scholarly journals The role of Myo2, a yeast class V myosin, in vesicular transport.

1995 ◽  
Vol 128 (6) ◽  
pp. 1055-1068 ◽  
Author(s):  
B Govindan ◽  
R Bowser ◽  
P Novick
Keyword(s):  
Mother Cell ◽  
Secretory Pathway ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Transit Times ◽  
Cargo Proteins ◽  
Section Electron ◽  
Synthetically Lethal ◽  
Actin Cables ◽  
Exocytic Pathway

Previous studies have shown that temperature-sensitive, myo2-66 yeast arrest as large, unbudded cells that accumulate vesicles within their cytoplasm (Johnston, G. C., J. A. Prendergast, and R. A. Singer. 1991. J. Cell Biol. 113:539-551). In this study we show that myo2-66 is synthetically lethal in combination with a subset of the late-acting sec mutations. Thin section electron microscopy shows that the post-Golgi blocked secretory mutants, sec1-1 and sec6-4, rapidly accumulate vesicles in the bud, upon brief incubations at the restrictive temperature. In contrast, myo2-66 cells accumulate vesicles predominantly in the mother cell. Double mutant analysis also places Myo2 function in a post-Golgi stage of the secretory pathway. Despite the accumulation of vesicles in myo2-66 cells, pulse-chase studies show that the transit times of several secreted proteins, including invertase and alpha factor, as well as the vacuolar proteins, carboxy-peptidase Y and alkaline phosphatase, are normal. Therefore the vesicles which accumulate in this mutant may function on an exocytic pathway that transports a set of cargo proteins that is distinct from those analyzed. Our observations are consistent with a role for Myo2 in transporting a class of secretory vesicles from the mother cell along actin cables into the bud.

scholarly journals Function of the ypt2 gene in the exocytic pathway of Schizosaccharomyces pombe.

1993 ◽  
Vol 4 (10) ◽  
pp. 1069-1076 ◽  
Author(s):  
M W Craighead ◽  
S Bowden ◽  
R Watson ◽  
J Armstrong
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Secretory Pathway ◽  
Normal Growth ◽  
Gene Replacement ◽  
Secretory Protein ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Last Stage ◽  
Exocytic Pathway ◽  
Mutant Cells

The ypt2 gene of the fission yeast Schizosaccharomyces pombe encodes a member of the ypt/rab family of small GTP-binding proteins, related in sequence to Sec4p of Saccharomyces cerevisiae but closer to mammalian rab8. We have introduced a mutation into the gene corresponding to a mutation identified in ypt1, in which a conserved valine residue was altered to asparagine. The mutated ypt2 gene was introduced into the S. pombe genome by gene replacement. The resulting strain was temperature-sensitive for growth. Normal growth was restored by introduction of a plasmid-borne wild-type ypt2 cDNA or by cDNA for rab8 but not by various other rab or ypt sequences. At restrictive temperature the mutant cells accumulated the secretory protein acid phosphatase in a form that appeared to be fully glycosylated and acquired a population of vesicles detectable by electron microscopy. Thus the ypt2 protein, and by inference rab8, appear to function in the last stage of the secretory pathway.

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

1999 ◽  
Vol 147 (4) ◽  
pp. 729-742 ◽  
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.

Mammalian GPI-anchored proteins require p24 proteins for their efficient transport from the ER to the plasma membrane

2007 ◽  
Vol 409 (2) ◽  
pp. 555-562 ◽  
Author(s):  
Satoshi Takida ◽  
Yusuke Maeda ◽  
Taroh Kinoshita
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Direct Evidence ◽  
Temperature Sensitive ◽  
Early Secretory Pathway ◽  
Cargo Proteins ◽  
Transport Vesicle ◽  
A Cell ◽  
P24 Proteins

The GPI (glycosylphosphatidylinositol) moiety is attached to newly synthesized proteins in the lumen of the ER (endoplasmic reticulum). The modified proteins are then directed to the PM (plasma membrane). Less well understood is how nascent mammalian GPI-anchored proteins are targeted from the ER to the PM. In the present study, we investigated mechanisms underlying membrane trafficking of the GPI-anchored proteins, focusing on the early secretory pathway. We first established a cell line that stably expresses inducible temperature-sensitive GPI-fused proteins as a reporter and examined roles of transport-vesicle constituents called p24 proteins in the traffic of the GPI-anchored proteins. We selectively suppressed one of the p24 proteins, namely p23, employing RNAi (RNA interference) techniques. The suppression resulted in pronounced delays of PM expression of the GPI-fused reporter proteins. Furthermore, maturation of DAF (decay-accelerating factor), one of the GPI-anchored proteins in mammals, was slowed by the suppression of p23, indicating delayed trafficking of DAF from the ER to the Golgi. Trafficking of non-GPI-linked cargo proteins was barely affected by p23 knockdown. This is the first to demonstrate direct evidence for the transport of mammalian GPI-anchored proteins being mediated by p24 proteins.

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

1998 ◽  
Vol 141 (5) ◽  
pp. 1107-1119 ◽  
Author(s):  
Susan M. VanRheenen ◽  
Xiaochun Cao ◽  
Vladimir V. Lupashin ◽  
Charles Barlowe ◽  
M. Gerard Waters
Keyword(s):  
Secretory Pathway ◽  
Genetic Relationships ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Dominant Form ◽  
Yeast Saccharomyces Cerevisiae ◽  
Vesicle Docking ◽  
Genes Encoding ◽  
A Cell ◽  
Cold Sensitive

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.

scholarly journals spp42, Identified as a Classical Suppressor of prp4-73, Which Encodes a Kinase Involved in Pre-mRNA Splicing in Fission Yeast, Is a Homologue of the Splicing Factor Prp8p

Genetics ◽  
1999 ◽  
Vol 153 (3) ◽  
pp. 1183-1191
Author(s):  
Henning Schmidt ◽  
Kathrin Richert ◽  
Robert A Drakas ◽  
Norbert F Käufer
Keyword(s):  
Mammalian Cells ◽  
Mrna Splicing ◽  
Splicing Factor ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Suppressor Genes ◽  
Bona Fide ◽  
Synthetically Lethal ◽  
Extragenic Suppressors ◽  
Chromosome I

Abstract We have identified two classical extragenic suppressors, spp41 and spp42, of the temperature sensitive (ts) allele prp4-73. The prp4+ gene of Schizosaccharomyces pombe encodes a protein kinase. Mutations in both suppressor genes suppress the growth and the pre-mRNA splicing defect of prp4-73ts at the restrictive temperature (36°). spp41 and spp42 are synthetically lethal with each other in the presence of prp4-73ts, indicating a functional relationship between spp41 and spp42. The suppressor genes were mapped on the left arm of chromosome I proximal to the his6 gene. Based on our mapping data we isolated spp42 by screening PCR fragments for functional complementation of the prp4-73ts mutant at the restrictive temperature. spp42 encodes a large protein (p275), which is the homologue of Prp8p. This protein has been shown in budding yeast and mammalian cells to be a bona fide pre-mRNA splicing factor. Taken together with other recent genetic and biochemical data, our results suggest that Prp4 kinase plays an important role in the formation of catalytic spliceosomes.

scholarly journals Sld2, Which Interacts with Dpb11 inSaccharomyces cerevisiae, Is Required for Chromosomal DNA Replication

1998 ◽  
Vol 18 (10) ◽  
pp. 6102-6109 ◽  
Author(s):  
Yoichiro Kamimura ◽  
Hiroshi Masumoto ◽  
Akio Sugino ◽  
Hiroyuki Araki
Keyword(s):  
Dna Replication ◽  
Temperature Shift ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Chromosomal Dna ◽  
Autonomously Replicating Sequence ◽  
Complementation Groups ◽  
Synthetically Lethal ◽  
Dna Polymerase Ii

ABSTRACT The DPB11 gene, which genetically interacts with DNA polymerase II (ɛ), one of three replicative DNA polymerases, is required for DNA replication and the S phase checkpoint inSaccharomyces cerevisiae. To identify factors interacting with Dbp11, we have isolated sld (synthetically lethal withdpb11-1) mutations which fall into six complementation groups (sld1 to -6). In this study, we characterized SLD2, encoding an essential 52-kDa protein. High-copy SLD2 suppressed the thermosensitive growth defect caused by dpb11-1. Conversely, high-copy DPB11suppressed the temperature-sensitive growth defect caused bysld2-6. The interaction between Sld2 and Dpb11 was detected in a two-hybrid assay. This interaction was evident at 25°C but not at 34°C when Sld2-6 or Dpb11-1 replaced its wild-type protein. No interaction between Sld2-6 and Dpb11-1 could be detected even at 25°C. Immunoprecipitation experiments confirmed that Dpb11 physically interacts with Sld2. sld2-6 cells were defective in DNA replication at the restrictive temperature, as were dpb11-1cells. Further, in dpb11-1 and sld2-6 cells, the bubble-shaped replication intermediates formed in the region of the autonomously replicating sequence reduced quickly after a temperature shift. These results strongly suggest the involvement of the Dpb11-Sld2 complex in a step close to the initiation of DNA replication.

scholarly journals Inhibition of Endosome Function in CHO Cells Bearing a Temperature-sensitive Defect in the Coatomer (COPI) Component ε-COP

1997 ◽  
Vol 139 (7) ◽  
pp. 1747-1759 ◽  
Author(s):  
Elizabeth Daro ◽  
David Sheff ◽  
Marie Gomez ◽  
Thomas Kreis ◽  
Ira Mellman
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Semliki Forest Virus ◽  
Temperature Shift ◽  
Endocytic Pathway ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Intact Cells ◽  
Early Endosomes ◽  
Mutant Phenotypes

Recent evidence has suggested that subunits of the coatomer protein (COPI) complexes are functionally associated with endosomes in mammalian cells. We now provide genetic evidence that COPI plays a role in endocytosis in intact cells. The ldlF mutant CHO cell line bears a temperature-sensitive defect in the COPI subunit ε-COP. In addition to exhibiting conditional defects in the secretory pathway, we find that the cells are also defective at mediating endosome-associated functions. As found for cells microinjected with anti-COPI antibodies, ldlF cells at the restrictive temperature could not be infected by vesicular stomatitis (VSV) or Semliki Forest virus (SFV) that require delivery to acidic endosomes to penetrate into the cytosol. Although there was no temperature-sensitive defect in the internalization of receptor-bound transferrin (Tfn), Tfn recycling and accumulation of HRP were markedly inhibited at the restrictive temperature. Sorting of receptor-bound markers such as EGF to lysosomes was also reduced, although delivery of fluid-phase markers was only partially inhibited. In addition, lysosomes redistributed from their typical perinuclear location to the tips of the ldlF cells. Mutant phenotypes began to emerge within 2 h of temperature shift, the time required for the loss of detectable ε-COP, suggesting that the endocytic defects were not secondary to a block in the secretory pathway. Importantly, the mutant phenotypes were also corrected by transfection of wild-type ε-COP cDNA demonstrating that they directly or indirectly reflected the ε-COP defect. Taken together, the results suggest that ε-COP acts early in the endocytic pathway, most likely inhibiting the normal sorting and recycling functions of early endosomes.

scholarly journals A subset of yeast vacuolar protein sorting mutants is blocked in one branch of the exocytic pathway

2002 ◽  
Vol 156 (2) ◽  
pp. 271-286 ◽  
Author(s):  
Edina Harsay ◽  
Randy Schekman
Keyword(s):  
Protein Sorting ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Vacuolar Protein Sorting ◽  
Sorting Receptor ◽  
Gradient Fractionation ◽  
Vacuolar Protein ◽  
Exocytic Pathway ◽  
Dense Vesicles

Exocytic vesicles that accumulate in a temperature-sensitive sec6 mutant at a restrictive temperature can be separated into at least two populations with different buoyant densities and unique cargo molecules. Using a sec6 mutant background to isolate vesicles, we have found that vacuolar protein sorting mutants that block an endosome-mediated route to the vacuole,including vps1, pep12, vps4, and a temperature-sensitive clathrin mutant, missort cargo normally transported by dense exocytic vesicles, such as invertase, into light exocytic vesicles, whereas transport of cargo specific to the light exocytic vesicles appears unaffected. Immunoisolation experiments confirm that missorting, rather than a changed property of the normally dense vesicles, is responsible for the altered density gradient fractionation profile. The vps41Δ and apl6Δmutants, which block transport of only the subset of vacuolar proteins that bypasses endosomes, sort exocytic cargo normally. Furthermore, avps10Δ sec6 mutant, which lacks the sorting receptor for carboxypeptidase Y (CPY), accumulates both invertase and CPY in dense vesicles. These results suggest that at least one branch of the yeast exocytic pathway transits through endosomes before reaching the cell surface. Consistent with this possibility, we show that immunoisolated clathrin-coated vesicles contain invertase.

scholarly journals Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection.

1987 ◽  
Vol 105 (4) ◽  
pp. 1587-1594 ◽  
Author(s):  
A P Newman ◽  
S Ferro-Novick
Keyword(s):  
Golgi Apparatus ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
New Genes ◽  
Complex Process ◽  
Complementation Groups ◽  
Thin Section Analysis

We have adapted a [3H]mannose suicide selection to identify mutations in additional genes which function in the early part of the yeast secretory pathway. Thus far this protocol has led to the identification of two new genes which are implicated in this process, as well as additional alleles of previously identified genes. The new mutants, bet1 and bet2, are temperature sensitive for growth and protein transport. Thin section analysis has revealed the accumulation of a network of endoplasmic reticulum (ER) at the restrictive temperature (37 degrees C). Precursors of exported proteins that accumulate in the cell at 37 degrees C are terminally core glycosylated. These observations suggest that the transport of precursors is blocked subsequent to translocation into the ER but before entry into the Golgi apparatus. The bet1 and bet2 mutants define two new complementation groups which have the same properties as previously identified ER-accumulating mutants. This and previous findings (Novick, P., C. Field, and R. Schekman, 1980, Cell, 21:205-215) suggest that protein exit from the ER and entry into the Golgi apparatus is a complex process requiring at least 11 genes.

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

1998 ◽  
Vol 18 (12) ◽  
pp. 7139-7146 ◽  
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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