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 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.

Trypanosoma brucei vacuolar protein sorting 41 (VPS41) is required for intracellular iron utilization and maintenance of normal cellular morphology

Parasitology ◽  
2007 ◽  
Vol 134 (11) ◽  
pp. 1639-1647 ◽  
Author(s):  
S. LU ◽  
T. SUZUKI ◽  
N. IIZUKA ◽  
S. OHSHIMA ◽  
Y. YABU ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Iron Metabolism ◽  
Protein Sorting ◽  
Tsetse Fly ◽  
Cellular Morphology ◽  
Yeast Cells ◽  
Intracellular Iron ◽  
Procyclic Form ◽  
Vacuolar Protein ◽  
Iron Utilization

SUMMARYProcyclic forms of Trypanosoma brucei brucei remain and propagate in the midgut of tsetse fly where iron is rich. Additional iron is also required for their growth in in vitro culture. However, little is known about the genes involved in iron metabolism and the mechanism of iron utilization in procyclic-form cells. Therefore, we surveyed the genes involved in iron metabolism in the T. b. brucei genome sequence database. We found a potential homologue of vacuole protein sorting 41 (VPS41), a gene that is required for high-affinity iron transport in Saccharomyces cerevisiae and cloned the full-length gene (TbVPS41). Complementation analysis of TbVPS41 in ΔScvps41 yeast cells showed that TbVPS41 could partially suppress the inability of ΔScvps41 yeast cells to grow on low-iron medium, but it could not suppress the fragmented vacuole phenotype. Further RNA interference (RNAi)-mediated gene knock-down in procyclic-form cells resulted in a significant reduction of growth in low-iron medium; however, no change in growth was observed in normal culture medium. Transmission electron microscopy showed that RNAi caused T. b. brucei cells to have larger numbers of small intracellular vesicles, similar to the fragmented vacuoles observed in ΔScvps41 yeast cells. The present study demonstrates that TbVPS41 plays an important role in the intracellular iron utilization system as well as in the maintenance of normal cellular morphology.

scholarly journals The product of HUM1, a novel yeast gene, is required for vacuolar Ca2+/H+ exchange and is related to mammalian Na+/Ca2+ exchangers.

1996 ◽  
Vol 16 (7) ◽  
pp. 3730-3741 ◽  
Author(s):  
T C Pozos ◽  
I Sekler ◽  
M S Cyert
Keyword(s):  
Mutant Strain ◽  
Critical Role ◽  
Ion Homeostasis ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Protein Product ◽  
Vacuolar Membrane ◽  
Yeast Cells ◽  
Vacuolar Acidification ◽  
High Concentrations

Calcineurin, or PP2B, plays a critical role in mediating Ca2+-dependent signaling in many cell types. In yeast cells, this highly conserved protein phosphatase regulates aspects of ion homeostasis and cell wall synthesis. We show that calcineurin mutants are sensitive to high concentrations of Mn2+ and identify two genes, CCC1 and HUM1, that, at high dosages, increase the Mn2+ tolerance of calcineurin mutants. CCC1 was previously identified by complementation of a Ca2+-sensitive (csg1) mutant. HUM1 (for "high copy number undoes manganese") is a novel gene whose predicted protein product shows similarity to mammalian Na+/Ca2+ exchangers. hum1 mutations confer Mn2+ sensitivity in some genetic backgrounds and exacerbate the Mn2+ sensitivity of calcineurin mutants. Furthermore, disruption of HUM1 in a calcineurin mutant strain results in a Ca2+-sensitive phenotype. We investigated the effect of disrupting HUM1 in other strains with defects in Ca2+ homeostasis. The Ca2+ sensitivity of pmc1 mutants, which lack a P-type ATPase presumed to transport Ca2+ into the vacuole, is exacerbated in a hum1 mutant strain background. Also, the Ca2+ content of hum1 pmc1 cells is less than that of pmc1 cells. In contrast, the Ca2+ sensitivity of vph1 mutants, which are specifically defective in vacuolar acidification, is not significantly altered by disruption of Hum1p function. These genetic interactions suggest that Hum1p may participate in vacuolar Ca2+/H+ exchange. Therefore, we prepared vacuolar membrane vesicles from wild-type and hum1 cells and compared their Ca2+ transport properties. Vacuolar membrane vesicles from hum1 mutants lack all Ca2+/H+ antiport activity, demonstrating that Hum1p catalyzes the exchange of Ca2+ for H+ across the yeast vacuolar membrane.

scholarly journals The plant vacuolar protein, phytohemagglutinin, is transported to the vacuole of transgenic yeast.

1987 ◽  
Vol 105 (5) ◽  
pp. 1971-1979 ◽  
Author(s):  
B W Tague ◽  
M J Chrispeels
Keyword(s):  
Signal Peptide ◽  
Yeast Cells ◽  
Cell Fractionation ◽  
Animal Cells ◽  
Yeast Vacuole ◽  
Transgenic Yeast ◽  
Protein Storage Vacuoles ◽  
Membrane Bound ◽  
Vacuolar Sorting ◽  
Vacuolar Protein

Phytohemagglutinin (PHA), the major seed lectin of the common bean, Phaseolus vulgaris, accumulates in the parenchyma cells of the cotyledons. It has been previously shown that PHA is cotranslationally inserted into the endoplasmic reticulum with cleavage of the NH2-terminal signal peptide. Two N-linked oligosaccharide side chains are added, one of which is modified to a complex type in the Golgi apparatus. PHA is then deposited in membrane-bound protein storage vacuoles which are biochemically and functionally equivalent to the vacuoles of yeast cells and the lysosomes of animal cells. We wished to determine whether yeast cells would recognize the vacuolar sorting determinant of PHA and target the protein to the yeast vacuole. We have expressed the gene for leukoagglutinating PHA (PHA-L) in yeast under control of the yeast acid phosphatase (PHO5) promoter. Under control of this promoter, PHA-L accumulates to 0.1% of the total yeast protein. PHA-L produced in yeast is glycosylated as expected for a yeast vacuolar glycoprotein. Cell fractionation studies show that PHA-L is efficiently transported to the yeast vacuole. This is the first demonstration that vacuolar targeting information is recognized between two highly divergent species. A small proportion of yeast PHA-L is secreted which may be due to inefficient recognition of the vacuolar sorting signal because of the presence of an uncleaved signal peptide on a subset of the PHA-L polypeptides. This system can now be used to identify the vacuolar sorting determinant of a plant vacuolar protein.

Sortilin: a new player in dementia and Alzheimer-type neuropathology

2018 ◽  
Vol 96 (5) ◽  
pp. 491-497 ◽  
Author(s):  
Shu-Yin Xu ◽  
Juan Jiang ◽  
Aihua Pan ◽  
Cai Yan ◽  
Xiao-Xin Yan
Keyword(s):  
Protein Sorting ◽  
Critical Role ◽  
Animal Experiments ◽  
Amyloid Plaque ◽  
Mortality Factor ◽  
Human Populations ◽  
The Past ◽  
Age Related ◽  
Intracellular Compartments ◽  
Vacuolar Protein

Age-related dementias are now a major mortality factor among most human populations in the world, with Alzheimer’s disease (AD) being the leading dementia-causing neurodegenerative disease. The pathogenic mechanism underlying dementia disorders, and AD in particular, remained largely unknown. Efforts to develop drugs targeting the disease’s hallmark lesions, such as amyloid plaque and tangle pathologies, have been unsuccessful so far. The vacuolar protein sorting 10p (Vps10p) family plays a critical role in membrane signal transduction and protein sorting and trafficking between intracellular compartments. Data emerging during the past few years point to an involvement of this family in the development of AD. Specifically, the Vps10p member sortilin has been shown to participate in amyloid plaque formation, tau phosphorylation, abnormal protein sorting and apoptosis. In this minireview, we update some latest findings from animal experiments and human brain studies suggesting that abnormal sortilin expression is associated with AD-type neuropathology, warranting further research that might lead to novel targets for the development of AD therapies.

Yeast Ypt51p and mammalian Rab5: counterparts with similar function in the early endocytic pathway

1995 ◽  
Vol 108 (11) ◽  
pp. 3509-3521 ◽  
Author(s):  
B. Singer-Kruger ◽  
H. Stenmark ◽  
M. Zerial
Keyword(s):  
Small Gtpase ◽  
Endocytic Pathway ◽  
Yeast Cells ◽  
Carboxypeptidase Y ◽  
Cell Fractionation ◽  
Animal Cells ◽  
Early Endosomes ◽  
Fractionation Analysis ◽  
Alpha Factor ◽  
Vacuolar Protein

Ypt51p, a small GTPase of Saccharomyces cerevisiae, has been previously identified as a structural homolog of mammalian Rab5. Although disruption analysis revealed that the protein is required for endocytic transport and for vacuolar protein sorting, the precise step controlled by Ypt51p was not determined. In this work we show that by heterologous expression in animal cells Ypt51p was targeted to Rab5-positive early endosomes and stimulated endocytosis. Furthermore, two Ypt51p mutants induced similar morphological alterations as the corresponding Rab5 mutants. Also in yeast cells Ypt51p was found to be required at an early step in endocytic membrane traffic, since alpha-factor accumulated in an early endocytic intermediate in the absence of Ypt51p. Cell fractionation analysis revealed cofractionation of Ypt51p with endocytic intermediates, while no association with the late Golgi compartment could be detected. Indirect immunofluorescence microscopy allowed us to morphologically identify the Ypt51p-containing compartment. Similar to the mammalian system larger Ypt51p-positive structures were revealed upon expression of Ypt51p Q66L. These structures were also positive for alpha-factor receptor and for carboxypeptidase Y, thus providing direct evidence for their endocytic nature and for the convergence of the vacuolar biosynthetic and endocytic pathways.

scholarly journals Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae.

1990 ◽  
Vol 10 (12) ◽  
pp. 6742-6754 ◽  
Author(s):  
P K Herman ◽  
S D Emr
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Sorting ◽  
Yeast Cells ◽  
Growth Defect ◽  
Cell Fractionation ◽  
Wild Type ◽  
Vacuolar Protein Sorting ◽  
Vacuolar Compartment ◽  
Dividing Cells ◽  
Vacuolar Protein

VPS34 gene function is required for the efficient localization of a variety of vacuolar proteins. We have cloned and sequenced the wild-type VPS34 gene in order to gain a better understanding of the role of its protein product in this intracellular sorting pathway. Interestingly, disruption of the VPS34 locus resulted in a temperature-sensitive growth defect, indicating that the VPS34 gene is essential for vegetative growth only at elevated growth temperatures. As with the original vps34 alleles, vps34 null mutants exhibited severe vacuolar protein sorting defects and possessed a morphologically normal vacuolar structure. The VPS34 gene DNA sequence identifies an open reading frame that could encode a hydrophilic protein of 875 amino acids. The predicted protein sequence lacks any apparent signal sequence or membrane-spanning domains, suggesting that Vps34p does not enter the secretory pathway. Results from immunoprecipitation experiments with antiserum prepared against a TrpE-Vps34 fusion protein were consistent with this prediction: a rare, unglycosylated protein of approximately 95,000 Da was detected in extracts of wild-type Saccharomyces cerevisiae cells. Cell fractionation studies indicated that a significant portion of the Vps34p is found associated with a particulate fraction of yeast cells. This particulate Vps34p was readily solubilized by treatment with 2 M urea but not with Triton X-100, suggesting that the presence of Vps34p in this pelletable structure is mediated by protein-protein interactions. vp34 mutant cells also exhibited a defect in the normal partitioning of the vacuolar compartment between mother and daughter cells during cell division. In more than 80% of the delta vps34 dividing cells examined, no vacuolar structures were observed in the newly emerging bud, whereas in wild-type dividing cells, more than 95% of the buds had a detectable vacuolar compartment. Our results suggest that the Vps34p may act as a component of a relatively large intracellular structure that functions to facilitate specific steps of the vacuolar protein delivery and inheritance pathways.

scholarly journals Analysis of the galactose signal transduction pathway in Saccharomyces cerevisiae: interaction between Gal3p and Gal80p.

1996 ◽  
Vol 16 (5) ◽  
pp. 2504-2508 ◽  
Author(s):  
T Suzuki-Fujimoto ◽  
M Fukuma ◽  
K I Yano ◽  
H Sakurai ◽  
A Vonika ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Critical Role ◽  
Constitutive Expression ◽  
Signal Transduction Pathway ◽  
Normal Function ◽  
Negative Regulator ◽  
Yeast Cells ◽  
Western Blots ◽  
Metabolizing Enzymes ◽  
Mutant Proteins

The GAL3 gene plays a critical role in galactose induction of the GAL genes that encode galactose- metabolizing enzymes in Saccharomyces cerevisiae. Defects in GAL3 result in a long delay in GAL gene induction, and overproduction of Gal3p causes constitutive expression of GAL. Here we demonstrate that concomitant overproduction of the negative regulator, Gal80p, and Gal3p suppresses this constitutive GAL expression. This interplay between Gal80p and Gal3p is direct, as tagged Gal3p coimmunoprecipitated with Gal80p. The amount of coprecipitated Gal80p increased when GAL80 yeast cells were grown in the presence of galactose. When both GAL80 and GAL3 were overexpressed, the amount of coprecipitated Gal80p was not affected by galactose. Tagged gal3 mutant proteins bound to purified Gal80p, but only poorly in comparison with the wild type, suggesting that formation of the Gal80p-Gal3p complex depends on the normal function of Gal3p. Gal3p appeared larger in Western blots (immunoblots) than predicted by the published nucleic acid sequence. Reexamination of the DNA sequence of GAL3 revealed several mistakes, including an extension at the 3' end of another predicted 97 amino acids.

scholarly journals Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae

1990 ◽  
Vol 10 (12) ◽  
pp. 6742-6754
Author(s):  
P K Herman ◽  
S D Emr
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Sorting ◽  
Yeast Cells ◽  
Growth Defect ◽  
Cell Fractionation ◽  
Wild Type ◽  
Vacuolar Protein Sorting ◽  
Vacuolar Compartment ◽  
Dividing Cells ◽  
Vacuolar Protein

VPS34 gene function is required for the efficient localization of a variety of vacuolar proteins. We have cloned and sequenced the wild-type VPS34 gene in order to gain a better understanding of the role of its protein product in this intracellular sorting pathway. Interestingly, disruption of the VPS34 locus resulted in a temperature-sensitive growth defect, indicating that the VPS34 gene is essential for vegetative growth only at elevated growth temperatures. As with the original vps34 alleles, vps34 null mutants exhibited severe vacuolar protein sorting defects and possessed a morphologically normal vacuolar structure. The VPS34 gene DNA sequence identifies an open reading frame that could encode a hydrophilic protein of 875 amino acids. The predicted protein sequence lacks any apparent signal sequence or membrane-spanning domains, suggesting that Vps34p does not enter the secretory pathway. Results from immunoprecipitation experiments with antiserum prepared against a TrpE-Vps34 fusion protein were consistent with this prediction: a rare, unglycosylated protein of approximately 95,000 Da was detected in extracts of wild-type Saccharomyces cerevisiae cells. Cell fractionation studies indicated that a significant portion of the Vps34p is found associated with a particulate fraction of yeast cells. This particulate Vps34p was readily solubilized by treatment with 2 M urea but not with Triton X-100, suggesting that the presence of Vps34p in this pelletable structure is mediated by protein-protein interactions. vp34 mutant cells also exhibited a defect in the normal partitioning of the vacuolar compartment between mother and daughter cells during cell division. In more than 80% of the delta vps34 dividing cells examined, no vacuolar structures were observed in the newly emerging bud, whereas in wild-type dividing cells, more than 95% of the buds had a detectable vacuolar compartment. Our results suggest that the Vps34p may act as a component of a relatively large intracellular structure that functions to facilitate specific steps of the vacuolar protein delivery and inheritance pathways.

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