scholarly journals Immunofluorescence localization of the unconventional myosin, Myo2p, and the putative kinesin-related protein, Smy1p, to the same regions of polarized growth in Saccharomyces cerevisiae.

1994 ◽  
Vol 125 (4) ◽  
pp. 825-842 ◽  
Author(s):  
S H Lillie ◽  
S S Brown
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Actin Binding ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Polarized Growth ◽  
Related Protein ◽  
Active Growth ◽  
Yeast Saccharomyces Cerevisiae ◽  
Double Labeling ◽  
Unconventional Myosin

Myo2 protein (Myo2p), an unconventional myosin in the budding yeast Saccharomyces cerevisiae, has been implicated in polarized growth and secretion by studies of the temperature-sensitive myo2-66 mutant. Overexpression of Smy1p, which by sequence is a kinesin-related protein, can partially compensate for defects in the myo2 mutant (Lillie, S. H. and S. S. Brown, 1992. Nature (Lond.). 356:358-361). We have now immunolocalized Smy1p and Myo2p. Both are concentrated in regions of active growth, as caps at incipient bud sites and on small buds, at the mother-bud neck just before cell separation, and in mating cells as caps on shmoo tips and at the fusion bridge of zygotes. Double labeling of cells with either Myo2p or Smy1p antibody plus phalloidin was used to compare the localization of Smy1p and Myo2p to actin, and by extrapolation, to each other. These studies confirmed that Myo2p and Smy1p colocalize, and are concentrated in the same general regions of the cell as actin spots. However, neither colocalizes with actin. We noted a correlation in the behavior of Myo2p, Smy1p, and actin, but not microtubules, under a number of circumstances. In cdc4 and cdc11 mutants, which produce multiple buds, Myo2p and Smy1p caps were found only in the subset of buds that had accumulations of actin. Mutations in actin or secretory genes perturb actin, Smy1p and Myo2p localization. The rearrangements of Myo2p and Smy1p correlate temporally with those of actin spots during the cell cycle, and upon temperature and osmotic shift. In contrast, microtubules are not grossly affected by these perturbations. Although wild-type Myo2p localization does not require Smy1p, Myo2p staining is brighter when SMY1 is overexpressed. The myo2 mutant, when shifted to restrictive temperature, shows a permanent loss in Myo2p localization and actin polarization, both of which can be restored by SMY1 overexpression. However, the lethality of MYO2 deletion is not overcome by SMY1 overexpression. We noted that the myo2 mutant can recover from osmotic shift (unlike actin mutants; Novick, P., and D. Botstein. 1985. Cell. 40:405-416). We have also determined that the myo2-66 allele encodes a Lys instead of a Glu at position 511, which lies at an actin-binding face in the motor domain.

scholarly journals The Saccharomyces cerevisiae MYO2 gene encodes an essential myosin for vectorial transport of vesicles.

1991 ◽  
Vol 113 (3) ◽  
pp. 539-551 ◽  
Author(s):  
G C Johnston ◽  
J A Prendergast ◽  
R A Singer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Motor ◽  
Actin Binding ◽  
Restrictive Temperature ◽  
Secretory Vesicles ◽  
Biosynthetic Activity ◽  
Yeast Saccharomyces Cerevisiae ◽  
Terminal Domain ◽  
Essential Form ◽  
Actin Cables

After the initiation of bud formation, cells of the yeast Saccharomyces cerevisiae direct new growth to the developing bud. We show here that this vectorial growth is facilitated by activity of the MYO2 gene. The wild-type MYO2 gene encodes an essential form of myosin composed of an NH2-terminal domain typical of the globular, actin-binding domain of other myosins. This NH2-terminal domain is linked by what appears to be a short alpha-helical domain to a novel COOH-terminal region. At the restrictive temperature the myo2-66 mutation does not impair DNA, RNA, or protein biosynthetic activity, but produces unbudded, enlarged cells. This phenotype suggests a defect in localization of cell growth. Measurements of cell size demonstrated that the continued development of initiated buds, as well as bud initiation itself, is inhibited. Bulk secretion continues in mutant cells, although secretory vesicles accumulate. The MYO2 myosin thus may function as the molecular motor to transport secretory vesicles along actin cables to the site of bud development.

An Essential Function of a Phosphoinositide-Specific Phospholipase C Is Relieved by Inhibition of a Cyclin-Dependent Protein Kinase in the Yeast Saccharomyces cerevisiae

Genetics ◽  
1998 ◽  
Vol 148 (1) ◽  
pp. 33-47 ◽  
Author(s):  
Jeffrey S Flick ◽  
Jeremy Thorner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Complete Medium ◽  
Restrictive Temperature ◽  
Dependent Protein Kinase ◽  
Yeast Saccharomyces Cerevisiae ◽  
Dependent Protein

Abstract The PLC1 gene product of Saccharomyces cerevisiae is a homolog of the δ isoform of mammalian phosphoinositide-specific phospholipase C (PI-PLC). We found that two genes (SPL1 and SPL2), when overexpressed, can bypass the temperature-sensitive growth defect of a plc1Δ cell. SPL1 is identical to the PHO81 gene, which encodes an inhibitor of a cyclin (Pho80p)-dependent protein kinase (Pho85p) complex (Cdk). In addition to overproduction of Pho81p, two other conditions that inactivate this Cdk, a cyclin (pho80Δ) mutation and growth on low-phosphate medium, also permitted growth of plc1Δ cells at the restrictive temperature. Suppression of the temperature sensitivity of plc1Δ cells by pho80Δ does not depend upon the Pho4p transcriptional regulator, the only known substrate of the Pho80p/Pho85p Cdk. The second suppressor, SPL2, encodes a small (17-kD) protein that bears similarity to the ankyrin repeat regions present in Pho81p and in other known Cdk inhibitors. Both pho81Δ and spl2Δ show a synthetic phenotype in combination with plc1Δ. Unlike single mutants, plc1Δ pho81Δ and plc1Δ spl2Δ double mutants were unable to grow on synthetic complete medium, but were able to grow on rich medium.

Motor proteins in Saccharomyces cerevisiae

1995 ◽  
Vol 73 (S1) ◽  
pp. 369-371 ◽  
Author(s):  
Susan S. Brown
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Key Words ◽  
Motor Proteins ◽  
Nucleotide Binding ◽  
Nucleotide Binding Site ◽  
Temperature Sensitive ◽  
Related Protein ◽  
Yeast Saccharomyces Cerevisiae

A number of myosins have been identified in yeast (Saccharomyces cerevisiae), an organism ideally suited to dissecting out their different functions. We have learned that a temperature-sensitive defect in one of these myosins (Myo2p) can be partially overcome by overexpression of a kinesin-like protein (Smy1p). This raises the possibility of the involvement of microtubules in the same function as Myo2p. However, we have been unable to demonstrate that this is the case, either using nocodazole to depolymerize microtubules or by altering the nucleotide-binding site of Smy1p. Key words: myosin, kinesin-related protein, cytoskeleton.

scholarly journals Specification of sites for polarized growth in Saccharomyces cerevisiae and the influence of external factors on site selection.

1992 ◽  
Vol 3 (9) ◽  
pp. 1025-1035 ◽  
Author(s):  
K Madden ◽  
M Snyder
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Types ◽  
Growth Conditions ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Polarized Growth ◽  
Mating Partner ◽  
Polarized Cell Growth ◽  
External Conditions

Many eucaryotic cell types exhibit polarized cell growth and polarized cell division at nonrandom sites. The sites of polarized growth were investigated in G1 arrested haploid Saccharomyces cerevisiae cells. When yeast cells are arrested during G1 either by treatment with alpha-factor or by shifting temperature-sensitive cdc28-1 cells to the restrictive temperature, the cells form a projection. Staining with Calcofluor reveals that in both cases the projection usually forms at axial sites (i.e., next to the previous bud scar); these are the same sites where bud formation is expected to occur. These results indicate that sites of polarized growth are specified before the end of G1. Sites of polarized growth can be influenced by external conditions. Cells grown to stationary phase and diluted into fresh medium preferentially select sites for polarized growth opposite the previous bud scar (i.e., distal sites). Incubation of cells in a mating mixture results in projection formation at nonaxial sites: presumably cells form projections toward their mating partner. These observations have important implications in understanding three aspects of cell polarity in yeast: 1) how yeast cell shape is influenced by growth conditions 2) how sites of polarized growth are chosen, and 3) the pathway by which polarity is affected and redirected during the mating process.

Mating-defective ste mutations are suppressed by cell division cycle start mutations in Saccharomyces cerevisiae

1982 ◽  
Vol 2 (9) ◽  
pp. 1052-1063
Author(s):  
J R Shuster
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Temperature Characteristic ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Gene Products ◽  
Wild Type ◽  
Mating Pheromone ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mating Pheromones

Temperature-sensitive mutants which arrest in the G1 phase of the cell cycle have been described for the yeast Saccharomyces cerevisiae. One class of these mutants (carrying cdc28, cdc36, cdc37, or cdc39) forms a shmoo morphology at restrictive temperature, characteristic of mating pheromone-arrested wild-type cells. Therefore, one hypothesis to explain the control of cell division by mating factors states that mating pheromones arrest wild-type cells by inactivating one or more of these CDC gene products. A class of mutants (carrying ste4, ste5, ste7, ste11, or ste12) which is insensitive to mating pheromone and sterile has also been described. One possible function of the STE gene products is the inactivation of the CDC gene products in the presence of a mating pheromone. A model incorporating these two hypotheses predicts that such STE gene products will not be required for mating in strains carrying an appropriate cdc lesion. This prediction was tested by assaying the mating abilities of double mutants for all of the pairwise combinations of cdc and ste mutations. Lesions in either cdc36 or cdc39 suppressed the mating defect due to ste4 and ste5. Allele specificity was observed in the suppression of both ste4 and ste5. The results indicate that the CDC36, CDC39, STE4, and STE5 gene products interact functionally or physically or both in the regulation of cell division mediated by the presence or absence of mating pheromones. The cdc36 and cdc39 mutations did not suppress ste7, ste11, or ste12. Lesions in cdc28 or cdc37 did not suppress any of the ste mutations. Other models of CDC and STE gene action which predicted that some of the cdc and ste mutations would be alleles of the same locus were tested. None of the cdc mutations was allelic to the ste mutations and, therefore, these models were eliminated.

A Novel Functional Domain of Cdc15 Kinase Is Required for Its Interaction With Tem1 GTPase in Saccharomyces cerevisiae

Genetics ◽  
2001 ◽  
Vol 157 (4) ◽  
pp. 1437-1450 ◽  
Author(s):  
Kazuhide Asakawa ◽  
Satoshi Yoshida ◽  
Fumiaki Otake ◽  
Akio Toh-e
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Kinase Domain ◽  
Binding Domain ◽  
Temperature Sensitive ◽  
Functional Domain ◽  
Restrictive Temperature ◽  
Cyclin Dependent Kinase ◽  
Yeast Saccharomyces Cerevisiae ◽  
Hybrid Screening

Abstract Exit from mitosis requires the inactivation of cyclin-dependent kinase (CDK) activity. In the budding yeast Saccharomyces cerevisiae, a number of gene products have been identified as components of the signal transduction network regulating inactivation of CDK (called the MEN, for the mitotic exit network). Cdc15, one of such components of the MEN, is an essential protein kinase. By the two-hybrid screening, we identified Cdc15 as a binding protein of Tem1 GTPase, another essential regulator of the MEN. Coprecipitation experiments revealed that Tem1 binds to Cdc15 in vivo. By deletion analysis, we found that the Tem1-binding domain resides near the conserved kinase domain of Cdc15. The cdc15-LF mutation, which was introduced into the Tem1-binding domain, reduced the interaction with Cdc15 and Tem1 and caused temperature-sensitive growth.The kinase activity of Cdc15 was not so much affected by the cdc15-LF mutation. However, Cdc15-LF failed to localize to the SPB at the restrictive temperature. Our data show that the interaction with Tem1 is important for the function of Cdc15 and that Cdc15 and Tem1 function in a complex to direct the exit from mitosis.

scholarly journals Isolation and characterization of temperature-sensitive mutations in the RAS2 and CYR1 genes of Saccharomyces cerevisiae.

Genetics ◽  
1989 ◽  
Vol 123 (4) ◽  
pp. 739-748 ◽  
Author(s):  
H Mitsuzawa ◽  
I Uno ◽  
T Oshima ◽  
T Ishikawa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carbon Sources ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Extra Copy ◽  
Yeast Saccharomyces Cerevisiae ◽  
Isolation And Characterization ◽  
Low Concentrations ◽  
Gal1 Promoter ◽  
Dependent Growth

Abstract The yeast Saccharomyces cerevisiae contains two ras homologues, RAS1 and RAS2, whose products have been shown to modulate the activity of adenylate cyclase encoded by the CYR1 gene. To isolate temperature-sensitive mutations in the RAS2 gene, we constructed a plasmid carrying a RAS2 gene whose expression is under the control of the galactose-inducible GAL1 promoter. A ras1 strain transformed with this plasmid was subjected to ethyl methanesulfonate mutagenesis and nystatin enrichment. Screening of approximately 13,000 mutagenized colonies for galactose-dependent growth at a high temperature (37 degrees) yielded six temperature-sensitive ras2 (ras2ts) mutations and one temperature-sensitive cyr1 (cyr1ts) mutation that can be suppressed by overexpression or increased dosage of RAS2. Some ras2ts mutations were shown to be suppressed by an extra copy of CYR1. Therefore increased dosage of either RAS2 or CYR1 can suppress the temperature sensitivity caused by a mutation in the other. ras1 ras2ts and ras1 cyr1ts mutants arrested in the G1 phase of the cell cycle at the restrictive temperature, and showed pleiotropic phenotypes to varying degrees even at a temperature permissive for growth (25 degrees), including slow growth, sporulation on rich media, increased accumulation of glycogen, impaired growth on nonfermentable carbon sources, heat-shock resistance, impaired growth on low concentrations of glucose, and lithium sensitivity. Of these, impaired growth on low concentrations of glucose and sensitivity to lithium are new phenotypes, which have not been reported for mutants defective in the cAMP pathway.

scholarly journals CDC42 and CDC43, two additional genes involved in budding and the establishment of cell polarity in the yeast Saccharomyces cerevisiae.

1990 ◽  
Vol 111 (1) ◽  
pp. 131-142 ◽  
Author(s):  
A E Adams ◽  
D I Johnson ◽  
R M Longnecker ◽  
B F Sloat ◽  
J R Pringle
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Surface ◽  
Actin Cytoskeleton ◽  
Cell Polarity ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Mitotic Spindles ◽  
Yeast Saccharomyces Cerevisiae ◽  
Continue Growth

Budding in the yeast Saccharomyces cerevisiae involves a polarized deposition of new cell surface material that is associated with a highly asymmetric disposition of the actin cytoskeleton. Mutants defective in gene CDC24, which are unable to bud or establish cell polarity, have been of great interest with regard to both the mechanisms of cellular morphogenesis and the mechanisms that coordinate cell-cycle events. To gain further insights into these problems, we sought additional mutants with defects in budding. We report here that temperature-sensitive mutants defective in genes CDC42 and CDC43, like cdc24 mutants, fail to bud but continue growth at restrictive temperature, and thus arrest as large unbudded cells. Nearly all of the arrested cells appear to begin nuclear cycles (as judged by the occurrence of DNA replication and the formation and elongation of mitotic spindles), and many go on to complete nuclear division, supporting the hypothesis that the events associated with budding and those of the nuclear cycle represent two independent pathways within the cell cycle. The arrested mutant cells display delocalized cell-surface deposition associated with a loss of asymmetry of the actin cytoskeleton. CDC42 maps distal to the rDNA on chromosome XII and CDC43 maps near lys5 on chromosome VII.

scholarly journals Mating-defective ste mutations are suppressed by cell division cycle start mutations in Saccharomyces cerevisiae.

1982 ◽  
Vol 2 (9) ◽  
pp. 1052-1063 ◽  
Author(s):  
J R Shuster
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Temperature Characteristic ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Gene Products ◽  
Wild Type ◽  
Mating Pheromone ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mating Pheromones

Temperature-sensitive mutants which arrest in the G1 phase of the cell cycle have been described for the yeast Saccharomyces cerevisiae. One class of these mutants (carrying cdc28, cdc36, cdc37, or cdc39) forms a shmoo morphology at restrictive temperature, characteristic of mating pheromone-arrested wild-type cells. Therefore, one hypothesis to explain the control of cell division by mating factors states that mating pheromones arrest wild-type cells by inactivating one or more of these CDC gene products. A class of mutants (carrying ste4, ste5, ste7, ste11, or ste12) which is insensitive to mating pheromone and sterile has also been described. One possible function of the STE gene products is the inactivation of the CDC gene products in the presence of a mating pheromone. A model incorporating these two hypotheses predicts that such STE gene products will not be required for mating in strains carrying an appropriate cdc lesion. This prediction was tested by assaying the mating abilities of double mutants for all of the pairwise combinations of cdc and ste mutations. Lesions in either cdc36 or cdc39 suppressed the mating defect due to ste4 and ste5. Allele specificity was observed in the suppression of both ste4 and ste5. The results indicate that the CDC36, CDC39, STE4, and STE5 gene products interact functionally or physically or both in the regulation of cell division mediated by the presence or absence of mating pheromones. The cdc36 and cdc39 mutations did not suppress ste7, ste11, or ste12. Lesions in cdc28 or cdc37 did not suppress any of the ste mutations. Other models of CDC and STE gene action which predicted that some of the cdc and ste mutations would be alleles of the same locus were tested. None of the cdc mutations was allelic to the ste mutations and, therefore, these models were eliminated.

scholarly journals The Saccharomyces cerevisiae gene SDS22 encodes a potential regulator of the mitotic function of yeast type 1 protein phosphatase.

1995 ◽  
Vol 15 (7) ◽  
pp. 3777-3785 ◽  
Author(s):  
S H MacKelvie ◽  
P D Andrews ◽  
M J Stark
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein A ◽  
Catalytic Subunit ◽  
Yeast Cells ◽  
Regulatory Subunit ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae

In higher eukaryotes, the activity and specificity of the type 1 protein serine-threonine phosphatase (PP1) catalytic subunit is thought to be controlled by its association with a number of regulatory or targeting subunits. Here we describe the characterization of a gene encoding one such potential polypeptide in the yeast Saccharomyces cerevisiae. The gene which we have isolated (termed SDS22) encodes a product with a high degree of sequence identity to the fission yeast sds22 protein, a known regulator of the mitotic function of PP1 in Schizosaccharomyces pombe. Using two different criteria, we have demonstrated that Sds22p and the catalytic subunit of PP1 (Glc7p) interact in yeast cells. We have also generated a temperature-sensitive allele of GLC7 (glc7-12) which causes a block to the completion of mitosis at the restrictive temperature. Additional copies of SDS22 lead to allele-specific suppression of the glc7-12 mutant, strongly suggesting that the interaction between the two proteins is of functional significance. Sds22p is therefore likely to be the second example of a PP1 regulatory subunit identified in S. cerevisiae.

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