scholarly journals Retrieval of Resident Late-Golgi Membrane Proteins from the Prevacuolar Compartment of Saccharomyces cerevisiae Is Dependent on the Function of Grd19p

1998 ◽  
Vol 140 (3) ◽  
pp. 577-590 ◽  
Author(s):  
Wolfgang Voos ◽  
Tom H. Stevens
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Localization ◽  
Transport Processes ◽  
Carboxypeptidase Y ◽  
Golgi Membrane ◽  
Cytosolic Domain ◽  
Prevacuolar Compartment ◽  
Vacuolar Protein

The dynamic vesicle transport processes at the late-Golgi compartment of Saccharomyces cerevisiae (TGN) require dedicated mechanisms for correct localization of resident membrane proteins. In this study, we report the identification of a new gene, GRD19, involved in the localization of the model late-Golgi membrane protein A-ALP (consisting of the cytosolic domain of dipeptidyl aminopeptidase A [DPAP A] fused to the transmembrane and lumenal domains of the alkaline phosphatase [ALP]), which localizes to the yeast TGN. A grd19 null mutation causes rapid mislocalization of the late-Golgi membrane proteins A-ALP and Kex2p to the vacuole. In contrast to previously identified genes involved in late-Golgi membrane protein localization, grd19 mutations cause only minor effects on vacuolar protein sorting. The recycling of the carboxypeptidase Y sorting receptor, Vps10p, between the TGN and the prevacuolar compartment is largely unaffected in grd19Δ cells. Kinetic assays of A-ALP trafficking indicate that GRD19 is involved in the process of retrieval of A-ALP from the prevacuolar compartment. GRD19 encodes a small hydrophilic protein with a predominantly cytosolic distribution. In a yeast mutant that accumulates an exaggerated form of the prevacuolar compartment (vps27), Grd19p was observed to localize to this compartment. Using an in vitro binding assay, Grd19p was found to interact physically with the cytosolic domain of DPAP A. We conclude that Grd19p is a component of the retrieval machinery that functions by direct interaction with the cytosolic tails of certain TGN membrane proteins during the sorting/budding process at the prevacuolar compartment.

The yeast VPS5/GRD2 gene encodes a sorting nexin-1-like protein required for localizing membrane proteins to the late Golgi

1997 ◽  
Vol 110 (9) ◽  
pp. 1063-1072 ◽  
Author(s):  
S.F. Nothwehr ◽  
A.E. Hindes
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Localization ◽  
Endocytic Pathway ◽  
Yeast Cells ◽  
Carboxypeptidase Y ◽  
Golgi Membrane ◽  
Sorting Nexin ◽  
Vacuolar Protein ◽  
Sorting Nexin 1

Genetic analysis of late Golgi membrane protein localization in Saccharomyces cerevisiae has uncovered a large number of genes (called GRD) that are required for retention of A-ALP, a model late Golgi membrane protein. Here we describe one of the GRD genes, VPSS/GRD2, that encodes a hydrophilic protein similar to human sorting nexin-1, a protein involved in trafficking of the epidermal growth factor receptor. In yeast cells containing a vps5 null mutation the late Golgi membrane proteins A-ALP and Kex2p were rapidly mislocalized to the vacuolar membrane. A-ALP was delivered to the vacuole in vps5 mutants in a manner independent of a block in the early endocytic pathway. vps5 null mutants also exhibited defects in both vacuolar morphology and in sorting of a soluble vacuolar protein, carboxypeptidase Y. The latter defect is apparently due to an inability to localize the carboxypeptidase Y sorting receptor, Vps10p, to the Golgi since it is rapidly degraded in the vacuole in vps5 mutants. Fractionation studies indicate that Vps5p is distributed between a free cytosolic pool and a particulate fraction containing Golgi, transport vesicles, and possibly endosomes, but lacking vacuolar membranes. Immunofluorescence microscopy experiments show that the membrane-associated pool of Vps5p localizes to an endosome-like organelle that accumulates in the class E vps27 mutant. These results support a model in which Vps5p is required for retrieval of membrane proteins from a prevacuolar/late endosomal compartment back to the late Golgi apparatus.

scholarly journals Selective and immediate effects of clathrin heavy chain mutations on Golgi membrane protein retention in Saccharomyces cerevisiae.

1992 ◽  
Vol 118 (3) ◽  
pp. 531-540 ◽  
Author(s):  
M Seeger ◽  
G S Payne
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Cell Surface ◽  
Heavy Chain ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Golgi Membrane ◽  
Alpha Factor ◽  
Mutant Cells

The role of clathrin in retention of Golgi membrane proteins has been investigated. Prior work showed that a precursor form of the peptide mating pheromone alpha-factor is secreted by Saccharomyces cerevisiae cells which lack the clathrin heavy chain gene (CHC1). This defect can be accounted for by the observation that the Golgi membrane protein Kex2p, which initiates maturation of alpha-factor precursor, is mislocalized to the cell surface of mutant cells. We have examined the localization of two additional Golgi membrane proteins, dipeptidyl aminopeptidase A (DPAP A) and guanosine diphosphatase (GDPase) in clathrin-deficient yeast strains. Our findings indicate that DPAP A is aberrantly transported to the cell surface but GDPase is not. In mutant cells carrying a temperature-sensitive allele of CHC1 (chc1-ts), alpha-factor precursor appears in the culture medium within 15 min, and Kex2p and DPAP A reach the cell surface within 30 min, after imposing the nonpermissive temperature. In contrast to these immediate effects, a growth defect is apparent only after 2 h at the nonpermissive temperature. Also, sorting of the vacuolar membrane protein, alkaline phosphatase, is not affected in chc1-ts cells until 2 h after the temperature shift. A temperature-sensitive mutation which blocks a late stage of the secretory pathway, sec1, prevents the appearance of mislocalized Kex2p at the cell surface of chc1-ts cells. We propose that clathrin plays a direct role in the retention of specific proteins in the yeast Golgi apparatus, thereby preventing their transport to the cell surface.

scholarly journals Golgi and vacuolar membrane proteins reach the vacuole in vps1 mutant yeast cells via the plasma membrane.

1995 ◽  
Vol 129 (1) ◽  
pp. 35-46 ◽  
Author(s):  
S F Nothwehr ◽  
E Conibear ◽  
T H Stevens
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Golgi Apparatus ◽  
Membrane Protein ◽  
Vacuolar Membrane ◽  
Yeast Cells ◽  
Permissive Temperature ◽  
Golgi Membrane ◽  
Prevacuolar Compartment ◽  
Mutant Cells

The Vps1 protein of Saccharomyces cerevisiae is an 80-kD GTPase associated with the Golgi apparatus. Vps1p appears to play a direct role in the retention of late Golgi membrane proteins, which are mislocalized to the vacuolar membrane in its absence. The pathway by which late Golgi and vacuolar membrane proteins reach the vacuole in vps1 delta mutants was investigated by analyzing transport of these proteins in vps1 delta cells that also contained temperature sensitive mutations in either the SEC4 or END4 genes, which are required for a late step in secretion and the internalization step of endocytosis, respectively. Not only was vacuolar transport of a Golgi membrane protein blocked in the vps1 delta sec4-ts and vps1 delta end4-ts double mutant cells at the non-permissive temperature but vacuolar delivery of the vacuolar membrane protein, alkaline phosphatase was also blocked in these cells. Moreover, both proteins expressed in the vps1 delta end4-ts cells at the elevated temperature could be detected on the plasma membrane by a protease digestion assay indicating that these proteins are transported to the vacuole via the plasma membrane in vps1 mutant cells. These data strongly suggest that a loss of Vps1p function causes all membrane traffic departing from the late Golgi normally destined for the prevacuolar compartment to instead be diverted to the plasma membrane. We propose a model in which Vps1p is required for formation of vesicles from the late Golgi apparatus that carry vacuolar and Golgi membrane proteins bound for the prevacuolar compartment.

scholarly journals The Yeast vps Class E Mutants: The Beginning of the Molecular Genetic Analysis of Multivesicular Body Biogenesis

2010 ◽  
Vol 21 (23) ◽  
pp. 4057-4060 ◽  
Author(s):  
Emily M. Coonrod ◽  
Tom H. Stevens
Keyword(s):  
Membrane Proteins ◽  
Molecular Genetic ◽  
Multivesicular Body ◽  
Molecular Genetic Analysis ◽  
Vacuolar Membrane ◽  
Multivesicular Bodies ◽  
Golgi Membrane ◽  
Cargo Proteins ◽  
Vacuolar Protein ◽  
Class E

In 1992, Raymond et al. published a compilation of the 41 yeast vacuolar protein sorting (vps) mutant groups and described a large class of mutants (class E vps mutants) that accumulated an exaggerated prevacuolar endosome-like compartment. Further analysis revealed that this “class E compartment” contained soluble vacuolar hydrolases, vacuolar membrane proteins, and Golgi membrane proteins unable to recycle back to the Golgi complex, yet these class E vps mutants had what seemed to be normal vacuoles. The 13 class E VPS genes were later shown to encode the proteins that make up the complexes required for formation of intralumenal vesicles in late endosomal compartments called multivesicular bodies, and for the sorting of ubiquitinated cargo proteins into these internal vesicles for eventual delivery to the vacuole or lysosome.

scholarly journals Immunoisolaton of the Yeast Golgi Subcompartments and Characterization of a Novel Membrane Protein, Svp26, Discovered in the Sed5-Containing Compartments

2005 ◽  
Vol 25 (17) ◽  
pp. 7696-7710 ◽  
Author(s):  
Hironori Inadome ◽  
Yoichi Noda ◽  
Hiroyuki Adachi ◽  
Koji Yoda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Golgi Apparatus ◽  
Membrane Protein ◽  
Considerable Portion ◽  
Marker Proteins ◽  
Evolutionarily Conserved ◽  
Golgi Compartment

ABSTRACT The Golgi apparatus consists of a set of vesicular compartments which are distinguished by their marker proteins. These compartments are physically separated in the Saccharomyces cerevisiae cell. To characterize them extensively, we immunoisolated vesicles carrying either of the SNAREs Sed5 or Tlg2, the markers of the early and late Golgi compartments, respectively, and analyzed the membrane proteins. The composition of proteins was mostly consistent with the position of each compartment in the traffic. We found six uncharacterized but evolutionarily conserved proteins and named them Svp26 (Sed5 compartment vesicle protein of 26 kDa), Tvp38, Tvp23, Tvp18, Tvp15 (Tlg2 compartment vesicle proteins of 38, 23, 18, and 15 kDa), and Gvp36 (Golgi vesicle protein of 36 kDa). The localization of Svp26 in the early Golgi compartment was confirmed by microscopic and biochemical means. Immunoprecipitation indicated that Svp26 binds to itself and a Golgi mannosyltransferase, Ktr3. In the absence of Svp26, a considerable portion of Ktr3 was mislocalized in the endoplasmic reticulum. Our data suggest that Svp26 has a novel role in retention of a subset of membrane proteins in the early Golgi compartments.

scholarly journals Vacuolar protein Tag1 and Atg1–Atg13 regulate autophagy termination during persistent starvation in S. cerevisiae

2021 ◽  
Vol 134 (4) ◽  
pp. jcs253682
Author(s):  
Shintaro Kira ◽  
Masafumi Noguchi ◽  
Yasuhiro Araki ◽  
Yu Oikawa ◽  
Tamotsu Yoshimori ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Membrane Protein ◽  
Nitrogen Starvation ◽  
Genetic Screen ◽  
Vacuolar Membrane ◽  
Phagophore Assembly Site ◽  
Vacuolar Protein ◽  
Assembly Site ◽  
Starvation Conditions

ABSTRACTUnder starvation conditions, cells degrade their own components via autophagy in order to provide sufficient nutrients to ensure their survival. However, even if starvation persists, the cell is not completely degraded through autophagy, implying the existence of some kind of termination mechanism. In the yeast Saccharomyces cerevisiae, autophagy is terminated after 10–12 h of nitrogen starvation. In this study, we found that termination is mediated by re-phosphorylation of Atg13 by the Atg1 protein kinase, which is also affected by PP2C phosphatases, and the eventual dispersion of the pre-autophagosomal structure, also known as the phagophore assembly site (PAS). In a genetic screen, we identified an uncharacterized vacuolar membrane protein, Tag1, as a factor responsible for the termination of autophagy. Re-phosphorylation of Atg13 and eventual PAS dispersal were defective in the Δtag1 mutant. The vacuolar luminal domain of Tag1 and autophagic progression are important for the behaviors of Tag1. Together, our findings reveal the mechanism and factors responsible for termination of autophagy in yeast.

scholarly journals The Sodium/Proton Exchanger Nhx1p Is Required for Endosomal Protein Trafficking in the YeastSaccharomyces cerevisiae

2000 ◽  
Vol 11 (12) ◽  
pp. 4277-4294 ◽  
Author(s):  
Katherine Bowers ◽  
Boaz P. Levi ◽  
Falguny I. Patel ◽  
Tom H. Stevens
Keyword(s):  
Protein Trafficking ◽  
Protein Sorting ◽  
Proton Exchange ◽  
Carboxypeptidase Y ◽  
Wild Type ◽  
Ion Balance ◽  
Sodium Proton Exchanger ◽  
Sodium Proton Exchange ◽  
Prevacuolar Compartment ◽  
Vacuolar Protein

We show that the vacuolar protein sorting gene VPS44is identical to NHX1, a gene that encodes a sodium/proton exchanger. The Saccharomyces cerevisiaeprotein Nhx1p shows high homology to mammalian sodium/proton exchangers of the NHE family. Nhx1p is thought to transport sodium ions into the prevacuole compartment in exchange for protons. Pulse-chase experiments show that ∼35% of the newly synthesized soluble vacuolar protein carboxypeptidase Y is missorted in nhx1Δ cells, and is secreted from the cell.nhx1Δ cells accumulate late Golgi, prevacuole, and lysosome markers in an aberrant structure next to the vacuole, and late Golgi proteins are proteolytically cleaved more rapidly than in wild-type cells. Our results show that efficient transport out of the prevacuolar compartment requires Nhx1p, and that nhx1Δ cells exhibit phenotypes characteristic of the “class E” group ofvps mutants. In addition, we show that Nhx1p is required for protein trafficking even in the absence of the vacuolar ATPase. Our analysis of Nhx1p provides the first evidence that a sodium/proton exchange protein is important for correct protein sorting, and that intraorganellar ion balance may be important for endosomal function in yeast.

scholarly journals The newly identified yeast GRD genes are required for retention of late-Golgi membrane proteins.

1996 ◽  
Vol 16 (6) ◽  
pp. 2700-2707 ◽  
Author(s):  
S F Nothwehr ◽  
N J Bryant ◽  
T H Stevens
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Alkaline Phosphatase ◽  
Membrane Proteins ◽  
Golgi Membrane ◽  
Golgi Retention ◽  
Complementation Groups ◽  
Aminopeptidase A ◽  
Dipeptidyl Aminopeptidase ◽  
Yeast Mutants ◽  
Kinetics Of

Processing of A-ALP, a late-Golgi membrane protein constructed by fusing the cytosolic domain of dipeptidyl aminopeptidase A to the transmembrane and lumenal domains of alkaline phosphatase (ALP), serves as a convenient assay for loss of retention of late-Golgi membrane proteins in Saccharomyces cerevisiae. In this study, a large group of novel grd (for Golgi retention defective) yeast mutants, representing 18 complementation groups, were identified on the basis of their mislocalization of A-ALP to the vacuole, where it was proteolytically processed and thus became enzymatically activated. All of the grd mutants exhibited significant mislocalization of A-ALP, as measured by determining the kinetics of A-ALP processing and by analyzing its

scholarly journals Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants.

1992 ◽  
Vol 3 (12) ◽  
pp. 1389-1402 ◽  
Author(s):  
C K Raymond ◽  
I Howald-Stevenson ◽  
C A Vater ◽  
T H Stevens
Keyword(s):  
Protein Sorting ◽  
Carboxypeptidase Y ◽  
Vacuolar Protein Sorting ◽  
Complementation Groups ◽  
Prevacuolar Compartment ◽  
Golgi Protein ◽  
Mother Cells ◽  
Vacuolar Protein ◽  
Morphological Phenotype

The collection of vacuolar protein sorting mutants (vps mutants) in Saccharomyces cerevisiae comprises of 41 complementation groups. The vacuoles in these mutant strains were examined using immunofluorescence microscopy. Most of the vps mutants were found to possess vacuolar morphologies that differed significantly from wild-type vacuoles. Furthermore, mutants representing independent vps complementation groups were found to share aberrant morphological features. Six distinct classes of vacuolar morphology were observed. Mutants from eight vps complementation groups were defective both for vacuolar segregation from mother cells into developing buds and for acidification of the vacuole. Another group of mutants, represented by 13 complementation groups, accumulated a novel organelle distinct from the vacuole that contained a late-Golgi protein, active vacuolar H(+)-ATPase complex, and soluble vacuolar hydrolases. We suggest that this organelle may represent an exaggerated endosome-like compartment. None of the vps mutants appeared to mislocalize significant amounts of the vacuolar membrane protein alkaline phosphatase. Quantitative immunoprecipitations of the soluble vacuolar hydrolase carboxypeptidase Y (CPY) were performed to determine the extent of the sorting defect in each vps mutant. A good correlation between morphological phenotype and the extent of the CPY sorting defect was observed.

Sign in / Sign up

Export Citation Format

Share Document