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 containing and integration of the was by PCR and DNA of the from was by integration of a amplified by PCR from at the of with was by was by of with expressing the for and for of containing The diploid was by its to from the or MND2 been (Table I) as as their were from the Saccharomyces The presence of the was in of these strains by PCR the cerevisiae strains in this is a of in the been Saccharomyces strains were all from and the to the Saccharomyces Saccharomyces strains were all from and the to the Saccharomyces Saccharomyces strains were all from and the to the Saccharomyces Saccharomyces strains were all from and the to the Saccharomyces Saccharomyces is a of in the been R. Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Saccharomyces strains were all from and the to the Saccharomyces in a cycle were in of as of was from a in to a of was to at a of M was from a in to a of cycle were by of the cells the desired for and large for and were from the and by a to at the desired cells were to a as described previously E. Cell. Full Text PDF PubMed Scopus Google Scholar). were the meiotic of yeast described M. R.W. Esposito Nat. 2000; PubMed Scopus Google Scholar). of was by by and all were from Science. and were in by to and to The was and enzyme of Yeast for the 11 previously identified APC subunits as as SWM1 and MND2 were amplified by PCR DNA and yeast DNA from as the enzyme to or The and of APC subunits containing an 3× FLAG or a in and translation The and of all and the of the were by DNA cells from were with and in of APC and containing and were at or were for with in a between for was by for at and a for at The was with of for were by for with of APC to a and an additional with of APC APC was by two at with of APC containing 3× FLAG were and APC was with of the an from was two equal APC was purified as described the other APC was purified an identical of that been by with of 3× FLAG in APC for the to the extract. the two preparations were from the were by and with were with a and to a were a mass were with and to the for protein by mass in this from the of S. cerevisiae a was by mass from the were to a mass to the of the in and translation were to the APC subunits, containing the 3× FLAG and the other containing a were coexpressed for at in containing of protein was by and of the was to with and The was a by and the was with of for at The was with of and specifically protein was with of 3× FLAG in at proteins were by and to and the presence of the protein was by with were and with to that the were of proteins in the was also by of the reaction. growth of strains of or MND2 as as the of were in of at were in and to at for growth at were to and for were to identical of for or for and at the points were were to cell for of growth from were at and for by cells from of were with of and in of at were with of and for at with and in with of cells were in of containing DNA was a of G1, S, and G2/M cells were We constructed a yeast the protein with a carboxyl-terminal 3× FLAG from its chromosomal for of the anaphase-promoting complex. In our preparations of APC from we identified all 11 known APC subunits by mass or as as two other that been previously described as APC subunits We also observed these two proteins associated with APC in preparations from a yeast containing a the APC be that the conditions for the of APC include a high salt at all the high salt of the APC. these proteins were specifically associated with our purified APC as to associated with the we a in the were with the 3× FLAG Both proteins were by the that their presence was to interaction with the APC. The two proteins, Mnd2 and Swm1, have been in meiosis, at (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). is known provide evidence that Mnd2 and Swm1 are subunits of the APC, we constructed yeast strains 3× of Mnd2 and Swm1 and We whole cell from these strains to the with in these preparations were identified from by mass In we identified of the 11 known APC subunits co-purifying with the epitope-tagged protein Apc11 and were these two small subunits for to the number of these results demonstrate that Mnd2 and Swm1 are associated with the core APC in haploid yeast cells. its the APC is a 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). Mnd2 and Swm1 are also associated with the APC cell cycle APC was from cell arrested in with in with and in M with Mnd2 and Swm1 were identified by mass spectrometry and were present in equal at all cell cycle with to the of the other APC subunits, from the of results the other 11 Mnd2 and Swm1 are associated with the APC and can be core the that of MND2 and SWM1 previously been associated with 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 a diploid expressing with of the FLAG to Mnd2 and Swm1 are components of the APC The diploid cells were to in a manner to a previously described E. Cell. Full Text PDF PubMed Scopus Google Scholar), and cells were M. R.W. Esposito Nat. 2000; PubMed Scopus Google Scholar). APC was purified from the meiotic cells our and the subunits were identified by mass spectrometry. Both Mnd2 and Swm1 were present with the other known APC we the that Mnd2 or Swm1 from the APC at a specific of to an our results the that they associated with the APC the We an interaction of two APC subunits, with a in an E. in vitro and translation system In this APC is as a with the 3× FLAG and the is as a with a the the containing the FLAG is purified and the presence or of the is by with in the is of a interaction between the two subunits that results in their We Mnd2 and Swm1 for with as many of the other APC subunits as including and with we by with a series of APC In this interacted with and The also a interaction with the was the other and in of the that was most of the other subunits and we that this a we were to a of expressing in the we a of APC subunits to its interaction In this interacted with and to the of these that the in the was of and with the the other the and were of and we can that Swm1 interacts with Cdc23 and We were to with Apc11 in this system consistently results we were to the or we that or in the system. we were to at a of the that are responsible for the of Swm1 and Mnd2 with the APC in in APC function in cell cycle in metaphase or a in of the cell cycle We the growth of swm1Δ and mnd2Δ haploid strains to strains containing of two other APC and as as the and swm1Δ all slow growth the growth was the slow growth phenotypes of and mnd2Δ were with the the of the swm1Δ growth was at to the of In an to the of the slow growth and cells from of the strains were the growth at and their DNA was by a and accumulation of G2/M cells with the and consistent with the anaphase previously for apc9Δ (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). an accumulation of G2/M cells that was comparable in with the apc9Δ that a in mitotic The accumulation of G2/M cells in was that or as as be its the of as as M or an cell a in the of G2/M cells with the The of for the and G2/M of and swm1Δ as as their in these strains with the our evidence that Swm1 is a of the APC, is to cells have a in mitosis from an APC that is in their slow growth and accumulation of G2/M cells in we the that Mnd2 Swm1 have cellular functions that growth and of cells and M our results are consistent with the that Mnd2 and Swm1 are constitutive components of the yeast APC that to APC of G2/M from and strains are with the of the and from at that are in C. were by in a strains are with the of the and from at that are in C. were by In this we have evidence that Mnd2 and Swm1 are constitutive core subunits of the budding yeast APC. Swm1 is to a protein that was previously observed in an APC (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) identified at the the total number of subunits in budding yeast APC to 13. to two previously unidentified subunits of the APC the of mass spectrometry as a and for and the of the 3× FLAG for of protein Another the of Mnd2 and Swm1 by mass spectrometry and the A. J.L. A.J. Curr. Biol. 2002; 12: Full Text Full Text PDF PubMed Scopus Google Scholar). allows a rapid preparation that can be in a single and high and as by the of and the to proteins by in of the include the small size of the 3× FLAG with other to protein and the of specific for allows the to be at high salt In to Mnd2 and Swm1 as components of the APC throughout the cell we have the that Mnd2 and Swm1 with the complex a interaction an E. in vitro and translation system of the E. system is the of APC that can with interaction eukaryotic In the of Mnd2 and Swm1, we have an important in the of the complex that be important for understanding the and function of the APC. We have that mnd2Δ and swm1Δ haploid strains have mitotic phenotypes consistent with an APC including growth and accumulation of G2/M cells these a of Mnd2 and Swm1 to the mitotic function of the APC, they are essential as are the of APC is in to diploid yeast in Mnd2 and Swm1 are essential for mnd2Δ diploid to meiotic division 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 a swm1Δ diploid in to (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). The for Mnd2 and Swm1 in mitosis for the APC functions and so many are two for the in mitotic and meiotic The most is that Mnd2 and Swm1 provide an essential function for the APC that is that evidence is that Mnd2 and Swm1 essential functions most of the APC subunits are essential for the of function the APC by and swm1Δ mitotic and meiotic phenotypes been previously the of APC in as as its in functions for APC subunits include recruitment of or of APC specific in response to or cellular for proper meiotic function of APC. yeast APC is known to with a substrate M.J. M. R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google Scholar). Mnd2 or Swm1 are involved in the of or recruitment of a yeast was identified as an of APC that division and S. J. Sci. 2001; PubMed Google Scholar), and a in budding yeast also been identified as a of APC M. P. S. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). is that Mnd2 or Swm1 be involved in the interaction of APC with these proteins as provide of APC function in mitosis meiosis, and may also to specific functions for the other APC subunits and in specific function for Mnd2 and Swm1 a the of their in The of mnd2Δ haploid yeast is and is that of Mnd2 in mitosis the of the complex to or some other of the APC. the slow growth of swm1Δ haploid yeast is at and comparable at consistent with a in complex that be at is to the that been to in complex at is for growth at to be almost entirely for growth at (5Zachariae W. Shin T.H. Galova M. Obermaier B. Nasmyth K. Science. 1996; 274: 1201-1204Crossref PubMed Scopus (233) Google Scholar). Mnd2 and Swm1 function may be specifically for the polyubiquitination of a substrate or of degradation is essential for only a in of ubiquitin ligase of APC Mnd2 or Swm1 their presence is specifically for or in some manner to APC function for Mnd2 and Swm1 a the of Mnd2 and Swm1 of is in 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, S. J. M. J. Science. 1998; PubMed Scopus Google Scholar), is (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, S. J. M. J. Science. 1998; PubMed Scopus Google Scholar). most of the APC subunits demonstrate a in as M. R.W. Esposito Nat. 2000; PubMed Scopus Google Scholar, S. J. M. J. Science. 1998; PubMed Scopus Google Scholar), that the of the complex are The for of the APC is We the components of meiotic APC of the complex from diploid cells and found that Mnd2 and Swm1 associated with the APC is consistent with the that an is responsible for the meiotic phenotypes in strains functional Mnd2 and Swm1. Mnd2 and Swm1 have (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) homology to subunits of the S. APC was A. J.L. A.J. Curr. Biol. 2002; 12: Full Text Full Text PDF PubMed Scopus Google Scholar), the of homology the that organisms may APC at the that the APC is essential for of some of the most fundamental cellular processes to all eukaryotic cells. is that have been to the ubiquitin ligase of the APC to function in other of cellular that are highly from Mnd2 and Swm1, budding yeast Apc9 (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) to have in other and a subunit, 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), that to have a in yeast. the large number of subunits in a large number of roles in the APC as a of enzyme complex with a catalytic core involving and Apc11 and a of other subunits to the ubiquitin ligase to specific at specific and the Another of the of Mnd2 and Swm1 is the that other subunits be unidentified in other organisms such as of be of other APC subunits in eukaryotes and understanding the roles of the numerous APC subunits that to this have known We Dr. Xiong for the and for the We and Dr. of the of for with and We are to the of for to the and We also Dr. Toshio Tsukiyama for of the p3FLAG-KanMX and the of Dr. and Dr. for yeast strains and
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