From the Research Institute of Molecular Pathology,
Dr.-Bohr Gasse 7, A-1030 Vienna, Austria and § Protein
Interaction Laboratory, University of Southern Denmark, Odense
University, Campusvej 55, DK-5230 Odense M, Denmark
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ABSTRACT |
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The anaphase-promoting complex/cyclosome (APC) is
a ubiquitin-protein ligase whose activity is essential for progression
through mitosis. The vertebrate APC is thought to be composed of 8 subunits, whereas in budding yeast several additional APC-associated
proteins have been identified, including a 33-kDa protein called Doc1
or Apc10. Here, we show that Doc1/Apc10 is a subunit of the yeast APC
throughout the cell cycle. Mutation of Doc1/Apc10 inactivates the APC
without destabilizing the complex. An ortholog of Doc1/Apc10, which we
call APC10, is associated with the APC in different vertebrates, including humans and frogs. Biochemical fractionation experiments and
mass spectrometric analysis of a component of the purified human APC
show that APC10 is a genuine APC subunit whose cellular levels or
association with the APC are not cell cycle-regulated. We have further
identified an APC10 homology region, which we propose to call the DOC
domain, in several protein sequences that also contain either cullin or
HECT domains. Cullins are present in several ubiquitination complexes
including the APC, whereas HECT domains represent the catalytic core of
a different type of ubiquitin-protein ligase. DOC domains may therefore
be important for reactions catalyzed by several types of
ubiquitin-protein ligases.
The initiation of anaphase and exit from mitosis depend on a
multi-subunit ubiquitination complex called the anaphase-promoting complex (APC)1 or cyclosome
(1-3) (for a recent review see Ref. 4). The APC ubiquitinates proteins
such as Pds1 in budding yeast and Cut2 in fission yeast whose
subsequent degradation by the 26 S proteasome is essential for the
initiation of sister chromatid separation at the metaphase-anaphase
transition (5, 6). Later in anaphase and telophase the APC promotes the
inactivation of the mitotic cyclin-dependent protein kinase
1 by ubiquitinating its activating subunit cyclin B (1-3, 7). The APC
also mediates the ubiquitin-dependent proteolysis of
several other mitotic regulators, including Polo-like kinases (8, 9),
spindle-associated proteins (10), the APC activating protein Cdc20 (9,
11), and inhibitors of DNA replication (12).
Like all known ubiquitination reactions, the assembly of polyubiquitin
chains on APC substrates depends on ubiquitin-activating and
ubiquitin-conjugating enzymes, also called E1 and E2 enzymes, respectively (13). E1 first activates ubiquitin by forming a ubiquitin
thioester and then transfers ubiquitin to an E2 enzyme, again forming a
thioester. Subsequently, ubiquitin is transferred from the E2 to the
substrate where polyubiquitin chains are assembled through isopeptide
bonds on the In the APC pathway, the APC fulfills the role of an E3, because it is
required for the transfer of ubiquitin from E2s to substrates. However,
it is not known whether the APC also functions as a covalent ubiquitin
carrier. A direct physical association of ubiquitin with APC has not so
far been detected (2), raising the possibility that the complex
facilitates ubiquitination reactions indirectly by bringing substrates
and E2s into close proximity and perhaps by altering their
conformation. The cloning of APC subunits has not revealed any
similarity with Ubr1 or HECT domain proteins (22-27). The APC2
subunit, however, is a distant member of another protein family
involved in ubiquitination, called cullins (26-28). The best studied
cullin is the budding yeast protein Cdc53, which is part of a
multi-subunit ubiquitin-protein ligase called Skp1-cullin-F-box protein
complex (SCF) (for review see Refs. 4 and 29).
SCF-dependent proteolysis is required for the initiation of
DNA replication at the G1-S transition but also has
multiple other physiologic roles. The E2 enzyme Cdc34 has been reported
to interact with a C-terminal domain in Cdc53 (30), which is conserved
in all cullins (28), including APC2. The conservation of this domain raises the possibility that also other cullins are part of
ubiquitin-protein ligases and interact with E2 enzymes.
SCF and APC differ from Ubr1 and HECT domain ubiquitin-protein ligases
with respect to their subunit complexity: SCF and APC are both
multi-subunit complexes, whereas Ubr1 and HECT proteins appear to be
functional as single polypeptide chain enzymes. The vertebrate APC was
believed to be composed of 8 constitutive subunits, 7 of which are
related to subunits of the budding yeast APC (22, 26). APC
immunoprecipitates from budding yeast contain five additional proteins,
which have so far not been identified in APC preparations from animal
cells (23, 27). Recently, a 33-kDa protein called Doc1 has been
identified in a genetic screen for mutants defective in the degradation
of mitotic cyclins (24). DOC1 deletion strains are barely
viable and temperature-sensitive mutations causing a defect in the
ubiquitination of mitotic cyclins in vitro could be assigned
to the DOC1 gene (31). The Doc1 protein was found to
cofractionate with other APC subunits suggesting that it is an APC
subunit (24). Indeed, DOC1 was found to encode the 33-kDa
protein Apc10 detected in the purified APC (27). We therefore refer to
the yeast protein as Doc1/Apc10. Recently, Kominami et al.
(32) reported the characterization of a fission yeast ortholog of
Doc1/Apc10. The fission yeast apc10+ gene is
essential and is required for the turnover of the mitotic cyclin Cdc13,
a known APC substrate. Based on the observation that an association
between fission yeast Apc10 and known APC subunits could only be
detected by coimmunoprecipitation but not in density gradient
centrifugation experiments, it has been suggested that this protein may
be a regulatory protein that only transiently associates with the
APC.
Here, we have further analyzed budding yeast Doc1/Apc10 and have for
the first time characterized its human ortholog APC10. Our results
demonstrate that these proteins are constitutively associated with the
APC in yeast and vertebrates, respectively, and suggest that Doc1/Apc10
has an important role in APC-dependent ubiquitination
reactions. We have further identified a conserved protein domain
homologous to the amino acid sequence of APC10 in several high
molecular weight protein sequences. This homology region, which we call
the DOC domain (from Doc1), is found in several putative cullins and
HECT domain proteins. This finding implies a conserved role for the
APC10 sequence in diverse types of ubiquitin-protein ligases that were
so far not known to have structural features in common.
Yeast Methods--
Strains were derivatives of W303.
CDC16-myc6 strains have been described (23). For
epitope-tagging of DOC1, a cassette containing an HA3 or
myc9 epitope tag and the Kluyveromyces lactis TRP1 gene was
amplified by polymerase chain reactions with DOC1-specific primers and integrated in front of the stop codon at the genomic locus.
The doc1 mutants used in this study have been isolated in
screens for mutants with a defect in the degradation of Clb2 (1, 7).
The doc1-22 mutation is identical to the previously characterized mutation cse1-22 (1, 7) and was renamed after finding that the mutation is in the DOC1 and not in the
CSE1 gene (31). For metabolic labeling, strains were grown
in synthetic complete medium lacking methionine. In a typical
experiment, 5 × 107 cells were resuspended in 0.5 ml
of medium containing 1 mCi of [35S]methionine and
[35S]cysteine and incubated at 25 °C. Fresh medium was
added after 60 min (0.5 ml) and 120 min (2 ml), and labeled cells were
harvested after 180 min. We were not able to arrest cells with
nocodazole in synthetic medium. For Fig. 1B, strains
(MATa bar1) were grown to exponential phase or arrested for
90 min with cDNAs, Recombinant Protein Expression, and Generation of
Antibodies--
Sequence analysis revealed that the human expressed
sequence tag (EST) clone AA234328 (GenBankTM nomenclature
of the National Center for Biotechnology Information) is composed of
843 base pairs and contains an open reading frame encoding APC10 (see
"Results"). The complete APC10 nucleotide and amino acid sequences
have been deposited at the National Center for Biotechnology
Information (GenBankTM accession number AF 132794). [35S]Methionine- and [35S]cysteine-labeled
APC10 protein was prepared from clone AA234328 by coupled
transcription-translation reactions in rabbit reticulocyte lysate
(Promega). For the generation of antibodies, cDNA fragments corresponding to amino acid residues 1-140 of APC10, to amino acid
residues 332-619 of human CDC16/APC6 (33), and to amino acid residues
257-591 of human CDC23/APC8 (26) were cloned into a pET28 expression
vector (Novagene). Recombinant protein containing N-terminal
hexa-histidine tags were expressed in Escherichia coli BL21(DE) cells (Novagene), purified on nickel-nitrilotriacetic acid
agarose (Qiagen) and used for the immunization of rabbits.
Peptide Sequencing by Mass Spectrometry--
The 24-kDa protein
present in immunoprecipitates of human APC was analyzed by
nanoelectrospray tandem mass spectrometry (34). Briefly, the
silver-stained protein band was excised from the gel, reduced and
S-alkylated and subjected to in-gel digestion with trypsin
(35, 36). After overnight digestion the supernatant was loaded on 50 nl
of reversed phase resin (POROS R2) in a capillary, washed with
acidified water and eluted with organic phase directly into a
nanoelectrospray needle (Protana, Odense, Denmark). Peptide sequencing
by tandem mass spectrometry was performed on a prototype quadrupole-time-of-flight instrument (PE-Sciex, Toronto, Canada). After
fragmentation, peptide sequence tags (37) were assigned in the
resulting spectra. The program PeptideSearch written by Mortensen and
Mann was used to retrieve protein sequences from both a nonredundant
protein data base and an EST data base. The retrieved data base hits
were compared with the complete fragmentation spectrum to verify the
complete peptide sequence.
Other Techniques--
Extracts from logarithmically growing HeLa
cells and from Xenopus eggs were prepared and fractionated
as described (38, 39). Resource Q column fractions obtained from HeLa
100,000 × g supernatant (S100) fractions were the kind
gift of I. Waizenegger, and extracts prepared from HeLa cells
synchronized by cell cycle inhibitors were the kind gift of E. R. Kramer (both of Research Institute of Molecular Pathology).
Immunoprecipitation, immunoblotting, and ubiquitination experiments
were done as described (38, 39).
Data Base Searches and Sequence Alignments--
Protein data
bases were searched by using the Gapped BLAST and the Psi-BLAST program
(40). Sequence alignments were performed by using Clustal_X (41) using
the Blosum 62 substitution matrix and were manually modified in the
GeneDOC program (42). The degree of identity between different amino
acid sequences was determined by the gap program of the Genetic
Computer Group.
Characterization of Budding Yeast Doc1/Apc10--
The
DOC1 gene was first identified in a genetic screen for
budding yeast mutants defective in the proteolysis of mitotic cyclins (24). DOC1 encodes a protein of 33 kDa that was shown to be associated with known APC subunits (24). To determine whether Doc1
represents one of the 12 proteins detected after purification of the
yeast APC (27), we constructed strains in which either Cdc16 or Doc1 or
both proteins carry epitope tags at their C termini. Strains were
metabolically labeled with [35S]methionine and
[35S]cysteine, and the APC was isolated by
immunoprecipitation of the Cdc16-myc6 protein with antibodies to the
Myc epitope (Fig. 1A). The APC
from CDC16-myc6 cells contains a 33-kDa protein called Apc10
(27). The mobility of this protein was significantly reduced when the
APC was isolated from CDC16-myc6 DOC1-HA3 cells,
demonstrating that the 33-kDa protein is encoded by the DOC1
gene. We therefore refer to this subunit as Doc1/Apc10. The same set of
proteins was coprecipitated with Doc1-myc9 and with Cdc16-myc6,
confirming that the association of Doc1/Apc10 with other APC subunits
is specific. Doc1/Apc10 was labeled less intensely than other subunits of higher molecular mass. This difference could be due to the lower
number of methionine and cysteine residues in Doc1/Apc10 than in larger
APC subunits. Alternatively, Doc1/Apc10 could bind to the APC in
substoichiometric amounts.
Most known APC subunits are part of the complex during all phases of
the cell cycle (22).2 In
contrast, two proteins that regulate APC activity, Cdc20 and Cdh1, are
predominantly associated with the APC in mitosis and G1
phase, respectively (38, 43-47). To analyze whether Doc1/Apc10 is a
constitutive or a cell cycle-regulated subunit, we compared the subunit
composition of the APC isolated from different cell cycle phases (Fig.
1B). APC was immunopurified from metabolically labeled
CDC16-myc6 cells that were either growing logarithmically or
that had been arrested in G1 by
Temperature-sensitive mutations in several budding and fission yeast
APC subunit genes (48, 49) and deletion of the budding yeast
CDC26 gene (27) have been shown to result in partial
disassembly of the complex at the restrictive temperature, suggesting
that these mutations and deletions affect APC function by complex
destabilization. To analyze whether mutation of the DOC1
gene also influences APC stability, we arrested metabolically labeled
CDC16-myc6 cells carrying two different
temperature-sensitive mutant alleles of the DOC1 gene at the
restrictive temperature. No differences in subunit composition were
apparent when we analyzed APC immunoprecipitates isolated from these
cells (Fig. 1C), suggesting that mutation of Doc1/Apc10 does
not inhibit APC function by destabilizing the complex.
Characterization of Human APC10 cDNAs--
To analyze whether
proteins related to budding yeast Doc1/Apc10 are associated with the
APC in vertebrates, we characterized EST clones and cDNAs obtained
by polymerase chain reactions that encode a related human protein.
Clone AA234328 contained an open reading frame that codes for a protein
of 185 amino acid residues with a predicted molecular mass of 21,300. This sequence is 29% identical to budding yeast Doc1/Apc10. Data base
searches identified several other closely related sequences in fission yeast (SPBC1E8.06/Apc10) (32), Caenorhabditis elegans
(Y48G1.Contiq49; St. Louis unfinished sequences from chromosome I) and
Drosophila (AA141768), which were 38, 38, and 56% identical
to the human sequence, respectively (Fig.
2). The high degrees of sequence similarity and our biochemical data (see below) suggest that these proteins are orthologs of budding yeast Doc1/Apc10. We therefore refer
to the human protein as APC10. Data base searches also identifed a
second closely related hypothetical C. elegans protein
(F15H10.3; GenBankTM accession number Z73972), which is
25% identical with human APC10.
The sequence of APC10 is similar in length to the related sequences in
fission yeast, Drosophila and C. elegans, but it
is considerably shorter than the sequence of budding yeast Doc1/Apc10, which contains 93 additional amino acid residues at its N terminus (24). To determine whether the APC10 cDNA contains a complete open
reading frame, we transcribed and translated the cDNA in reticulocyte lysates and estimated its molecular mass by SDS-PAGE and
phosphorimaging (Fig. 3A). The
resulting in vitro translation product comigrated with a
24-kDa polypeptide band in HeLa cell extracts that was specifically
recognized by antibodies raised against a recombinant fragment of APC10
(Fig. 3A), indicating that the APC10 cDNA contains a
complete open reading frame.
Identification of APC10 as a Subunit of the APC in
Vertebrates--
To address whether the protein detected by APC10
antibodies is associated with the APC, we immunoprecipitated HeLa cell
extracts with antibodies specific for various APC subunits and analyzed the precipitates by immunoblotting with APC10 antibodies (Fig. 3,
A and B). The APC10 band coprecipitated with
antibodies to the constitutive subunits CDC16, CDC23, CDC27, and APC7
but not with the respective preimmune antibodies. APC10 could also be coimmunoprecipitated with antibodies to CDC20 (Fig. 3C), a
protein whose physical association with the APC is required for mitotic APC activation (38, 44). APC10 antibodies were able to
immunoprecipitate in vitro translated APC10 but did not
precipitate APC (data not shown), suggesting that the epitope
recognized by these antibodies is inaccessible in the native complex. A
protein similar in electrophoretic mobility to APC10 was recognized by
APC10 antibodies in APC immunoprecipitates isolated from mouse and
bovine cells, and two polypeptide bands were recognized in APC
preparations obtained from Xenopus egg extracts (Fig.
3B). We conclude that APC10 is specifically associated with
the APC in different vertebrates.
To analyze whether the association of APC10 with the APC is cell
cycle-regulated, we synchronized logarithmically growing HeLa cells by
drug treatment and determined the presence of APC10 in cell extracts
and in APC immunoprecipitates by immunoblotting. Similar amounts of
APC10 were present in cell extracts and in APC immunoprecipitates in
all cell cycle phases (Fig. 3D and data not shown),
indicating that APC10 is a constitutive APC subunit.
When we immunoprecipitated APC from human HeLa and mouse myeloma cells
and analyzed its subunit composition by SDS-PAGE and silver staining,
we observed a 24-kDa polypeptide band that could not be detected in
immunoprecipitates obtained with preimmune sera (indicated by an
arrow in Fig. 4A).
To analyze whether this protein represents APC10, we isolated tryptic
peptides from this band and sequenced them by nanoelectrospray tandem
mass spectrometry (50) using a novel hybrid quadrupole-time-of-flight
mass spectrometer instrument (51). The unseparated peptide mixture
resulted in the mass spectrum shown in Fig. 4B. The main
detected peaks correspond to trypsin autolysis products (T) due to low
amounts of protein on the gel. Several smaller peaks in the spectrum at
a mass to charge ratio of 480.92, 574.97, 661.84, 671.34, 845.67, and
921.44.were fragmented and found not to belong to any entry in the
protein data base. As an example, Fig. 4C shows that the
peptide with m/z 661.84 was sequenced completely,
revealing the sequence IYTPVEESSIGK. In total, 6 peptides were
sequenced (Table I). Data base searches with these peptide sequences identified a total of 11 ESTs, all of
which encode parts of APC10. The 24-kDa band present in APC preparations does therefore represent APC10.
To determine what fraction of APC10 is associated with the APC, we
separated extracts from logarithmically growing HeLa cells by Q column
anion exchange chromatography and by linear sucrose density
centrifugation (Fig. 5). The column and
gradient fractions were analyzed by immunoblotting for the presence of
APC10 and the APC subunits CDC16 and CDC27 (Fig. 5, B and
C). In addition, individual Q column fractions were analyzed
for their ability to ubiquitinate a radiolabeled N-terminal fragment of
cyclin B in a reconstituted reaction containing purified E1 and E2
enzymes (Fig. 5A). In both experiments, APC10 cofractionated
with the peak of CDC16 and CDC27 proteins, whereas there was no APC10
detectable in other fractions. The presence of APC10 correlated closely
with the ability of the Q column fractions to ubiquitinate cyclin B. However, the elution and sedimentation behavior of APC10 was not identical to that of CDC16 and CDC27; the latter two proteins could be
detected in multiple fractions in both separation experiments, but
APC10 could only be detected in the APC peak fractions (as defined by
the peak of cyclin B ubiquitination activity and CDC16 and CDC27
concentration), even after prolonged exposure times during the
immunoblotting procedure. These experiments suggest that the large
majority if not all APC10 molecules are tightly associated with the
APC, whereas CDC16 and CDC27 may either be more loosely associated or
may be present in multiple complexes or aggregates.
Identification of an APC10 Homology Domain--
Data base searches
with the APC10 sequence not only identified putative APC10 orthologs in
different species, but also several high molecular weight proteins that
contain regions of significant homology to APC10 (Figs. 2 and
6). Two human cDNAs called KIAA0076 (32, 52) and KIAA0708 (53) code for proteins with predicted molecular
masses of 191,000 and >196,600, respectively (the precise predicted
mass of the KIAA0708 polypeptide is not known because the corresponding
cDNA may not be complete at the 5' end). The two amino acid
sequences are 57% identical to each other. Both contain a region
homologous to APC10, which is 90% identical between KIAA0076 and
KIAA0708 (Fig. 2), and a more C-terminal region homologous to cullins
(28). The cullin domain of KIAA0076 (amino acid residue 1187-1576) is
most closely related to human CUL1, whereas the cullin domain of
KIAA0708 (amino acid residue 782-1065) is similar to both human CUL4B
and CUL1 (as determined by the BLAST 2.0 program). In addition,
KIAA0708 contains a zinc-binding RING finger motif (54) at amino acid
positions 1472-1501.
An APC10 homology domain was also found in a 527-kDa mouse protein
called RJS (runty-jerky-sterile) (55) and its human ortholog HERC2
(56). Both proteins contain a HECT domain in the C-terminal region (at
position 4459-4796 of RJS). A fourth mammalian protein containing a
region of less pronounced but still significant homology to APC10 is a
510-kDa protein associated with the
transcription factor Myc (PAM) (57). The sequence of PAM
does not contain cullin or HECT domains, but numerous other sequence
motifs have been recognized previously (57), including a subtype of the RING finger, called RING-H2 finger (54), which is also present in the
budding yeast APC subunit Apc11 (27) and in Ubr1 (58). In addition, PAM
contains a putative protein binding domain originally identified in the
RCC1 protein (regulator of chromosome
condensation 1) where this domain has recently
been shown to form a seven-bladed propeller structure (59). Three
domains containing RCC1 repeats are also present in RJS/HERC2 (55, 56).
The genome of C. elegans contains an open reading frame
(C01B7.6) that codes for a protein whose sequence is 32% identical to
PAM (57) and accordingly also contains a region related to APC10.
The sequence similarity between APC10 and the related regions in
KIAA0076, KIAA0708, RJS/HERC2, PAM, and the PAM-related C. elegans sequence suggest that parts of these proteins are derived from a common ancestral protein domain (Figs. 2 and 6). Based on the
original name of APC10 in budding yeast, Doc1, we propose to call this
homology region the DOC domain.
From our results, we conclude that budding yeast Doc1/Apc10 and
its vertebrate ortholog APC10 represent an evolutionary conserved subunit of the APC. Kominami et al. (32) have recently
proposed that fission yeast Apc10 may be loosely associated with the
APC and may therefore be a regulator of the complex. However, our data
suggest that this protein is tightly and constitutively associated with
the APC, at least in budding yeast and human cells.
An association of Doc1/Apc10 with the budding yeast APC had already
been reported previously (24), but APC10 had so far not been recognized
as a subunit of the vertebrate APC (22, 26). We suspect that APC10 was
not detected in previous analyses of the vertebrate APC because the
24-kDa APC10 polypeptide band is less intensely stained by silver than
other APC subunits (Fig. 4A). We presently do not know
whether these differences in staining intensity are due to
substoichiometric amounts of APC10 or whether this protein is less
agyrophilic than other APC subunits. In either case, our
characterization of human APC10 and the evolutionary conservation
between most known APC subunits suggest that orthologs of other
proteins, which so far were only identified as subunits of the budding
yeast APC, may remain to be discovered in the vertebrate APC. This view
is supported by the observation that a protein related to the budding
yeast APC subunit Cdc26 is associated with the APC in fission yeast
(49) and in human cells3 and
that human cDNAs encoding proteins closely related to budding yeast
Apc11 exist (27).
Like the function of other APC subunits, the function of APC10 within
the APC is unknown. Our result that mutation of budding yeast
Doc1/Apc10 abolishes APC function without disrupting the complex (Fig.
1) may indicate that Doc1/Apc10 has a rather direct role in the
ubiquitination function of APC. This notion is consistent with our
observation that in biochemical fractionation experiments the abundance
of APC10 correlates with the ability of human APC to ubiquitinate
cyclin B in vitro (Fig. 5).
We noticed that the sequences of several high molecular weight proteins
found in the data bases contain a region of significant homology to
APC10, which we propose to call the DOC domain (from Doc1/Apc10).
During preparation of this manuscript, Kominami et al. (32)
also reported that fission yeast Apc10 is similar to a portion of one
of these proteins, KIAA0076. The presence of either cullin or HECT
domains in three of these proteins, KIAA0076, KIAA0708, and RJS/HERC2,
implies that these proteins may be ubiquitin-protein ligases or
subunits of such enzymes. KIAA0076, KIAA0708, and RJS/HERC2 may
therefore participate in ubiquitination reactions or perhaps in related
reactions in which ubiquitin-like proteins are conjugated to other proteins.
These observations have several interesting implications. First, the
presence of DOC domains in the APC and in high molecular weight cullin
and HECT domain proteins suggests that the function of the DOC domain
may be linked to ubiquitination reactions, although the function of
more DOC domain proteins will need to be examined to test this
hypothesis. The DOC domain appears to be able to perform its function
either as a single subunit in trans (in the case of APC10)
or as a protein domain in cis (in the case of KIAA0076, KIAA0708, and RJS/HERC2). Second, the presence of the DOC domain in the
cullins KIAA0076 and KIAA0708 raises the possibility that within the
APC the subunit APC10 interacts structurally or functionally with the
cullin protein APC2. Third, the presence of a DOC domain in the 510-kDa
protein PAM, which has been identified via association with
the short-lived transcription factor Myc (57), raises the possibility
that also this protein and its C. elegans homolog may have
some role in ubiquitination reactions.
Finally, the occurrence of the DOC domain in conjunction with either
cullin or HECT domains suggests a modular structure for ubiquitin-protein ligases. This notion is further supported by the
observation that also the RING finger motif and protein repeats of the
WD40 and the RCC1 type are found in a number of proteins that are
connected with the ubiquitin system (although the occurrence of these
motifs is by no means restricted to proteins of the ubiquitin system).
Recent crystallographic analyses have shown that both WD40 and RCC1
repeats form seven-bladed propeller-like structures (59-61). Because
the WD40 repeats of the SCF subunit Cdc4 have been shown to be required
for the recognition and subsequent ubiquitination of substrates (62),
it will be interesting to analyze if seven-bladed propeller structures
are used by different ubiquitin-protein ligases for the interaction
with substrates. These observations and our finding that the DOC domain
is found either in association with a cullin domain or in association
with a HECT domain raises the interesting possibility that structural
domains are shared between different types of ubiquitin-protein ligases
such as Ubr1, HECT enzymes, and cullins that were so far not thought to
have any features in common.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino side chains of lysine residues. The latter
reaction usually depends on a third component, called ubiquitin-protein
ligase or E3. The first E3s that were molecularly characterized were a
yeast protein called Ubr1 (14) and a human protein called E6-AP (15).
Ubr1 functions in the N-end rule pathway in which substrates are
recognized based on the identity of their N-terminal amino acid residue
(16), whereas E6-AP was identified as a protein required for the
ubiquitination of the tumor suppressor protein p53 in human cells
infected with oncogenic papilloma viruses (17). E6-AP and possibly also
Ubr1 function as ubiquitin carriers that transfer the ubiquitin residue from the E2 to the substrate by forming a ubiquitin thioester intermediate (16, 18). The C-terminal domain of E6-AP where thioester
formation takes place is also found in a number of other proteins (19,
20). Several of these proteins with homology to the
E6-AP C-terminus (HECT domain
proteins) have been shown to form ubiquitin thioesters and are
therefore likely to function as ubiquitin-protein ligases (19, 21).
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-factor (0.5 µg/ml) or nocodazole (15 µg/ml) in rich
medium at 30 °C. As judged by microscopic examination, approximately
95% of cells were arrested in G1 (unbudded cells) or
mitosis (large-budded cells), respectively. Cells were labeled (3 mCi/ml) for 150 min in 1 ml of YEPD medium.
-Factor or nocodazole
was added to maintain cell cycle arrests. Preparation of extracts and
immunoprecipitations were performed as described (27). Cells were
broken in 0.25 ml of buffer B60 containing bovine serum albumin (5 mg/ml) and extract from an unlabeled
pep4 strain (5 mg/ml) grown under similar conditions. In Fig. 3C, the yeast
extract was omitted from the breakage buffer. Myc-tagged proteins were
immunoprecipitated with the monoclonal antibody 9E10. Proteins were
separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and detected by fluorography.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Characterization of Doc1/Apc10 as a subunit
of the budding yeast APC. Wild type cells (WT) and
cells expressing epitope-tagged versions of the APC subunit Cdc16 and
of the Doc1/Apc10 protein were metabolically labeled with
[35S]methionine and [35S]cysteine. Protein
extracts were prepared and immunoprecipitated with an antibody to the
Myc epitope. Bound proteins were detected by fluorography.
A, identification of the 33-kDa APC subunit as the
DOC1 gene product. Cells were labeled for 180 min at
25 °C in synthetic medium. B, association of Doc1/Apc10
with the APC at different cell cycle stages. Wild type or
CDC16-myc6 cells were grown in rich medium at 30 °C and
arrested in G1 with -factor (
-fact) or in
mitosis with nocodazole (noc). Growing (cyc) and
arrested cells were labeled for 150 min. C, subunit
composition of the APC in extracts from different doc1
mutant strains. Cells were grown in synthetic medium and labeled for 80 min at 24 °C and then for 100 min at the restrictive temperature
(36 °C). Polypeptide bands that bound nonspecifically to the
antibody beads are marked by stars.
-factor or in mitosis by
nocodazole treatment. No significant changes in the abundance of
Doc1/Apc10 or of other APC subunits could be observed, indicating that
the association of Doc1/Apc10 with the APC is not cell
cycle-regulated.
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Fig. 2.
Alignment of the amino acid sequences of
APC10 orthologs from various species and of the DOC domain found in
several high molecular weight proteins. DOC domain proteins were
identified by Psi-BLAST using the human APC10 sequence as a query.
Amino acid residues that are identical or similar (according to the
Blosum 62 substitution matrix) in at least six sequences are
highlighted by black boxes (because the DOC domains of
KIAA0076 and KIAA0708 are 90% identical, a weight of only 0.5 instead
of 1.0 was assigned to these two sequences). A serine residue that is
conserved in all DOC domains and whose mutation to phenylalanine has
previously been shown to inactivate budding yeast Doc1/Apc10 (24) is
marked by a black dot. Amino acid residues are given in the
one-letter code. The number of the first and the last amino acid that
is shown for each sequence is indicated at the beginning and the end of
each sequence, respectively. The sequence of Y48G1.Contiq49 was deduced
from genomic C. elegans sequence using the program fgenen
(63) except for the last putative exon (amino acids 163-216), which
was selected based on homology with other APC10 relatives. Note that
only for Hs APC10, Dm AA141768 and Sp Apc10, the full-length sequences
are shown. Ce, C. elegans; Dm,
Drosophila melanogaster; Hs, Homo
sapiens; Mm, Mus musculus; Sc,
Saccharomyces cerevisiae; Sp,
Schizosaccharomyces cerevisiae.
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Fig. 3.
Characterization of APC10 as a subunit of the
vertebrate APC. A, in vitro translated
35S-labeled human APC10 was synthesized in
transcription-translation mixtures containing T7 RNA polymerase
(35S IVT T7) and was separated by SDS-PAGE and
analyzed either by phosphorimaging (left panel) or by
Western blot (WB) using antibodies raised against APC10
(right panel). As negative controls,
transcription-translation mixtures containing the human APC10 cDNA
and T3 RNA polymerase (35S IVT T3) and rabbit
reticulocyte lysate not containing any cDNA (RRL) were
analyzed side by side. T7 and T3 RNA polymerases synthesize "sense"
and "antisense" mRNA from the human APC10 cDNA,
respectively. Whole cell extracts from logarithmically growing HeLa
cells (HeLa xt) and immunoprecipitates obtained from these
extracts either with CDC16 antibodies (IP a-CDC16) or with
the corresponding preimmune antibodies (IP pre-CDC16) were
also analyzed by immunoblotting. B, whole cell extracts from
logarithmically growing HeLa cells (HeLa), from mouse
myeloma Ag8.653 cells (Mm), and from Xenopus
interphase egg extracts (Xl) were analyzed by
immunoprecipitation with antibodies against CDC23, CDC27, CDC16, and
APC7 or with preimmune antibodies corresponding to the CDC23 antibodies
(pre-CDC23). All immunoprecipitates (IP) were
analyzed by SDS-PAGE and Western blot (WB) with CDC16 and
APC10 antibodies. C, CDC20 and APC7 immunoprecipitates
obtained from whole cell extracts of HeLa cells arrested in mitosis
with nocodazole were analyzed by immunoblotting with CDC20 and APC10
antibodies. D, whole cell extracts of logarithmically
growing HeLa cells (log) or from cells enriched in different
cell cycle phases were analyzed by immunoblotting with CDC16 and APC10
antibodies. Cells were either enriched in G1 by lovastatin
(LOVA), in S-phase by hydroxyurea (HU), or by
mimosin (MIM), in G2 by nitrogen mustard
(NM), or in mitosis by colcemid (COL). Cell cycle
synchronization was monitored by flow cytometry (see Ref. 38, and data
not shown). The APC10 polypeptide band detected by phosphorimaging and
by immunoblotting is marked by an arrow in A and
D.
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Fig. 4.
Sequencing of human APC10 by nanoelectrospray
tandem mass spectrometry. A, immunoprecipitates
obtained with CDC27 antibodies (a-CDC27) from whole cell
extracts of logarithmically growing mouse Ag8.653 (Mm) or
human HeLa cells were analyzed by SDS-PAGE and silver staining. As a
negative control, immunoprecipitates obtained with the corresponding
CDC27 preimmune antibodies (pre-CDC27) from HeLa extracts
were analyzed side by side. The position of the subunits APC1, APC2,
CDC27, APC4, APC5, CDC16, APC7, and APC8 was determined by
immunoblotting (data not shown) and has been indicated by bars on the
right side. The 24-kDa APC10 polypeptide band is marked by an
arrow. Bars on the left indicate the position of marker
proteins whose molecular masses are listed in kDa. B, mass
spectrum of the unseparated peptide mixture of the 24-kDa band present
in human APC immunoprecipitates separated by SDS-PAGE as in
A. The peaks marked with T correspond to trypsin autolysis
products. All peaks marked by their m/z value
were sequenced by nanoelectrospray mass spectrometry. C,
tandem mass spectrum of the peak at m/z 661.84. The high resolution and mass accuracy of the quadrupole-time-of-flight
instrument allowed complete sequence assignment as IYTPVEESSIGK, which
maps to a tryptic peptide of APC10.
Tryptic APC10 peptides sequenced by nanoelectrospray tandem mass
spectrometry
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Fig. 5.
Cofractionation of human APC10 with the
cyclin B ubiquitination activity of the APC and with the APC subunits
CDC27 and CDC16. A, a S100 fraction from
logarithmically growing HeLa cells was separated by Resource Q anion
exchange column chromatography, and the resulting column fractions were
analyzed for their ability to ubiquitinate a radiolabeled fragment of
cyclin B ([125I]CycB) in a reconstituted system.
B, protein fractions obtained as in A were
analyzed by SDS-PAGE and immunoblotting with antibodies against CDC27,
CDC16, or APC10. C, a whole cell extract from
logarithmically growing HeLa cells was separated by centrifugation in a
10-40% sucrose density gradient, and the resulting fractions were
analyzed by immunoblotting as in B.
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Fig. 6.
Schematic representation of protein domains
that are shared between APC and SCF subunits and DOC domain
proteins. For the APC and SCF, only those subunits are shown that
contain cullin or DOC domains. The length of the amino acid
(aa) sequences of the DOC domain proteins is shown on the
right side.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We are grateful to E. Kramer, E. Vorlaufer, and I. Waizenegger for providing protein fractions and antibodies, to L. Huber for critical comments on the manuscript, to R. Kurzbauer and G. Schaffner for DNA sequencing, and to S. Tugendreich and P. Hieter for the human CDC16/APC6 cDNA.
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FOOTNOTES |
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* This research was supported by Grant FFF 800207 from the Austrian Industrial Research Promotion Fund (to J.-M. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF132794.
¶ To whom correspondence should be addressed. Tel.: 43-1-797-30-886; Fax: 43-1-798-7153; E-mail: peters{at}nt.imp.univie.ac.at.
2 W. Zachariae, unpublished observations.
3 C. Gieffers, A. V. Podtelejnikov, M. Mann, and J.-M. Peters, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: APC, anaphase-promoting complex; HECT, homology to E6-AP C terminus; PAM, protein-associated with Myc; RJS, runty-jerky-sterile protein; SCF, Skp1-cullin-F-box protein complex; PAGE, polyacrylamide gel electrophoresis; EST, expressed sequence tag.
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REFERENCES |
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