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Role for Drs2p, a P-type ATPase and potential aminophospholipid translocase, in yeast late Golgi function.

Chen CY, Ingram MF, Rosal PH, Graham TR - J. Cell Biol. (1999)

Bottom Line: Consistent with these genetic analyses, we found that the drs2Delta mutant exhibits late Golgi defects that may result from a loss of clathrin function at this compartment.Subcellular fractionation and immunofluorescence analysis indicate that Drs2p localizes to late Golgi membranes containing Kex2p.These observations indicate a novel role for a P-type ATPase in late Golgi function and suggest a possible link between membrane asymmetry and clathrin function at the Golgi complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA.

ABSTRACT
ADP-ribosylation factor appears to regulate the budding of both COPI and clathrin-coated transport vesicles from Golgi membranes. An arf1Delta synthetic lethal screen identified SWA3/DRS2, which encodes an integral membrane P-type ATPase and potential aminophospholipid translocase (or flippase). The drs2 allele is also synthetically lethal with clathrin heavy chain (chc1) temperature-sensitive alleles, but not with mutations in COPI subunits or other SEC genes tested. Consistent with these genetic analyses, we found that the drs2Delta mutant exhibits late Golgi defects that may result from a loss of clathrin function at this compartment. These include a defect in the Kex2-dependent processing of pro-alpha-factor and the accumulation of abnormal Golgi cisternae. Moreover, we observed a marked reduction in clathrin-coated vesicles that can be isolated from the drs2Delta cells. Subcellular fractionation and immunofluorescence analysis indicate that Drs2p localizes to late Golgi membranes containing Kex2p. These observations indicate a novel role for a P-type ATPase in late Golgi function and suggest a possible link between membrane asymmetry and clathrin function at the Golgi complex.

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Related in: MedlinePlus

The drs2Δ mutant exhibits a cold-sensitive defect in pro–α-factor processing. Wild-type, drs2Δ (hereafter used to represent SEY6210 and the isogenic strain 6210 drs2Δ, respectively), and clc1Δ (LSY93.1-10A) strains were labeled for 10 min at 15°C, chased for the times indicated, and converted to spheroplasts. Intracellular (I) and extracellular (E) portions were separated by centrifugation and subjected to immunoprecipitation with antiserum to α-factor.
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Figure 2: The drs2Δ mutant exhibits a cold-sensitive defect in pro–α-factor processing. Wild-type, drs2Δ (hereafter used to represent SEY6210 and the isogenic strain 6210 drs2Δ, respectively), and clc1Δ (LSY93.1-10A) strains were labeled for 10 min at 15°C, chased for the times indicated, and converted to spheroplasts. Intracellular (I) and extracellular (E) portions were separated by centrifugation and subjected to immunoprecipitation with antiserum to α-factor.

Mentions: One of the more striking phenotypes of yeast clathrin mutants is the mislocalization of several late Golgi (TGN) proteins that are required for pro–α-factor proteolytic processing. This results in the secretion of fully glycosylated pro–α-factor rather than the mature peptide (Payne and Schekman 1989). If clathrin function at the TGN is perturbed in the drs2Δ mutant, we would expect this mutant to secrete pro–α-factor. To test this, wild-type, drs2Δ, and clc1Δ (clathrin light chain ) strains were grown at 30°C, and after shifting to 20°C for 1 h, were metabolically labeled and chased at this nonpermissive temperature for drs2Δ. Aliquots of cells were removed at the chase times indicated in Fig. 2, converted to spheroplasts, and then centrifuged to separate intracellular (I) from extracellular (E) fractions. α-Factor was then recovered from each sample by immunoprecipitation.


Role for Drs2p, a P-type ATPase and potential aminophospholipid translocase, in yeast late Golgi function.

Chen CY, Ingram MF, Rosal PH, Graham TR - J. Cell Biol. (1999)

The drs2Δ mutant exhibits a cold-sensitive defect in pro–α-factor processing. Wild-type, drs2Δ (hereafter used to represent SEY6210 and the isogenic strain 6210 drs2Δ, respectively), and clc1Δ (LSY93.1-10A) strains were labeled for 10 min at 15°C, chased for the times indicated, and converted to spheroplasts. Intracellular (I) and extracellular (E) portions were separated by centrifugation and subjected to immunoprecipitation with antiserum to α-factor.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2168089&req=5

Figure 2: The drs2Δ mutant exhibits a cold-sensitive defect in pro–α-factor processing. Wild-type, drs2Δ (hereafter used to represent SEY6210 and the isogenic strain 6210 drs2Δ, respectively), and clc1Δ (LSY93.1-10A) strains were labeled for 10 min at 15°C, chased for the times indicated, and converted to spheroplasts. Intracellular (I) and extracellular (E) portions were separated by centrifugation and subjected to immunoprecipitation with antiserum to α-factor.
Mentions: One of the more striking phenotypes of yeast clathrin mutants is the mislocalization of several late Golgi (TGN) proteins that are required for pro–α-factor proteolytic processing. This results in the secretion of fully glycosylated pro–α-factor rather than the mature peptide (Payne and Schekman 1989). If clathrin function at the TGN is perturbed in the drs2Δ mutant, we would expect this mutant to secrete pro–α-factor. To test this, wild-type, drs2Δ, and clc1Δ (clathrin light chain ) strains were grown at 30°C, and after shifting to 20°C for 1 h, were metabolically labeled and chased at this nonpermissive temperature for drs2Δ. Aliquots of cells were removed at the chase times indicated in Fig. 2, converted to spheroplasts, and then centrifuged to separate intracellular (I) from extracellular (E) fractions. α-Factor was then recovered from each sample by immunoprecipitation.

Bottom Line: Consistent with these genetic analyses, we found that the drs2Delta mutant exhibits late Golgi defects that may result from a loss of clathrin function at this compartment.Subcellular fractionation and immunofluorescence analysis indicate that Drs2p localizes to late Golgi membranes containing Kex2p.These observations indicate a novel role for a P-type ATPase in late Golgi function and suggest a possible link between membrane asymmetry and clathrin function at the Golgi complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA.

ABSTRACT
ADP-ribosylation factor appears to regulate the budding of both COPI and clathrin-coated transport vesicles from Golgi membranes. An arf1Delta synthetic lethal screen identified SWA3/DRS2, which encodes an integral membrane P-type ATPase and potential aminophospholipid translocase (or flippase). The drs2 allele is also synthetically lethal with clathrin heavy chain (chc1) temperature-sensitive alleles, but not with mutations in COPI subunits or other SEC genes tested. Consistent with these genetic analyses, we found that the drs2Delta mutant exhibits late Golgi defects that may result from a loss of clathrin function at this compartment. These include a defect in the Kex2-dependent processing of pro-alpha-factor and the accumulation of abnormal Golgi cisternae. Moreover, we observed a marked reduction in clathrin-coated vesicles that can be isolated from the drs2Delta cells. Subcellular fractionation and immunofluorescence analysis indicate that Drs2p localizes to late Golgi membranes containing Kex2p. These observations indicate a novel role for a P-type ATPase in late Golgi function and suggest a possible link between membrane asymmetry and clathrin function at the Golgi complex.

Show MeSH
Related in: MedlinePlus