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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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Nucleotide and amino acid sequence of GRD19 gene  region. (A) Underlined nucleotide sequence represents the sequence of the oligonucleotides used for cloning the full-length  GRD19 gene. The region was identified in the yeast genome database as ORF YOR357c. (B) Schematic drawing showing proteins related to Grd19p and the relative position of the conserved  PX domain. (C) Sequence comparison between the PX domains  of several proteins that were aligned by the ClustalW program.  The sequence data are available from EMBL/DDBJ/Genbank  under accession numbers: Mvp1p (U16137), Vps5p (U84735),  SNX1 (U53225), and Grd19p (AF016101).
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Figure 1: Nucleotide and amino acid sequence of GRD19 gene region. (A) Underlined nucleotide sequence represents the sequence of the oligonucleotides used for cloning the full-length GRD19 gene. The region was identified in the yeast genome database as ORF YOR357c. (B) Schematic drawing showing proteins related to Grd19p and the relative position of the conserved PX domain. (C) Sequence comparison between the PX domains of several proteins that were aligned by the ClustalW program. The sequence data are available from EMBL/DDBJ/Genbank under accession numbers: Mvp1p (U16137), Vps5p (U84735), SNX1 (U53225), and Grd19p (AF016101).

Mentions: GRD19 encodes a small hydrophilic protein of 162 amino acids (Fig. 1 A) with a predicted molecular mass of 18.7 kD. The amino acid sequence shows no obvious modification motifs or targeting signals. Protein sequence comparisons (BLAST at NCBI) revealed significant homology to several other yeast and mammalian proteins (Fig. 1 B). Interestingly, the most similar protein found in the database is Mvp1p, which is also implicated in vesicle transport processes between the Golgi compartment and the vacuole (Ekena and Stevens, 1995). The homology between Grd19p and Mvp1p is mainly restricted to a sequence, ∼100 amino acids long, near the carboxy terminus of Grd19p. A similar homologous region was found in Vps5p (Horazdovsky et al., 1997), which was also identified as Grd2p (Nothwehr and Hindes, 1997). Grd2p/Vps5p is also involved in vacuolar sorting processes and is required for the localization of A-ALP, Kex2p, and Vps10p. An extensive set of experiments was performed to determine whether there are any genetic interactions between the GRD19, VPS5, and MVP1 genes. The Golgi localization defects of grd19Δ cells could not be suppressed by overexpression of VPS5 or MVP1. Additionally, analysis of the double deletion mutants grd19Δ vps5Δ and grd19Δ mvp1Δ did not reveal any synthetic phenotypes concerning growth, CPY secretion, or A-ALP localization (data not shown).


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)

Nucleotide and amino acid sequence of GRD19 gene  region. (A) Underlined nucleotide sequence represents the sequence of the oligonucleotides used for cloning the full-length  GRD19 gene. The region was identified in the yeast genome database as ORF YOR357c. (B) Schematic drawing showing proteins related to Grd19p and the relative position of the conserved  PX domain. (C) Sequence comparison between the PX domains  of several proteins that were aligned by the ClustalW program.  The sequence data are available from EMBL/DDBJ/Genbank  under accession numbers: Mvp1p (U16137), Vps5p (U84735),  SNX1 (U53225), and Grd19p (AF016101).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140161&req=5

Figure 1: Nucleotide and amino acid sequence of GRD19 gene region. (A) Underlined nucleotide sequence represents the sequence of the oligonucleotides used for cloning the full-length GRD19 gene. The region was identified in the yeast genome database as ORF YOR357c. (B) Schematic drawing showing proteins related to Grd19p and the relative position of the conserved PX domain. (C) Sequence comparison between the PX domains of several proteins that were aligned by the ClustalW program. The sequence data are available from EMBL/DDBJ/Genbank under accession numbers: Mvp1p (U16137), Vps5p (U84735), SNX1 (U53225), and Grd19p (AF016101).
Mentions: GRD19 encodes a small hydrophilic protein of 162 amino acids (Fig. 1 A) with a predicted molecular mass of 18.7 kD. The amino acid sequence shows no obvious modification motifs or targeting signals. Protein sequence comparisons (BLAST at NCBI) revealed significant homology to several other yeast and mammalian proteins (Fig. 1 B). Interestingly, the most similar protein found in the database is Mvp1p, which is also implicated in vesicle transport processes between the Golgi compartment and the vacuole (Ekena and Stevens, 1995). The homology between Grd19p and Mvp1p is mainly restricted to a sequence, ∼100 amino acids long, near the carboxy terminus of Grd19p. A similar homologous region was found in Vps5p (Horazdovsky et al., 1997), which was also identified as Grd2p (Nothwehr and Hindes, 1997). Grd2p/Vps5p is also involved in vacuolar sorting processes and is required for the localization of A-ALP, Kex2p, and Vps10p. An extensive set of experiments was performed to determine whether there are any genetic interactions between the GRD19, VPS5, and MVP1 genes. The Golgi localization defects of grd19Δ cells could not be suppressed by overexpression of VPS5 or MVP1. Additionally, analysis of the double deletion mutants grd19Δ vps5Δ and grd19Δ mvp1Δ did not reveal any synthetic phenotypes concerning growth, CPY secretion, or A-ALP localization (data not shown).

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