scholarly journals Role of vacuolar acidification in protein sorting and zymogen activation: a genetic analysis of the yeast vacuolar proton-translocating ATPase.

1990 ◽  
Vol 10 (7) ◽  
pp. 3737-3749 ◽  
Author(s):  
C T Yamashiro ◽  
P M Kane ◽  
D F Wolczyk ◽  
R A Preston ◽  
T H Stevens
Keyword(s):  
Null Allele ◽  
Protein Sorting ◽  
Normal Growth ◽  
Zymogen Activation ◽  
Cellular Processes ◽  
Flow Cytometric ◽  
Vacuolar Acidification ◽  
Vacuolar Protein ◽  
A Minor ◽  
Proton Translocating Atpase

Vacuolar acidification has been proposed to play a key role in a number of cellular processes, including protein sorting, zymogen activation, and maintenance of intracellular pH. We investigated the significance of vacuolar acidification by cloning and mutagenizing the gene for the yeast vacuolar proton-translocating ATPase 60-kilodalton subunit (VAT2). Cells carrying a vat2 null allele were viable; however, these cells were severely defective for growth in medium buffered at neutral pH. Vacuoles isolated from cells bearing the vat2 null allele were completely devoid of vacuolar ATPase activity. The pH of the vacuolar lumen of cells bearing the vat2 mutation was 7.1, compared with the wild-type pH of 6.1, as determined by a flow cytometric pH assay. These results indicate that the vacuolar proton-translocating ATPase complex is essential for vacuolar acidification and that the low-pH state of the vacuole is crucial for normal growth. The vacuolar acidification-defective vat2 mutant exhibited normal zymogen activation but displayed a minor defect in vacuolar protein sorting.

Role of vacuolar acidification in protein sorting and zymogen activation: a genetic analysis of the yeast vacuolar proton-translocating ATPase

1990 ◽  
Vol 10 (7) ◽  
pp. 3737-3749
Author(s):  
C T Yamashiro ◽  
P M Kane ◽  
D F Wolczyk ◽  
R A Preston ◽  
T H Stevens
Keyword(s):  
Null Allele ◽  
Protein Sorting ◽  
Normal Growth ◽  
Zymogen Activation ◽  
Cellular Processes ◽  
Flow Cytometric ◽  
Vacuolar Acidification ◽  
Vacuolar Protein ◽  
A Minor ◽  
Proton Translocating Atpase

Vacuolar acidification has been proposed to play a key role in a number of cellular processes, including protein sorting, zymogen activation, and maintenance of intracellular pH. We investigated the significance of vacuolar acidification by cloning and mutagenizing the gene for the yeast vacuolar proton-translocating ATPase 60-kilodalton subunit (VAT2). Cells carrying a vat2 null allele were viable; however, these cells were severely defective for growth in medium buffered at neutral pH. Vacuoles isolated from cells bearing the vat2 null allele were completely devoid of vacuolar ATPase activity. The pH of the vacuolar lumen of cells bearing the vat2 mutation was 7.1, compared with the wild-type pH of 6.1, as determined by a flow cytometric pH assay. These results indicate that the vacuolar proton-translocating ATPase complex is essential for vacuolar acidification and that the low-pH state of the vacuole is crucial for normal growth. The vacuolar acidification-defective vat2 mutant exhibited normal zymogen activation but displayed a minor defect in vacuolar protein sorting.

scholarly journals Acidification of the lysosome-like vacuole and the vacuolar H+-ATPase are deficient in two yeast mutants that fail to sort vacuolar proteins.

1989 ◽  
Vol 109 (1) ◽  
pp. 93-100 ◽  
Author(s):  
J H Rothman ◽  
C T Yamashiro ◽  
C K Raymond ◽  
P M Kane ◽  
T H Stevens
Keyword(s):  
Protein Sorting ◽  
Critical Role ◽  
Normal Function ◽  
Yeast Cells ◽  
Zymogen Activation ◽  
Animal Cells ◽  
Vacuolar Acidification ◽  
Vacuolar Protein ◽  
Vacuolar Membranes ◽  
Yeast Mutants

Organelle acidification plays a demonstrable role in intracellular protein processing, transport, and sorting in animal cells. We investigated the relationship between acidification and protein sorting in yeast by treating yeast cells with ammonium chloride and found that this lysosomotropic agent caused the mislocalization of a substantial fraction of the newly synthesized vacuolar (lysosomal) enzyme proteinase A (PrA) to the cell surface. We have also determined that a subset of the vpl mutants, which are deficient in sorting of vacuolar proteins (Rothman, J. H., and T. H. Stevens. 1986. Cell. 47:1041-1051; Rothman, J. H., I. Howald, and T. H. Stevens. EMBO [Eur. Mol. Biol. Organ.] J. In press), failed to accumulate the lysosomotropic fluorescent dye quinacrine within their vacuoles, mimicking the phenotype of wild-type cells treated with ammonium. The acidification defect of vpl3 and vpl6 mutants correlated with a marked deficiency in vacuolar ATPase activity, diminished levels of two immunoreactive subunits of the protontranslocating ATPase (H+-ATPase) in purified vacuolar membranes, and accumulation of the intracellular portion of PrA as the precursor species. Therefore, some of the VPL genes are required for the normal function of the yeast vacuolar H+-ATPase complex and may encode either subunits of the enzyme or components required for its assembly and targeting. Collectively, these findings implicate a critical role for acidification in vacuolar protein sorting and zymogen activation in yeast, and suggest that components of the yeast vacuolar acidification system may be identified by examining mutants defective in sorting of vacuolar proteins.

scholarly journals The Saccharomyces cerevisiae MVP1 gene interacts with VPS1 and is required for vacuolar protein sorting.

1995 ◽  
Vol 15 (3) ◽  
pp. 1671-1678 ◽  
Author(s):  
K Ekena ◽  
T H Stevens
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Interaction ◽  
Protein Sorting ◽  
Dominant Negative ◽  
Yeast Cells ◽  
Vacuolar Protein Sorting ◽  
Cellular Processes ◽  
Golgi Membranes ◽  
Multicopy Suppressors ◽  
Vacuolar Protein

The VPS1 gene of Saccharomyces cerevisiae encodes an 80-kDa GTPase that associates with Golgi membranes and is required for the sorting of proteins to the yeast vacuole. Vps1p is a member of a growing family of high-molecular-weight GTPases that are found in a number of organisms and are involved in a variety of cellular processes. Vps1p is most similar to mammalian dynamin and the Drosophila Shibire protein, both of which have been shown to play a role in an early step of endocytosis. To identify proteins that interact with Vps1p, a genetic screen was designed to isolate multicopy suppressors of dominant-negative vps1 mutations. One such suppressor, MVP1, that exhibits genetic interaction with VPS1 and is itself required for vacuolar protein sorting has been isolated. Overproduction of Mvp1p will suppress several dominant alleles of VPS1, and suppression is dependent on the presence of wild-type Vps1p. MVP1 encodes a 59-kDa hydrophilic protein, Mvp1p, which appears to colocalize with Vps1p in vps1d and vps27 delta yeast cells. We therefore propose that Mvp1p and Vps1p act in concert to promote membrane traffic to the vacuole.

scholarly journals Saccharomyces cerevisiae Is Dependent on Vesicular Traffic between the Golgi Apparatus and the Vacuole When Inositolphosphorylceramide Synthase Aur1 Is Inactivated

2015 ◽  
Vol 14 (12) ◽  
pp. 1203-1216 ◽  
Author(s):  
Natalia S. Voynova ◽  
Carole Roubaty ◽  
Hector M. Vazquez ◽  
Shamroop K. Mallela ◽  
Christer S. Ejsing ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Protein Transport ◽  
Reactive Oxygen ◽  
Cytosolic Calcium ◽  
Minor Role ◽  
Vesicular Traffic ◽  
Vacuolar Acidification ◽  
Aureobasidin A ◽  
Vacuolar Protein ◽  
A Minor

ABSTRACTInositolphosphorylceramide (IPC) and its mannosylated derivatives are the only complex sphingolipids of yeast. Their synthesis can be reduced by aureobasidin A (AbA), which specifically inhibits the IPC synthase Aur1. AbA reportedly, by diminishing IPC levels, causes endoplasmic reticulum (ER) stress, an increase in cytosolic calcium, reactive oxygen production, and mitochondrial damage leading to apoptosis. We found that when Aur1 is gradually depleted by transcriptional downregulation, the accumulation of ceramides becomes a major hindrance to cell survival. Overexpression of the alkaline ceramidaseYPC1rescues cells under this condition. We established hydroxylated C26fatty acids as a reliable hallmark of ceramide hydrolysis. Such hydrolysis occurs only whenYPC1is overexpressed. In contrast, overexpression ofYPC1has no beneficial effect when Aur1 is acutely repressed by AbA. A high-throughput genetic screen revealed that vesicle-mediated transport between Golgi apparatus, endosomes, and vacuole becomes crucial for survival when Aur1 is repressed, irrespective of the mode of repression. In addition, vacuolar acidification becomes essential when cells are acutely stressed by AbA, and quinacrine uptake into vacuoles shows that AbA activates vacuolar acidification. The antioxidantN-acetylcysteine does not improve cell growth on AbA, indicating that reactive oxygen radicals induced by AbA play a minor role in its toxicity. AbA strongly induces the cell wall integrity pathway, but osmotic support does not improve the viability of wild-type cells on AbA. Altogether, the data support and refine current models of AbA-mediated cell death and add vacuolar protein transport and acidification as novel critical elements of stress resistance.

scholarly journals An Interorganellar Bidding War: Vps13 Localization by Competitive Organelle-Specific Adaptors

Contact ◽  
2018 ◽  
Vol 1 ◽  
pp. 251525641881462
Author(s):  
Samantha K. Dziurdzik ◽  
Björn D.M. Bean ◽  
Elizabeth Conibear
Keyword(s):  
Endoplasmic Reticulum ◽  
Recent Work ◽  
Protein Sorting ◽  
Repeat Region ◽  
Vacuolar Protein Sorting ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Prospore Membrane ◽  
Membrane Contact ◽  
Vacuolar Protein

Membrane contact sites are regulated through the controlled recruitment of constituent proteins. Yeast vacuolar protein sorting 13 (Vps13) dynamically localizes to membrane contact sites at endosomes, vacuoles, mitochondria, and the endoplasmic reticulum under different cellular conditions and is recruited to the prospore membrane during meiosis. Prior to our recent work, the mechanism for localization at contact sites was largely unknown. We identified Ypt35 as a novel Vps13 adaptor for endosomes and the nucleus-vacuole junction. Furthermore, we discovered a conserved recruitment motif in Ypt35 and found related motifs in the prospore membrane and mitochondrial adaptors, Spo71 and Mcp1, respectively. All three adaptors compete for binding to a six-repeat region of Vps13, suggesting adaptor competition regulates Vps13 localization. Here, we summarize and discuss the implications of our work, highlighting key outstanding questions.

Heat shock factor is required for growth at normal temperatures in the fission yeast Schizosaccharomyces pombe

1993 ◽  
Vol 13 (2) ◽  
pp. 749-761
Author(s):  
G J Gallo ◽  
H Prentice ◽  
R E Kingston
Keyword(s):  
Heat Shock ◽  
Schizosaccharomyces Pombe ◽  
Normal Growth ◽  
Growth Conditions ◽  
Heat Shock Factor ◽  
Binding Ability ◽  
Lower Growth ◽  
General Role ◽  
Functional Conservation ◽  
Cellular Processes

Schizosaccharomyces pombe is becoming an increasingly useful organism for the study of cellular processes, since in certain respects, such as the cell cycle and splicing, it is similar to metazoans. Previous biochemical studies have shown that the DNA binding ability of S. pombe heat shock factor (HSF) is fully induced only under stressed conditions, in a manner similar to that of Drosophila melanogaster and humans but differing from the constitutive binding by HSF in the budding yeasts. We report the isolation of the cDNA and gene for the HSF from S. pombe. S. pombe HSF has a domain structure that is more closely related to the structure of human and D. melanogaster HSFs than to the structure of the budding yeast HSFs, further arguing that regulation of HSF in S. pombe is likely to reflect regulation in metazoans. Surprisingly, the S. pombe HSF gene is required for growth at normal temperatures. We show that the S. pombe HSF gene can be replaced by the D. melanogaster HSF gene and that strains containing either of these genes behave similarly to transiently heat-shocked strains with respect to viability and the level of heat-induced transcripts from heat shock promoters. Strains containing the D. melanogaster HSF gene, however, have lower growth rates and show altered morphology at normal growth temperatures. These data demonstrate the functional conservation of domains of HSF that are required for response to heat shock. They further suggest a general role for HSF in growth of eukaryotic cells under normal (nonstressed) growth conditions.

Sign in / Sign up

Export Citation Format

Share Document