).

C-peptide was assayed by RIA (MYRIA; Technogenetics, Milan, Italy).

Serum NEFA was measured spectrophotometrically.


The 24 h ISR was estimated by using a previously proposed mathematical model of glucose-NEFA comodulation [19].

Model equations are reported and briefly explained in the appendix.

The model allows the computation of the ISR from the time-course of glucose and NEFA plasma concentrations.

To analyse the C-peptide data, the ISR model was complemented by the equations for the whole body C-peptide kinetics.

We have used the validated two- compartment model [30, 31], calculating the standard parameters of C-peptide kinetics for each individual as proposed by van Cauter et al. [ 31].


The ISR model parameters were estimated for each individual by weighted least-squares fitting of plasma C-peptide concentration data [19].

The least-squares index was minimised by means of a constrained Levenberg- Marquardt routine of the MATLAB library and the SDs of the estimates of individual parameters were evaluated by the Jackknife method [19].

From the parameter esti- mates, the 24 h individual ISR profile was derived.

For comparison, the ISR profile was also reconstructed from the plasma C-peptide concentration by the deconvolution method, according to van Cauter et al. [ 31].

The β-oxidation rate was computed for each individual by means of Eq. 3 in the appendix.


All data are expressed as means±SE, unless otherwise specified.

The Wilcoxon paired-sample test and the ANOVA test for repeated measurements, followed by a Tukey test were used for intragroup and intergroup comparisons, respectively.

Two-sided p<0.05 was considered significant.


The duration (ranging from 6 months to 10 years before the operation) and the degree of compensation of diabetes (HbA 1c ) were used as covariates in a multiple regression analysis where the dependent variable was the change in beta cell glucose sensitivity after BPD.


A non-significant weight loss (6.7±3.3 kg [mean±SD]) was observed 1 week after BPD.

The FFM after BPD was 80.9± 15.7 kg and was not significantly different from FFM before BPD.

The 24 h average concentrations of glucose before and after BPD (13.6±5.6 vs 7.8±2.6 mmol/l) were significantly different (p<0.01), and the glucose concentration after BPD was not significantly different from that found in controls (5.2±1.0 mmol/l).

The 24 h average concentration of C-peptide after BPD (1.2±0.7 nmol/l) was significantly (p<0.05) lower than that before BPD (1.7±0.9 nmol/l) and did not differ significantly from the average concentration in controls (0.7±0.3 nmol/l).

The 24 h average concen- trations of NEFA before and after BPD (0.64±0.08 vs 0.65±0.30 mmol/l) were not significantly different and were larger than in controls (0.31±0.08 mmol/l, p<0.05).

The time-course of NEFA plasma level over 24 h before and after BPD is reported in Fig. 1.


The estimates of model parameters are reported in Table 1.

For all individuals, the SD of the individual parameter estimates was consistently <20%.

The parameter p 1 , which represents the 24 h average beta cell sensitivity to both glucose and NEFA, was doubled 1 week following BPD (p<0.01).

Nevertheless, this parameter did not reach the values observed in healthy controls.


The parameter p 2 , which is related to the rate of long-chain acyl-CoA (LC- CoA) use in the cytosol, recovered completely after BPD, being substantially equal to the values found in controls.

Also p 3 , which expresses the efficiency of LC-CoA transport into the mitochondria, where LC-CoAs are 1278 Diabetologia (2008) 51:1276-1284 β-oxidised, reverted to normality.

Thus the ratio p 3 /p 2 , which represents the balance between LC-CoA β-oxidation and its use in the cytosol [19], was fully normalised early after the operation.


The integral over 24 h of the insulin secretion rate (ISR; cumulative insulin secretion, AUC ISR ) decreased from 277.1±144.4 to 198.0±107.6 nmol/m 2 (p< 0.02) and did not differ from that found in controls (135.0± 30.0 nmol/m 2 ).

The amplitude δ and the initial phase ϕ of the ISR circadian rhythm did not change significantly after BPD.

Whereas the amplitude was significantly higher in controls (p<0.001), the phase was not statistically different from before/after BPD.


The rate of β-oxidation in the beta cell was found to be completely normalised after the operation, from 0.032±0.012×10 −12 to 0.103±0.031× 10 −12 mmol/min per cell, having assumed a beta cell volume of 1.376×10 −12 l [32].

For the controls we found 0.098±0.052×10 −12 mmol/min per cell.


Figure 2a-c shows the model fitting of the 24 h C-peptide plasma concentration before and after the opera- tion in a representative diabetic patient.

The time-course of the ISR, estimated both by the model and by the deconvo- lution method, is also reported.

The cumulative 24 h insulin secretion attained the same value when computed by the mathematical model or by the deconvolution.

The peaks corresponding to the meals were reduced after the operation, shifting towards the values observed in controls (Fig. 2d,e).


The relationship between the static component of the ISR (denoted as S, see Eq. 1 in Appendix) and NEFA concentration is shown in Fig. 3a for controls and type 2 diabetic patients before and after BPD.

The curves are plotted at the glucose level (G) indicated, using the parameters p 1 , p 2 and p 3 of Table 1.

The points mark the 24 h average NEFA/ISR value with the average glucose concentration found in each group of individuals.

The variability of the curve with glucose level is shown only for controls.


Figure 3b shows the rate of β-oxidation per cell (B) as a function of plasma glucose concentration, computed by the model (Eq. 3 in appendix) in diabetic patients before and after BPD and in controls.

At least to our knowledge, no data are available in the literature on fatty acid oxidation in islets from obese diabetic humans or in healthy individuals.

Therefore we plotted in Fig. 3b, as a reference, the values of palmitate oxidation in Zucker lean rat islets, as deduced by the data reported in Nolan et al. [ 23] assuming that the average beta cellular protein content is 200 fg.


The time-courses of glucose and insulin concentration during the OGTT of diabetic patients and controls are shown in Fig. 4.

Both the fasting glucose and insulin plasma levels and their values at 120 min were dramatically reduced after BPD.

The ADA criteria to define diabetes mellitus (fasting plasma glucose ≥7 mmol/l or an OGTT with the 2 h postload value ≥11.1 mmol/l) were no longer fulfilled after the operation, as shown in Fig. 4, so the


The major results of the present study may be stated as follows:


In diabetic patients who underwent malabsorptive bariatric surgery, the beta cell glucose/NEFA sensitivity was doubled early after the operation, before a significant weight loss was achieved.


The shorter the time interval between diagnosis of type 2 diabetes and bariatric operation and the lower the severity of diabetes, the better was the recovery of beta cell function.


Fatty acid oxidation in the beta cell, as calculated by the mathematical model, was fully normalised after BPD, suggesting that the impairment of pancreatic fatty acid β-oxidation is a reversible phenomenon.


4. Insulin secretion was reduced mainly as a consequence of the normalisation of insulin sensitivity.

Circulating NEFA was indeed slightly, although not significantly, increased after BPD, probably as a consequence of the increased fat mobilisation by hormone-sensitive lipase, which is activated when the circulating insulin level drops.

The lack of a significant increase in NEFA concentration may be attributable to a higher insulin sensitivity of adipose tissue, which attenuates the potential rise in NEFA.


Points 1-4 are discussed in detail in the following.


One of the first pieces of evidence of diabetes resolution after bariatric surgery was reported by Pories et al. [ 33].

Although the surgical technique used in that study was the gastric bypass, whereas BPD was used in the present study, in our series also diabetes was reversed early after the operation when body weight was quite unchanged.


Insulin sensitivity exerts a modulating effect on the beta cell function, so that beta cells are initially able to compensate for insulin resistance, thus maintaining glucose homeostasis.

In fact, insulin sensitivity and beta cell function are inversely related, whereby the product of these two parameters is a constant referred to as the disposition index [34].

But insulin hypersecretion is no longer able to fully compensate for increasing insulin resistance when beta cell function is impaired, so that type 2 diabetes mellitus develops [35-40].

Individuals with impaired glucose tolerance and overt type 2 diabetes are character- ised by moderate to severe insulin resistance [40, 41].

For instance, Haffner et al. [ 42] reported that insulin resistance was present in >85% of a cohort of diabetic individuals.

It is interesting to note that Ferrannini et al. [ 41] have shown that a halving of beta cell glucose sensitivity or a halving of insulin sensitivity predicts a similar rise in the 2 h glycaemia after glucose challenge as well as in the fasting plasma glucose levels, suggesting that either the beta cell glucose sensitivity or the insulin resistance can equally contribute to hyperglycaemia in type 2 diabetic patients.


In our series, a reduction of insulin secretion was found after BPD in all individuals whereas the insulin sensitivity was significantly increased, suggesting that the improvement of insulin sensitivity may induce a corresponding improvement in beta cell glucose/NEFA sensitivity.


The effect of bariatric surgery in terms of recovery of beta cell function (Δp 1 ) was maximal in those individuals whose diabetes was diagnosed shortly before the operation, while the effect decreased linearly with the increase of diabetes duration.

The UKPDS data [43] clearly indicate that there is a progressive deterioration of pancreatic beta cell function as type 2 diabetes progresses.

In fact, type 2 diabetic patients at diagnosis had about 50% reduction in pancreatic beta cell function and were insulin resistant, whereas over time there was a reduction (about 4%/year) in the beta cell function regardless of treatment, without change in insulin resistance.


In agreement with our results Schauer et al. [ 44] found, in a cohort of 1,160 patients who underwent laparoscopic Roux-en-Y gastric bypass, that the factors that negatively correlated with type 2 diabetes mellitus resolution included the preoperative duration of diabetes, high levels of HbA 1c and insulin therapy, which are all measures of diabetes severity.

In contrast, the weight loss was positively associated with diabetes resolution.

Our data are not affected by the weight loss variable, since our patients were studied very early after the operation when no significant weight loss was attained.


On the basis of the present observations, it may be hypothesised that in patients with long-standing type 2 diabetes the defects in beta cell mass predominate, whereas the patients with a short duration of diabetes primarily exhibit an impairment in beta cell function.


Beta cell fatty acid oxidation A β-oxidation impairment in the beta cells has been described by Frigerio et al. [ 45].

The anti-epileptic drug topiramate exerts a protective effect against INS-1E beta cell lipotoxicity induced by prolonged exposure to high levels of fatty acids, by enhancing the cellular capacity of lipid clearance.

In fact, this drug enhances the β-oxidation rate, suggesting that a protective mechanism against lipotoxicity could be the inhibition of lipid accumulation in the cytoplasm through increased fatty acid oxidation.

Furthermore, fa/fa rat islets, a useful replica of the human phenotype of adipogenic type 2 diabetes, exhibit a markedly increased lipogenic capacity and a decreased oxidative capacity [46]; unused NEFA in islets are esterified and over time an excessive quantity of triacygly- cerol is deposited [46].


Our observation that fatty acid oxidation rate in beta cells is normalised a few days after BPD suggests that the impairment of fatty acid oxidation is reversible and that it is not related to the fatty acid overloading of beta cells.

In fact, circulating NEFA levels were not statisti- cally changed from before BPD, when their oxidation rate was calculated to be three times lower than in healthy controls.


At least to our knowledge this is the first evidence of a defect in beta cell fatty acid oxidation in type 2 diabetes in humans.

This defect is completely reversed shortly after BPD even before complete normalisation of beta cell glucose sensitivity.

It is likely that this prompt reversibility might depend on the removal of some competitive inhibitory action exerted on carnitine palmitoyltransferase 1 (CPT-1).


It has been reported that in type 2 diabetic patients the insulin pulses related to meals are decreased in amplitude and disorganised in their temporal pattern [47, 48] and that, in most diabetic patients, these abnormal patterns did not improve with weight loss [49].


In our post-BPD patients, although insulin secretion was reduced, it was still sufficient to provide a normalisation of the insulin-dependent glucose disposal.

This effect occurred in spite of the non-significant reduction in circulating NEFA levels, suggesting that other mechanisms should intervene.

In particular we hypothesise that insulin sensi- tivity normalisation may be the first to occur and that, secondarily, the need for insulin secretion diminishes.