The cdc2+ gene product p34cdc2 is located immunocytochemically in both the nucleus and cytoplasm of human cells. It is uniformly distributed throughout the cytoplasm and is irregularly distributed in the nucleus. Part of p34cdc2 is associated with the centrosome and centrosomal staining increases late in the cell cycle and at the onset of mitosis. This distribution is corroborated by cell fractionation which also indicates that slower migrating forms of p34cdc2 are found in isolated centrosomes and in Triton-insoluble fractions. We propose that one role of the p34Cdc2 protein kinase is to modify the centrosome bringing about formation of the mitotic spindle. At anaphase p34cdc2 becomes associated with vesicles in the middle of the cell between the reforming nuclei. A similar location is found for pl3SUCl and we suggest that the vesicular localization plays a role in p34cdc2 kinase inactivation at the end of mitosis.
Entry of a cell into mitosis induces a series of structural and functional changes including arrest of intracellular transport. Knowledge of how the mitotic cycle is driven progressed substantially with the identification of the p34cdc2 protein kinase as a subunit of maturation-promoting factor, the universal regulating component of the mitotic cycle. Activation of the kinase at the onset of mitosis is thought to trigger the important mitotic events by phosphorylating key proteins. Small guanine nucleotide-binding proteins have been implicated in regulating transport pathways. For instance, two small Ras-related GTP-binding proteins, Sec4p and Ypt1p, control distinct stages of the secretory pathway in budding yeast. The GTP-binding proteins of the Rab family in rats and humans display strong homologies with Sec4p and Ypt1p, and might therefore also be involved in regulating intracellular transport. Indeed, distinct Rab proteins are located in the exocytotic and endocytotic compartments. Interruption of vesicular transport during mitosis might involve modification of these proteins. We now present biochemical evidence for a mitosis-specific p34cdc2 phosphorylation of Rab1Ap and Rab4p. By contrast, Rab2p and Rab6p are not phosphorylated. We also show that the distribution of Rab1Ap and Rab4p between cytosolic and membrane-bound forms is different in interphase and mitotic cells. This may provide a clue to the mechanism by which phosphorylation could affect membrane traffic during mitosis.
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 inbibitor Pds1. Stabilization of Pds1 is partially responsible for the metaphase arrest phenotype of cks1 mutants because deletion of PDS1 partially releaves the metaphase block in these mutants.
The mitotic cyclin Clb2 plays a major role in promoting M-phase in budding yeast, despite its functional redundancy with three closely related cyclins Clb1, Clb3 and Clb4. Here, we further investigate the mechanisms controlling the cellular distribution of Clb2 in living cells. In agreement with observations recently made by Hood et al. [Hood, J. K., Hwang, W. W. and Silver, P. A. (2001) J. Cell Sci. 114, 589-597], we find that GFP-tagged Clb2 expressed from its natural promoter localizes to various cellular compartments, including the nucleus, the mitotic spindle, the spindle pole bodies as well as the mother-bud neck. The neck localization is specific to Clb2 as Clb1, Clb3 and Clb4 are never observed there, even when over-expressed. Mutational analysis identifies a central region of Clb2, comprising residues 213-255 and a phylogenetically conserved hydrophobic patch, as an essential cis-acting determinant. Clb2 co-localizes with the bud site selection protein Bud3. Consistent with a role of Bud3 in targeting Clb2 to the bud neck, we report a two-hybrid interaction between these proteins. Furthermore, Clb2 is shown to be specifically delocalized in Δbud3 cells and in a bud3 mutant deleted for its C-terminal Clb2-interacting domain (bud3Δ1221), but not in a Δbud10 mutant. Correlating with this phenotype, bud3Δ1221 cells exhibit a pronounced (15-30 minutes) delay in cytokinesis and/or cell separation, suggesting an unanticipated function of Clb2 in these late mitotic events. Taken together, our data uncover a new role for Bud3 in cytokinesis that correlates with its capacity to target Clb2 at the neck, independently of its well established cell-type-specific function in bud site selection.
By selectively eliminating ubiquitin-conjugated proteins, the 26S proteasome plays a pivotal role in a large variety of cellular regulatory processes, particularly in the control of cell cycle transitions. Access of ubiquitinated substrates to the inner catalytic chamber within the 20S core particle is mediated by the 19S regulatory particle (RP), whose subunit composition in budding yeast has been recently elucidated. In this study, we have investigated the cell cycle defects resulting from conditional inactivation of one of these RP components, the essential non-ATPase Rpn3/Sun2 subunit. Using temperature-sensitive mutant alleles, we show that rpn3 mutations do not prevent the G 1 /S transition but cause a metaphase arrest, indicating that the essential Rpn3 function is limiting for mitosis. rpn3 mutants appear severely compromised in the ubiquitin-dependent proteolysis of several physiologically important proteasome substrates. Thus, RPN3 function is required for the degradation of the G 1 -phase cyclin Cln2 targeted by SCF; the S-phase cyclin Clb5, whose ubiquitination is likely to involve a combination of E3 (ubiquitin protein ligase) enzymes; and anaphase-promoting complex targets, such as the B-type cyclin Clb2 and the anaphase inhibitor Pds1. Our results indicate that the Pds1 degradation defect of the rpn3 mutants most likely accounts for the metaphase arrest phenotype observed. Surprisingly, but consistent with the lack of a G 1 arrest phenotype in thermosensitive rpn3 strains, the Cdk inhibitor Sic1 exhibits a short half-life regardless of the RPN3 genotype. In striking contrast, Sic1 turnover is severely impaired by a temperature-sensitive mutation in RPN12/NIN1, encoding another essential RP subunit. While other interpretations are possible, these data strongly argue for the requirement of distinct RP subunits for efficient proteolysis of specific cell cycle regulators. The potential implications of these data are discussed in the context of possible Rpn3 function in multiubiquitin-protein conjugate recognition by the 19S proteasomal regulatory particle.
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