Cdc28 activates exit from mitosis in budding yeast

The activity of the cyclin-dependent kinase 1 (Cdk1), Cdc28, inhibits the transition from anaphase to G1 in budding yeast. CDC28-T18V, Y19F (CDC28-VF), a mutant that lacks inhibitory phosphorylation sites, delays the exit from mitosis and is hypersensitive to perturbations that arrest cells in mitosis. Surprisingly, this behavior is not due to a lack of inhibitory phosphorylation or increased kinase activity, but reflects reduced activity of the anaphase-promoting complex (APC), a defect shared with other mutants that lower Cdc28/Clb activity in mitosis. CDC28-VF has reduced Cdc20- dependent APC activity in mitosis, but normal Hct1- dependent APC activity in the G1 phase of the cell cycle. The defect in Cdc20-dependent APC activity in CDC28-VF correlates with reduced association of Cdc20 with the APC. The defects of CDC28-VF suggest that Cdc28 activity is required to induce the metaphase to anaphase transition and initiate the transition from anaphase to G1 in budding yeast.     

Cdc15 is required for spore morphogenesis independently of Cdc14 in Saccharomyces cerevisiae

In Saccharomyces cerevisiae exit from mitosis requires the Cdc14 phosphatase to reverse CDK-mediated phosphorylation. Cdc14 is released from the nucleolus by the Cdc14 early anaphase release (FEAR) and mitotic exit network (MEN) pathways. In meiosis, the FEAR pathway is essential for exit from anaphase I. The MEN component Cdc15 is required for the formation of mature spores. To analyze the role of Cdc15 during sporulation, a conditional mutant in which CDC15 expression was controlled by the CLB2 promoter was used. Cdc15-depleted cells proceeded normally through the meiotic divisions but were unable to properly disassemble meiosis II spindles. The morphology of the prospore membrane was aberrant and failed to capture the nuclear lobes. Cdc15 was not required for Cdc14 release from the nucleoli, but it was essential to maintain Cdc14 released and for its nucleo-cytoplasmic transport. However, cells carrying a CDC14 allele with defects in nuclear export (Cdc14-DeltaNES) were able to disassemble the spindle and to complete spore formation, suggesting that the Cdc14 nuclear export defect was not the cause of the phenotypes observed in cdc15 mutants.     

Saccharomyces cerevisiae Mob1p is required for cytokinesis and mitotic exit

The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved set of genes that mediate the transition from mitosis to G(1) by regulating mitotic cyclin degradation and the inactivation of cyclin-dependent kinase (CDK). Here, we demonstrate that, in addition to mitotic exit, S. cerevisiae MEN gene MOB1 is required for cytokinesis and cell separation. The cytokinesis defect was evident in mob1 mutants under conditions in which there was no mitotic-exit defect. Observation of live cells showed that yeast myosin II, Myo1p, was present in the contractile ring at the bud neck but that the ring failed to contract and disassemble. The cytokinesis defect persisted for several mitotic cycles, resulting in chains of cells with correctly segregated nuclei but with uncontracted actomyosin rings. The cytokinesis proteins Cdc3p (a septin), actin, and Iqg1p/ Cyk1p (an IQGAP-like protein) appeared to correctly localize in mob1 mutants, suggesting that MOB1 functions subsequent to actomyosin ring assembly. We also examined the subcellular distribution of Mob1p during the cell cycle and found that Mob1p first localized to the spindle pole bodies during mid-anaphase and then localized to a ring at the bud neck just before and during cytokinesis. Localization of Mob1p to the bud neck required CDC3, MEN genes CDC5, CDC14, CDC15, and DBF2, and spindle pole body gene NUD1 but was independent of MYO1. The localization of Mob1p to both spindle poles was abolished in cdc15 and nud1 mutants and was perturbed in cdc5 and cdc14 mutants. These results suggest that the MEN functions during the mitosis-to-G(1) transition to control cyclin-CDK inactivation and cytokinesis.     

Physical interaction of Cdc28 with Cdc37 in Saccharomyces cerevisiae

The Cdc37 protein in Saccharomyces cerevisiae is thought to be a kinase-targeting subunit of the chaperone Hsp90. In a genetic screen, four protein kinases were identified as interacting with Cdc37 - Cdc5, Cdc7, Cdc15 and Cak1. This result underlines the importance of Cdc37 for the folding of protein kinases. In addition, we showed that Ydj1, a yeast DnaJ homolog belonging to the Hsp40 family of chaperones, genetically interacts with Cdc37. No physical interaction has so far been detected between Cdc37 and Cdc28, although genetic interactions (synthetic lethality and mutation suppression), and biochemical studies have suggested that these two proteins functionally interact. We found that, when separately expressed, the N-terminal lobe of Cdc28 interacted strongly with the C-terminal moiety of Cdc37 in a two-hybrid system. This was not the case for the full-length Cdc28 protein. We present models to explain these results.     

Tyrosine kinase activity may be necessary but is not sufficient for c-erbB1-mediated tissue-specific tumorigenicity.

Expression of mutant avian c-erbB1 genes results in tissue-specific transformation in chickens. Site-directed mutagenesis was used to generate kinase-defective mutants of several tissue-specific v-erbB transforming mutants by replacement of the ATP-binding lysine residue in the kinase domain with an arginine residue. These kinase-defective v-erbB mutants were analyzed for their in vitro and in vivo transforming potentials. Specifically, kinase-defective mutants of erythroleukemogenic, hemangioma-inducing, and sarcomagenic v-erbB genes were assessed for their oncogenic potential. In vitro transformation potential was assessed by soft-agar colony formation in primary cultures of chick embryo fibroblasts (CEF). In vivo transformation potential was determined by infection of 1-day-old line 0 chicks with concentrated recombinant retrovirus and then monitoring of birds for tumor formation. These transformation assays demonstrate that kinase activity is absolutely essential for transformation by tissue-specific transforming mutants of the avian c-erbB1 gene. Since all of the tissue-specific v-erbB mutants characterized to date exhibit tyrosine kinase activity in vitro but do not transform all tissues in which they are expressed, we conclude that v-erbB-associated tyrosine kinase activity may be necessary but is not sufficient to induce tumor formation.     

NDT80, a meiosis-specific gene required for exit from pachytene in Saccharomyces cerevisiae.

We describe the identification of a new meiosis-specific gene of Saccharomyces cerevisiae, NDT80. The ndt80 null and point mutants arrest at the pachytene stage of meiosis, with homologs connected by full-length synaptonemal complexes and spindle pole bodies duplicated but unseparated. Meiotic recombination in an ndt80 delta mutant is relatively normal, although commitment to heteroallelic recombination is elevated two- to threefold and crossing over is decreased twofold compared with those of the wild type. ndt80 arrest is not alleviated by mutations in early recombination genes, e.g., SPO11 or RAD50, and thus cannot be attributed to an intermediate block in prophase chromosome metabolism like that observed in several other mutants. The ndt80 mutant phenotype during meiosis most closely resembles that of a cdc28 mutant, which contains a thermolabile p34, the catalytic subunit of maturation-promoting factor. Cloning and molecular analysis reveal that the NDT80 gene maps on the right arm of chromosome VIII between EPT1 and a Phe-tRNA gene, encodes a 627-amino-acid protein which exhibits no significant homology to other known proteins, and is transcribed specifically during middle meiotic prophase. The NDT80 gene product could be a component of the cell cycle regulatory machinery involved in the transition out of pachytene, a participant in an unknown aspect of meiosis sensed by a pachytene checkpoint, or a SPO11- and RAD50-independent component of meiotic chromosomes that is the target of cell cycle signaling.     

Invariant phosphorylation of the Saccharomyces cerevisiae Cdc28 protein kinase.

The phosphorylation level of the Saccharomyces cerevisiae Cdc28 protein remained invariant under conditions that resulted in cell cycle arrest in the G1 phase and loss of Cdc28-specific protein kinase activity when the activity was assayed in vitro. These results are in contrast to the proposed regulation of the homologous Cdc2 protein kinase of Schizosaccharomyces pombe.     

DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae.

We have used a set of seven temperature-sensitive mutants in the DNA polymerase I gene of Saccharomyces cerevisiae to investigate the role of DNA polymerase I in various aspects of DNA synthesis in vivo. Previously, we showed that DNA polymerase I is required for mitotic DNA replication. Here we extend our studies to several stages of meiosis and repair of X-ray-induced damage. We find that sporulation is blocked in all of the DNA polymerase temperature-sensitive mutants and that premeiotic DNA replication does not occur. Commitment to meiotic recombination is only 2% of wild-type levels. Thus, DNA polymerase I is essential for these steps. However, repair of X-ray-induced single-strand breaks is not defective in the DNA polymerase temperature-sensitive mutants, and DNA polymerase I is therefore not essential for repair of such lesions. These results suggest that DNA polymerase II or III or both, the two other nuclear yeast DNA polymerases for which roles have not yet been established, carry out repair in the absence of DNA polymerase I, but that DNA polymerase II and III cannot compensate for loss of DNA polymerase I in meiotic replication and recombination. These results do not, however, rule out essential roles for DNA polymerase II or III or both in addition to that for DNA polymerase I.     

Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus.

The human immunodeficiency virus productively infects and integrates into cells that have been arrested in the cell cycle with either gamma irradiation or aphidicolin. Integration by oncoretroviruses such as the murine leukemia virus (MuLV), on the other hand, depends on cell proliferation. Although the entire cell cycle is not necessary for MuLV infection, it is essential that the infected cells pass through mitosis. The long terminal repeat circle junction, a marker for nuclear entry, is first observed in MuLV-infected cells immediately after mitosis. These results suggest that mitosis is necessary for nuclear entry of MuLV, but not human immunodeficiency virus, unintegrated proviral DNA.     

Fatty acylation is important but not essential for Saccharomyces cerevisiae RAS function.

Two proteins in the yeast Saccharomyces cerevisiae that are encoded by the genes RAS1 and RAS2 are structurally and functionally homologous to proteins of the mammalian ras oncogene family. We examined the role of fatty acylation in the maturation of yeast RAS2 protein by creating mutants in the putative palmitate addition site located at the carboxyl terminus of the protein. Two mutations, Cys-318 to an opal termination codon and Cys-319 to Ser-319, were created in vitro and substituted in the chromosome in place of the normal RAS2 allele. These changes resulted in a failure of RAS2 protein to be acylated with palmitate and a failure of RAS2 protein to be localized to a membrane fraction. The mutations yielded a Ras2- phenotype with respect to the ability of the resultant mutants to grow on nonfermentable carbon sources and to complement ras1- mutants. However, overexpression of the ras2Ser-319 product yielded a Ras+ phenotype without a corresponding association of the mutant protein with the membrane fraction. We conclude that the presence of a fatty acyl moiety is important for localizing RAS2 protein to the membrane where it is active but that the fatty acyl group is not an absolute requirement of RAS2 protein function.     

Inhibition of Cdc42 during mitotic exit is required for cytokinesis

The role of Cdc42 and its regulation during cytokinesis is not well understood. Using biochemical and imaging approaches in budding yeast, we demonstrate that Cdc42 activation peaks during the G₁/S transition and during anaphase but drops during mitotic exit and cytokinesis. Cdc5/Polo kinase is an important upstream cell cycle regulator that suppresses Cdc42 activity. Failure to down-regulate Cdc42 during mitotic exit impairs the normal localization of key cytokinesis regulators—Iqg1 and Inn1—at the division site, and results in an abnormal septum. The effects of Cdc42 hyperactivation are largely mediated by the Cdc42 effector p21-activated kinase Ste20. Inhibition of Cdc42 and related Rho guanosine triphosphatases may be a general feature of cytokinesis in eukaryotes.     

Nin1p, a regulatory subunit of the 26S proteasome, is necessary for activation of Cdc28p kinase of Saccharomyces cerevisiae.

The nin1-1 mutant of Saccharomyces cerevisiae cannot perform the G1/S and G2/M transitions at restrictive temperatures. At such temperatures, nin1-1 strains fail to activate histone H1 kinase after release from alpha factor-imposed G1 block and after release from hydroxyurea-imposed S block. The nin1-1 mutation shows synthetic lethality with certain cdc28 mutant alleles such as cdc28-IN. Two lines of evidence indicate that Nin1p is a component of the 26S proteasome complex: (i) Nin1p, as well as the known component of the 26S proteasome, shifted to the 26S proteasome peak in the glycerol density gradient after preincubation of crude extract with ATP-Mg2+, and (ii) nin1-1 cells accumulated polyubiquitinated proteins under restrictive conditions. These results suggest that activation of Cdc28p kinase requires proteolysis. We have cloned a human cDNA encoding a regulatory subunit of the 26S proteasome, p31, which was found to be a homolog of Nin1p.     

The role of Cdc55 in the spindle checkpoint is through regulation of mitotic exit in Saccharomyces cerevisiae

Cdc55, a B-type regulatory subunit of protein phosphatase 2A, has been implicated in mitotic spindle checkpoint activity and maintenance of sister chromatid cohesion during metaphase. The spindle checkpoint is composed of two independent pathways, one leading to inhibition of the metaphase-to-anaphase transition by checkpoint proteins, including Mad2, and the other to inhibition of mitotic exit by Bub2. We show that Cdc55 is a negative regulator of mitotic exit. A cdc55 mutant, like a bub2 mutant, prematurely releases Cdc14 phosphatase from the nucleolus during spindle checkpoint activation, and premature exit from mitosis indirectly leads to loss of sister chromatid cohesion and inviability in nocodazole. The role of Cdc55 is separable from Bub2 and inhibits release of Cdc14 through a mechanism independent of the known negative regulators of mitotic exit. Epistasis experiments indicate Cdc55 acts either downstream or independent of the mitotic exit network kinase Cdc15. Interestingly, the B-type cyclin Clb2 is partially stable during premature activation of mitotic exit in a cdc55 mutant, indicating mitotic exit is incomplete.     

Multiple telophase arrest bypassed (tab) mutants alleviate the essential requirement for Cdc15 in exit from mitosis in S. cerevisiae

Background  

The Mitotic Exit Network (MEN) proteins – including the protein kinase Cdc15 and the protein phosphatase Cdc14 – are essential for exit from mitosis in Saccharomyces cerevisiae. To identify downstream targets of the MEN, we sought telophase arrest bypassed (tab) mutations that bypassed the essential requirement for CDC15. Previous studies identified net1 ^tab2-1 and CDC14 ^TAB6-1 as mutations in the RENT complex subunits Net1 and Cdc14, respectively, and revealed that the MEN acts by promoting release of Cdc14 from its nucleolar Net1 anchor during anaphase. However, the remaining tab mutants were not characterized.     

Thioredoxin is required for vacuole inheritance in Saccharomyces cerevisiae

The vacuole of Saccharomyces cerevisiae projects a stream of tubules a and vesicles (a segregation structure) into the bud in early S phase. We have described an in vitro reaction, requiring physiological temperature, ATP, and cytosol, in which isolated vacuoles form segregation structures and fuse. This in vitro reaction is defective when reaction components are prepared from vac mutants that are defective in this process in vivo, Fractionation of the cytosol reveals at least three components, each of which can support the vacuole fusion reaction, and two stimulatory fractions. Purification of one low molecular weight activity (LMA1) yields a heterodimeric protein with a thioredoxin subunit. Most of the thioredoxin of yeast is in this complex rather than the well-studied monomer. A deletion of both S. cerevisiae thioredoxin genes causes a striking vacuole inheritance defect in vivo, establishing a role for thioredoxin as a novel factor in this trafficking reaction.     

The role of MEN (mitosis exit network) proteins in the cytokinesis of Saccharomyces cerevisiae.

At the latest stages of their cell cycle, cells carry out crucial processes for the correct segregation of their genetic and cytoplasmic material. In this work, we provide evidence demonstrating that the cell cycle arrest of some MEN (mitosis exit network) mutants in the anaphase-telophase transition is bypassed. In addition, the ability of cdc15 diploid mutant strains to develop non-septated chains of cells, supported by nuclear division, is shown. This phenotype is also displayed by haploid cdc15 mutant strains when cell lysis is prevented by osmotic protection, and shared by other MEN mutants. By contrast, anaphase-telophase arrest is strictly observed in double MEN-FEAR (fourteen early anaphase release) mutants. In this context, the overexpression of a FEAR component, SPO12, in a MEN mutant background enhances the ability of MEN mutants to bypass cell cycle arrest. Taken together, these data suggest a critical role of Cdc15 and other MEN proteins in cytokinesis, allowing a new model for their cellular function to be proposed.     

The actin cytoskeleton is required for selective types of autophagy, but not nonspecific autophagy, in the yeast Saccharomyces cerevisiae

Autophagy is a catabolic multitask transport route that takes place in all eukaryotic cells. During starvation, cytoplasmic components are randomly sequestered into huge double-membrane vesicles called autophagosomes and delivered into the lysosome/vacuole where they are destroyed. Cells are able to modulate autophagy in response to their needs, and under certain circumstances, cargoes such as aberrant protein aggregates, organelles and bacteria can be selectively and exclusively incorporated into autophagosomes. In the yeast Saccharomyces cerevisiae, for example, double-membrane vesicles are used to transport the Ape1 protease into the vacuole, or for the elimination of superfluous peroxisomes. In the present study we reveal that in this organism, actin plays a role in these two types of selective autophagy but not in the nonselective, bulk process. In particular, we show that precursor Ape1 is not correctly recruited to the PAS, the putative site of double-membrane vesicle biogenesis, and superfluous peroxisomes are not degraded in a conditional actin mutant. These phenomena correlate with a defect in Atg9 trafficking from the mitochondria to the PAS.     

Mitochondrial ferredoxin is required for heme A synthesis in Saccharomyces cerevisiae.

Heme A is a prosthetic group of all eukaryotic and some prokaryotic cytochrome oxidases. This heme differs from heme B (protoheme) at two carbon positions of the porphyrin ring. The synthesis of heme A begins with farnesylation of the vinyl group at carbon C-2 of heme B. The heme O product of this reaction is then converted to heme A by a further oxidation of a methyl to a formyl group on C-8. In a previous study (Barros, M. H., Carlson, C. G., Glerum, D. M., and Tzagoloff, A. (2001) FEBS Lett. 492, 133-138) we proposed that the formyl group is formed by an initial hydroxylation of the C-8 methyl by a three-component monooxygenase consisting of Cox15p, ferredoxin, and ferredoxin reductase. In the present study three lines of evidence confirm a requirement of ferredoxin in heme A synthesis. 1) Temperature-conditional yah1 mutants grown under restrictive conditions display a decrease in heme A relative to heme B. 2) The incorporation of radioactive delta-aminolevulinic acid into heme A is reduced in yah1 ts but not in the wild type after the shift to the restrictive temperature; and 3) the overexpression of Cox15p in cytochrome oxidase mutants that accumulate heme O leads to an increased mitochondrial concentration of heme A. The increase in heme A is greater in mutants that overexpress Cox15p and ferredoxin. These results are consistent with a requirement of ferredoxin and indirectly of ferredoxin reductase in hydroxylation of heme O.     

Dephosphorylation of Cdc20 is required for its C-box-dependent activation of the APC/C

The anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase is tightly regulated to ensure programmed proteolysis in cells. The activity of the APC/C is positively controlled by cyclin-dependent kinase (CDK), but a second level of control must also exist because phosphorylation inactivates Cdc20, a mitotic APC/C co-activator. How Cdc20 is dephosphorylated specifically, when CDK is high, has remained unexplained. Here, we show that phosphatases are crucial to activate the APC/C. Cdc20 is phosphorylated at six conserved residues (S50/T64/T68/T79/S114/S165) by CDK in Xenopus egg extracts. When all the threonine residues are phosphorylated, Cdc20 binding to and activation of the APC/C are inhibited. Their dephosphorylation is regulated depending on the sites and protein phosphatase 2A, active in mitosis, is essential to dephosphorylate the threonine residues and activate the APC/C. Consistently, most of the Cdc20 bound to the APC/C in anaphase evades phosphorylation at T79. Furthermore, we show that the 'activation domain' of Cdc20 associates with the Apc6 and Apc8 core subunits. Our data suggest that dephosphorylation of Cdc20 is required for its loading and activation of the APC/C ubiquitin ligase.