Cdc50p, a protein required for polarized growth, associates with the Drs2p P-type ATPase implicated in phospholipid translocation in Saccharomyces cerevisiae

Cdc50p, a transmembrane protein localized to the late endosome, is required for polarized cell growth in yeast. Genetic studies suggest that CDC50 performs a function similar to DRS2, which encodes a P-type ATPase of the aminophospholipid translocase (APT) subfamily. At low temperatures, drs2Delta mutant cells exhibited depolarization of cortical actin patches and mislocalization of polarity regulators, such as Bni1p and Gic1p, in a manner similar to the cdc50Delta mutant. Both Cdc50p and Drs2p were localized to the trans-Golgi network and late endosome. Cdc50p was coimmunoprecipitated with Drs2p from membrane protein extracts. In cdc50Delta mutant cells, Drs2p resided on the endoplasmic reticulum (ER), whereas Cdc50p was found on the ER membrane in drs2Delta cells, suggesting that the association on the ER membrane is required for transport of the Cdc50p-Drs2p complex to the trans-Golgi network. Lem3/Ros3p, a homolog of Cdc50p, was coimmunoprecipitated with another APT, Dnf1p; Lem3p was required for exit of Dnf1p out of the ER. Both Cdc50p-Drs2p and Lem3p-Dnf1p were confined to the plasma membrane upon blockade of endocytosis, suggesting that these proteins cycle between the exocytic and endocytic pathways, likely performing redundant functions. Thus, phospholipid asymmetry plays an important role in the establishment of cell polarity; the Cdc50p/Lem3p family likely constitute potential subunits specific to unique P-type ATPases of the APT subfamily.     

Pxl1p, a paxillin-like protein in Saccharomyces cerevisiae, may coordinate Cdc42p and Rho1p functions during polarized growth

Rho-family GTPases Cdc42p and Rho1p play critical roles in the budding process of the yeast Saccharomyces cerevisiae. However, it is not clear how the functions of these GTPases are coordinated temporally and spatially during this process. Based on its ability to suppress cdc42-Ts mutants when overexpressed, a novel gene PXL1 was identified. Pxl1p resembles mammalian paxillin, which is involved in integrating various signaling events at focal adhesion. Both proteins share amino acid sequence homology and structural organization. When expressed in yeast, chicken paxillin localizes to the sites of polarized growth as Pxl1p does. In addition, the LIM domains in both proteins are the primary determinant for targeting the proteins to the cortical sites in their native cells. These data strongly suggest that Pxl1p is the "ancient paxillin" in yeast. Deletion of PXL1 does not produce any obvious phenotype. However, Pxl1p directly binds to Rho1p-GDP in vitro, and inhibits the growth of rho1-2 and rho1-3 mutants in a dosage-dependent manner. The opposite effects of overexpressed Pxl1p on cdc42 and rho1 mutants suggest that the functions of Cdc42p and Rho1p may be coordinately regulated during budding and that Pxl1p may be involved in this coordination.     

Saccharomyces cerevisiae Cdc42p localizes to cellular membranes and clusters at sites of polarized growth

The Cdc42p GTPase controls polarized growth and cell cycle progression in eukaryotes from yeasts to mammals, and its precise subcellular localization is essential for its function. To examine the cell cycle-specific targeting of Cdc42p in living yeast cells, a green fluorescent protein (GFP)-Cdc42 fusion protein was used. In contrast to previous immunolocalization data, GFP-Cdc42p was found at the plasma membrane around the entire cell periphery and at internal vacuolar and nuclear membranes throughout the cell cycle, and it accumulated or clustered at polarized growth sites, including incipient bud sites and mother-bud neck regions. These studies also showed that C-terminal CAAX and polylysine domains were sufficient for membrane localization but not for clustering. Time-lapse fluorescence microscopy showed that GFP-Cdc42p clustered at the incipient bud site prior to bud emergence and at the mother-bud neck region postanaphase as a diffuse, single band and persisted as two distinct bands on mother and daughter cells following cytokinesis and cell separation. Initial clustering occurred immediately prior to actomyosin ring contraction and persisted postcontraction. These results suggest that Cdc42p targeting occurs through a novel mechanism of membrane localization followed by cell cycle-specific clustering at polarized growth sites.     

Zds2p regulates Swe1p-dependent polarized cell growth in Saccharomyces cerevisiae via a novel Cdc55p interaction domain

Deletion of the paralogs ZDS1 and ZDS2 in the budding yeast Saccharomyces cerevisiae causes a mis-regulation of polarized cell growth. Here we show a function for these genes as regulators of the Swe1p (Wee1p) kinase-dependent G2/M checkpoint. We identified a conserved domain in the C-terminus of Zds2p consisting of amino acids 813-912 (hereafter referred to as ZH4 for Zds homology 4) that is required for regulation of Swe1p-dependent polarized bud growth. ZH4 is shown by protein affinity assays to be necessary and sufficient for interaction with Cdc55p, a regulatory subunit of protein phosphatase 2A (PP2A). We hypothesized that the Zds proteins are in a pathway that negatively regulates the Swe1p-dependent G2/M checkpoint via Cdc55p. Supporting this model, deletion of CDC55 rescues the aberrant bud morphology of a zds1Δzds2Δ strain. We also show that expression of ZDS1 or ZDS2 from a strong galactose-inducible promoter can induce mitosis even when the Swe1p-dependent G2/M checkpoint is activated by mis-organization of the actin cytoskeleton. This negative regulation requires the CDC55 gene. Together these data indicate that the Cdc55p/Zds2p module has a function in the regulation of the Swe1p-dependent G2/M checkpoint.     

The Glc7p-interacting protein Bud14p attenuates polarized growth,pheromone response,and filamentous growth in Saccharomyces cerevisiae

A genetic selection in Saccharomyces cerevisiae for mutants that stimulate the mating pathway uncovered a mutant that had a hyperactive pheromone response pathway and also had hyperpolarized growth. Cloning and segregation analysis demonstrated that BUD14 was the affected gene. Disruption of BUD14 in wild-type cells caused mild stimulation of pheromone response pathway reporters, an increase in sensitivity to mating factor, and a hyperelongated shmoo morphology. The bud14 mutant also had hyperfilamentous growth. Consistent with a role in the control of cell polarity, a Bud14p-green fluorescent protein fusion was localized to sites of polarized growth in the cell. Bud14p shared morphogenetic functions with the Ste20p and Bni1p proteins as well as with the type 1 phosphatase Glc7p. The genetic interactions between BUD14 and GLC7 suggested a role for Glc7p in filamentous growth, and Glc7p was found to have a positive function in filamentous growth in yeast.     

Cdc48p interacts with Ufd3p, a WD repeat protein required for ubiquitin-mediated proteolysis in Saccharomyces cerevisiae.

A library of random 10 residue peptides fused to the N-terminus of a reporter protein was screened in the yeast Saccharomyces cerevisiae for sequences that can target the reporter for degradation by the N-end rule pathway, a ubiquitin (Ub)-dependent proteolytic system that recognizes potential substrates through binding to their destabilizing N-terminal residues. One of the N-terminal sequences identified by this screen was used in a second screen for mutants incapable of degrading the corresponding reporter fusion. A mutant thus identified had an abnormally low content of free Ub. This mutant was found to be allelic to a previously isolated mutant in a Ub-dependent proteolytic system distinct from the N-end rule pathway. We isolated the gene involved, termed UFD3, which encodes an 80 kDa protein containing tandem repeats of a motif that is present in many eukaryotic proteins and called the WD repeat. Both co-immunoprecipitation and two-hybrid assays demonstrated that Ufd3p is an in vivo ligand of Cdc48p, an essential ATPase required for the cell cycle progression and the fusion of endoplasmic reticulum membranes. Further, we showed that, similarly to Ufd3p, Cdc48p is also required for the Ub-dependent proteolysis of test substrates. The discovery of the Ufd3p--Cdc48p complex and the finding that this complex is a part of the Ub system open up a new direction for studies of the function of Ub in the cell cycle and membrane dynamics.     

The Saccharomyces cerevisiae protein Ccz1p interacts with components of the endosomal fusion machinery

The yeast protein Ccz1p is necessary for vacuolar protein trafficking and biogenesis. In a complex with Mon1p, it mediates fusion of transport intermediates with the vacuole membrane by activating the small GTPase Ypt7p. Additionally, genetic data suggest a role of Ccz1p in earlier transport steps, in the Golgi. In a search for further proteins interacting with Ccz1p, we identified the endosomal soluble N -ethylmaleimide-sensitive factor attachment protein receptor Pep12p as an interaction partner of Ccz1p. Combining the ccz1 Δ mutation with deletions of PEP12 or other genes encoding components of the endosomal fusion machinery, VPS21, VPS9 or VPS45 , results in synthetic growth phenotypes. The genes MON1 and YPT7 also interact genetically with PEP12 . These results suggest that the Ccz1p–Mon1p–Ypt7p complex is involved in fusion of transport vesicles to multiple target membranes in yeast cells.     

Role of actin and Myo2p in polarized secretion and growth of Saccharomyces cerevisiae

We examined the role of the actin cytoskeleton in secretion in Saccharomyces cerevisiae with the use of several quantitative assays, including time-lapse video microscopy of cell surface growth in individual living cells. In latrunculin, which depolymerizes filamentous actin, cell surface growth was completely depolarized but still occurred, albeit at a reduced level. Thus, filamentous actin is necessary for polarized secretion but not for secretion per se. Consistent with this conclusion, latrunculin caused vesicles to accumulate at random positions throughout the cell. Cortical actin patches cluster at locations that correlate with sites of polarized secretion. However, we found that actin patch polarization is not necessary for polarized secretion because a mutant, bee1Delta(las17Delta), which completely lacks actin patch polarization, displayed polarized growth. In contrast, a mutant lacking actin cables, tpm1-2 tpm2Delta, had a severe defect in polarized growth. The yeast class V myosin Myo2p is hypothesized to mediate polarized secretion. A mutation in the motor domain of Myo2p, myo2-66, caused growth to be depolarized but with only a partial decrease in the level of overall growth. This effect is similar to that of latrunculin, suggesting that Myo2p interacts with filamentous actin. However, inhibition of Myo2p function by expression of its tail domain completely abolished growth.     

The PXL1 gene of Saccharomyces cerevisiae encodes a paxillin-like protein functioning in polarized cell growth

The Saccharomyces cerevisiae open reading frame YKR090w encodes a predicted protein displaying similarity in organization to paxillin, a scaffolding protein that organizes signaling and actin cytoskeletal regulating activities in many higher eucaryotic cell types. We found that YKR090w functions in a manner analogous to paxillin as a mediator of polarized cell growth; thus, we have named this gene PXL1 (Paxillin-like protein 1). Analyses of pxl1Delta strains show that PXL1 is required for the selection and maintenance of polarized growth sites during vegetative growth and mating. Genetic analyses of strains lacking both PXL1 and the Rho GAP BEM2 demonstrate that such cells display pronounced growth defects in response to different conditions causing Rho1 pathway activation. PXL1 also displays genetic interactions with the Rho1 effector FKS1. Pxl1p may therefore function as a modulator of Rho-GTPase signaling. A GFP::Pxl1 fusion protein localizes to sites of polarized cell growth. Experiments mapping the localization determinants of Pxl1p demonstrate the existence of localization mechanisms conserved between paxillin and Pxl1p and indicate an evolutionarily ancient and conserved role for LIM domain proteins in acting to modulate cell signaling and cytoskeletal organization during polarized growth.     

Subcellular localization of Cdc42p, a Saccharomyces cerevisiae GTP-binding protein involved in the control of cell polarity.

The Saccharomyces cerevisiae Cdc42 protein, a member of the Ras superfamily of low-molecular-weight GTP-binding proteins, is involved in the control of cell polarity during the yeast cell cycle. This protein has a consensus sequence (CAAX) for geranylgeranyl modification and is likely to be associated, at least in part, with cell membranes. Using cell fractionation and immunolocalization techniques, we have investigated the subcellular localization of Cdc42p. Cdc42p was found in both soluble and particulate pools, and neither its abundance nor its distribution varied through the cell cycle. The particulate form of Cdc42p could be solubilized with detergents but not with NaCl or urea, suggesting that it is tightly associated with membranes. An increase in soluble Cdc42p was observed in a geranylgeranyltransferase mutant strain (cdc43-2ts) grown at the restrictive temperature. In addition, Cdc42p from a cdc42C188S mutant strain (that has an alteration at the prenylation consensus site) was almost exclusively in the soluble fraction, suggesting that membrane localization is dependent on geranylgeranyl modification at Cys-188. Immunofluorescence and immunoelectron microscopy experiments demonstrated that Cdc42p localizes to the plasma membrane in the vicinity of secretory vesicles that were found at the site of bud emergence, at the tips and sides of enlarging buds, and within mating projections (shmoo tips) in alpha-factor-arrested cells. These results indicate that Cdc42p is localized to the bud site early in the cell cycle and suggest that this localization is critical for the selection of the proper site for bud emergence and for polarized cell growth.     

Highly conserved toxicity of Saccharomyces cerevisiae Rap1p

Budding yeast ( Saccharomyces cerevisiae ) Rap1p has been expressed in fission yeast ( Schizosaccharo-myces pombe ) under the control of the regulatable fructose bisphosphatase ( fbp ) promoter. When the fbp promoter was derepressed, cells containing the complete RAP1 gene failed to show any significant growth, suggesting that Rap1p is toxic. A derivative of Rap1p that has a temperature-sensitive mutation in the DNA-binding domain was not toxic in cells grown at 37°C, a temperature at which DNA binding by rap1p ^ts is severely inhibited. Removal of a short region downstream of the DNA-binding domain, including a region previously shown to be essential for Rap1p toxicity in budding yeast, also abolished the toxic effect. The toxic effect of Rap1p has therefore been conserved between two distantly related yeasts. In budding yeast, overexpression of Rap1p also caused changes to the lengths of the telomeric repeats. No effects on telomeres were detected in fission yeast.     

Pie1, a protein interacting with Mec1, controls cell growth and checkpoint responses in Saccharomyces cerevisiae

In eukaryotes, the ATM and ATR family proteins play a critical role in the DNA damage and replication checkpoint controls. These proteins are characterized by a kinase domain related to the phosphatidylinositol 3-kinase, but they have the ability to phosphorylate proteins. In budding yeast, the ATR family protein Mec1/Esr1 is essential for checkpoint responses and cell growth. We have isolated the PIE1 gene in a two-hybrid screen for proteins that interact with Mec1, and we show that Pie1 interacts physically with Mec1 in vivo. Like MEC1, PIE1 is essential for cell growth, and deletion of the PIE1 gene causes defects in the DNA damage and replication block checkpoints similar to those observed in mec1Delta mutants. Rad53 hyperphosphorylation following DNA damage and replication block is also decreased in pie1Delta cells, as in mec1Delta cells. Pie1 has a limited homology to fission yeast Rad26, which forms a complex with the ATR family protein Rad3. Mutation of the region in Pie1 homologous to Rad26 results in a phenotype similar to that of the pie1Delta mutation. Mec1 protein kinase activity appears to be essential for checkpoint responses and cell growth. However, Mec1 kinase activity is unaffected by the pie1Delta mutation, suggesting that Pie1 regulates some essential function other than Mec1 kinase activity. Thus, Pie1 is structurally and functionally related to Rad26 and interacts with Mec1 to control checkpoints and cell proliferation.     

Inp1p is a peroxisomal membrane protein required for peroxisome inheritance in Saccharomyces cerevisiae

Cells have evolved molecular mechanisms for the efficient transmission of organelles during cell division. Little is known about how peroxisomes are inherited. Inp1p is a peripheral membrane protein of peroxisomes of Saccharomyces cerevisiae that affects both the morphology of peroxisomes and their partitioning during cell division. In vivo 4-dimensional video microscopy showed an inability of mother cells to retain a subset of peroxisomes in dividing cells lacking the INP1 gene, whereas cells overexpressing INP1 exhibited immobilized peroxisomes that failed to be partitioned to the bud. Overproduced Inp1p localized to both peroxisomes and the cell cortex, supporting an interaction of Inp1p with specific structures lining the cell periphery. The levels of Inp1p vary with the cell cycle. Inp1p binds Pex25p, Pex30p, and Vps1p, which have been implicated in controlling peroxisome division. Our findings are consistent with Inp1p acting as a factor that retains peroxisomes in cells and controls peroxisome division. Inp1p is the first peroxisomal protein directly implicated in peroxisome inheritance.     

Recombination protein Tid1p controls resolution of cohesin-dependent linkages in meiosis in Saccharomyces cerevisiae

Sister chromatid cohesion and interhomologue recombination are coordinated to promote the segregation of homologous chromosomes instead of sister chromatids at the first meiotic division. During meiotic prophase in Saccharomyces cerevisiae, the meiosis-specific cohesin Rec8p localizes along chromosome axes and mediates most of the cohesion. The mitotic cohesin Mcd1p/Scc1p localizes to discrete spots along chromosome arms, and its function is not clear. In cells lacking Tid1p, which is a member of the SWI2/SNF2 family of helicase-like proteins that are involved in chromatin remodeling, Mcd1p and Rec8p persist abnormally through both meiotic divisions, and chromosome segregation fails in the majority of cells. Genetic results indicate that the primary defect in these cells is a failure to resolve Mcd1p-mediated connections. Tid1p interacts with recombination enzymes Dmc1p and Rad51p and has an established role in recombination repair. We propose that Tid1p remodels Mcd1p-mediated cohesion early in meiotic prophase to facilitate interhomologue recombination and the subsequent segregation of homologous chromosomes.     

酿酒酵母Afr1p调控septin蛋白Cdc12p的定位

AFR1最初被鉴定为在过量表达的情况下,可以使细胞产生α因子抗性,同时对融合过程中融合突起的形成起重要作用。Afr1p还具有调节细胞壁完整性途径中的MAPK Mpk1p的定位及活性的功能。该文通过对蛋白定位的观察发现,半乳糖诱导GAL-AFR1过量表达破坏了在出芽过程中Cdc12p的定位;缺失AFR1也会导致Cdc12p定位异常。Western blot结果显示,在营养生长过程中Afr1p稳定表达。这说明,稳定表达的AFR1有调节septin Cdc12p定位的功能,从而对维持septin的结构起到一定的作用。     

Expression and subcellular localization of a membrane protein related to Hsp30p in Saccharomyces cerevisiae

The Saccharomyces cerevisiae YDR033w gene product is homologous to Hsp30p and Yro2p, both of which are induced during heat shock. To investigate the subcellular localization of the YDR033w gene product, hemagglutinin (HA) epitope-tagged protein was expressed, detected on immunoblots, and localized by immunofluorescence to cell membranes, primarily the plasma membrane. A punctuate immunofluorescence pattern was observed within cell buds. The nuclear envelope, but not the vacuole or mitochondrial membranes, were also immunostained. We refer to YDR033w as MRH1 to denote that it encodes a membrane protein related to Hsp30p.     

Itt1p, a novel protein inhibiting translation termination in Saccharomyces cerevisiae

Background

Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRFl and eRF3. eRFl recognizes nonsense codons UAA, UAG and UGA, while eRF3 stimulates polypeptide release from the ribosome in a GTP- and eRFl – dependent manner. Recent studies has shown that proteins interacting with these release factors can modulate the efficiency of nonsense codon readthrough.

Results

We have isolated a nonessential yeast gene, which causes suppression of nonsense mutations, being in a multicopy state. This gene encodes a protein designated Itt1p, possessing a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes. Overexpression of Itt1p decreases the efficiency of translation termination, resulting in the readthrough of all three types of nonsense codons. Itt1p interacts in vitro with both eRFl and eRF3. Overexpression of eRFl, but not of eRF3, abolishes the nonsense suppressor effect of overexpressed Itt1p.

Conclusions

The data obtained demonstrate that Itt1p can modulate the efficiency of translation termination in yeast. This protein possesses a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes, and this is a first observation of such protein being involved in translation.     

Cell Cycle Regulation of the Saccharomyces cerevisiae Polo-Like Kinase Cdc5p

Progression through and completion of mitosis require the actions of the evolutionarily conserved Polo kinase. We have determined that the levels of Cdc5p, a Saccharomyces cerevisiae member of the Polo family of mitotic kinases, are cell cycle regulated. Cdc5p accumulates in the nuclei of G₂/M-phase cells, and its levels decline dramatically as cells progress through anaphase and begin telophase. We report that Cdc5p levels are sensitive to mutations in key components of the anaphase-promoting complex (APC). We have determined that Cdc5p-associated kinase activity is restricted to G₂/M and that this activity is posttranslationally regulated. These results further link the actions of the APC to the completion of mitosis and suggest possible roles for Cdc5p during progression through and completion of mitosis.