Phosphorylation of the mitotic regulator Pds1/securin by Cdc28 is required for efficient nuclear localization of Esp1/separase.
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Sister chromatid separation at the metaphase-to-anaphase transition is induced by the proteolytic cleavage of one of the cohesin complex subunits. This process is mediated by a conserved protease called separase. Separase is associated with its inhibitor, securin, until the time of anaphase initiation, when securin is degraded in an anaphase-promoting complex/cyclosome (APC/C)-dependent manner. In budding yeast securin/Pds1 not only inhibits separase/Esp1, but also promotes its nuclear localization. The molecular mechanism and regulation of this nuclear targeting are presently unknown. Here we show that Pds1 is a substrate of the cyclin-dependent kinase Cdc28. Phosphorylation of Pds1 by Cdc28 is important for efficient binding of Pds1 to Esp1 and for promoting the nuclear localization of Esp1. Our results uncover a previously unknown mechanism for regulating the Pds1-Esp1 interaction and shed light on a novel role for Cdc28 in promoting the metaphase-to-anaphase transition in budding yeast. (+info)
Transferable domain in the G(1) cyclin Cln2 sufficient to switch degradation of Sic1 from the E3 ubiquitin ligase SCF(Cdc4) to SCF(Grr1).
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Degradation of Saccharomyces cerevisiae G(1) cyclins Cln1 and Cln2 is mediated by the ubiquitin-proteasome pathway and involves the SCF E3 ubiquitin-ligase complex containing the F-box protein Grr1 (SCF(Grr1)). Here we identify the domain of Cln2 that confers instability and describe the signals in Cln2 that result in binding to Grr1 and rapid degradation. We demonstrate that mutants of Cln2 that lack a cluster of four Cdc28 consensus phosphorylation sites are highly stabilized and fail to interact with Grr1 in vivo. Since one of the phosphorylation sites lies within the Cln2 PEST motif, a sequence rich in proline, aspartate or glutamate, serine, and threonine residues found in many unstable proteins, we fused various Cln2 C-terminal domains containing combinations of the PEST and the phosphoacceptor motifs to stable reporter proteins. We show that fusion of the Cln2 domain to a stabilized form of the cyclin-dependent kinase inhibitor Sic1 (Delta N-Sic1), a substrate of SCF(Cdc4), results in degradation in a phosphorylation-dependent manner. Fusion of Cln2 degradation domains to Delta N-Sic1 switches degradation of Sic1 from SCF(Cdc4) to SCF(Grr1). Delta N-Sic1 fused with a Cln2 domain containing the PEST motif and four phosphorylation sites binds to Grr1 and is unstable and ubiquitinated in vivo. Interestingly, the phosphoacceptor domain of Cln2 binds to Grr1 but is not ubiquitinated and is stable. In summary, we have identified a small transferable domain in Cln2 that can redirect a stabilized SCF(Cdc4) target for SCF(Grr1)-mediated degradation by the ubiquitin-proteasome pathway. (+info)
The Rho-GAP Bem2p plays a GAP-independent role in the morphogenesis checkpoint.
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The Saccharomyces cerevisiae morphogenesis checkpoint delays mitosis in response to insults that impair actin organization and/or bud formation. The delay is due to accumulation of the inhibitory kinase Swe1p, which phosphorylates the cyclin-dependent kinase Cdc28p. Having screened through a panel of yeast mutants with defects in cell morphogenesis, we report here that the polarity establishment protein Bem2p is required for the checkpoint response. Bem2p is a Rho-GTPase activating protein (GAP) previously shown to act on Rho1p, and we now show that it also acts on Cdc42p, the GTPase primarily responsible for establishment of cell polarity in yeast. Whereas the morphogenesis role of Bem2p required GAP activity, the checkpoint role of Bem2p did not. Instead, this function required an N-terminal Bem2p domain. Thus, this single protein has a GAP-dependent role in promoting cell polarity and a GAP-independent role in responding to defects in cell polarity by enacting the checkpoint. Surprisingly, Swe1p accumulation occurred normally in bem2 cells, but they were nevertheless unable to promote Cdc28p phosphorylation. Therefore, Bem2p defines a novel pathway in the morphogenesis checkpoint. (+info)
An essential function of yeast cyclin-dependent kinase Cdc28 maintains chromosome stability.
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Multiple surveillance pathways maintain genomic integrity in yeast during mitosis. Although the cyclin-dependent kinase Cdc28 is a well established regulator of mitotic progression, evidence for a direct role in mitotic surveillance has been lacking. We have now implicated a conserved sequence in the Cdc28 carboxyl terminus in maintaining chromosome stability through mitosis. Six temperature-sensitive mutants were isolated via random mutagenesis of 13 carboxyl-terminal residues. These mutants identify a Cdc28 domain necessary for proper mitotic arrest in the face of kinetochore defects or microtubule inhibitors. These chromosome stability-defective cdc28(CST) mutants inappropriately continue mitosis when the mitotic spindle is disrupted at 23 degrees C, display high rates of spontaneous chromosome loss at 30 degrees C, and suffer catastrophic aneuploidy at 35 degrees C. A dosage suppression screen identified Cak1, a kinase known to phosphorylate and activate Cdc28, as a specific high copy suppressor of cdc28(CST) temperature sensitivity and chromosome instability. Suppression is independent of the kinase activity of Cak1, suggesting that Cak1 may bind to the carboxyl terminus to serve a non-catalytic role in assembly and/or stabilization of active Cdc28 complexes. Significantly, these studies implicate Cdc28 and Cak1 in an essential surveillance function required to maintain genetic stability through mitosis. (+info)
Determinants of Swe1p degradation in Saccharomyces cerevisiae.
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Swe1p, the sole Wee1-family kinase in Saccharomyces cerevisiae, is synthesized during late G1 and is then degraded as cells proceed through the cell cycle. However, Swe1p degradation is halted by the morphogenesis checkpoint, which responds to insults that perturb bud formation. The Swe1p stabilization promotes cell cycle arrest through Swe1p-mediated inhibitory phosphorylation of Cdc28p until the cells can recover from the perturbation and resume bud formation. Swe1p degradation involves the relocalization of Swe1p from the nucleus to the mother-bud neck, and neck targeting requires the Swe1p-interacting protein Hsl7p. In addition, Swe1p degradation is stimulated by its substrate, cyclin/Cdc28p, and Swe1p is thought to be a target of the ubiquitin ligase SCF(Met30) acting with the ubiquitin-conjugating enzyme Cdc34p. The basis for regulation of Swe1p degradation by the morphogenesis checkpoint remains unclear, and in order to elucidate that regulation we have dissected the Swe1p degradation pathway in more detail, yielding several novel findings. First, we show here that Met30p (and by implication SCF(Met30)) is not, in fact, required for Swe1p degradation. Second, cyclin/Cdc28p does not influence Swe1p neck targeting, but can directly phosphorylate Swe1p, suggesting that it acts downstream of neck targeting in the Swe1p degradation pathway. Third, a screen for functional but nondegradable mutants of SWE1 identified two small regions of Swe1p that are key to its degradation. One of these regions mediates interaction of Swe1p with Hsl7p, showing that the Swe1p-Hsl7p interaction is critical for Swe1p neck targeting and degradation. The other region did not appear to affect interactions with known Swe1p regulators, suggesting that other as-yet-unknown regulators exist. (+info)
Phosphorylation of the septin cdc3 in g1 by the cdc28 kinase is essential for efficient septin ring disassembly.
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The septins constitute a family of filament-forming proteins ubiquitous in eukaryotic species. We demonstrate here that the Saccharomyces cerevisiae septin, Cdc3, is a substrate of the cell cycle regulatory cyclin-dependent kinase (Cdk), Cdc28. Two serines near the C-terminus of Cdc3 are phosphorylated in a Cdc28-dependent manner. Analysis of a mutant allele that cannot be phosphorylated at these sites revealed an effect of Cdc28 phosphorylation of Cdc3 at the time of budding. Immunofluorescence analysis of wild-type and mutant Cdc3 indicated that prevention of phosphorylation at Cdc28-dependent sites impairs the disassembly of the old septin ring, which is inherited at mitosis but which usually disappears immediately prior to assembly of a new ring. Furthermore, immuno-fluorescence analysis of septin ring dynamics in a G1 cyclin (Cln) mutant suggests that G1 cyclin function is required for efficient ring disassembly. Thus, phosphorylation of Cdc3 by the Cdc28 kinase at the end of G1 may facilitate initiation of a new cell cycle by promoting disassembly of the obsolete septin ring from the previous cell cycle. (+info)
Spatial regulation of the guanine nucleotide exchange factor Lte1 in Saccharomyces cerevisiae.
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In budding yeast, activation of the small Ras-like GTPase Tem1 triggers exit from mitosis and cytokinesis. Tem1 is regulated by Bub2/Bfa1, a two-component GTPase-activating protein (GAP), and by Lte1, a putative guanine nucleotide exchange factor. Lte1 is confined to the bud cortex, and its spatial separation from Tem1 at the spindle pole body (SPB) is important to prevent untimely exit from mitosis. The pathways contributing to Lte1 asymmetry have not been elucidated. Here we show that establishment of Lte1 at the cortex occurs by an actin-independent mechanism, which requires activation of Cdc28/Cln kinase at START and Cdc42, a key regulator of cell polarity and cytoskeletal organisation. This defines a novel role for Cdc42 in late mitotic events. In turn, dissociation of Lte1 from the cortex in telophase depends on activation of the Cdc14 phosphatase. Ectopic expression of Cdc14 at metaphase results in premature dephosphorylation of Lte1 coincident with its release from the cortex. In vitro phosphatase assays confirm that Lte1 is a direct substrate for Cdc14. Our results suggest that the asymmetry in Lte1 localisation is imposed by Cdc28-dependent phosphorylation. Finally, we report a mutational analysis undertaken to investigate intrinsic Lte1 determinants for localisation. Our data suggest that an intrameric interaction between the N-and C-terminal regions of Lte1 is important for cortex association. (+info)
Rpn6p, a proteasome subunit from Saccharomyces cerevisiae, is essential for the assembly and activity of the 26 S proteasome.
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We report the functional characterization of RPN6, an essential gene from Saccharomyces cerevisiae encoding the proteasomal subunit Rpn6p. For this purpose, conditional mutants that are able to grow on galactose but not on glucose were obtained. When these mutants are shifted to glucose, Rpn6p depletion induces several specific phenotypes. First, multiubiquitinated proteins accumulate, indicating a defect in proteasome-mediated proteolysis. Second, mutant yeasts are arrested as large budded cells with a single nucleus and a 2C DNA content; in addition, the spindle pole body is duplicated, indicating a general cell cycle defect related to the turnover of G(2)-cyclins after DNA synthesis. Clb2p and Pds1p, but not Sic1p, accumulate in the arrested cells. Depletion of Rpn6p affects both the structure and the peptidase activity of proteasomes in the cell. These results implicate Rpn6p function in the specific recognition of a subset of substrates and point to a role in maintaining the correct quaternary structure of the 26 S proteasome. (+info)