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INTRODUCTION |
The eukaryotic cell division cycle involves the replication
of chromosomal DNA and its equal distribution to daughter cells in a
highly regulated series of events. Failure to faithfully duplicate and
segregate chromosomes can have dire consequences, such as the onset of
cancer, in multicellular organisms. One of the essential regulatory
components of chromosome segregation in eukaryotes is a large
multisubunit enzyme termed the anaphase-promoting complex
(APC),1 or cyclosome
(recently reviewed in Refs. 1 and 2). The APC is an E3 ubiquitin ligase
responsible for initiating the metaphase to anaphase transition once
chromosomes are attached and aligned at the metaphase plate and
promoting mitotic exit once chromosome segregation is complete. The APC
targets numerous substrate proteins involved in mitosis, meiosis, and
other cellular processes for degradation by the proteasome by
catalyzing their polyubiquitination (1) and is regulated by checkpoint
signaling pathways that monitor DNA and chromosome integrity (3,
4).
Eleven constitutive core subunits of the APC have been identified in
the budding yeast, Saccharomyces cerevisiae (5-7), and in
vertebrates (8-11). Homologs of most of the subunits have been found
in other model systems as well, including Schizosaccharomyces pombe, Caenorhabditis elegans, and
Drosophila melanogaster (reviewed in Ref. 2). Ten
of the 11 known APC subunits of budding yeast have human homologs, with
yeast Apc9 being the only exception. The extensive homology between
APCs of organisms as diverse as humans and yeasts points to an ancient
evolutionary origin and reflects the importance of the APC in
controlling some of the most fundamental cell cycle events in eukaryotes.
The presence of so many subunits makes the APC an unusual E3 enzyme in
terms of its size and complexity. The actual catalytic reaction
involving transfer of ubiquitin from an E2 enzyme to a substrate
protein is intrinsic to a single small RING finger subunit, Apc11 (11,
12). Another subunit, Apc2, containing a highly conserved cullin domain
present in other E3 ubiquitin ligases interacts with Apc11 (13) and is
also believed to be important for catalyzing ubiquitin transfer (14).
The specific functions of the remaining subunits are almost entirely
unknown. Candidate roles for these components include substrate
recruitment and specificity, cellular localization, or interaction with
and response to regulatory proteins such as
cyclin-dependent kinases and spindle assembly checkpoint
proteins (2). Some subunits may function in a purely structural manner
by forming a scaffold that allows proper complex assembly.
In our efforts to purify the APC from budding yeast extracts, we
consistently observed two previously unidentified proteins co-purifying
under high salt conditions with the core complex. Here, we provide
evidence that these two proteins, Mnd2 and Swm1, are, in fact,
constitutive and functional components of the core APC, bringing the
total number of identified subunits in budding yeast to 13. We believe
that Swm1 is identical to Apc13, a small protein observed previously in
APC preparations that was never identified (7). We discuss the
significance of these identifications, the previously described meiotic
defects associated with MND2 and SWM1 mutations
(15, 16), and the mitotic phenotypes we have observed in
mnd2
and swm1
strains for understanding
aspects of APC function in all organisms.
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EXPERIMENTAL PROCEDURES |
Yeast Methods and Strain Construction--
Yeast strains
expressing Cdc27, Swm1, and Mnd2 (Table I) containing carboxyl-terminal
3× FLAG epitopes were constructed by integration of PCR products
amplified from the template p3FLAG-KanMX (gift from Dr. Toshio
Tsukiyama; Fred Hutchinson Cancer Center) at the desired location as
described (17). Integrants were selected on YPD agar containing 500 µg/ml G418, and correct integration of the cassette was confirmed by
PCR and DNA sequencing. Deletion of the BAR1 gene from
W1588-4c was achieved by integration of a URA3 cassette
amplified by PCR from pRS406 at the BAR1 locus. Replacement
of BAR1 with URA3 was confirmed by PCR. Diploid
strain YKA180 was created by transformation of YKA151 with
YCp50::HO expressing the wild-type HO endonuclease, selecting
for transformants on medium lacking uracil and then
counterselecting for loss of YCp50::HO on medium
containing 5-fluoroorotic acid. The diploid strain was confirmed
by its ability to sporulate. Strains from which the APC9,
APC10, SWM1, or MND2 genes had been
deleted (Table I) as well as their parent
strain, BY4741, were from the Saccharomyces Genome Deletion
Project, available through ResGen. The presence of the correct deletion
was confirmed in each of these strains by PCR using primers flanking
the appropriate open reading frame.
Cell cycle arrests were performed in midlog phase cultures of strain
YKA155 as follows. For G1 arrest,
-factor peptide
(University of North Carolina peptide synthesis facility) was added
from a 5 mg/ml stock in ethanol to a final concentration of 50 µg/liter. For S arrest, hydroxyurea (Sigma) powder was added directly
to cultures at a final concentration of 10 mg/ml. For M arrest,
nocodazole (Sigma) was added from a 1.5 mg/ml stock in dimethyl
sulfoxide to a final concentration of 15 µg/ml. Cell cycle arrests
were monitored by phase-contrast microscopy until >90% of the cells had achieved the desired morphology (unbudded for G1 and
large budded for S and M). Samples were removed from the cultures and analyzed by flow cytometry on a FACScan flow cytometer (Becton Dickinson) to confirm arrest at the desired stage. Diploid cells were
induced to enter a synchronous meiosis exactly as described previously
(18). Cells were harvested midway through meiosis based on the meiotic
progression of yeast strain W303 described recently (19). Induction of
sporulation was confirmed by monitoring spore formation by microscopy.
RTS Expression Constructs--
Expression plasmid pIVEX-2.3d and
all Rapid Translation System (RTS) reagents were from Roche
Applied Science. To construct pIVEX-FLAG, oligonucleotides
5'-CATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGGGCGGAGGAGC-3' and 5'-GGCCGCTCCTCCGCCCTTGTCATCGTCATCCTTGTAATCGA
TGTCATGATCTTTATAATCACCGTCATGGTCTTTGTAGTC-3' were annealed in 10 mM Tris-HCl, pH 8.5, by heating to 95 °C and slowly
cooling to 30 °C. The annealed product was ligated into the
NcoI and NotI restriction enzyme sites of
pIVEX-2.3d.
Yeast open reading frames for the 11 previously identified APC subunits
as well as SWM1 and MND2 were amplified by PCR
using Platinum Pfx DNA polymerase (Invitrogen) and yeast
genomic DNA from strain YPH499 (Stratagene) as the template.
Oligonucleotide primers contained restriction enzyme sites to
facilitate ligation into either pIVEX-FLAG or pIVEX-2.3d. The resulting
pIVEX-FLAG and pIVEX-2.3d constructs allow expression of APC subunits
containing an N-terminal 3× FLAG epitope or a C-terminal
His6 sequence, respectively, using RTS in vitro
transcription and translation reactions. The 5' and 3' junctions of all
clones and the identities of the cloned open reading frames were
confirmed by DNA sequencing.
Purification of APC--
Approximately 1011 cells
from late log phase cultures were washed with H2O and
resuspended in 200 ml of cold (4 °C) APC buffer (25 mM
HEPES-NaOH, pH 7.5, 400 mM NaCl, 10% glycerol, 0.1%
Triton X-100, 0.5 mM dithiothreitol, 25 mM NaF,
25 mM
-glycerophosphate, and 1 mM activated
sodium orthovanadate) containing freshly added complete protease
inhibitor tablets (Roche Applied Science) and 0.5 mM
phenylmethylsulfonyl fluoride. All subsequent steps were performed at
4 °C or on ice. Cells were disrupted six times for 1.5 min with
0.5-mm glass beads in a bead beater (Biospec Products), allowing 5 min
between pulses for cooling. Extract (~200 ml) was precleared by
centrifugation for 30 min at 35,000 × g and cleared a
second time for 1 h at 92,000 × g. The soluble
extract (5-15 mg/ml protein) was incubated with 100 µl of
pre-equilibrated EZview anti-FLAG M2 antibody-coupled agarose resin
(Sigma) for 2 h. Beads were collected by centrifugation, washed
four times for 10 min with 25 ml of APC buffer, transferred to a
microcentrifuge tube, and washed an additional three times with 1 ml of
APC buffer. APC was eluted by two sequential 30-min incubations at
30 °C with 200 µl of APC buffer containing 500 µg/ml 3× FLAG
peptide (Sigma). Elutions were pooled, and APC was precipitated with 6 volumes of acetone.
For the peptide block control, an extract from YKA151 was split into
two equal volumes. From one half, APC was purified as described above.
From the other half, APC was purified using an identical volume of
anti-FLAG affinity resin that had been blocked by incubation with 1 ml
of 500 µg/ml 3× FLAG peptide in APC buffer for 1 h before the
addition to the extract. Otherwise, the two preparations were performed identically.
Identification of SDS-PAGE Gel Bands by Mass Spectrometry
(MS)--
Proteins eluted from the anti-FLAG affinity resin were
separated by SDS-PAGE on 4-12% gradient NuPAGE gels (Invitrogen) and stained with Coomassie Brilliant Blue R-250 (Bio-Rad). Individual gel
bands were carefully excised with a razor and subjected to trypsin
proteolysis using a ProGest automated digester (Genomic Solutions).
Extracted tryptic peptides were analyzed on a Reflex III
matrix-assisted laser desorption ionization time-of-flight mass
spectrometer (Bruker Daltonics). Data were internally calibrated with
trypsin autoproteolysis peaks and submitted to the MASCOT database
search engine (Matrix Science) for protein identification by peptide
mass fingerprinting. All identifications in this study represent
statistically significant matches from the database of S. cerevisiae proteins. When a statistically significant match was
not obtained by peptide mass fingerprinting, individual peptides from
the spectrum were subjected to nanoelectrospray tandem MS on a QStar
mass spectrometer (Applied Biosystems) to confirm the identity of the protein.
APC Subunit Interaction Assay--
RTS in vitro
transcription and translation reactions were performed according to the
supplied instructions. Two APC subunits, one containing the 3× FLAG
epitope and the other containing a His6 tag were
coexpressed for 5 h at 30 °C in 50-µl reactions containing
~0.3 µg of each expression plasmid. Insoluble protein was removed
by centrifugation, and 40 µl of the soluble material was diluted to
500 µl with RTS buffer (25 mM HEPES-NaOH, pH 7.5, 150 mM sodium acetate, 10% glycerol, 0.1% Nonidet P-40, and
0.5 mM dithiothreitol). The sample was cleared a second
time by centrifugation, and the supernatant was incubated with 10 µl
of pre-equilibrated anti-FLAG affinity resin for 1 h at 4 °C.
The resin was washed three times with 1 ml of RTS buffer, and
specifically bound protein was eluted with 40 µl of 500 µg/ml 3×
FLAG peptide in RTS buffer overnight at 4 °C. Eluted proteins were
separated by SDS-PAGE and transferred to polyvinylidene difluoride
membranes, and the presence of the His6-tagged protein was
evaluated by Western blot with anti-His6 polyclonal
antibody (Covance). Membranes were stripped and reprobed with anti-FLAG
M2 monoclonal antibody (Sigma) to ensure that the immunoaffinity
purifications were successful. Expression of both proteins in the
reactions was also confirmed by Western blot using 5 µl of the
original reaction.
Growth Curves and Flow Cytometry--
To measure relative growth
rates of strains harboring deletions of APC9,
APC10, SWM1, or MND2 as well as the
isogenic wild-type strain, three individual colonies of each strain
were grown overnight in 5 ml of YPD at 30 °C. Cultures were diluted
20-fold in YPD and allowed to grow at 30 °C for 3 h. For growth
curves at 37 °C, cultures were transferred to 37 °C and incubated
for 1 h before beginning measurements. Cultures were diluted to
identical starting densities of 3 × 105 cells/ml for
30 °C experiments or 5 × 105 cells/ml for 37 °C
experiments, and at the indicated time points OD660
measurements were taken. OD660 values were converted to cell density for graphical display of growth curves. Samples from each
culture were removed at OD660 ~0.5 and prepared for
analysis by flow cytometry.
For flow cytometry, cells from 0.5 ml of culture were washed with 1 ml
of H2O and fixed overnight in 1 ml of 70% ethanol at 4 °C. Cells were rinsed twice with 1 ml of 50 mM
Tris-HCl, pH 8.0, and incubated for 2-3 h at 50 °C with 2 µg/ml
RNase A and 1 mg/ml proteinase K in 50 mM Tris-HCl. After
washing with 1 ml of FC buffer (200 mM Tris-HCl, pH 8.0, 200 mM NaCl, 78 mM MgCl2), cells
were resuspended in 500 µl of FC buffer containing 5 µM Sytox Green (Molecular Probes, Inc., Eugene, OR). DNA content was
measured on a FACScan instrument. Percentages of G1, S, and G2/M cells were calculated using ModFit LT software (Verity
Software House, Inc.).
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RESULTS |
Identification of Two Unknown Proteins Co-purifying with Yeast
APC--
We constructed a yeast strain, YKA151, which produces the
Cdc27 protein with a carboxyl-terminal 3× FLAG epitope tag from its
natural chromosomal locus for immunoaffinity purification of the
anaphase-promoting complex. In our preparations of APC from YKA151, we
identified all 11 known APC subunits by peptide mass fingerprinting or
tandem MS as well as two other bands that had not been previously
described as APC subunits (Fig.
1A, second lane). We also observed these two proteins associated with
APC in preparations from a yeast strain containing a 6-Myc epitope tag
on the Cdc16 APC subunit (data not shown). It should be noted that the
conditions used for the affinity purification of APC include a high
salt concentration (425 mM Na+) at all steps,
demonstrating the high salt stability of the APC. To determine whether
these proteins were specifically associated with our purified APC as
opposed to nonspecifically associated with the anti-FLAG affinity
resin, we performed a control purification in which the anti-FLAG
affinity beads were preblocked with the antigenic 3× FLAG peptide
(Fig. 1A, first lane). Both proteins were effectively competed away by the blocking peptide, suggesting that
their presence was due to direct interaction with the APC. The two
proteins, Mnd2 and Swm1, have both been implicated in meiosis, but at
different stages (15, 16). Little else is known about them.

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Fig. 1.
Mnd2 and Swm1 copurify with yeast APC.
Immunoaffinity purifications of APC from yeast whole cell extracts were
separated by SDS-PAGE as described under "Experimental Procedures"
and stained with Coomassie Blue. Each lane represents the
material obtained from ~1011 cells. Individual bands were
excised and digested with trypsin, and the proteins were identified by
mass spectrometry. All labeled proteins represent statistically
significant scores obtained by the MASCOT search engine. A,
APC was prepared from strain YKA151, which produces 3× FLAG
epitope-tagged Cdc27. The samples in both lanes were treated
identically except that the anti-FLAG beads in the first sample were
preblocked with 3× FLAG peptide before incubation with the cell
extract. B, APC was purified from strain YKA152, which
produces 3× FLAG epitope-tagged Mnd2, and strain YKA153, which
produces 3× FLAG epitope-tagged Swm1. Bands labeled with an
asterisk in each preparation were identified as mouse IgG.
Most of the visible but faint unlabeled bands on these gels were
identified as proteolytic fragments of APC subunits.
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Known APC Subunits Co-purify with FLAG Epitope-tagged Mnd2 and
Swm1--
To provide more convincing evidence that Mnd2 and Swm1 are
true subunits of the APC, we constructed yeast strains producing 3×
FLAG-tagged versions of Mnd2 and Swm1 (YKA152 and YKA153,
respectively). We subjected whole cell extracts from these strains to
the same stringent immunoaffinity purification protocol used with
YKA151. Proteins in these preparations were identified from
Coomassie-stained polyacrylamide gels by peptide mass fingerprinting.
In both cases, we identified 9 of the 11 known APC subunits
co-purifying with the epitope-tagged protein (Fig. 1B).
Apc11 and Cdc26 were not identified; however, these two small subunits
generally required tandem MS for identification due to the low number
of tryptic peptides generated. Regardless, these results conclusively
demonstrate that Mnd2 and Swm1 are stably associated with the core APC
in haploid yeast cells.
Mnd2 and Swm1 Are Constitutive Components of the APC during the
Cell Cycle--
Although its activity fluctuates, the APC is a stable
complex that is present throughout the cell cycle (8, 10). To determine whether Mnd2 and Swm1 are also associated with the APC during different
cell cycle stages, APC was immunoaffinity-purified from cell cultures
arrested in G1 phase with
-mating factor, in S phase
with hydroxyurea, and in M phase with nocodazole (Fig.
2, A-C). Mnd2 and Swm1 were
identified by mass spectrometry and were present in approximately equal
abundance at all three cell cycle stages with respect to the abundance
of the other APC subunits, judging from the intensity of
Coomassie-stained gel bands. These results suggest that, like the other
11 components, Mnd2 and Swm1 are constitutively associated with the APC
and can therefore be considered core subunits.

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Fig. 2.
Mnd2 and Swm1 are constitutive components of
yeast APC. APC was immunoaffinity-purified as described under
"Experimental Procedures" from haploid yeast cell cultures arrested
in G1 phase with -factor peptide (A), S phase
with hydroxyurea (B), or M phase with nocodazole
(C) or from diploid cells induced to undergo meiosis
(D). The purified APC in each case was separated by
SDS-PAGE, and proteins were visualized by staining with Coomassie Blue.
The presence of Mnd2 and Swm1 in each of the four preparations was
confirmed by mass spectrometry. A small sample of cells from each of
the haploid cultures was analyzed by flow cytometry to confirm arrest
at the desired cell cycle stage. Histograms of the DNA content in
these samples are displayed below the corresponding
preparation. 1n, one copy of the genome; 2n, two
copies of the genome. The two asterisks in the
diploid preparation (D) indicate prominent bands that were
not apparent in the haploid preparations. The larger of the two
proteins was identified as 6-phosphofructo-2-kinase, which happens to
contain a consensus FLAG epitope within its amino acid sequence. This
protein was also observed in our haploid preparations at lower levels.
The smaller of the asterisk-labeled
bands was identified as Ach1p, which is involved in acetate
metabolism and is probably a nonspecific contaminant that appears
because the sporulating cells use acetate as a carbon source.
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Given the fact that lack of MND2 and SWM1 has
previously been associated with severe meiotic defects (15, 16), we
created a diploid strain expressing CDC27 with three copies
of the FLAG epitope to determine whether Mnd2 and Swm1 are components
of the APC during meiosis. The diploid cells were induced to sporulate in a synchronous manner according to a previously described method (18), and cells were harvested midway through meiosis (19). APC was
purified from the meiotic cells using our standard purification procedure (Fig. 2D), and the subunits were identified by
mass spectrometry. Both Mnd2 and Swm1 were present along with the other known APC subunits. Although we cannot rule out the possibility that
Mnd2 or Swm1 dissociates transiently from the APC at a specific stage
of meiosis to carry out an APC-independent function, our results
support the conclusion that they remain associated with the APC during
the sporulation program.
Identification of Subunit-Subunit Contacts Involving Mnd2 and
Swm1--
We established an interaction assay based on coexpression of
two APC subunits, each with a different affinity tag, in an E. coli-derived in vitro transcription and translation
system (see "Experimental Procedures"). In this assay, one APC
subunit is expressed as a fusion with the 3× FLAG epitope, and the
second subunit is expressed as a fusion with a His6 tag.
Following the reaction, the subunit containing the FLAG epitope is
purified using anti-FLAG antibody-coupled beads, and the presence or
absence of the second subunit is monitored by Western blot with
anti-His6 antibody. A positive signal in the
anti-His6 Western blot is indicative of a physical
interaction between the two subunits that results in their copurification.
We screened Mnd2 and Swm1 for interactions with as many of the other
APC subunits as possible, including homodimeric interactions and
interactions with each other. First, we tested Mnd2-His6 by coexpression with a series of FLAG-tagged APC subunits. In this assay,
Mnd2-His6 interacted with Apc1-FLAG, Apc5-FLAG, and
Cdc23-FLAG (Fig. 3A). The data
also suggested a weak interaction with Apc2-FLAG. However, the signal
was substantially weaker than the other three interactions, and in
light of the fact that Apc2-FLAG expression was greater than most of
the other subunits (Fig. 3A and data not shown), we cannot
definitively conclude that this result represents a bona
fide interaction.

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Fig. 3.
Subunit-subunit interactions involving Mnd2
and Swm1. A, Mnd2-His6 protein was
coexpressed with each of the indicated 3× FLAG epitope-tagged APC
subunits in an E. coli-based in vitro
transcription and translation reaction as described under
"Experimental Procedures." Expression of each protein was confirmed
by Western blot using either anti-His6 polyclonal or
anti-FLAG monoclonal antibodies (Input). The
Mnd2-His6 band in the Input blot appears as a
doublet, but the upper band is a cross-reacting protein from the
E. coli lysate. FLAG-tagged subunits were purified using
anti-FLAG affinity resin and specifically eluted with 3× FLAG peptide.
Co-purification of Mnd2-His6 indicative of a physical
interaction was determined by Western blot of the eluted sample.
B, the same in vitro transcription and
translation reactions were used to coexpress 3× FLAG epitope-tagged
Swm1 with the indicated APC subunits containing a His6 tag.
Expression of each protein was confirmed by Western blot
(Input). Swm1-FLAG was purified using anti-FLAG affinity
resin and eluted with 3× FLAG peptide, and the presence of
co-purifying APC subunits was detected by Western blot with
anti-His6 antibody. There was no detectable expression of
Apc4-His6 in this experiment. C,
Apc10-His6, Cdc23-His6, and
Apc5-His6 were coexpressed with Swm1-FLAG or expressed
alone. Reactions were subjected to the same anti-FLAG affinity
purification and elution performed in A and B.
Expression of each subunit (Input) and the presence of the
His6-tagged proteins in the elution were monitored by
Western blot with anti-His6 antibody. To conserve space,
only the full-length FLAG-tagged proteins are displayed in A
and B to confirm expression, although in many cases numerous
unfinished translation products or proteolytic fragments were also
visible on the Western blots because the 3× FLAG epitope is present at
the N terminus of each subunit. Therefore, the relative expression
levels of each FLAG-tagged protein cannot be accurately compared from
this figure. Also, it is important to note that the
intensity of bands in the anti-His6 versus
anti-FLAG Western blots cannot be used to compare protein quantity.
Therefore, there is no reliable way to compare the actual amount of
each of the two subunits produced in a given reaction. Although not
shown, we reprobed all gels containing the purified samples with
anti-FLAG antibody to ensure that the immunoaffinity purifications were
successful.
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Since we were unable to make a construct capable of expressing
Swm1-His6 in the RTS system, we screened Swm1-FLAG against a collection of His6-tagged APC subunits to identify its
interaction partners. In this experiment, Swm1-FLAG interacted with
Apc10-His6, Cdc23-His6, and
Apc5-His6 (Fig. 3B). Control reactions to
evaluate the specificity of these apparent interactions (Fig.
3C) revealed that the Apc10-His6 signal in the
elution was not dependent on coexpression of Swm1-FLAG and probably
reflected nonspecific association with the antibody resin. On the other
hand, the Cdc23-His6 and Apc5-His6 signals were
dependent on coexpression of Swm1-FLAG, and we can conclude that Swm1
physically interacts with Cdc23 and Apc5.
We were unable to evaluate potential interactions with Apc11, because
Apc11 expressed in this system consistently gave artifactual results
(not shown). Also, we were unable to evaluate the potential interactions Mnd2-Cdc27, Swm1-Swm1, or Swm1-Apc1 because we could not
generate clones that would express Cdc27-FLAG, Swm1-His6, or Apc1-His6 in the RTS system. Nonetheless, we were able
to identify at least a portion of the subunit contacts that are
probably responsible for the stable association of Swm1 and Mnd2 with
the APC in vivo.
Deletion of MND2 or SWM1 Results in Slow Growth Associated with
Accumulation of G2/M Cells--
Defects in APC function
generally result in cell cycle arrest in metaphase or a delay in
progression of the cell cycle through mitosis. We compared the growth
rates of swm1
and mnd2
haploid strains to
strains containing deletions of two other nonessential APC genes,
apc9
and apc10
, as well as the isogenic
wild-type strain. At 30 °C, apc9
, mnd2
,
and swm1
all exhibited modest but statistically
significant slow growth phenotypes, whereas the growth defect of
apc10
was much more acute (Fig.
4A). At 37 °C, the slow
growth phenotypes of apc9
, and mnd2
were
slightly more severe compared with the parental strain. However, the
severity of the swm1
growth defect was greatly increased
at 37 °C, nearly to the level of apc10
(Fig.
4B).

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Fig. 4.
Effect of MND2 and SWM1
deletions on yeast cell growth and the cell cycle. Relative
growth rates of haploid yeast strains harboring deletions of
APC9 ( ), APC10 ( ), MND2 ( ),
and SWM1 ( ) and their isogenic parent strain BY4741 ( )
were monitored at 30 °C (A) or 37 °C (B),
starting from identical cell densities. Three independent cultures of
each strain were used in a single experiment. The graphs
depict a typical single experiment, with data points representing the
average of the three cultures. The error bars
indicate S.D. C, samples from each strain grown at 37 °C
(B) were removed at OD660 = 0.5 and analyzed by
flow cytometry as described under "Experimental Procedures." ModFit
LT analysis software was used to extract the percentages of cells from
each strain in G1, S, and G2/M phases.
Solid areas represent the computer-generated fit
for G1 (1n) and G2/M
(2n), and the striped area represents
the fit for S phase. The numerical values for the G2/M
peaks from the computer fitting are displayed in Table II.
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In an effort to pinpoint the cause of the slow growth phenotype of
swm1
and mnd2
, cells from each of the five
strains were taken during the growth curve experiment at 37 °C, and
their DNA content was analyzed by flow cytometry. The
apc9
strain exhibited a slight but noticeable and
statistically significant accumulation of G2/M cells
compared with the wild-type strain (Fig. 4C and Table
II), consistent with the delayed anaphase
entry reported previously for apc9
(7). The
mnd2
strain exhibited an accumulation of G2/M
cells that was comparable in magnitude with the apc9
strain, suggesting that it too has a similar delay in mitotic progression. The accumulation of G2/M cells in the
apc10
strain was more pronounced than that of
apc9
or mnd2
, although perhaps not as much
as would be expected given its severely retarded growth. Perhaps the
lack of Apc10 impairs progression through G1 as well as M
phase or has an independent effect on overall cell growth. Finally, the
swm1
strain exhibited a dramatic increase in the percentage of G2/M cells compared with the wild-type parent
strain (63% versus 29%, Table II). The greater
coefficients of variation for the G1 and G2/M
peaks of apc10
and swm1
(Fig.
4C) as well as their forward scatter data (not shown)
suggest significant morphological changes in these strains compared
with the parental strain. Considering our convincing evidence that Swm1
is a component of the APC, it is reasonable to conclude that
swm1
cells have a substantial delay in progression
through mitosis resulting from an APC defect that is reflected in their
slow growth and dramatic accumulation of G2/M cells
illustrated in Fig. 4. Although we cannot rule out the possibility that
Mnd2 and/or Swm1 have APC-independent cellular functions that affect
vegetative growth and progression of cells through G2 and M
phase, our results are consistent with the conclusion that Mnd2 and
Swm1 are constitutive components of the yeast APC that contribute to
normal APC activity during mitosis.
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Table II
Quantitative analysis of G2/M content from wild-type and
deletion strains
All strains are isogenic with the exception of the noted gene
deletions. Percentages represent averages and S.D. values from three
cultures grown at 37 °C that are depicted in Fig. 4, B
and C. Data values were generated by ModFit LT flow
cytometry analysis software.
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DISCUSSION |
In this report, we have provided strong evidence that Mnd2 and
Swm1 are constitutive core subunits of the budding yeast APC. Swm1 is
probably equivalent to a 19-kDa protein labeled Apc13 that was
previously observed in an APC immunoaffinity purification (7) but not
identified at the time. These findings bring the total number of
confirmed subunits in budding yeast APC to 13. Our ability to identify
two previously unidentified subunits of the APC emphasizes the power of
mass spectrometry as a sensitive and accurate analytical tool for
biological research and the utility of the 3× FLAG epitope for
immunoaffinity purification of protein complexes. Another group
recently achieved the same identification of Mnd2 and Swm1 by coupling
mass spectrometry and the tandem affinity purification method (20). Our
protocol allows a rapid one-step preparation that can be completed in a
single day and provides high yield and purity as evidenced by the lack
of nonspecific contaminants and the ability to see proteins by
Coomassie staining in Fig. 1A. Further advantages of the
FLAG-based immunoaffinity purification include the small size of the
3× FLAG epitope (less than 3 kDa) compared with other commonly used
affinity tags, making it less likely to impair normal protein function,
and the lack of specific buffer requirements, which, for example,
allows the purification to be performed at high salt concentrations if desired.
In addition to identifying Mnd2 and Swm1 as components of the APC
throughout the cell cycle, we have defined the subunit-subunit interactions that mediate Mnd2 and Swm1 association with the complex using a powerful interaction assay based on an E. coli
in vitro transcription and translation system (Fig. 3). A
key advantage of the E. coli-based system is the lack of
endogenous APC subunit homologs that can potentially interfere with
interaction assays using eukaryotic expression systems. In identifying
the subunit contacts of Mnd2 and Swm1, we have taken an important first
step in defining the organization of the complex that will be important for understanding the structure and function of the APC.
We have demonstrated that mnd2
and swm1
haploid strains have mitotic phenotypes consistent with an APC defect,
including retarded growth rates and accumulation of G2/M
cells (Fig. 4). Although these data suggest a significant contribution
of Mnd2 and Swm1 to the mitotic function of the APC, they are clearly not essential as are the majority of APC subunits. This is in contrast
to diploid yeast in which Mnd2 and Swm1 are essential for progression
through meiosis. An mnd2
diploid strain arrests prior to
meiotic nuclear division (16), and a swm1
diploid strain
arrests late in meiosis prior to spore wall formation (15). The
different requirement for Mnd2 and Swm1 in mitosis versus meiosis has interesting implications for how the APC functions and why
it has so many subunits. There are two possible general explanations
for the difference in mitotic and meiotic phenotypes. The most likely
is that Mnd2 and Swm1 provide an essential function for the APC during
meiosis that is not required during mitosis. A second possibility that
currently lacks supporting evidence is that Mnd2 and Swm1 perform
essential APC-independent functions during meiosis.
Because most of the APC subunits are essential for viability, the
separation of function within the APC suggested by the
mnd2
and swm1
mitotic and meiotic
phenotypes has not been previously described. Furthermore, the role of
APC in meiosis has not received as much attention as its role in
mitosis. Specific functions for APC subunits during meiosis could
include recruitment of meiosis-specific substrates, activation or
inhibition of APC toward specific substrates in response to
meiosis-specific signals, or mediating cellular localization required
for proper meiotic function of APC. Budding yeast APC is known to
interact with a meiosis-specific substrate activator protein, Ama1
(21). Perhaps Mnd2 or Swm1 are involved in the binding of Ama1 or
recruitment of Ama1-specific substrates during meiosis. Similarly, a
fission yeast protein, Mfr1, was identified as an activator of APC
during meiosis that coordinates nuclear division and sporulation (22),
and a meiosis-specific kinase in budding yeast has also been identified
as a negative regulator of APC activity (23). It is conceivable that
Mnd2 or Swm1 might be involved in the interaction of APC with these proteins as well. Future research will hopefully provide insight into
details of APC function in mitosis versus meiosis, and it may also prove interesting to examine possible specific functions for
the other nonessential APC subunits Apc9, Apc10, and Cdc26 in meiosis.
A specific function for Mnd2 and Swm1 during meiosis raises a question
about the nature of their nonessential role in mitosis. The phenotype
of mnd2
haploid yeast is mild, and it is possible that
lack of Mnd2 in mitosis affects the stability of the complex enough to
indirectly impair catalysis or some other property of the APC.
Similarly, the slow growth phenotype of swm1
haploid yeast is severe at 37 °C but mild and comparable with
mnd2
at 30 °C, consistent with a defect in complex
stability that would be exacerbated at elevated temperatures. This is
similar to the Cdc26 subunit that has been suggested to aid in complex
stability at elevated temperatures because it is required for growth at 37 °C but seems to be almost entirely unnecessary for normal growth at 30 °C (5). Alternatively, Mnd2 and Swm1 function may be required
specifically for the polyubiquitination of a substrate or set of
substrates whose degradation is essential for progression through
meiosis but plays only a minor role in mitosis. Analysis of
substrate-specific ubiquitin ligase activity of APC lacking Mnd2 or
Swm1 might help determine whether their presence is required specifically for certain substrates or contributes generally in some
manner to basic APC activity.
An APC-independent function for Mnd2 and Swm1 during meiosis remains a
formal possibility. This would probably require the existence of free
Mnd2 and Swm1 after initiation of sporulation. MND2
expression is moderately up-regulated in meiosis (16, 24), whereas
SWM1 expression is greatly increased (15, 24). However, most
of the APC subunits demonstrate a sharp increase in expression during
meiosis as well (19, 24), suggesting that the levels of the entire
complex are increased. The reason for increased expression of the APC
during meiosis is not known. We examined the components of meiotic APC
after purification of the complex from sporulating diploid cells and
found that Mnd2 and Swm1 remain associated with the APC during meiosis.
This result is more consistent with the notion that an APC-specific
defect is responsible for the severe meiotic phenotypes in strains
lacking functional Mnd2 and Swm1.
Mnd2 and Swm1 do not have any obvious mammalian homologs (15) (data not
shown). Weak homology to subunits of the S. pombe APC was
recently suggested (20), but the lack of strong homology suggests the
possibility that different organisms may contain species-specific APC
subunits. This possibility seems unlikely at first, given the fact that
the APC is essential for execution of some of the most fundamental
cellular processes common to all eukaryotic cells. It is possible that
during evolution, species have been able to recruit the
ubiquitin ligase activity of the APC to function in other types of
cellular transactions that are not necessarily highly conserved. Aside
from Mnd2 and Swm1, budding yeast Apc9 (7) appears to have no homolog
in other organisms, and metazoans contain a subunit, APC7 (9), that
does not appear to have a homolog in yeast. Perhaps the large number of
subunits reflects, in part, a large number of biological roles in which the APC acts as a type of modular enzyme complex with a catalytic core
involving Apc2 and Apc11 and a host of other subunits to direct the
ubiquitin ligase activity to specific substrates at specific times and
locations within the cell. Another ramification of the identification
of Mnd2 and Swm1 is the likelihood that other subunits might still be
unidentified in other organisms such as humans. A major focus of future
research will no doubt be focused on identification of other APC
subunits in higher eukaryotes and understanding the roles of the
numerous APC subunits that to this date have no known functions.