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Retrieval of resident late-Golgi membrane proteins from the prevacuolar compartment of Saccharomyces cerevisiae is dependent on the function of Grd19p.

Voos W, Stevens TH - J. Cell Biol. (1998)

Bottom Line: In contrast to previously identified genes involved in late-Golgi membrane protein localization, grd19 mutations cause only minor effects on vacuolar protein sorting.In a yeast mutant that accumulates an exaggerated form of the prevacuolar compartment (vps27), Grd19p was observed to localize to this compartment.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229, USA.

ABSTRACT
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 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 grd19Delta 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.

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Sorting of CPY and the cycling of Vps10p are normal in  grd19Δ cells. (A) Wild-type (SNY36), grd19Δ (WVY4), and  vps10Δ (AACY30) cells were labeled for the indicated times and  analyzed for sorting of CPY by immunoprecipitation from intracellular (I) and extracellular (E) fractions as described. Indicated  are the mature protein (m), the ER/early-Golgi precursor form  (p1) and the fully glycosylated form (p2). (B) The same strains as  in Fig. 2 B were analyzed for the stability of Vps10p by pulse– chase labeling and immunoprecipitation using antibodies against  Vps10p. Indicated are the mature form (m) and the vacuolar degradation product (d). (C) Colony overlay assay with wild-type cells  carrying pAH37 (2μ-VPS5) show relative rates of CPY secretion.
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Figure 3: Sorting of CPY and the cycling of Vps10p are normal in grd19Δ cells. (A) Wild-type (SNY36), grd19Δ (WVY4), and vps10Δ (AACY30) cells were labeled for the indicated times and analyzed for sorting of CPY by immunoprecipitation from intracellular (I) and extracellular (E) fractions as described. Indicated are the mature protein (m), the ER/early-Golgi precursor form (p1) and the fully glycosylated form (p2). (B) The same strains as in Fig. 2 B were analyzed for the stability of Vps10p by pulse– chase labeling and immunoprecipitation using antibodies against Vps10p. Indicated are the mature form (m) and the vacuolar degradation product (d). (C) Colony overlay assay with wild-type cells carrying pAH37 (2μ-VPS5) show relative rates of CPY secretion.

Mentions: Since many of the recently identified grd mutants also exhibit Vps− phenotypes (Nothwehr et al., 1996), we tested whether a deletion of GRD19 also affects CPY sorting using pulse–chase labeling and immunoprecipitation experiments. Newly synthesized CPY was immunoprecipitated from internal and external fractions of grd19Δ and wild-type cells, as well as cells carrying a wild-type copy of GRD19 on a centromere-based plasmid. As a control for a strain with severe defects in CPY sorting, the experiment included a vps10Δ strain. As shown in Fig. 3 A, grd19Δ cells exhibited only a minor defect in the sorting of CPY. grd19Δ cells missorted ∼16% of the Golgi-modified (p2) form of CPY to the cell surface, compared to 6% in wild-type cells. By constrast, vps10 cells secreted 86% of CPY, and virtually none of the CPY reached the vacuole.


Retrieval of resident late-Golgi membrane proteins from the prevacuolar compartment of Saccharomyces cerevisiae is dependent on the function of Grd19p.

Voos W, Stevens TH - J. Cell Biol. (1998)

Sorting of CPY and the cycling of Vps10p are normal in  grd19Δ cells. (A) Wild-type (SNY36), grd19Δ (WVY4), and  vps10Δ (AACY30) cells were labeled for the indicated times and  analyzed for sorting of CPY by immunoprecipitation from intracellular (I) and extracellular (E) fractions as described. Indicated  are the mature protein (m), the ER/early-Golgi precursor form  (p1) and the fully glycosylated form (p2). (B) The same strains as  in Fig. 2 B were analyzed for the stability of Vps10p by pulse– chase labeling and immunoprecipitation using antibodies against  Vps10p. Indicated are the mature form (m) and the vacuolar degradation product (d). (C) Colony overlay assay with wild-type cells  carrying pAH37 (2μ-VPS5) show relative rates of CPY secretion.
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Related In: Results  -  Collection

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Figure 3: Sorting of CPY and the cycling of Vps10p are normal in grd19Δ cells. (A) Wild-type (SNY36), grd19Δ (WVY4), and vps10Δ (AACY30) cells were labeled for the indicated times and analyzed for sorting of CPY by immunoprecipitation from intracellular (I) and extracellular (E) fractions as described. Indicated are the mature protein (m), the ER/early-Golgi precursor form (p1) and the fully glycosylated form (p2). (B) The same strains as in Fig. 2 B were analyzed for the stability of Vps10p by pulse– chase labeling and immunoprecipitation using antibodies against Vps10p. Indicated are the mature form (m) and the vacuolar degradation product (d). (C) Colony overlay assay with wild-type cells carrying pAH37 (2μ-VPS5) show relative rates of CPY secretion.
Mentions: Since many of the recently identified grd mutants also exhibit Vps− phenotypes (Nothwehr et al., 1996), we tested whether a deletion of GRD19 also affects CPY sorting using pulse–chase labeling and immunoprecipitation experiments. Newly synthesized CPY was immunoprecipitated from internal and external fractions of grd19Δ and wild-type cells, as well as cells carrying a wild-type copy of GRD19 on a centromere-based plasmid. As a control for a strain with severe defects in CPY sorting, the experiment included a vps10Δ strain. As shown in Fig. 3 A, grd19Δ cells exhibited only a minor defect in the sorting of CPY. grd19Δ cells missorted ∼16% of the Golgi-modified (p2) form of CPY to the cell surface, compared to 6% in wild-type cells. By constrast, vps10 cells secreted 86% of CPY, and virtually none of the CPY reached the vacuole.

Bottom Line: In contrast to previously identified genes involved in late-Golgi membrane protein localization, grd19 mutations cause only minor effects on vacuolar protein sorting.In a yeast mutant that accumulates an exaggerated form of the prevacuolar compartment (vps27), Grd19p was observed to localize to this compartment.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229, USA.

ABSTRACT
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 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 grd19Delta 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.

Show MeSH
Related in: MedlinePlus