SWM1, a developmentally regulated gene, is required for spore wall assembly in Saccharomyces cerevisiae. (1/853)

Meiosis in Saccharomyces cerevisiae is followed by encapsulation of haploid nuclei within multilayered spore walls. Formation of this spore-specific wall requires the coordinated activity of enzymes involved in the biosynthesis of its components. Completion of late events in the sporulation program, leading to spore wall formation, requires the SWM1 gene. SWM1 is expressed at low levels during vegetative growth but its transcription is strongly induced under sporulating conditions, with kinetics similar to those of middle sporulation-specific genes. Homozygous swm1Delta diploids proceed normally through both meiotic divisions but fail to produce mature asci. Consistent with this finding, swm1Delta mutant asci display enhanced sensitivity to enzymatic digestion and heat shock. Deletion of SWM1 specifically affects the expression of mid-late and late sporulation-specific genes. All of the phenotypes observed are similar to those found for the deletion of SPS1 or SMK1, two putative components of a sporulation-specific MAP kinase cascade. However, epistasis analyses indicate that Swm1p does not form part of the Sps1p-Smk1p-MAP kinase pathway. We propose that Swm1p, a nuclear protein, would participate in a different signal transduction pathway that is also required for the coordination of the biochemical and morphological events occurring during the last phase of the sporulation program.  (+info)

Inhibitory phosphorylation of the APC regulator Hct1 is controlled by the kinase Cdc28 and the phosphatase Cdc14. (2/853)

BACKGROUND: Exit from mitosis requires inactivation of mitotic cyclin-dependent kinases (CDKs). A key mechanism of CDK inactivation is ubiquitin-mediated cyclin proteolysis, which is triggered by the late mitotic activation of a ubiquitin ligase known as the anaphase-promoting complex (APC). Activation of the APC requires its association with substoichiometric activating subunits termed Cdc20 and Hct1 (also known as Cdh1). Here, we explore the molecular function and regulation of the APC regulatory subunit Hct1 in Saccharomyces cerevisiae. RESULTS: Recombinant Hct1 activated the cyclin-ubiquitin ligase activity of APC isolated from multiple cell cycle stages. APC isolated from cells arrested in G1, or in late mitosis due to the cdc14-1 mutation, was more responsive to Hct1 than APC isolated from other stages. We found that Hct1 was phosphorylated in vivo at multiple CDK consensus sites during cell cycle stages when activity of the cyclin-dependent kinase Cdc28 is high and APC activity is low. Purified Hct1 was phosphorylated in vitro at these sites by purified Cdc28-cyclin complexes, and phosphorylation abolished the ability of Hct1 to activate the APC in vitro. The phosphatase Cdc14, which is known to be required for APC activation in vivo, was able to reverse the effects of Cdc28 by catalyzing Hct1 dephosphorylation and activation. CONCLUSIONS: We conclude that Hct1 phosphorylation is a key regulatory mechanism in the control of cyclin destruction. Phosphorylation of Hct1 provides a mechanism by which Cdc28 blocks its own inactivation during S phase and early mitosis. Following anaphase, dephosphorylation of Hct1 by Cdc14 may help initiate cyclin destruction.  (+info)

The schizosaccharomyces pombe dim1(+) gene interacts with the anaphase-promoting complex or cyclosome (APC/C) component lid1(+) and is required for APC/C function. (3/853)

The Schizosaccharomyces pombe dim1(+) gene is required for entry into mitosis and for chromosome segregation during mitosis. To further understand dim1p function, we undertook a synthetic lethal screen with the temperature-sensitive dim1-35 mutant and isolated lid (for lethal in dim1-35) mutants. Here, we describe the temperature-sensitive lid1-6 mutant. At the restrictive temperature of 36 degrees C, lid1-6 mutant cells arrest with a "cut" phenotype similar to that of cut4 and cut9 mutants. An epitope-tagged version of lid1p is a component of a multiprotein approximately 20S complex; the presence of lid1p in this complex depends upon functional cut9(+). lid1p-myc coimmunoprecipitates with several other proteins, including cut9p and nuc2p, and the presence of cut9p in a 20S complex depends upon the activity of lid1(+). Further, lid1(+) function is required for the multiubiquitination of cut2p, an anaphase-promoting complex or cyclosome (APC/C) target. Thus, lid1p is a component of the S. pombe APC/C. In dim1 mutants, the abundances of lid1p and the APC/C complex decline significantly, and the ubiquitination of an APC/C target is abolished. These data suggest that at least one role of dim1p is to maintain or establish the steady-state level of the APC/C.  (+info)

Reconstitution of G1 cyclin ubiquitination with complexes containing SCFGrr1 and Rbx1. (4/853)

Control of cyclin levels is critical for proper cell cycle regulation. In yeast, the stability of the G1 cyclin Cln1 is controlled by phosphorylation-dependent ubiquitination. Here it is shown that this reaction can be reconstituted in vitro with an SCF E3 ubiquitin ligase complex. Phosphorylated Cln1 was ubiquitinated by SCF (Skp1-Cdc53-F-box protein) complexes containing the F-box protein Grr1, Rbx1, and the E2 Cdc34. Rbx1 promotes association of Cdc34 with Cdc53 and stimulates Cdc34 auto-ubiquitination in the context of Cdc53 or SCF complexes. Rbx1, which is also a component of the von Hippel-Lindau tumor suppressor complex, may define a previously unrecognized class of E3-associated proteins.  (+info)

ROC1, a homolog of APC11, represents a family of cullin partners with an associated ubiquitin ligase activity. (5/853)

We have identified two highly conserved RING finger proteins, ROC1 and ROC2, that are homologous to APC11, a subunit of the anaphase-promoting complex. ROC1 and ROC2 commonly interact with all cullins while APC11 specifically interacts with APC2, a cullin-related APC subunit. YeastROC1 encodes an essential gene whose reduced expression resulted in multiple, elongated buds and accumulation of Sic1p and Cln2p. ROC1 and APC11 immunocomplexes can catalyze isopeptide ligations to form polyubiquitin chains in an E1- and E2-dependent manner. ROC1 mutations completely abolished their ligase activity without noticeable changes in associated proteins. Ubiquitination of phosphorylated I kappa B alpha can be catalyzed by the ROC1 immunocomplex in vitro. Hence, combinations of ROC/APC11 and cullin proteins proteins potentially constitute a wide variety of ubiquitin ligases.  (+info)

Characterization of the DOC1/APC10 subunit of the yeast and the human anaphase-promoting complex. (6/853)

The anaphase-promoting complex/cyclosome (APC) is a ubiquitin-protein ligase whose activity is essential for progression through mitosis. The vertebrate APC is thought to be composed of 8 subunits, whereas in budding yeast several additional APC-associated proteins have been identified, including a 33-kDa protein called Doc1 or Apc10. Here, we show that Doc1/Apc10 is a subunit of the yeast APC throughout the cell cycle. Mutation of Doc1/Apc10 inactivates the APC without destabilizing the complex. An ortholog of Doc1/Apc10, which we call APC10, is associated with the APC in different vertebrates, including humans and frogs. Biochemical fractionation experiments and mass spectrometric analysis of a component of the purified human APC show that APC10 is a genuine APC subunit whose cellular levels or association with the APC are not cell cycle-regulated. We have further identified an APC10 homology region, which we propose to call the DOC domain, in several protein sequences that also contain either cullin or HECT domains. Cullins are present in several ubiquitination complexes including the APC, whereas HECT domains represent the catalytic core of a different type of ubiquitin-protein ligase. DOC domains may therefore be important for reactions catalyzed by several types of ubiquitin-protein ligases.  (+info)

Cyclin-dependent kinase and Cks/Suc1 interact with the proteasome in yeast to control proteolysis of M-phase targets. (7/853)

Cell cycle-specific proteolysis is critical for proper execution of mitosis in all eukaryotes. Ubiquitination and subsequent proteolysis of the mitotic regulators Clb2 and Pds1 depend on the cyclosome/APC and the 26S proteasome. We report here that components of the cell cycle machinery in yeast, specifically the cell cycle regulatory cyclin-dependent kinase Cdc28 and a conserved associated protein Cks1/Suc1, interact genetically, physically, and functionally with components of the 26S proteasome. A mutation in Cdc28 (cdc28-1N) that interferes with Cks1 binding, or inactivation of Cks1 itself, confers stabilization of Clb2, the principal mitotic B-type cyclin in budding yeast. Surprisingly, Clb2-ubiquitination in vivo and in vitro is not affected by mutations in cks1, indicating that Cks1 is not essential for cyclosome/APC activity. However, mutant Cks1 proteins no longer physically interact with the proteasome, suggesting that Cks1 is required for some aspect of proteasome function during M-phase-specific proteolysis. We further provide evidence that Cks1 function is required for degradation of the anaphase inhibitor Pds1. Stabilization of Pds1 is partially responsible for the metaphase arrest phenotype of cks1 mutants because deletion of PDS1 partially relieves the metaphase block in these mutants.  (+info)

Sister chromatid separation and chromosome re-duplication are regulated by different mechanisms in response to spindle damage. (8/853)

In yeast, anaphase entry depends on Pds1 proteolysis, while chromosome re-duplication in the subsequent S-phase involves degradation of mitotic cyclins such as Clb2. Sequential proteolysis of Pds1 and mitotic cyclins is mediated by the anaphase-promoting complex (APC). Lagging chromosomes or spindle damage are detected by surveillance mechanisms (checkpoints) which block anaphase onset, cytokinesis and DNA re-replication. Until now, the MAD and BUB genes implicated in this regulation were thought to function in a single pathway that blocks APC activity. We show that spindle damage blocks sister chromatid separation solely by inhibiting APCCdc20-dependent Pds1 proteolysis and that this process requires Mad2. Blocking APCCdh1-mediated Clb2 proteolysis and chromosome re-duplication does not require Mad2 but a different protein, Bub2. Our data imply that Mad1, Mad2, Mad3 and Bub1 regulate APCCdc20, whereas Bub2 regulates APCCdh1.  (+info)

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