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.     

Nalidixic acid causes a transient G1 arrest in the yeast Saccharomyces cerevisiae.

Summary The addition of nalidixic acid to growing cells of the yeast Saccharomyces cerevisiae resulted in a transient depression in the rate of ribosomal precursor RNA production and a transient arrest of cells in G1. Protein synthesis rates were less affected. Lower concentrations of nalidixic acid also affected RNA synthesis and progression through G1 but had no effect on protein synthesis rates. We suggest that nalidixic acid has a primary effect on RNA synthesis leading to a G1 arrest.     

Characterization of nuclease-dependent functions of Exo1p in Saccharomyces cerevisiae

Exo1p is a member of the Rad2p family of structure-specific nucleases that contain conserved N and I nuclease domains. Exo1p has been implicated in numerous DNA metabolic processes, such as recombination, double-strand break repair and DNA mismatch repair (MMR). In this report, we describe in vitro and in vivo characterization of full-length wild-type and mutant forms of Exo1p. Herein, we demonstrate that full-length yeast Exo1p possesses an intrinsic 5'-3' exonuclease activity as reported previously, but also possesses a flap-endonuclease activity. Our study indicates that Exo1p shares similar, but not identical structure-function relationships to other characterized members of the Rad2p family in the N and I nuclease domains. The two exo1p mutants we examined, showed deficiencies for both double-stranded DNA (dsDNA) 5'-3' exonuclease and flap-endonuclease activities. Examining the genetic interaction of these two exo1 mutations with rad27Delta suggest that the Exo1p flap-endonuclease activity and not the dsDNA 5'-3' exonuclease is redundant to Rad27p for viability. In addition, our in vivo results also indicate that many exo1Delta phenotypes are dependent on the complete catalytic activities of Exo1p. Finally, our findings plus those of other investigators suggest that Exo1p functions both in a catalytic and a structural capacity during DNA MMR.     

Characterization and complementation of a Fus3/Kss1 type MAPK from Tuber borchii, TBMK

Mitogen-activated protein kinases (MAPK) are used by organisms to transduce extra cellular signals from the environment in cellular events such as proliferation and differentiation. In the present study, we have characterized the first MAPK from the ectomycorrhizal fungus Tuber borchii (TBMK) which belongs to the YERK1 (yeast extra cellular regulated kinase) subfamily. TBMK is present as a single copy in the genome and the codified protein was phosphorylated during the interaction with the host plant, Tilia americana. Complementation studies showed that TBMK restores pheromone signaling in Saccharomyces cerevisiae and partially restores invasive growth of Fusarium oxysporum that lack the fmk1 gene. This suggests a protein kinase activity and its involvement in the infection processes. Hence, TBMK could play an important role during the pre-symbiotic phase of T. borchii with its host plant in the modulation of genes necessary for the establishment of symbiosis leading to the synthesis of functional ectomycorrhizae.     

G1/S cyclin-dependent kinase regulates small GTPase Rho1p through phosphorylation of RhoGEF Tus1p in Saccharomyces cerevisiae

Rho1p is an essential small GTPase that plays a key role in the morphogenesis of Saccharomyces cerevisiae. We show here that the activation of Rho1p is regulated by a cyclin-dependent kinase (CDK). Rho1p is activated at the G1/S transition at the incipient-bud sites by the Cln2p (G1 cyclin) and Cdc28p (CDK) complex, in a process mediated by Tus1p, a guanine nucleotide exchange factor for Rho1p. Tus1p interacts physically with Cln2p/Cdc28p and is phosphorylated in a Cln2p/Cdc28p-dependent manner. CDK phosphorylation consensus sites in Tus1p are required for both Cln2p-dependent activation of Rho1p and polarized organization of the actin cytoskeleton. We propose that Cln2p/Cdc28p-dependent phosphorylation of Tus1p is required for appropriate temporal and spatial activation of Rho1p at the G1/S transition.     

Grr1 functions in the ubiquitin pathway in Saccharomyces cerevisiae through association with Skp1

Cdc34, a ubiquitin-conjugating enzyme in Saccharomyces cerevisiae, is required for cell cycle progression. Sic1, an S-phase cyclin-dependent kinase (CDK) inhibitor, is a critical target of Cdc34-mediated ubiquitination. Other essential target protein(s) could be defined since cdc34 sic1 double mutants still arrest in G2 phase. To identify proteins which function in the Cdc34-dependent ubiquitin pathway, a series of extragenic suppressors of the cdc34-1 sic1 double mutations was isolated. One of them was found to be defective in GRR1, which is involved not only in glucose repression but also in G1 cyclin destabilization. However, neither lack of glucose repression nor stabilization of G1 cyclin caused the suppression of cdc34-1 sic1. Conversely, Grr1 overproduction in cdc34-1 sic1 cells impaired colony formation, even at the permissive temperature. A multicopy suppressor, MGO1, which rescued the growth defect associated with Grr1 overproduction was isolated, and found to be identical to SKP1. Furthermore, Grr1 bound Skp1 directly in vitro. These results strongly suggest that Grr1 functions in the ubiquitin pathway through association with Skp1.     

Rapamycin-mediated G1 arrest involves regulation of the Cdk inhibitor Sic1 in Saccharomyces cerevisiae

The rapamycin-sensitive (TOR) signalling pathway in Saccharomyces cerevisiae controls growth and cell proliferation in response to nutrient availability. Rapamycin treatment causes cells to arrest growth in G1 phase. The mechanism by which the inhibition of the TOR pathway regulates cell cycle progression is not completely understood. Here we show that rapamycin causes G1 arrest by a dual mechanism that comprises downregulation of the G1-cyclins Cln1-3 and upregulation of the Cdk inhibitor protein Sic1. The increase of Sic1 level is mostly independent of the downregulation of the G1 cyclins, being unaffected by ectopic CLN2 expression, but requires Sic1 phosphorylation of Thr173, because it is lost in cells expressing Sic1(T173A). Rapamycin-mediated Sic1 upregulation involves nuclear accumulation of a more stable, non-ubiquitinated protein. Either SIC1 deletion or CLN3 overexpression results in non-cell-cycle-specific arrest upon rapamycin treatment and makes cells sensitive to a sublethal dose of rapamycin and to nutrient starvation. In conclusion, our data indicate that Sic1 is involved in rapamycin-induced G1 arrest and that deregulated entrance into S phase severely decreases the ability of a cell to cope with starvation conditions induced by nutrient depletion or which are mimicked by rapamycin treatment.     

Recovery of Saccharomyces cerevisiae mating-type a cells from G1 arrest by alpha factor.

Mating-type a cells of the yeast Saccharomyces cerevisiae that had been specifically arrested in the G1 phase of the cell cycle by alpha factor, an oligopeptide pheromone made by alpha cells, recovered and resumed cell division after a period of inhibition which was dependent on the concentration of alpha factor used. These treated a cells were more resistant to alpha factor than untreated a cells, but lost their resistance upon further cell division. However, cells arrested for 6 h were no more resistant to alpha factor than cells arrested for only 2.5 h. Mating-type a strains could inactivate or remove alpha factor from the culture fluid, but two a sterile (nonmating) mutants and an a/alpha diploid strain could not. These results suggest that a cells have a mechanism, which may involve uptake or inactivation of alpha factor, for recovering from alpha factor arrest. However, the results do not distinguish between a recovery mechanism which is constitutive and one which is induced by alpha factor. The loss of alpha factor activity during recovery appeared to be primarily cell contact mediated, although an extracellular, diffusible inhibitor of alpha factor that is labile or that functions stoichiometrically could not be ruled out.     

The RHO1-GAPs SAC7, BEM2 and BAG7 control distinct RHO1 functions in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the small GTPase RHO1 plays an essential role in the control of cell wall synthesis and organization of the actin cytoskeleton. Several regulators for RHO1 are known, including the GTPase-activating proteins (GAPs) SAC7 and BEM2. Here we show that BAG7, identified as the closest homologue of SAC7, also acts as a GAP for RHO1 in vitro and in vivo. Furthermore, we find that BAG7, SAC7, and BEM2 are functionally different in vivo. Overexpression of BAG7 or SAC7,but not BEM2, suppresses the cold sensitivity of a sac7 mutation and the lethality of RHO1 hyperactivation in response to cell wall damage. In contrast, overexpression of BEM2 or SAC7, but not BAG7, downregulates the RHO1-controlled PKC1-MPK1 pathway, and disruption of BEM2 or SAC7, but not BAG7, results in increased MPK1 activation. We conclude that BEM2 and SAC7, but not BAG7, are involved in the control of the RHO1-mediated activation of MPK1, whereas BAG7 and SAC7, but not BEM2, are involved in the regulation of other RHO1 functions. This suggests that different RHO1GAPs control different RHO1 effector pathways, thus ensuring their individual regulation at the appropriate place and time.     

Afr1p regulates the Saccharomyces cerevisiae alpha-factor receptor by a mechanism that is distinct from receptor phosphorylation and endocytosis.

The alpha-factor pheromone receptor activates a G protein signaling pathway that induces the conjugation of the yeast Saccharomyces cerevisiae. Our previous studies identified AFR1 as a gene that regulates this signaling pathway because overexpression of AFR1 promoted resistance to alpha-factor. AFR1 also showed an interesting genetic relationship with the alpha-factor receptor gene, STE2, suggesting that the receptor is regulated by Afr1p. To investigate the mechanism of this regulation, we tested AFR1 for a role in the two processes that are known to regulate receptor signaling: phosphorylation and down-regulation of ligand-bound receptors by endocytosis. AFR1 overexpression diminished signaling in a strain that lacks the C-terminal phosphorylation sites of the receptor, indicating that AFR1 acts independently of phosphorylation. The effects of AFR1 overexpression were weaker in strains that were defective in receptor endocytosis. However, AFR1 overexpression did not detectably influence receptor endocytosis or the stability of the receptor protein. Instead, gene dosage studies showed that the effects of AFR1 overexpression on signaling were inversely proportional to the number of receptors. These results indicate that AFR1 acts independently of endocytosis, and that the weaker effects of AFR1 in strains that are defective in receptor endocytosis were probably an indirect consequence of their increased receptor number caused by the failure of receptors to undergo ligand-stimulated endocytosis. Analysis of the ligand binding properties of the receptor showed that AFR1 overexpression did not alter the number of cell-surface receptors or the affinity for alpha-factor. Thus, Afr1p prevents alpha-factor receptors from activating G protein signaling by a mechanism that is distinct from other known pathways.     

Saccharomyces cerevisiae Rab-GDI displacement factor ortholog Yip3p forms distinct complexes with the Ypt1 Rab GTPase and the reticulon Rtn1p

Rab GTPases are crucial regulators of organelle biogenesis, maintenance, and transport. Multiple Rabs are expressed in all cells, and each is localized to a distinct set of organelles, but little is known regarding the mechanisms by which Rabs are targeted to their resident organelles. Integral membrane proteins have been postulated to serve as receptors that recruit Rabs from the cytosol in a complex with the Rab chaperone, GDI, to facilitate the dissociation of Rab and GDI, hence facilitating loading of Rabs on membranes. We show here that the yeast (Saccharomyces cerevisiae) Golgi Rab GTPase Ypt1p can be copurified with the integral membrane protein Yip3p from detergent cell extracts. In addition, a member of the highly conserved reticulon protein family, Rtn1p, is also associated with Yip3p in vivo. However, Ypt1p did not copurify with Rtn1p, indicating that Yip3p is a component of at least two different protein complexes. Yip3p and Rtn1p are only partially colocalized in cells, with Yip3p localized predominantly to the Golgi and secondarily to the endoplasmic reticulum, whereas Rtn1p is localized predominantly to the endoplasmic reticulum and secondarily to the Golgi. Surprisingly, the intracellular localization of Rabs was not perturbed in yip3Delta or rtn1Delta mutants, suggesting that these proteins do not play a role in targeting Rabs to intracellular membranes. These data indicate that Yip3p may have multiple functions and that its interaction with Rabs is not critical for their recruitment to organelle membranes.     

Far3 and five interacting proteins prevent premature recovery from pheromone arrest in the budding yeast Saccharomyces cerevisiae

In budding yeast, diffusible mating pheromones initiate a signaling pathway that culminates in several responses, including cell cycle arrest. Only a handful of genes required for the interface between pheromone response and the cell cycle have been identified, among them FAR1 and FAR3; of these, only FAR1 has been extensively characterized. In an effort to learn about the mechanism by which Far3 acts, we used the two-hybrid method to identify interacting proteins. We identified five previously uncharacterized open reading frames, dubbed FAR7, FAR8, FAR9, FAR10, and FAR11, that cause a far3-like pheromone arrest defect when disrupted. Using two-hybrid and coimmunoprecipitation analysis, we found that all six Far proteins interact with each other. Moreover, velocity sedimentation experiments suggest that Far3 and Far7 to Far11 form a complex. The phenotype of a sextuple far3far7-far11 mutant is no more severe than any single mutant. Thus, FAR3 and FAR7 to FAR11 all participate in the same pathway leading to G1 arrest. These mutants initially arrest in response to pheromone but resume budding after 10 h. Under these conditions, wild-type cells fail to resume budding even after several days whereas far1 mutant cells resume budding within 1 h. We conclude that the FAR3-dependent arrest pathway is functionally distinct from that which employs FAR1.     

The reconstructed ancestral subunit a functions as both V-ATPase isoforms Vph1p and Stv1p in Saccharomyces cerevisiae

The vacuolar-type, proton-translocating ATPase (V-ATPase) is a multisubunit enzyme responsible for organelle acidification in eukaryotic cells. Many organisms have evolved V-ATPase subunit isoforms that allow for increased specialization of this critical enzyme. Differential targeting of the V-ATPase to specific subcellular organelles occurs in eukaryotes from humans to budding yeast. In Saccharomyces cerevisiae, the two subunit a isoforms are the only difference between the two V-ATPase populations. Incorporation of Vph1p or Stv1p into the V-ATPase dictates the localization of the V-ATPase to the vacuole or late Golgi/endosome, respectively. A duplication event within fungi gave rise to two subunit a genes. We used ancestral gene reconstruction to generate the most recent common ancestor of Vph1p and Stv1p (Anc.a) and tested its function in yeast. Anc.a localized to both the Golgi/endosomal network and vacuolar membrane and acidified these compartments as part of a hybrid V-ATPase complex. Trafficking of Anc.a did not require retrograde transport from the late endosome to the Golgi that has evolved for retrieval of the Stv1p isoform. Rather, Anc.a localized to both structures through slowed anterograde transport en route to the vacuole. Our results suggest an evolutionary model that describes the differential localization of the two yeast V-ATPase isoforms.     

Multiple functions of the vacuolar sorting protein Ccz1p in Saccharomyces cerevisiae

The CCZ1 (YBR131w) gene encodes a protein required for fusion of various transport intermediates with the vacuole. Ccz1p, in a complex with Mon1p, is a close partner of Ypt7p in the processes of fusion of endosomes to vacuoles and homotypic vacuole fusion. In this work, we exploited the Ca(2+)-sensitivity of the ccz1Delta mutant to identify genes specifically interacting with CCZ1, basing on functional multicopy suppression of calcium toxicity. The presented results indicate that Ccz1p functions in the cell either in association with Mon1p and Ypt7p in fusion at the vacuolar membrane, or--separately--with Arl1p at early steps of vacuolar transport. We also show that suppression of calcium toxicity by the calcium pumps Pmr1p and Pmc1p is restricted only to the subset of mutants defective in vacuole morphology. The mechanisms of Ca(2+)-pump-mediated suppression also differ from each other, since the action of Pmr1p, but not Pmc1p, appears to require Arl1p function.     

Saccharomyces cerevisiae Pra1p/Yip3p interacts with Yip1p and Rab proteins.

The regulation of membrane traffic involves the Rab family of Ras-related GTPases, of which there are a total of 11 members in the yeast Saccharomyces cerevisiae. Previous work has identified PRA1 as a dual prenylated Rab GTPase and VAMP2 interacting protein [Martinic et al. (1999) J. Biol. Chem. 272, 26991-26998]. In this study we demonstrate that the yeast counterpart of PRA1 interacts with Rab proteins and with Yip1p, a membrane protein of unknown function that has been reported to interact specifically with the Rab proteins Ypt1p and Ypt31p. Yeast Pra1p/Yip3p is a factor capable of biochemical interaction with a panel of different Rab proteins and does not show in vitro specificity for any particular Rab. The interactions between Pra1p/Yip3p and Rab proteins are dependent on the presence of the Rab protein C-terminal cysteines and require C-terminal prenylation.     

Phosphorylation and localization of Kss1, a MAP kinase of the Saccharomyces cerevisiae pheromone response pathway.

Kss1 protein kinase, and the homologous Fus3 kinase, are required for pheromone signal transduction in Saccharomyces cerevisiae. In MATa haploids exposed to alpha-factor, Kss1 was rapidly phosphorylated on both Thr183 and Tyr185, and both sites were required for Kss1 function in vivo. De novo protein synthesis was required for sustained pheromone-induced phosphorylation of Kss1. Catalytically inactive Kss1 mutants displayed alpha-factor-induced phosphorylation on both residues, even in kss1 delta cells; hence, autophosphorylation is not obligatory for these modifications. In kss1 delta fus3 delta double mutants, Kss1 phosphorylation was elevated even in the absence of pheromone; thus, cross-phosphorylation by Fus3 is not responsible for Kss1 activation. In contrast, pheromone-induced Kss1 phosphorylation was eliminated in mutants deficient in two other protein kinases, Ste11 and Ste7. A dominant hyperactive allele of STE11 caused a dramatic increase in the phosphorylation of Kss1, even in the absence of pheromone stimulation, but required Ste7 for this effect, suggesting an order of function: Ste11-->Ste7-->Kss1. When overproduced, Kss1 stimulated recovery from pheromone-imposed G1 arrest. Catalytic activity was essential for Kss1 function in signal transmission, but not for its recovery-promoting activity. Kss1 was found almost exclusively in the particulate material and its subcellular fractionation was unaffected by pheromone treatment. Indirect immunofluorescence demonstrated that Kss1 is concentrated in the nucleus and that its distribution is not altered detectably during signaling.