Sec35p, a Novel Peripheral Membrane Protein, Is Required for ER to Golgi Vesicle Docking

SEC35 was identified in a novel screen for temperature-sensitive mutants in the secretory pathway of the yeast Saccharomyces cerevisiae (Wuestehube et al., 1996. Genetics. 142:393–406). At the restrictive temperature, the sec35-1 strain exhibits a transport block between the ER and the Golgi apparatus and accumulates numerous vesicles. SEC35 encodes a novel cytosolic protein of 32 kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec35-1 is efficiently suppressed by YPT1, which encodes the rab-like GTPase required early in the secretory pathway, or by SLY1-20, which encodes a dominant form of the ER to Golgi target -SNARE–associated protein Sly1p. Weaker suppression is evident upon overexpression of genes encoding the vesicle-SNAREs SEC22, BET1, or YKT6. The cold-sensitive lethality that results from deleting SEC35 is suppressed by YPT1 or SLY1-20. These genetic relationships suggest that Sec35p acts upstream of, or in conjunction with, Ypt1p and Sly1p as was previously found for Uso1p. Using a cell-free assay that measures distinct steps in vesicle transport from the ER to the Golgi, we find Sec35p is required for a vesicle docking stage catalyzed by Uso1p. These genetic and biochemical results suggest Sec35p acts with Uso1p to dock ER-derived vesicles to the Golgi complex.

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Sec34p, a Protein Required for Vesicle Tethering to the Yeast Golgi Apparatus, Is in a Complex with Sec35p

The Journal of Cell Biology ◽

10.1083/jcb.147.4.729 ◽

1999 ◽

Vol 147 (4) ◽

pp. 729-742 ◽

Cited By ~ 98

Author(s):

Susan M. VanRheenen ◽

Xiaochun Cao ◽

Stephanie K. Sapperstein ◽

Elbert C. Chiang ◽

Vladimir V. Lupashin ◽

...

Keyword(s):

Golgi Complex ◽

Secretory Pathway ◽

Rab Gtpase ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Dominant Form ◽

Vesicle Tethering ◽

Protein Receptor ◽

Complex Target

A screen for mutants of Saccharomyces cerevisiae secretory pathway components previously yielded sec34, a mutant that accumulates numerous vesicles and fails to transport proteins from the ER to the Golgi complex at the restrictive temperature (Wuestehube, L.J., R. Duden, A. Eun, S. Hamamoto, P. Korn, R. Ram, and R. Schekman. 1996. Genetics. 142:393–406). We find that SEC34 encodes a novel protein of 93-kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec34-2 is suppressed by the rab GTPase Ypt1p that functions early in the secretory pathway, or by the dominant form of the ER to Golgi complex target-SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor)–associated protein Sly1p, Sly1-20p. Weaker suppression is evident upon overexpression of genes encoding the vesicle tethering factor Uso1p or the vesicle-SNAREs Sec22p, Bet1p, or Ykt6p. This genetic suppression profile is similar to that of sec35-1, a mutant allele of a gene encoding an ER to Golgi vesicle tethering factor and, like Sec35p, Sec34p is required in vitro for vesicle tethering. sec34-2 and sec35-1 display a synthetic lethal interaction, a genetic result explained by the finding that Sec34p and Sec35p can interact by two-hybrid analysis. Fractionation of yeast cytosol indicates that Sec34p and Sec35p exist in an ∼750-kD protein complex. Finally, we describe RUD3, a novel gene identified through a genetic screen for multicopy suppressors of a mutation in USO1, which suppresses the sec34-2 mutation as well.

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A Link between Secretion and Pre-mRNA Processing Defects in Saccharomyces cerevisiae and the Identification of a Novel Splicing Gene, RSE1

Molecular and Cellular Biology ◽

10.1128/mcb.18.12.7139 ◽

1998 ◽

Vol 18 (12) ◽

pp. 7139-7146 ◽

Cited By ~ 17

Author(s):

Esther J. Chen ◽

Alison R. Frand ◽

Elizabeth Chitouras ◽

Chris A. Kaiser

Keyword(s):

Saccharomyces Cerevisiae ◽

Secretory Pathway ◽

Small Gtpase ◽

Mrna Splicing ◽

Secretory Proteins ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Golgi Transport ◽

Pathway Function ◽

A Cell

ABSTRACT Secretory proteins in eukaryotic cells are transported to the cell surface via the endoplasmic reticulum (ER) and the Golgi apparatus by membrane-bounded vesicles. We screened a collection of temperature-sensitive mutants of Saccharomyces cerevisiaefor defects in ER-to-Golgi transport. Two of the genes identified in this screen were PRP2, which encodes a known pre-mRNA splicing factor, and RSE1, a novel gene that we show to be important for pre-mRNA splicing. Both prp2-13 andrse1-1 mutants accumulate the ER forms of invertase and the vacuolar protease CPY at restrictive temperature. The secretion defect in each mutant can be suppressed by increasing the amount ofSAR1, which encodes a small GTPase essential for COPII vesicle formation from the ER, or by deleting the intron from theSAR1 gene. These data indicate that a failure to spliceSAR1 pre-mRNA is the specific cause of the secretion defects in prp2-13 and rse1-1. Moreover, these data imply that Sar1p is a limiting component of the ER-to-Golgi transport machinery and suggest a way that secretory pathway function might be coordinated with the amount of gene expression in a cell.

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Casein kinase I-like protein kinases encoded by YCK1 and YCK2 are required for yeast morphogenesis.

Molecular and Cellular Biology ◽

10.1128/mcb.13.5.2870 ◽

1993 ◽

Vol 13 (5) ◽

pp. 2870-2881 ◽

Cited By ~ 83

Author(s):

L C Robinson ◽

M M Menold ◽

S Garrett ◽

M R Culbertson

Keyword(s):

Protein Kinase ◽

Eukaryotic Cell ◽

Casein Kinase ◽

Temperature Sensitive ◽

Protein Kinase Activity ◽

Restrictive Temperature ◽

Loss Of Function ◽

Casein Kinase I ◽

Yeast Saccharomyces Cerevisiae

Casein kinase I is an acidotropic protein kinase class that is widely distributed among eukaryotic cell types. In the yeast Saccharomyces cerevisiae, the casein kinase I isoform encoded by the gene pair YCK1 and YCK2 is a 60- to 62-kDa membrane-associated form. The Yck proteins perform functions essential for growth and division; either alone supports growth, but loss of function of both is lethal. We report here that casein kinase I-like activity is associated with a soluble Yck2-beta-galactosidase fusion protein in vitro and that thermolabile protein kinase activity is exhibited by a protein encoded by fusion of a temperature-sensitive yck2 allele with lacZ. Cells carrying the yck2-2ts allele arrest at restrictive temperature with multiple, elongated buds containing multiple nuclei. This phenotype suggests that the essential functions of the Yck proteins include roles in bud morphogenesis, possibly in control of cell growth polarity, and in cytokinesis or cell separation. Further, a genetic relationship between the yck2ts allele and deletion of CDC55 indicates that the function of Yck phosphorylation may be related to that of protein phosphatase 2A activity.

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Cell cycle arrest of cdc mutants and specificity of the RAD9 checkpoint.

Genetics ◽

10.1093/genetics/134.1.63 ◽

1993 ◽

Vol 134 (1) ◽

pp. 63-80 ◽

Cited By ~ 9

Author(s):

T A Weinert ◽

L H Hartwell

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Dna Replication ◽

Cell Cycle Control ◽

Dna Lesions ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

A Cell ◽

G2 Phase ◽

Rad Mutants

Abstract In eucaryotes a cell cycle control called a checkpoint ensures that mitosis occurs only after chromosomes are completely replicated and any damage is repaired. The function of this checkpoint in budding yeast requires the RAD9 gene. Here we examine the role of the RAD9 gene in the arrest of the 12 cell division cycle (cdc) mutants, temperature-sensitive lethal mutants that arrest in specific phases of the cell cycle at a restrictive temperature. We found that in four cdc mutants the cdc rad9 cells failed to arrest after a shift to the restrictive temperature, rather they continued cell division and died rapidly, whereas the cdc RAD cells arrested and remained viable. The cell cycle and genetic phenotypes of the 12 cdc RAD mutants indicate the function of the RAD9 checkpoint is phase-specific and signal-specific. First, the four cdc RAD mutants that required RAD9 each arrested in the late S/G2 phase after a shift to the restrictive temperature when DNA replication was complete or nearly complete, and second, each leaves DNA lesions when the CDC gene product is limiting for cell division. Three of the four CDC genes are known to encode DNA replication enzymes. We found that the RAD17 gene is also essential for the function of the RAD9 checkpoint because it is required for phase-specific arrest of the same four cdc mutants. We also show that both X- or UV-irradiated cells require the RAD9 and RAD17 genes for delay in the G2 phase. Together, these results indicate that the RAD9 checkpoint is apparently activated only by DNA lesions and arrests cell division only in the late S/G2 phase.

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Distinct Roles for the Yeast Phosphatidylinositol 4-Kinases, Stt4p and Pik1p, in Secretion, Cell Growth, and Organelle Membrane Dynamics

Molecular Biology of the Cell ◽

10.1091/mbc.11.8.2673 ◽

2000 ◽

Vol 11 (8) ◽

pp. 2673-2689 ◽

Cited By ~ 254

Author(s):

Anjon Audhya ◽

Michelangelo Foti ◽

Scott D. Emr

Keyword(s):

Cell Growth ◽

Membrane Trafficking ◽

Secretory Pathway ◽

Membrane Dynamics ◽

Small Gtpase ◽

Effector Proteins ◽

Restrictive Temperature ◽

Yeast Saccharomyces Cerevisiae ◽

Cytoskeleton Organization ◽

Yeast Viability

The yeast Saccharomyces cerevisiae possesses two genes that encode phosphatidylinositol (PtdIns) 4-kinases,STT4 and PIK1. Both gene products phosphorylate PtdIns at the D-4 position of the inositol ring to generate PtdIns(4)P, which plays an essential role in yeast viability because deletion of either STT4 orPIK1 is lethal. Furthermore, although both enzymes have the same biochemical activity, increased expression of either kinase cannot compensate for the loss of the other, suggesting that these kinases regulate distinct intracellular functions, each of which is required for yeast cell growth. By the construction of temperature-conditional single and double mutants, we have found that Stt4p activity is required for the maintenance of vacuole morphology, cell wall integrity, and actin cytoskeleton organization. In contrast, Pik1p is essential for normal secretion, Golgi and vacuole membrane dynamics, and endocytosis. Strikingly,pik1tscells exhibit a rapid defect in secretion of Golgi-modified secretory pathway cargos, Hsp150p and invertase, whereas stt4tscells exhibit no detectable secretory defects. Both single mutants reduce PtdIns(4)P by ∼50%; however,stt4ts/pik1tsdouble mutant cells produce more than 10-fold less PtdIns(4)P as well as PtdIns(4,5)P2. The aberrant Golgi morphology found in pik1tsmutants is strikingly similar to that found in cells lacking the function of Arf1p, a small GTPase that is known to regulate multiple membrane trafficking events throughout the cell. Consistent with this observation, arf1 mutants exhibit reduced PtdIns(4)P levels. In contrast, diminished levels of PtdIns(4)P observed in stt4tscells at restrictive temperature result in a dramatic change in vacuole size compared with pik1tscells and persistent actin delocalization. Based on these results, we propose that Stt4p and Pik1p act as the major, if not the only, PtdIns 4-kinases in yeast and produce distinct pools of PtdIns(4)P and PtdIns(4,5)P2that act on different intracellular membranes to recruit or activate as yet uncharacterized effector proteins.

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CDC68, a yeast gene that affects regulation of cell proliferation and transcription, encodes a protein with a highly acidic carboxyl terminus

Molecular and Cellular Biology ◽

10.1128/mcb.11.11.5718-5726.1991 ◽

1991 ◽

Vol 11 (11) ◽

pp. 5718-5726

Author(s):

A Rowley ◽

R A Singer ◽

G C Johnston

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Cycle Performance ◽

Carboxyl Terminus ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Negative Effects ◽

Yeast Saccharomyces Cerevisiae ◽

Acid Protein ◽

Mutant Cells

The cell cycle of the budding yeast Saccharomyces cerevisiae has been investigated through the study of conditional cdc mutations that specifically affect cell cycle performance. Cells bearing the cdc68-1 mutation (J. A. Prendergast, L. E. Murray, A. Rowley, D. R. Carruthers, R. A. Singer, and G. C. Johnston, Genetics 124:81-90, 1990) are temperature sensitive for the performance of the G1 regulatory event, START. Here we describe the CDC68 gene and present evidence that the CDC68 gene product functions in transcription. CDC68 encodes a 1,035-amino-acid protein with a highly acidic and serine-rich carboxyl terminus. The abundance of transcripts from several unrelated genes is decreased in cdc68-1 mutant cells after transfer to the restrictive temperature, while at least one transcript, from the HSP82 gene, persists in an aberrant fashion. Thus, the cdc68-1 mutation has both positive and negative effects on gene expression. Our findings complement those of Malone et al. (E. A. Malone, C. D. Clark, A. Chiang, and F. Winston, Mol. Cell. Biol. 11:5710-5717, 1991), who have independently identified the CDC68 gene (as SPT16) as a transcriptional suppressor of delta-insertion mutations. Among transcripts that rapidly become depleted in cdc68-1 mutant cells are those of the G1 cyclin genes CLN1, CLN2, and CLN3/WHI1/DAF1, whose activity has been previously shown to be required for the performance of START. The decreased abundance of cyclin transcripts in cdc68-1 mutant cells, coupled with the suppression of cdc68-1-mediated START arrest by the CLN2-1 hyperactive allele of CLN2, shows that the CDC68 gene affects START through cyclin gene expression.

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Fission yeast genes nda1+ and nda4+, mutations of which lead to S-phase block, chromatin alteration and Ca2+ suppression, are members of the CDC46/MCM2 family.

Molecular Biology of the Cell ◽

10.1091/mbc.4.10.1003 ◽

1993 ◽

Vol 4 (10) ◽

pp. 1003-1015 ◽

Cited By ~ 44

Author(s):

S Miyake ◽

N Okishio ◽

I Samejima ◽

Y Hiraoka ◽

T Toda ◽

...

Keyword(s):

Fission Yeast ◽

Restrictive Temperature ◽

Permissive Temperature ◽

Positive Role ◽

A Cell ◽

Cold Sensitive ◽

Single Nucleus ◽

Yeast Genes ◽

Cell Cycle Block ◽

Mutant Cells

Fission yeast cold-sensitive mutants nda1-376 and nda4-108 display a cell cycle block phenotype at the restrictive temperature (cell elongation with the single nucleus) accompanied by an alteration in the nuclear chromatin region. DNA content analysis shows that the onset of DNA synthesis is blocked or greatly delayed in both mutant cells, the block being reversible in nda4-108. Upon release to the permissive temperature, nda4-108 cells resumed replicating DNA, followed by mitosis and cytokinesis. The nda4 phenotype was partly rescued by the addition of Ca2+ to the medium; Ca2+ plays a positive role in the nda4+ function. The predicted protein sequences of nda1+ and nda4+ isolated by complementation are similar to each other and also, respectively, to those of the budding yeast, MCM2 and CDC46, both of which are members of the gene family required for the initiation of DNA replication. The central domains of these proteins are conserved, whereas the NH2- and COOH- domains are distinct. Results of the disruption of the nda1+ and nda4+ genes demonstrates that they are essential for viability.

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Mitochondrial GrpE modulates the function of matrix Hsp70 in translocation and maturation of preproteins.

Molecular and Cellular Biology ◽

10.1128/mcb.15.12.7098 ◽

1995 ◽

Vol 15 (12) ◽

pp. 7098-7105 ◽

Cited By ~ 55

Author(s):

S Laloraya ◽

P J Dekker ◽

W Voos ◽

E A Craig ◽

N Pfanner

Keyword(s):

Proteolytic Processing ◽

Mitochondrial Proteins ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Wild Type ◽

Yeast Saccharomyces Cerevisiae ◽

Conditional Mutant ◽

The Matrix ◽

Matrix Heat

Mitochondrial GrpE (Mge1p) is a mitochondrial cochaperone essential for viability of the yeast Saccharomyces cerevisiae. To study the role of Mge1p in the biogenesis of mitochondrial proteins, we isolated a conditional mutant allele of MGE1 which conferred a temperature-sensitive growth phenotype and led to the accumulation of mitochondrial preproteins after shifting of the cells to the restrictive temperature. The mutant Mge1 protein was impaired in its interaction with the matrix heat shock protein mt-Hsp70. The mutant mitochondria showed a delayed membrane translocation of preproteins, and the maturation of imported proteins was impaired, as evidenced by the retarded second proteolytic processing of a preprotein in the matrix. Moreover, the aggregation of imported proteins was decreased in the mutant mitochondria. The mutant Mge1p differentially modulated the interaction of mt-Hsp70 with preproteins compared with the wild type, resulting in decreased binding to preproteins in membrane transit and enhanced binding to fully imported proteins. We conclude that the interaction of Mge1p with mt-Hsp70 promotes the progress of the Hsp70 reaction cycle, which is essential for import and maturation of mitochondrial proteins.

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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 ◽

10.1093/genetics/148.1.33 ◽

1998 ◽

Vol 148 (1) ◽

pp. 33-47 ◽

Cited By ~ 2

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.

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