Stress and developmental regulation of the yeast C-type cyclin Ume3p (Srb11p/Ssn8p).

The ume3-1 allele was identified as a mutation that allowed the aberrant expression of several meiotic genes (e.g. SPO11, SPO13) during mitotic cell division in Saccharomyces cerevisiae. Here we report that UME3 is also required for the full repression of the HSP70 family member SSA1. UME3 encodes a non-essential C-type cyclin (Ume3p) whose levels do not vary through the mitotic cell cycle. However, Ume3p is destroyed during meiosis or when cultures are subjected to heat shock. Ume3p mutants resistant to degradation resulted in a 2-fold reduction in SPO13 mRNA levels during meiosis, indicating that the down-regulation of this cyclin is important for normal meiotic gene expression. Mutational analysis identified two regions (PEST-rich and RXXL) that mediate Ume3p degradation. A third destruction signal lies within the highly conserved cyclin box, a region that mediates cyclin-cyclin-dependent kinase (Cdk) interactions. However, the Cdk activated by Ume3p (Ume5p) is not required for the rapid destruction of this cyclin. Finally, Ume3p destruction was not affected in mutants defective for ubiquitin-dependent proteolysis. These results support a model in which Ume3p, when exposed to heat shock or sporulation conditions, is targeted for destruction to allow the expression of genes necessary for the cell to respond correctly to these environmental cues.     

The G1 cyclin Cln3p controls vacuolar biogenesis in Saccharomyces cerevisiae

How organelle biogenesis and inheritance is linked to cell division is poorly understood. In the budding yeast Saccharomyces cerevisiae the G(1) cyclins Cln1,2,3p control initiation of cell division. Here we show that Cln3p controls vacuolar (lysosomal) biogenesis and segregation. First, loss of Cln3p, but not Cln1p or Cln2p, resulted in vacuolar fragmentation. Although the vacuoles of cln3delta cells were fragmented, together they occupied a large space, which accounted for a significant fraction of the overall cell size increase in cln3delta cells. Second, cytosol prepared from cells lacking Cln3p had reduced vacuolar homotypic fusion activity in cell-free assays. Third, vacuolar segregation was perturbed in cln3delta cells. Our findings reveal a novel role for a eukaryotic G(1) cyclin in cytoplasmic organelle biogenesis and segregation.     

Involvement of Saccharomyces cerevisiae Avo3p/Tsc11p in maintaining TOR complex 2 integrity and coupling to downstream signaling

Target-of-rapamycin proteins (TORs) are Ser/Thr kinases serving a central role in cell growth control. TORs function in two conserved multiprotein complexes, TOR complex 1 (TORC1) and TORC2; the mechanisms underlying their actions and regulation are not fully elucidated. Saccharomyces TORC2, containing Tor2p, Avo1p, Avo2p, Avo3p/Tsc11p, Bit61p, and Lst8p, regulates cell integrity and actin organization. Two classes of avo3 temperature-sensitive (avo3(ts)) mutants that we previously identified display cell integrity and actin defects, yet one is suppressed by AVO1 while the other is suppressed by AVO2 or SLM1, defining two TORC2 downstream signaling mechanisms, one mediated by Avo1p and the other by Avo2p/Slm1p. Employing these mutants, we explored Avo3p functions in TORC2 structure and signaling. By observing binary protein interactions using coimmunoprecipitation, we discovered that the composition of TORC2 and its recruitment of the downstream effectors Slm1p and Slm2p were differentially affected in different avo3(ts) mutants. These molecular defects can be corrected only by expressing AVO3, not by expressing suppressors, highlighting the role of Avo3p as a structural and signaling scaffold for TORC2. Phenotypic modifications of avo3(ts) mutants by deletion of individual Rho1p-GTPase-activating proteins indicate that two TORC2 downstream signaling branches converge on Rho1p activation. Our results also suggest that Avo2p/Slm1p-mediated signaling, but not Avo1p-mediated signaling, links to Rho1p activation specifically through the Rho1p-guanine nucleotide exchange factor Tus1p.     

The Saccharomyces cerevisiae Sln1p-Ssk1p two-component system mediates response to oxidative stress and in an oxidant-specific fashion

Aerobic organisms experience oxidative stress due to generation of reactive oxygen species during normal aerobic metabolism. In addition, environmental gamma and UV radiation, as well as several chemicals also generate reactive oxygen species, which induce oxidative stress. Thus oxidative stress constitutes a major threat to organisms living in aerobic environments. Oxidative stress induces the expression of several genes in yeast Saccharomyces cerevisiae. However, the primary sensor(s) that trigger the response is unknown. This study demonstrates that primary sensors of osmotic stress, the Sln1p-Ssk1p two-component proteins, are involved in sensing oxidative stress specifically induced by hydrogen peroxide and diamide, but not by other oxidants used in the study. Wild type and sln1-ssk1 mutant were treated with hydrogen peroxide, diamide, menadione, UV, and gamma-radiation. Results show that sln1-ssk1 mutant is only sensitive to hydrogen peroxide and diamide but not to other oxidants. S. cerevisiae contains an additional cell surface osmosensor, Sho1p, that targets the osmotic signal to Hog1p. Data is presented that shows Sho1 and Hog1 proteins are also involved in signaling oxidant-specific cellular damage. Furthermore, it is demonstrated that expression of the mammalian homolog of Hog1p provides protection from oxidative stress induced by hydrogen peroxide. These results suggest that Sln1p-Ssk1p and Sho1p signal transduction pathways participate in oxidative stress response. However, this response to oxidative stress is limited to specific oxidants.     

GSK-3beta-dependent destabilization of cyclin D1 mediates replicational stress-induced arrest of cell cycle

Chemotherapeutic agents are well known to induce growth arrest of cancerous cells by inducing DNA damage/replicational stress and engaging cellular apoptotic machinery. Our studies on hydroxyurea (HU) recognized cyclin D1 destabilization as the initiator of growth arrest at G(1)/S-phase independent of other cell cycle regulators. Cyclin D1 degradation was associated with its phosphorylation at Thr286 by glycogen synthase kinase-3beta and inactivation of Akt kinase. Overexpression of the cyclin D1(T286A) mutant, or constitutively active Akt, conferred stability to cyclin D1 and helped bypass cell cycle arrest. Thus, growth arrest by HU seems to involve destabilization of cyclin D1 in addition to its well-established role as ribonucleotide reductase inhibitor.     

Compartmentalization of Spo11p in vegetative cells of yeast Saccharomyces cerevisiae

Homologous recombination is initiated in meiotic eukaryotic cells at DNA double-strand breaks, which are generated by several proteins, Spo11p playing a key role. The protein products of SPO11 orthologs are highly conserved, are found in most eukaryotes from plants to human, and are structurally similar to subunit A of archaeal DNA topoisomerase VI. Saccharomyces cerevisiae SPO11 is expressed in meiotic prophase I. Spo11p acts as topoisomerase II and is presumably active as a dimer. Experimental data on Spo11p compartmentalization in vegetative yeast cells are unavailable. The SPO11 coding region and its fragments were fused in frame with the egfp reporter and expressed in vegetative yeast cells. The Spo11p-EGFP fusion was localized in the nucleus, while cytoplasmic localization was observed for Spo11Δ-EGFP devoid of the 25 N-terminal residues. N-terminal Spo11p region 7–25 fused with EGFP ensured the nuclear targeting of the reporter protein and was assumed to harbor the nuclear localization signal.     

Compartmentalization of Spo11p in vegetative cells of yeast Saccharomyces cerevisiae

Double-stranded DNA breaks are currently thought to initiate homologous DNA recombination during meiosis. These breaks are mediated by several proteins, the key protein is Spol1p. Spo11 proteins being encoded by the highly conserved orthologs of SPO11 are present in most eukaryotes ranging from plants to man and are structurally similar to the subunit A of the archaea topoisomerase VI. The SPO11 of S. cerevisiae is currently known to be expressed during prophase I. It encodes a topoisomerase II that is apparently active as a dimer. Neither its localization in the native cells nor its nuclear localisation signals have been described in the literature. We report the expression of the coding region of SPO11 and its truncated variants C-terminally tagged by the egfp reporter in yeast. As judged by the EGFP fluorescence, the Spo11 p-EGFP fusion was localized in vegetative yeast nuclei whereas Spo11pdelta-EGFP lacking 25 N-terminal amino acids of Spollp was localized in cytoplasm. Nineteen N-terminal amino acids of Spo11p fused to EGFP made some reporter to be localized in the nucleus. Thus, we conclude that N-terminal part of Spo11p is a nuclear localization signal that is not specific for prophase I and is used to import proteins in vegetative yeast cells.