Budding yeast RSI1/APC2, a novel gene necessary for initiation of anaphase, encodes an APC subunit.

SIC1 is a non-essential gene encoding a CDK inhibitor of Cdc28-Clb kinase activity. Sic1p is involved in both mitotic exit and the timing of DNA synthesis. To identify other genes involved in controlling Clb-kinase activity, we have undertaken a genetic screen for mutations which render SIC1 essential. Here we describe a gene we have identified by this means, RSI1/APC2. Temperature-sensitive rsi1/apc2 mutants arrest in metaphase and are unable to degrade Clb2p, suggesting that Rsi1p/Apc2p is associated with the anaphase promoting complex (APC). This is an E3 ubiquitin-ligase that controls anaphase initiation through degradation of Pds1p and mitotic exit via degradation of Clb cyclins. Indeed, the anaphase block in rsi1/apc2 temperature-sensitive mutants is overcome by removal of PDS1, consistent with Rsi1p/Apc2p being part of the APC. In addition, like our rsi1/apc2 mutations, cdc23-1, encoding a known APC subunit, is also lethal with sic1Delta. Thus SIC1 clearly becomes essential when APC function is compromised. Finally, we find that Rsi1p/Apc2p co-immunoprecipitates with Cdc23p. Taken together, our results suggest that RSI1/APC2 is a subunit of APC.     

Doc1 mediates the activity of the anaphase-promoting complex by contributing to substrate recognition

The anaphase-promoting complex (APC) is a multisubunit E3 ubiquitin ligase that targets specific cell cycle-related proteins for degradation, regulating progression from metaphase to anaphase and exit from mitosis. The APC is regulated by binding of the coactivator proteins Cdc20p and Cdh1p, and by phosphorylation. We have developed a purification strategy that allowed us to purify the budding yeast APC to near homogeneity and identify two novel APC-associated proteins, Swm1p and Mnd2p. Using an in vitro ubiquitylation system and a native gel binding assay, we have characterized the properties of wild-type and mutant APC. We show that both the D and KEN boxes contribute to substrate recognition and that coactivator is required for substrate binding. APC lacking Apc9p or Doc1p/Apc10 have impaired E3 ligase activities. However, whereas Apc9p is required for structural stability and the incorporation of Cdc27p into the APC complex, Doc1p/Apc10 plays a specific role in substrate recognition by APC-coactivator complexes. These results imply that Doc1p/Apc10 may play a role to regulate the binding of specific substrates, similar to that of the coactivators.     

Mnd2, an essential antagonist of the anaphase-promoting complex during meiotic prophase

Meiotic cohesin serves in sister chromatid linkage and DNA repair until its subunit Rec8 is cleaved by separase. Separase is activated when its inhibitor, securin, is polyubiquitinated by the Cdc20 regulated anaphase-promoting complex (APC(Cdc20)) and consequently degraded. Differently regulated APCs (APC(Cdh1), APC(Ama1)) have not been implicated in securin degradation at meiosis I. We show that Mnd2, a factor known to associate with APC components, prevents premature securin degradation in meiosis by APC(Ama1). mnd2Delta cells lack linear chromosome axes and exhibit precocious sister chromatid separation, but deletion of AMA1 suppresses these defects. Besides securin, Sgo1, a protein essential for protection of centromeric cohesion during anaphase I, is also destabilized in mnd2delta cells. Mnd2's disappearance prior to anaphase II may activate APC(Ama1). Human oocytes may spend many years in meiotic prophase before maturation. Inhibitors of meiotic APC variants could prevent loss of chiasmata also in these cells, thereby guarding against aberrant chromosome segregation.     

Swm1/Apc13 is an evolutionarily conserved subunit of the anaphase-promoting complex stabilizing the association of Cdc16 and Cdc27

The anaphase-promoting complex (APC/C) is a large ubiquitin-protein ligase which controls progression through anaphase by triggering the degradation of cell cycle regulators such as securin and B-type cyclins. The APC/C is an unusually complex ligase containing at least 10 different, evolutionarily conserved components. In contrast to APC/C's role in cell cycle regulation little is known about the functions of individual subunits and how they might interact with each other. Here, we have analyzed Swm1/Apc13, a small subunit recently identified in the budding yeast complex. Database searches revealed proteins related to Swm1/Apc13 in various organisms including humans. Both the human and the fission yeast homologues are associated with APC/C subunits, and they complement the phenotype of an SWM1 deletion mutant of budding yeast. Swm1/Apc13 promotes the stable association with the APC/C of the essential subunits Cdc16 and Cdc27. Accordingly, Swm1/Apc13 is required for ubiquitin ligase activity in vitro and for the timely execution of APC/C-dependent cell cycle events in vivo.     

Novel interaction between Apc5p and Rsp5p in an intracellular signaling pathway in Saccharomyces cerevisiae

The ubiquitin-targeting pathway is evolutionarily conserved and critical for many cellular functions. Recently, we discovered a role for two ubiquitin-protein ligases (E3s), Rsp5p and the Apc5p subunit of the anaphase-promoting complex (APC), in mitotic chromatin assembly in Saccharomyces cerevisiae. In the present study, we investigated whether Rsp5p and Apc5p interact in an intracellular pathway regulating chromatin remodeling. Our genetic studies strongly suggest that Rsp5p and Apc5p do interact and that Rsp5p acts upstream of Apc5p. Since E3 enzymes typically require the action of a ubiquitin-conjugating enzyme (E2), we screened E2 mutants for chromatin assembly defects, which resulted in the identification of Cdc34p and Ubc7p. Cdc34p is the E2 component of the SCF (Skp1p/Cdc53p/F-box protein). Therefore, we analyzed additional SCF mutants for chromatin assembly defects. Defective chromatin assembly extracts generated from strains harboring a mutation in the Cdc53p SCF subunit or a nondegradable SCF target, Sic1(Deltaphos), confirmed that the SCF was involved in mitotic chromatin assembly. Furthermore, we demonstrated that Ubc7p physically and genetically interacts with Rsp5p, suggesting that Ubc7p acts as an E2 for Rsp5p. However, rsp5CA and Deltaubc7 mutations had opposite genetic effects on apc5CA and cdc34-2 phenotypes. Therefore, the antagonistic interplay between Deltaubc7 and rsp5CA, with respect to cdc34-2 and apc5CA, indicates that the outcome of Rsp5p's interaction with Cdc34p and Apc5p may depend on the E2 interacting with Rsp5p.     

Apc10 and Ste9/Srw1, two regulators of the APC-cyclosome, as well as the CDK inhibitor Rum1 are required for G1 cell-cycle arrest in fission yeast.

Many eukaryotic cells arrest the cell cycle at G1 phase upon nutrient deprivation. In fission yeast, during nitrogen starvation, cells divide twice and arrest at G1. We have isolated a novel type of sterile mutant, which undergoes one additional S phase upon starvation and, as a result, arrests at G2. Three loci (apc10, ste9/srw1 and rum1) were identified. The apc10 mutants, previously unidentified, show, in addition to sterility, temperature-sensitive growth with defects in chromosome segregation. apc10(+) is essential for viability, encodes a conserved protein (a homologue of budding yeast Apc10/Doc1) and is required for ubiquitination and degradation of mitotic B-type cyclins. Apc10 does not co-sediment with the 20S APC-cyclosome, a ubiquitin ligase for B-type cyclins, and in the apc10 mutant the 20S complex is intact, suggesting that it is a novel regulator for this complex. A subpopulation of Apc10 does co-immunoprecipitate with the anaphase-promoting complex (APC). A second gene, ste9(+)/srw1(+), encodes a member of the fizzy-related family, also regulators of the APC. Finally, Rum1 is a cyclin-dependent kinase (CDK) inhibitor which exists only in G1. The results suggest that dual downregulation of CDK, one via the APC and the other via the CDK inhibitor, is a universal mechanism that is used to arrest cell cycle progression at G1.     

Genome-wide studies of histone demethylation catalysed by the fission yeast homologues of mammalian LSD1

In order to gain a more global view of the activity of histone demethylases, we report here genome-wide studies of the fission yeast SWIRM and polyamine oxidase (PAO) domain homologues of mammalian LSD1. Consistent with previous work we find that the two S. pombe proteins, which we name Swm1 and Swm2 (after SWIRM1 and SWIRM2), associate together in a complex. However, we find that this complex specifically demethylates lysine 9 in histone H3 (H3K9) and both up- and down-regulates expression of different groups of genes. Using chromatin-immunoprecipitation, to isolate fragments of chromatin containing either H3K4me2 or H3K9me2, and DNA microarray analysis (ChIP-chip), we have studied genome-wide changes in patterns of histone methylation, and their correlation with gene expression, upon deletion of the swm1(+) gene. Using hyper-geometric probability comparisons we uncover genetic links between lysine-specific demethylases, the histone deacetylase Clr6, and the chromatin remodeller Hrp1. The data presented here demonstrate that in fission yeast the SWIRM/PAO domain proteins Swm1 and Swm2 are associated in complexes that can remove methyl groups from lysine 9 methylated histone H3. In vitro, we show that bacterially expressed Swm1 also possesses lysine 9 demethylase activity. In vivo, loss of Swm1 increases the global levels of both H3K9me2 and H3K4me2. A significant accumulation of H3K4me2 is observed at genes that are up-regulated in a swm1 deletion strain. In addition, H3K9me2 accumulates at some genes known to be direct Swm1/2 targets that are down-regulated in the swm1Delta strain. The in vivo data indicate that Swm1 acts in concert with the HDAC Clr6 and the chromatin remodeller Hrp1 to repress gene expression. In addition, our in vitro analyses suggest that the H3K9 demethylase activity requires an unidentified post-translational modification to allow it to act. Thus, our results highlight complex interactions between histone demethylase, deacetylase and chromatin remodelling activities in the regulation of gene expression.     

Mnd1/Hop2 facilitates Dmc1-dependent interhomolog crossover formation in meiosis of budding yeast

During meiosis, each chromosome must pair with its homolog and undergo meiotic crossover recombination in order to segregate properly at the first meiotic division. Recombination in meiosis in Saccharomyces cerevisiae relies on two Escherichia coli recA homologs, Rad51 and Dmc1, as well as the more recently discovered heterodimer Mnd1/Hop2. Meiotic recombination in S. cerevisiae mnd1 and hop2 single mutants is initiated via double-strand breaks (DSBs) but does not progress beyond this stage; heteroduplex DNA, joint molecules, and crossovers are not detected. Whereas hop2 and mnd1 single mutants are profoundly recombination defective, we show that mnd1 rad51, hop2 rad51, and mnd1 rad17 double mutants are able to carry out crossover recombination. Interestingly, noncrossover recombination is absent, indicating a role for Mnd1/Hop2 in the designation of DSBs for noncrossover recombination. We demonstrate that in the rad51 mnd1 double mutant, recombination is more likely to occur between repetitive sequences on nonhomologous chromosomes. Our results support a model in which Mnd1/Hop2 is required for DNA-DNA interactions that help ensure Dmc1-mediated stable strand invasion between homologous chromosomes, thereby preserving genomic integrity.     

Mitotic phosphorylation of the anaphase-promoting complex inhibitory subunit Mnd2 is necessary for efficient progression through meiosis i

The yeast anaphase-promoting complex (APC) subunit Mnd2 is necessary for maintaining sister chromatid cohesion in prophase I of meiosis by inhibiting premature ubiquitination and subsequent degradation of substrates by the APC(Ama1) ubiquitin ligase. In a proteomics screen for post-translational modifications on the APC, we discovered that Mnd2 is phosphorylated during mitosis in a cell cycle-dependent manner. We identified and characterized the sites of mitotic Mnd2 phosphorylation during the cell cycle. Collective mutation of Mnd2 phosphorylation sites to alanine had no effect on vegetative growth but a striking effect (>85% reduction) on the percentage of tetrad-forming cells compared with the wild type strain. Similar to the MND2 deletion strain, cells harboring the alanine mutant that did not form spores arrested after premeiotic S phase with a single undivided nucleus and low levels of the APC(Ama1) meiotic substrate, Clb5, relative to wild type cells. In contrast, collective mutation of Mnd2 phosphorylation sites to aspartic acid resulted in partial suppression of the sporulation defect. No differences were observed in the binding between each Mnd2 isoform and the APC in vitro. However, in vivo, we observed a gradient in the abundance of APC-associated Mnd2 in each strain that was proportional to the observed differences in sporulation and Clb5 levels. Taken together, these data suggest that mitotic phosphorylation of Mnd2 is necessary for APC-mediated progression beyond the first meiotic nuclear division.     

Deletion of Swm2p selectively impairs trimethylation of snRNAs by trimethylguanosine synthase (Tgs1p)

The 5′ cap trimethylation of small nuclear (snRNAs) and several nucleolar RNAs (snoRNAs) by trimethylguanosine synthase 1 (Tgs1p) is required for efficient pre-mRNA splicing. The previously uncharacterised protein Swm2p interacted with Tgs1p in yeast two-hybrid screens. In the present study we show that Swm2p interacts with the N-terminus of Tgs1p and its deletion impairs pre-mRNA splicing and pre-rRNA processing. The trimethylation of spliceosomal snRNAs and the U3 snoRNA, but not other snoRNAs, was abolished in the absence of Swm2p, indicating that Swm2p is required for a substrate-specific activity of Tgs1p.

Structured summary

MINT-7949608: p53 (uniprotkb:P02340) physically interacts (MI:0915) with large T-antigen (uniprotkb:P03070) by two-hybrid (MI:0018)MINT-7949574: swm2 (uniprotkb:P40342) physically interacts (MI:0915) with swm2 (uniprotkb:P40342) by pull down (MI:0096)MINT-7949556: swm2 (uniprotkb:P40342) physically interacts (MI:0915) with TGS1 (uniprotkb:Q12052) by pull down (MI:0096)MINT-7949587: swm2 (uniprotkb:P40342) physically interacts (MI:0915) with tgs1 (uniprotkb:Q12052) by two-hybrid (MI:0018)MINT-7949641: nop1 (uniprotkb:P15646) colocalizes (MI:0403) with TGS1 (uniprotkb:Q12052) by fluorescence microscopy (MI:0416)MINT-7949627: swm2 (uniprotkb:P40342) and nop1 (uniprotkb:P15646) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7949540: swm2 (uniprotkb:P40342) physically interacts (MI:0915) with TGS1 (uniprotkb:Q12052) by tandem affinity purification (MI:0676)     

The APC11 RING-H2 finger mediates E2-dependent ubiquitination

Polyubiquitination marks proteins for degradation by the 26S proteasome and is carried out by a cascade of enzymes that includes ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). The anaphase-promoting complex or cyclosome (APC/C) comprises a multisubunit ubiquitin ligase that mediates mitotic progression. Here, we provide evidence that the Saccharomyces cerevisiae RING-H2 finger protein Apc11 defines the minimal ubiquitin ligase activity of the APC. We found that the integrity of the Apc11p RING-H2 finger was essential for budding yeast cell viability, Using purified, recombinant proteins we showed that Apc11p interacted directly with the Ubc4 ubiquitin conjugating enzyme (E2). Furthermore, purified Apc11p was capable of mediating E1- and E2-dependent ubiquitination of protein substrates, including Clb2p, in vitro. The ability of Apc11p to act as an E3 was dependent on the integrity of the RING-H2 finger, but did not require the presence of the cullin-like APC subunit Apc2p. We suggest that Apc11p is responsible for recruiting E2s to the APC and for mediating the subsequent transfer of ubiquitin to APC substrates in vivo.     

Smt3/SUMO and Ubc9 are required for efficient APC/C-mediated proteolysis in budding yeast

Ubiquitin-mediated proteolysis triggered by the anaphase-promoting complex/cyclosome (APC/C) is essential for sister chromatid separation and the mitotic exit. Like ubiquitylation, protein modification with the small ubiquitin-related modifier SUMO appears to be important during mitosis, because yeast cells impaired in the SUMO-conjugating enzyme Ubc9 were found to be blocked in mitosis and defective in cyclin degradation. Here, we analysed the role of SUMOylation in the metaphase/anaphase transition and in APC/C-mediated proteolysis in Saccharomyces cerevisiae. We show that cells depleted of Ubc9 or Smt3, the yeast SUMO protein, mostly arrested with undivided nuclei and with high levels of securin Pds1. This metaphase block was partially relieved by a deletion of PDS1. The absence of Ubc9 or Smt3 also resulted in defects in chromosome segregation. Temperature-sensitive ubc9-2 mutants were delayed in proteolysis of Pds1 and of cyclin Clb2 during mitosis. The requirement of SUMOylation for APC/C-mediated degradation was tested more directly in G1-arrested cells. Both ubc9-2 and smt3-331 mutants were defective in efficient degradation of Pds1 and mitotic cyclins, whereas proteolysis of unstable proteins that are not APC/C substrates was unaffected. We conclude that SUMOylation is needed for efficient proteolysis mediated by APC/C in budding yeast.     

Avl9p, a member of a novel protein superfamily, functions in the late secretory pathway

The branching of exocytic transport routes in both yeast and mammalian cells has complicated studies of the late secretory pathway, and the mechanisms involved in exocytic cargo sorting and exit from the Golgi and endosomes are not well understood. Because cargo can be sorted away from a blocked route and secreted by an alternate route, mutants defective in only one route do not exhibit a strong secretory phenotype and are therefore difficult to isolate. In a genetic screen designed to isolate such mutants, we identified a novel conserved protein, Avl9p, the absence of which conferred lethality in a vps1Delta apl2Delta strain background (lacking a dynamin and an adaptor-protein complex 1 subunit). Depletion of Avl9p in this strain resulted in secretory defects as well as accumulation of Golgi-like membranes. The triple mutant also had a depolarized actin cytoskeleton and defects in polarized secretion. Overexpression of Avl9p in wild-type cells resulted in vesicle accumulation and a post-Golgi defect in secretion. Phylogenetic analysis indicated evolutionary relationships between Avl9p and regulators of membrane traffic and actin function.     

Contribution of CAF-I to anaphase-promoting-complex-mediated mitotic chromatin assembly in Saccharomyces cerevisiae

The anaphase-promoting complex (APC) is required for mitotic progression and genomic stability. Recently, we demonstrated that the APC is also required for mitotic chromatin assembly and longevity. Here, we investigated the role the APC plays in chromatin assembly. We show that apc5(CA) mutations genetically interact with the CAF-I genes as well as ASF1, HIR1, and HIR2. When present in multiple copies, the individual CAF-I genes, CAC1, CAC2, and MSI1, suppress apc5(CA) phenotypes in a CAF-1- and Asf1p-independent manner. CAF-I and the APC functionally overlap, as cac1delta cac2delta msi1delta (caf1delta) cells expressing apc5(CA) exhibit a phenotype more severe than that of apc5(CA) or caf1delta. The Ts- phenotypes observed in apc5(CA) and apc5(CA) caf mutants may be rooted in compromised histone metabolism, as coexpression of histones H3 and H4 suppressed the Ts- defects. Synthetic genetic interactions were also observed in apc5(CA) asf1delta cells. Furthermore, increased expression of genes encoding Asf1p, Hir1p, and Hir2p suppressed the apc5(CA) Ts- defect in a CAF-I-dependent manner. Together, these results suggest the existence of a complex molecular mechanism controlling APC-dependent chromatin assembly. Our data suggest the APC functions with the individual CAF-I subunits, Asf1p, and the Hir1p and Hir2p proteins. However, Asf1p and an intact CAF-I complex are dispensable for CAF-I subunit suppression, whereas CAF-I is necessary for ASF1, HIR1, and HIR2 suppression of apc5(CA) phenotypes. We discuss the implications of our observations.     

Overexpression ofapc10 + in fission yeast can suppress the temperature sensitivity ofnuc2-663 mutant but not its sterility

One of the key cell cycle regulators, the anaphase promoting complex (APC) or cyclosome, plays a dual role during mitotic exit. By destroying anaphase inhibitors it promotes sister chromatid separation, and by destroying B-type cyclins it promotes cytokinesis and removes the replication block. Under unfavorable growth conditions, most eukaryotic cells, including the fission yeastSchizosaccharomyces pombe exit mitosis normally but are arrested in G₁ and do not enter the S phase. InS. pombe, mutations in two APC/cyclosome subunits,nuc2-663 andapc10 ^ts, cause mitotic defects at 36°C, and under nitrogen starvation at 25°C they lead to inability of stopping in G₁ and hence to sterility. To gain more insight into the mechanisms regulating APC/cyclosome activity during normal growth and under nitrogen starvation, we screened a genomic library to identify high-copy suppressors of the temperature sensitivity ofnuc2-663. Here we show that overexpression ofapc10 ⁺ allows this strain to grow at 32°C and rescues it from sensitivity to the protease inhibitor N-tosyl-L-phenylalanine chloromethyl ketone at 25°C. These observations are consistent with the proposed role for Apc10p as a positive regulator of the APC/cyclosome. However,apc10 ⁺ does not suppress the sterility ofnuc2-663 mutant cells, suggesting that it plays a specific role in APC regulation (e.g., in substrate recognition) rather than in general APC activation.     

The role of Cdh1p in maintaining genomic stability in budding yeast

Cdh1p, a substrate specificity factor for the cell cycle-regulated ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C), promotes exit from mitosis by directing the degradation of a number of proteins, including the mitotic cyclins. Here we present evidence that Cdh1p activity at the M/G(1) transition is important not only for mitotic exit but also for high-fidelity chromosome segregation in the subsequent cell cycle. CDH1 showed genetic interactions with MAD2 and PDS1, genes encoding components of the mitotic spindle assembly checkpoint that acts at metaphase to prevent premature chromosome segregation. Unlike cdh1delta and mad2delta single mutants, the mad2delta cdh1delta double mutant grew slowly and exhibited high rates of chromosome and plasmid loss. Simultaneous deletion of PDS1 and CDH1 caused extensive chromosome missegregation and cell death. Our data suggest that at least part of the chromosome loss can be attributed to kinetochore/spindle problems. Our data further suggest that Cdh1p and Sic1p, a Cdc28p/Clb inhibitor, have overlapping as well as nonoverlapping roles in ensuring proper chromosome segregation. The severe growth defects of both mad2delta cdh1delta and pds1delta cdh1dDelta strains were rescued by overexpressing Swe1p, a G(2)/M inhibitor of the cyclin-dependent kinase, Cdc28p/Clb. We propose that the failure to degrade cyclins at the end of mitosis leaves cdh1delta mutant strains with abnormal Cdc28p/Clb activity that interferes with proper chromosome segregation.     

Orthologues of the anaphase-promoting complex/cyclosome coactivators Cdc20p and Cdh1p are important for mitotic progression and morphogenesis in Candida albicans

The conserved anaphase-promoting complex/cyclosome (APC/C) system mediates protein degradation during mitotic progression. Conserved coactivators Cdc20p and Cdh1p regulate the APC/C during early to late mitosis and G(1) phase. Candida albicans is an important fungal pathogen of humans, and it forms highly polarized cells when mitosis is blocked through depletion of the polo-like kinase Cdc5p or other treatments. However, the mechanisms governing mitotic progression and associated polarized growth in the pathogen are poorly understood. In order to gain insights into these processes, we characterized C. albicans orthologues of Cdc20p and Cdh1p. Cdc20p-depleted cells were blocked in early or late mitosis with elevated levels of Cdc5p and the mitotic cyclin Clb2p, suggesting that Cdc20p is essential and has some conserved functions during mitosis. However, the yeast cells formed highly polarized buds in contrast to the large doublets of S. cerevisiae cdc20 mutants, implying a distinct role in morphogenesis. In comparison, cdh1Δ/cdh1Δ cells were viable but showed enrichment of Clb2p and Cdc5p, suggesting that Cdh1p may influence mitotic exit. The cdh1Δ/cdh1Δ phenotype was pleiotropic, consisting of normal or enlarged yeast, pseudohyphae, and some elongated buds, whereas S. cerevisiae cdh1Δ yeast cells were reduced in size. Thus, C. albicans Cdh1p may have some distinct functions. Finally, absence of Cdh1p or Cdc20p had a minor or no effect on hyphal development, respectively. Overall, the results suggest that Cdc20p and Cdh1p may be APC/C activators that are important for mitosis but also morphogenesis in C. albicans. Their novel features imply additional variations in function and underscore rewiring in the emerging mitotic regulatory networks of the pathogen.