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• W2014505382 abstract "The anaphase-promoting complex (APC) is a multisubunit E3 ubiquitin ligase that regulates the metaphase-anaphase transition and exit from mitosis in eukaryotic cells. Eleven subunits have been previously identified in APC from budding yeast. We have identified two additional subunits, Mnd2 and Swm1, by mass spectrometry. Both Mnd2 and Swm1 were found specifically associated with a highly purified preparation of APC from haploid yeast whole cell extract. Moreover, the APC co-purified with epitope-tagged Mnd2 and Swm1. Both proteins were present in APC preparations from haploid cells arrested in G1, S, and M phases and from meiotic diploid cells, indicating that they are constitutive components of the complex throughout the yeast cell cycle. Mnd2 interacted strongly with Cdc23, Apc5, and Apc1 when coexpressed in an in vitro transcription/translation reaction. Swm1 also interacted with Cdc23 and Apc5 in this system. Previous studies described meiotic defects for mutations in MND2 and SWM1. Here, we show that mnd2Δ and swm1Δ haploid strains exhibit slow growth and accumulation of G2/M cells comparable with that seen in apc9Δ orapc10Δ strains and consistent with an APC defect. Taken together, these results demonstrate that Swm1 and Mnd2 are functional components of the yeast APC. The anaphase-promoting complex (APC) is a multisubunit E3 ubiquitin ligase that regulates the metaphase-anaphase transition and exit from mitosis in eukaryotic cells. Eleven subunits have been previously identified in APC from budding yeast. We have identified two additional subunits, Mnd2 and Swm1, by mass spectrometry. Both Mnd2 and Swm1 were found specifically associated with a highly purified preparation of APC from haploid yeast whole cell extract. Moreover, the APC co-purified with epitope-tagged Mnd2 and Swm1. Both proteins were present in APC preparations from haploid cells arrested in G1, S, and M phases and from meiotic diploid cells, indicating that they are constitutive components of the complex throughout the yeast cell cycle. Mnd2 interacted strongly with Cdc23, Apc5, and Apc1 when coexpressed in an in vitro transcription/translation reaction. Swm1 also interacted with Cdc23 and Apc5 in this system. Previous studies described meiotic defects for mutations in MND2 and SWM1. Here, we show that mnd2Δ and swm1Δ haploid strains exhibit slow growth and accumulation of G2/M cells comparable with that seen in apc9Δ orapc10Δ strains and consistent with an APC defect. Taken together, these results demonstrate that Swm1 and Mnd2 are functional components of the yeast APC. anaphase-promoting complex rapid translation system mass spectrometry ubiquitin-conjugating enzyme ubiquitin-protein ligase 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. 1Peters J.M. Mol. Cell. 2002; 9: 931-943Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar and 2Harper J.W. Burton J.L. Solomon M.J. Genes Dev. 2002; 16: 2179-2206Crossref PubMed Scopus (425) Google Scholar). 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 (1Peters J.M. Mol. Cell. 2002; 9: 931-943Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar) and is regulated by checkpoint signaling pathways that monitor DNA and chromosome integrity (3Musacchio A. Hardwick K.G. Nat. Rev. Mol. Cell. Biol. 2002; 3: 731-741Crossref PubMed Scopus (474) Google Scholar,4Foiani M. Pellicioli A. Lopes M. Lucca C. Ferrari M. Liberi G. Muzi Falconi M. Plevani P. Mutat. Res. 2000; 451: 187-196Crossref PubMed Scopus (102) Google Scholar). Eleven constitutive core subunits of the APC have been identified in the budding yeast, Saccharomyces cerevisiae (5Zachariae W. Shin T.H. Galova M. Obermaier B. Nasmyth K. Science. 1996; 274: 1201-1204Crossref PubMed Scopus (233) Google Scholar, 6Hwang L.H. Murray A.W. Mol. Biol. Cell. 1997; 8: 1877-1887Crossref PubMed Scopus (77) Google Scholar, 7Zachariae W. Shevchenko A. Andrews P.D. Ciosk R. Galova M. Stark M.J.R. Mann M. Nasmyth K. Science. 1998; 279: 1216-1219Crossref PubMed Scopus (297) Google Scholar), and in vertebrates (8Peters J.-M. King R.W. Höög C. Kirschner M.W. Science. 1996; 274: 1199-1201Crossref PubMed Scopus (175) Google Scholar, 9Yu H. Peters J.-M. King R.W. Page A.M. Hieter P. Kirschner M.W. Science. 1998; 279: 1219-1222Crossref PubMed Scopus (204) Google Scholar, 10Grossberger R. Gieffers C. Zachariae W. Podtelejnikov A. Schleiffer A. Nasmyth K. Mann M. Peters J.-M. J. Biol. Chem. 1999; 274: 14500-14507Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 11Gmachl M. Gieffers C. Podtelejnikov A.V. Mann M. Peters J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8973-8978Crossref PubMed Scopus (157) Google Scholar). Homologs of most of the subunits have been found in other model systems as well, including Schizosaccharomyces pombe, Caenorhabditis elegans, andDrosophila melanogaster (reviewed in Ref. 2Harper J.W. Burton J.L. Solomon M.J. Genes Dev. 2002; 16: 2179-2206Crossref PubMed Scopus (425) Google Scholar). 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 (11Gmachl M. Gieffers C. Podtelejnikov A.V. Mann M. Peters J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8973-8978Crossref PubMed Scopus (157) Google Scholar,12Leverson J.D. Joazeiro C.A.P. Page A.M. Huang H. Hieter P. Hunter T. Mol. Biol. Cell. 2000; 11: 2315-2325Crossref PubMed Scopus (158) Google Scholar). Another subunit, Apc2, containing a highly conserved cullin domain present in other E3 ubiquitin ligases interacts with Apc11 (13Ohta T. Michel J.J. Schottelius A.J. Xiong Y. Mol. Cell. 1999; 3: 535-541Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar) and is also believed to be important for catalyzing ubiquitin transfer (14Tang Z. Li B. Bharadwaj R. Zhu H. Ozkan E. Hakala K. Deisenhofer J. Yu H. Mol. Biol. Cell. 2001; 12: 3839-3851Crossref PubMed Scopus (152) Google Scholar). 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 (2Harper J.W. Burton J.L. Solomon M.J. Genes Dev. 2002; 16: 2179-2206Crossref PubMed Scopus (425) Google Scholar). 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 (7Zachariae W. Shevchenko A. Andrews P.D. Ciosk R. Galova M. Stark M.J.R. Mann M. Nasmyth K. Science. 1998; 279: 1216-1219Crossref PubMed Scopus (297) Google Scholar). We discuss the significance of these identifications, the previously described meiotic defects associated with MND2 and SWM1 mutations (15Ufano S. San-Segundo P. del Rey F. Vázguez de Aldana C.R. Mol. Cell. Biol. 1999; 19: 2118-2129Crossref PubMed Scopus (26) Google Scholar, 16Rabitsch K.P. Tóth A. Gálova M. Schleiffer A. Schaffner G. Aigner E. Rupp C. Penkner A.M. Moreno-Borchart A.C. Primig M. Easton Esposito R. Klein F. Knop M. Nasmyth K. Curr. Biol. 2001; 11: 1001-1009Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar), and the mitotic phenotypes we have observed inmnd2Δ and swm1Δ strains for understanding aspects of APC function in all organisms. 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 (17Gelbart M.E. Rechsteiner T. Richmond T.J. Tsukiyama T. Mol. Cell. Biol. 2001; 21: 2098-2106Crossref PubMed Scopus (143) Google Scholar). Integrants were selected on YPD agar containing 500 ॖg/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.Table IS. cerevisiae strains used in this studyStrainRelevant genotypeSourceW1588-4c1-aW1588-4c is a derivative of W303 in which the weakrad5 mutation has been repaired (25)MATa ade2–1 can1–100 His3–11,15 leu2–3, 112 trp1–1 ura3–1R. RothsteinYKA151CDC27–3FLAG:KanMX4This studyYKA152MND2–3FLAG:KanMX4This studyYKA153SWM1–3FLAG:KanMX4This studyYKA155CDC27–3FLAG:KanMX4 bar1Δ::URA3This studyBY4741MATaHis3D1 leu2D0 met15D0 ura3D0SGDP1-bSGDP, Saccharomyces Genome Deletion Project.27131-cThese strains were all derived from BY4741, and the values correspond to the Saccharomyces Genome Deletion Project record number.apc9Δ::KanMX4SGDP1-bSGDP, Saccharomyces Genome Deletion Project.46071-cThese strains were all derived from BY4741, and the values correspond to the Saccharomyces Genome Deletion Project record number.apc10Δ::KanMX4SGDP1-bSGDP, Saccharomyces Genome Deletion Project.59601-cThese strains were all derived from BY4741, and the values correspond to the Saccharomyces Genome Deletion Project record number.mnd2Δ::KanMX4SGDP1-bSGDP, Saccharomyces Genome Deletion Project.36191-cThese strains were all derived from BY4741, and the values correspond to the Saccharomyces Genome Deletion Project record number.swm1Δ::KanMX4SGDP1-bSGDP, Saccharomyces Genome Deletion Project.YPH499MATa ura3–52 lys2–801 ade2–101 trp1-Δ63 his3-Δ200 leu2-Δ1StratageneYKA180MATa/α CDC27–3FLAG:KanMX4This study1-a W1588-4c is a derivative of W303 in which the weakrad5 mutation has been repaired (25Zhao X. Muller E.G. Rothstein R. Mol. Cell. 1998; 2: 329-340Abstract Full Text Full Text PDF PubMed Scopus (606) Google Scholar)1-b SGDP, Saccharomyces Genome Deletion Project.1-c These strains were all derived from BY4741, and the values correspond to the Saccharomyces Genome Deletion Project record number. Open table in a new tab Cell cycle arrests were performed in midlog phase cultures of strain YKA155 as follows. For G1 arrest, α-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 ॖg/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 ॖg/ml. Cell cycle arrests were monitored by phase-contrast microscopy until >907 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 (18Cao L. Alani E. Kleckner N. Cell. 1990; 61: 1089-1101Abstract Full Text PDF PubMed Scopus (537) Google Scholar). Cells were harvested midway through meiosis based on the meiotic progression of yeast strain W303 described recently (19Primig M. Williams R.M. Winzeler E.A. Tevadze G.G. Conway A.R. Hwang S.Y. Davis R.W. Esposito R.E. Nat. Genet. 2000; 26: 415-423Crossref PubMed Scopus (368) Google Scholar). Induction of sporulation was confirmed by monitoring spore formation by microscopy. 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 theNcoI 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 vitrotranscription 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. Approximately 1011 cells from late log phase cultures were washed with H2O and resuspended in 200 ml of cold (4 °C) APC buffer (25 mmHEPES-NaOH, pH 7.5, 400 mm NaCl, 107 glycerol, 0.17 Triton X-100, 0.5 mm dithiothreitol, 25 mm NaF, 25 mm औ-glycerophosphate, and 1 mm activated sodium orthovanadate) containing freshly added complete protease inhibitor tablets (Roche Applied Science) and 0.5 mmphenylmethylsulfonyl 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 ॖl 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 ॖl of APC buffer containing 500 ॖg/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 ॖg/ml 3× FLAG peptide in APC buffer for 1 h before the addition to the extract. Otherwise, the two preparations were performed identically. Proteins eluted from the anti-FLAG affinity resin were separated by SDS-PAGE on 4–127 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. RTS in vitrotranscription 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-ॖl reactions containing ∼0.3 ॖg of each expression plasmid. Insoluble protein was removed by centrifugation, and 40 ॖl of the soluble material was diluted to 500 ॖl with RTS buffer (25 mm HEPES-NaOH, pH 7.5, 150 mm sodium acetate, 107 glycerol, 0.17 Nonidet P-40, and 0.5 mm dithiothreitol). The sample was cleared a second time by centrifugation, and the supernatant was incubated with 10 ॖl 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 ॖl of 500 ॖg/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 ॖl of the original reaction. 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 OD660measurements 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 707 ethanol at 4 °C. Cells were rinsed twice with 1 ml of 50 mmTris-HCl, pH 8.0, and incubated for 2–3 h at 50 °C with 2 ॖg/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 ॖl of FC buffer containing 5 ॖmSytox 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.). 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 (15Ufano S. San-Segundo P. del Rey F. Vázguez de Aldana C.R. Mol. Cell. Biol. 1999; 19: 2118-2129Crossref PubMed Scopus (26) Google Scholar, 16Rabitsch K.P. Tóth A. Gálova M. Schleiffer A. Schaffner G. Aigner E. Rupp C. Penkner A.M. Moreno-Borchart A.C. Primig M. Easton Esposito R. Klein F. Knop M. Nasmyth K. Curr. Biol. 2001; 11: 1001-1009Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Little else is known about them. 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. Although its activity fluctuates, the APC is a stable complex that is present throughout the cell cycle (8Peters J.-M. King R.W. Höög C. Kirschner M.W. Science. 1996; 274: 1199-1201Crossref PubMed Scopus (175) Google Scholar, 10Grossberger R. Gieffers C. Zachariae W. Podtelejnikov A. Schleiffer A. Nasmyth K. Mann M. Peters J.-M. J. Biol. Chem. 1999; 274: 14500-14507Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). 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 α-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. Given the fact that lack of MND2 and SWM1 has previously been associated with severe meiotic defects (15Ufano S. San-Segundo P. del Rey F. Vázguez de Aldana C.R. Mol. Cell. Biol. 1999; 19: 2118-2129Crossref PubMed Scopus (26) Google Scholar, 16Rabitsch K.P. Tóth A. Gálova M. Schleiffer A. Schaffner G. Aigner E. Rupp C. Penkner A.M. Moreno-Borchart A.C. Primig M. Easton Esposito R. Klein F. Knop M. Nasmyth K. Curr. Biol. 2001; 11: 1001-1009Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar), 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 (18Cao L. Alani E. Kleckner N. Cell. 1990; 61: 1089-1101Abstract Full Text PDF PubMed Scopus (537) Google Scholar), and cells were harvested midway through meiosis (19Primig M. Williams R.M. Winzeler E.A. Tevadze G.G. Conway A.R. Hwang S.Y. Davis R.W. Esposito R.E. Nat. Genet. 2000; 26: 415-423Crossref PubMed Scopus (368) Google Scholar). 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. 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. 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. 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 swm1Δ and mnd2Δ haploid strains to strains containing deletions of two other nonessential APC genes,apc9Δ and apc10Δ, as well as the isogenic wild-type strain. At 30 °C, apc9Δ, mnd2Δ, and swm1Δ all exhibited modest but statistically significant slow growth phenotypes, whereas the growth defect ofapc10Δ was much more acute (Fig.4A). At 37 °C, the slow growth phenotypes of apc9Δ, and mnd2Δ were slightly more severe compared with the parental strain. However, the severity of the swm1Δ growth defect was greatly increased at 37 °C, nearly to the level of apc10Δ (Fig.4B). In an effort to pinpoint the cause of the slow growth phenotype ofswm1Δ and mnd2Δ, 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. Theapc9Δ strain exhibited a slight but noticeable and statistically significant accumulation of G2/M cells compared with the wild-type strain (Fig. 4C and TableII), consistent with the delayed anaphase entry reported previously for ap" @default.
• W2014505382 created "2016-06-24" @default.
• W2014505382 creator A5028128648 @default.
• W2014505382 creator A5057238214 @default.
• W2014505382 creator A5060991052 @default.
• W2014505382 creator A5064651221 @default.
• W2014505382 date "2003-05-01" @default.
• W2014505382 modified "2023-10-16" @default.
• W2014505382 title "Mnd2 and Swm1 Are Core Subunits of the Saccharomyces cerevisiae Anaphase-promoting Complex" @default.
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