scholarly journals A Role for Tlg1p in the Transport of Proteins within the Golgi Apparatus ofSaccharomyces cerevisiae

1999 ◽  
Vol 10 (7) ◽  
pp. 2407-2423 ◽  
Author(s):  
John G. S. Coe ◽  
Anthony C. B. Lim ◽  
Jing Xu ◽  
Wanjin Hong
Keyword(s):  
Golgi Apparatus ◽  
Snare Complex ◽  
Growth Defect ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Dependent Manner ◽  
Nonpermissive Temperature ◽  
Vacuolar Protein

Members of the syntaxin protein family participate in the docking–fusion step of several intracellular vesicular transport events. Tlg1p has been identified as a nonessential protein required for efficient endocytosis as well as the maintenance of normal levels of trans-Golgi network proteins. In this study we independently describe Tlg1p as an essential protein required for cell viability. Depletion of Tlg1p in vivo causes a defect in the transport of the vacuolar protein carboxypeptidase Y through the early Golgi. Temperature-sensitive (ts) mutants of Tlg1p also accumulate the endoplasmic reticulum/cis-Golgi form of carboxypeptidase Y at the nonpermissive temperature (38°C) and exhibit underglycosylation of secreted invertase. Overexpression of Tlg1p complements the growth defect of vti1-11 at the nonpermissive temperature, whereas incomplete complementation was observed with vti1-1, further suggesting a role for Tlg1p in the Golgi apparatus. Overexpression of Sed5p decreases the viability of tlg1 ts mutants compared with wild-type cells, suggesting that tlg1 ts mutants are more susceptible to elevated levels of Sed5p. Tlg1p is able to bind His6-tagged Sec17p (yeast α-SNAP) in a dose-dependent manner and enters into a SNARE complex with Vti1p, Tlg2p, and Vps45p. Morphological analyses by electron microscopy reveal that cells depleted of Tlg1p or tlg1 ts mutants incubated at the restrictive temperature accumulate 40- to 50-nm vesicles and experience fragmentation of the vacuole.

scholarly journals Characterization of a Novel Yeast SNARE Protein Implicated in Golgi Retrograde Traffic

1997 ◽  
Vol 8 (12) ◽  
pp. 2659-2676 ◽  
Author(s):  
Vladimir V. Lupashin ◽  
Irina D. Pokrovskaya ◽  
James A. McNew ◽  
M. Gerard Waters
Keyword(s):  
Protein Trafficking ◽  
Sequence Similarity ◽  
Integral Membrane Protein ◽  
Growth Defect ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Snare Protein ◽  
Yeast Viability ◽  
Vacuolar Protein

The protein trafficking machinery of eukaryotic cells is employed for protein secretion and for the localization of resident proteins of the exocytic and endocytic pathways. Protein transit between organelles is mediated by transport vesicles that bear integral membrane proteins (v-SNAREs) which selectively interact with similar proteins on the target membrane (t-SNAREs), resulting in a docked vesicle. A novelSaccharomyces cerevisiae SNARE protein, which has been termed Vti1p, was identified by its sequence similarity to known SNAREs. Vti1p is a predominantly Golgi-localized 25-kDa type II integral membrane protein that is essential for yeast viability. Vti1p can bind Sec17p (yeast SNAP) and enter into a Sec18p (NSF)-sensitive complex with the cis-Golgi t-SNARE Sed5p. This Sed5p/Vti1p complex is distinct from the previously described Sed5p/Sec22p anterograde vesicle docking complex. Depletion of Vti1p in vivo causes a defect in the transport of the vacuolar protein carboxypeptidase Y through the Golgi. Temperature-sensitive mutants of Vti1p show a similar carboxypeptidase Y trafficking defect, but the secretion of invertase and gp400/hsp150 is not significantly affected. The temperature-sensitive vti1 growth defect can be rescued by the overexpression of the v-SNARE, Ykt6p, which physically interacts with Vti1p. We propose that Vti1p, along with Ykt6p and perhaps Sft1p, acts as a retrograde v-SNARE capable of interacting with the cis-Golgi t-SNARE Sed5p.

scholarly journals Yeast carboxypeptidase Y can be translocated and glycosylated without its amino-terminal signal sequence.

1987 ◽  
Vol 104 (5) ◽  
pp. 1183-1191 ◽  
Author(s):  
E Blachly-Dyson ◽  
T H Stevens
Keyword(s):  
Secretory Pathway ◽  
Signal Sequence ◽  
Mutant Gene ◽  
Carboxypeptidase Y ◽  
Dependent Manner ◽  
Hydrophobic Core ◽  
Amino Terminal ◽  
Type Gene ◽  
Vacuolar Protein

We have constructed a series of mutations in the signal sequence of the yeast vacuolar protein carboxypeptidase Y (CPY), and have used pulse-chase radiolabeling and immunoprecipitation to examine the in vivo effects of these mutations on the entry of the mutant CPY proteins into the secretory pathway. We find that introduction of a negatively charged residue, aspartate, into the hydrophobic core of the signal sequence has no apparent effect on signal sequence function. In contrast, internal in-frame deletions within the signal sequence cause CPY to be synthesized as unglycosylated precursors. These are slowly and inefficiently converted to glycosylated precursors that are indistinguishable from the glycosylated forms produced from the wild-type gene. These precursors are converted to active CPY in a PEP4-dependent manner, indicating that they are correctly localized to the vacuole. Surprisingly, a deletion mutation that removes the entire CPY signal sequence has a similar effect: unglycosylated precursor accumulates in cells carrying this mutant gene, and greater than 10% of it is posttranslationally glycosylated. Thus, the amino-terminal signal sequence of CPY, while important for translocation efficiency, is not absolutely required for the translocation of this protein.

scholarly journals A yeast protein related to a mammalian Ras-binding protein, Vps9p, is required for localization of vacuolar proteins.

1996 ◽  
Vol 16 (5) ◽  
pp. 2369-2377 ◽  
Author(s):  
C G Burd ◽  
P A Mustol ◽  
P V Schu ◽  
S D Emr
Keyword(s):  
Intracellular Transport ◽  
Protein Sorting ◽  
Growth Defect ◽  
Carboxypeptidase Y ◽  
Rab Gtpase ◽  
Temperature Sensitive ◽  
Electron Microscopic ◽  
Yeast Saccharomyces Cerevisiae ◽  
Vacuolar Protein Sorting ◽  
Vacuolar Protein

In the yeast Saccharomyces cerevisiae, mutations in vacuolar protein sorting (VPS) genes result in secretion of proteins normally localized to the vacuole. Characterization of the VPS pathway has provided considerable insight into mechanisms of protein sorting and vesicle-mediated intracellular transport. We have cloned VPS9 by complementation of the vacuolar protein sorting defect of vps9 cells, characterized its gene product, and investigated its role in vacuolar protein sorting. Cells with a vps9 disruption exhibit severe vacuolar protein sorting defects and a temperature-sensitive growth defect at 38 degrees C. Electron microscopic examination of delta vps9 cells revealed the appearance of novel reticular membrane structures as well as an accumulation of 40- to 50-nm-diameter vesicles, suggesting that Vps9p may be required for the consumption of transport vesicles containing vacuolar protein precursors. A temperature-conditional allele of vps9 was constructed and used to investigate the function of Vps9p. Immediately upon shifting of temperature-conditional vps9 cells to the nonpermissive temperature, newly synthesized carboxypeptidase Y was secreted, indicating that Vps9p function is directly required in the VPS pathway. Antibodies raised against Vps9p immunoprecipitate a rare 52-kDa protein that fractionates with cytosolic proteins following cell lysis and centrifugation. Analysis of the VPS9 DNA sequence predicts that Vps9p is related to human proteins that bind Ras and negatively regulate Ras-mediated signaling. We term the related regions of Vps9p and these Ras-binding proteins a GTPase binding homology domain and suggest that it defines a family of proteins that bind monomeric GTPases. Vps9p may bind and serve as an effector of a rab GTPase, like Vps2lp, required for vacuolar protein sorting.

scholarly journals Vam7p, a SNAP-25-Like Molecule, and Vam3p, a Syntaxin Homolog, Function Together in Yeast Vacuolar Protein Trafficking

1998 ◽  
Vol 18 (9) ◽  
pp. 5308-5319 ◽  
Author(s):  
Trey K. Sato ◽  
Tamara Darsow ◽  
Scott D. Emr
Keyword(s):  
Protein Trafficking ◽  
Protein Transport ◽  
Coiled Coil ◽  
Snare Complex ◽  
Temperature Sensitive ◽  
Physical Interaction ◽  
Nonpermissive Temperature ◽  
Coiled Coil Domain ◽  
Px Domain ◽  
Vacuolar Protein

ABSTRACT A genetic screen to isolate gene products required for vacuolar morphogenesis in the yeast Saccharomyces cerevisiaeidentified VAM7, a gene which encodes a protein containing a predicted coiled-coil domain homologous to the coiled-coil domain of the neuronal t-SNARE, SNAP-25 (Y. Wada and Y. Anraku, J. Biol. Chem. 267:18671–18675, 1992; T. Weimbs, S. H. Low, S. J. Chapin, K. E. Mostov, P. Bucher, and K. Hofmann, Proc. Natl. Acad. Sci. USA 94:3046–3051, 1997). Analysis of a temperature-sensitive-for-function (tsf) allele ofVAM7 (vam7tsf ) demonstrated that the VAM7 gene product directly functions in vacuolar protein transport. vam7tsf mutant cells incubated at the nonpermissive temperature displayed rapid defects in the delivery of multiple proteins that traffic to the vacuole via distinct biosynthetic pathways. Examination ofvam7tsf cells at the nonpermissive temperature by electron microscopy revealed the accumulation of aberrant membranous compartments that may represent unfused transport intermediates. A fraction of Vam7p was localized to vacuolar membranes. Furthermore,VAM7 displayed genetic interactions with the vacuolar syntaxin homolog, VAM3. Consistent with the genetic results, Vam7p physically associated in a complex containing Vam3p, and this interaction was enhanced by inactivation of the yeast NSF (N-ethyl maleimide-sensitive factor) homolog, Sec18p. In addition to the coiled-coil domain, Vam7p also contains a putative NADPH oxidase p40phox (PX) domain. Changes in two conserved amino acids within this domain resulted in synthetic phenotypes when combined with the vam3tsf mutation, suggesting that the PX domain is required for Vam7p function. This study provides evidence for the functional and physical interaction between Vam7p and Vam3p at the vacuolar membrane, where they function as part of a t-SNARE complex required for the docking and/or fusion of multiple transport intermediates destined for the vacuole.

scholarly journals Structure-Function Analysis of Yeast mRNA Cap Methyltransferase and High-Copy Suppression of Conditional Mutants by AdoMet Synthase and the Ubiquitin Conjugating Enzyme Cdc34p

Genetics ◽  
2000 ◽  
Vol 155 (4) ◽  
pp. 1561-1576
Author(s):  
Beate Schwer ◽  
Nayanendu Saha ◽  
Xiangdong Mao ◽  
Hsiao-Wang Chen ◽  
Stewart Shuman
Keyword(s):  
Function Analysis ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Mrna Levels ◽  
Ubiquitin Conjugating Enzyme ◽  
Ubiquitin Conjugation ◽  
Yeast Genes ◽  
Conjugating Enzyme

Abstract Here we present a genetic analysis of the yeast cap-methylating enzyme Abd1p. To identify individual amino acids required for Abd1p function, we introduced alanine mutations at 35 positions of the 436-amino acid yeast protein. Two new recessive lethal mutations, F256A and Y330A, were identified. Alleles F256L and Y256L were viable, suggesting that hydrophobic residues at these positions sufficed for Abd1p function. Conservative mutations of Asp-178 established that an acidic moiety is essential at this position (i.e., D178E was viable whereas D178N was not). Phe-256, Tyr-330, and Asp-178 are conserved in all known cellular cap methyltransferases. We isolated temperature-sensitive abd1 alleles and found that abd1-ts cells display a rapid shut-off of protein synthesis upon shift to the restrictive temperature, without wholesale reduction in steady-state mRNA levels. These in vivo results are consistent with classical biochemical studies showing a requirement for the cap methyl group in cap-dependent translation. We explored the issue of how cap methylation might be regulated in vivo by conducting a genetic screen for high-copy suppressors of the ts growth defect of abd1 mutants. The identification of the yeast genes SAM2 and SAM1, which encode AdoMet synthase, as abd1 suppressors suggests that Abd1p function can be modulated by changes in the concentration of its substrate AdoMet. We also identified the ubiquitin conjugating enzyme Cdc34p as a high-copy abd1 suppressor. We show that mutations of Cdc34p that affect its ubiquitin conjugation activity or its capacity to interact with the E3-SCF complex abrogate its abd1 suppressor function. Moreover, the growth defect of abd1 mutants is exacerbated by cdc34-2. These findings suggest a novel role for Cdc34p in gene expression and engender a model whereby cap methylation or cap utilization is negatively regulated by a factor that is degraded when Cdc34p is overexpressed.

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 Vesicle-mediated protein transport: regulatory interactions between the Vps15 protein kinase and the Vps34 PtdIns 3-kinase essential for protein sorting to the vacuole in yeast.

1995 ◽  
Vol 129 (2) ◽  
pp. 321-334 ◽  
Author(s):  
J H Stack ◽  
D B DeWald ◽  
K Takegawa ◽  
S D Emr
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Protein Sorting ◽  
Intracellular Localization ◽  
Kinase Domain ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Vacuolar Protein Sorting ◽  
Vacuolar Protein

A membrane-associated complex composed of the Vps15 protein kinase and the Vps34 phosphatidylinositol 3-kinase (PtdIns 3-kinase) is essential for the delivery of proteins to the yeast vacuole. An active Vps15p is required for the recruitment of Vps34p to the membrane and subsequent stimulation of Vps34p PtdIns 3-kinase activity. Consistent with this, mutations altering highly conserved residues in the lipid kinase domain of Vps34p lead to a dominant-negative phenotype resulting from titration of activating Vps15 proteins. In contrast, catalytically inactive Vps15p mutants do not produce a dominant mutant phenotype because they are unable to associate with Vps34p in a wild-type manner. These data indicate that an intact Vps15p protein kinase domain is necessary for the association with and activation of Vps34p, and they demonstrate that a functional Vps15p-Vps34p complex is absolutely required for the efficient delivery of proteins to the vacuole. Analysis of a temperature-conditional allele of VPS15, in which a shift to the nonpermissive temperature leads to a decrease in cellular PtdIns(3)P levels, indicates that the loss of Vps15p function leads to a defect in activation of Vps34p. In addition, characterization of a temperature-sensitive allele of VPS34 demonstrates that inactivation of Vps34p leads to the immediate missorting of soluble vacuolar proteins (e.g., carboxypeptidase Y) without an apparent defect in the sorting of the vacuolar membrane protein alkaline phosphatase. This rapid block in vacuolar protein sorting appears to be the result of loss of PtdIns 3-kinase activity since cellular PtdIns(3)P levels decrease dramatically in vps34 temperature-sensitive mutant cells that have been incubated at the nonpermissive temperature. Finally, analysis of the defects in cellular PtdIns(3)P levels in various vps15 and vsp34 mutant strains has led to additional insights into the importance of PtdIns(3)P intracellular localization, as well as the roles of Vps15p and Vps34p in vacuolar protein sorting.

The Mechanism of Replication of Single-Stranded DNA. VI. Requirements for Ribonucleoside Triphosphates and DNA Polymerase III

1973 ◽  
Vol 51 (12) ◽  
pp. 1588-1597 ◽  
Author(s):  
David T. Denhardt ◽  
Makoto Iwaya ◽  
Grant McFadden ◽  
Gerald Schochetman
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerases ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Dna Polymerase Iii ◽  
Single Stranded Dna ◽  
E Coli ◽  
Polymerase Iii

Evidence is presented that in Escherichia coli made permeable to nucleotides by exposure to toluene, the synthesis of a DNA chain complementary to the infecting single-stranded DNA of bacteriophage [Formula: see text] requires ATP as well as the four deoxyribonucleoside triphosphates. This synthesis results in the formation of the parental double-stranded replicative-form (RF) molecule. The ATP is not required simply to prevent degradation of the ribonucleoside or deoxyribonucleoside triphosphates; it can be partially substituted for by other ribonucleoside triphosphates.No single one of the known E. coli DNA polymerases appears to be uniquely responsible in vivo for the formation of the parental RF. Since [Formula: see text] replicates well in strains lacking all, or almost all, of the in-vitro activities of DNA polymerases I and II, neither of these two enzymes would seem essential; and in a temperature-sensitive E. coli mutant (dnaEts) deficient in DNA polmerase-I activity and possessing a temperature-sensitive DNA polymerase III, the viral single-stranded DNA is efficiently incorporated into an RF molecule at the restrictive temperature. In contrast, both RF replication and progeny single-stranded DNA synthesis are dependent upon DNA polymerase III activity.

scholarly journals A High Copy Suppressor Screen Reveals Genetic Interactions Between BET3 and a New Gene: Evidence for a Novel Complex in ER-to-Golgi Transport

Genetics ◽  
1998 ◽  
Vol 149 (2) ◽  
pp. 833-841
Author(s):  
Yu Jiang ◽  
Al Scarpa ◽  
Li Zhang ◽  
Shelly Stone ◽  
Ed Feliciano ◽  
...  
Keyword(s):  
Growth Defect ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Specific Suppression ◽  
Golgi Transport ◽  
New Gene ◽  
Insight Into ◽  
Suppressor Screen

Abstract The BET3 gene in the yeast Saccharomyces cerevisiae encodes a 22-kD hydrophilic protein that is required for vesicular transport between the ER and Golgi complex. To gain insight into the role of Bet3p, we screened for genes that suppress the growth defect of the temperature-sensitive bet3 mutant at 34°. This high copy suppressor screen resulted in the isolation of a new gene, called BET5. BET5 encodes an essential 18-kD hydrophilic protein that in high copy allows growth of the bet3-1 mutant, but not other ER accumulating mutants. This strong and specific suppression is consistent with the fact that Bet3p and Bet5p are members of the same complex. Using PCR mutagenesis, we generated a temperature-sensitive mutation in BET5 (bet5-1) that blocks the transport of carboxypeptidase Y to the vacuole and prevents secretion of the yeast pheromone α-factor at 37°. The precursor forms of these proteins that accumulate in this mutant are indicative of a block in membrane traffic between the ER and Golgi apparatus. High copy suppressors of the bet5-1 mutant include several genes whose products are required for ER-to-Golgi transport (BET1, SEC22, USO1 and DSS4) and the maintenance of the Golgi (ANP1). These findings support the hypothesis that Bet5p acts in conjunction with Bet3p to mediate a late stage in ER-to-Golgi transport. The identification of mammalian homologues of Bet3p and Bet5p implies that the Bet3p/Bet5p complex is highly conserved in evolution.

Identification of temperature-sensitive DNA- mutants of Chinese hamster cells affected in cellular and viral DNA synthesis

1986 ◽  
Vol 6 (12) ◽  
pp. 4594-4601
Author(s):  
J J Dermody ◽  
B E Wojcik ◽  
H Du ◽  
H L Ozer
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Chinese Hamster ◽  
Human Adenovirus ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Dependent Manner ◽  
Viral Dna ◽  
Cell Functions

We described a strategy which facilitates the identification of cell mutants which are restricted in DNA synthesis in a temperature-dependent manner. A collection of over 200 cell mutants temperature-sensitive for growth was isolated in established Chinese hamster cell lines (CHO and V79) by a variety of selective and nonselective techniques. Approximately 10% of these mutants were identified as ts DNA- based on differential inhibition of macromolecular synthesis at the restrictive temperature (39 degrees C) as assessed by incorporation of [3H]thymidine and [35S]methionine. Nine such mutants, selected for further study, demonstrated rapid shutoff of DNA replication at 39 degrees C. Infections with two classes of DNA viruses extensively dependent on host-cell functions for their replication were used to distinguish defects in DNA synthesis itself from those predominantly affecting other aspects of DNA replication. All cell mutants supported human adenovirus type 2 (Ad2) and mouse polyomavirus DNA synthesis at the permissive temperature. Five of the nine mutants (JB3-B, JB3-O, JB7-K, JB8-D, and JB11-J) restricted polyomavirus DNA replication upon transfection with viral sequences at 33 degrees C and subsequent shift to 39 degrees C either before or after the onset of viral DNA synthesis. Only one of these mutants (JB3-B) also restricted Ad2 DNA synthesis after virion infection under comparable conditions. No mutant was both restrictive for Ad2 and permissive for polyomavirus DNA synthesis at 39 degrees C. The differential effect of these cell mutants on viral DNA synthesis is expected to assist subsequent definition of the biochemical defect responsible.

Sign in / Sign up

Export Citation Format

Share Document