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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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The drs2Δ mutant exhibits a cold-sensitive kinetic defect in CPY transport to the vacuole. Wild-type, drs2Δ, and arf1Δ (all isogenic to SEY6210) strains were labeled for 10 min at 15° or 30°C, and then chased for the times indicated. CPY was recovered from each sample by immunoprecipitation and subjected to SDS-PAGE.
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Figure 4: The drs2Δ mutant exhibits a cold-sensitive kinetic defect in CPY transport to the vacuole. Wild-type, drs2Δ, and arf1Δ (all isogenic to SEY6210) strains were labeled for 10 min at 15° or 30°C, and then chased for the times indicated. CPY was recovered from each sample by immunoprecipitation and subjected to SDS-PAGE.

Mentions: These studies indicated that the endocytic defect observed in drs2Δ cells was attributable to a defect in the endosome-to-vacuole pathway rather than clathrin-dependent endocytosis from the plasma membrane. If so, CPY transport should be affected as well since this protein follows a TGN to endosome to vacuole delivery route. To test this, we examined the transport of CPY to the vacuole in the drs2Δ mutant. CPY is synthesized in the ER as the p1 precursor form and is modified on N-linked oligosaccharides by the Golgi α1,3 mannosyltransferase (Mnn1p) to form the p2 precursor. p2 CPY is sorted from secreted proteins in the TGN and ultimately processed to the mature form in the vacuole (Stevens et al. 1982). Wild-type, drs2Δ, and arf1Δ cells were pulse-labeled and chased at either the permissive or nonpermissive temperature of drs2Δ. Aliquots of cells were removed at the chase times indicated and CPY was recovered by immunoprecipitation. As shown in Fig. 4, drs2Δ cells displayed near wild-type CPY transport kinetics at 30°C, while at 15°C the transport of CPY in drs2Δ mutants was significantly delayed relative to that in the wild-type cells, and was similar to the defect observed in arf1Δ cells at either temperature (approximately threefold slower transport kinetics). The partial glycosylation defect observed in drs2Δ (and arf1Δ cells) prevented the formation of a p2 CPY form that could be resolved from the p1 form in SDS-polyacrylamide gels. Thus, the kinetics of ER-to-Golgi transport could not be assessed for CPY. However, the ER-to-Golgi transport kinetics for α-factor and invertase in drs2Δ cells was found to be nearly wild type at the nonpermissive temperature, as scored by disappearance of the ER core form (Fig. 2, and data not shown). Therefore, it is unlikely that ER-to-Golgi transport for CPY is disturbed in drs2Δ cells. These data are most consistent with the interpretation that protein transport from the late Golgi or endosomes to the vacuole is perturbed by the drs2Δ mutation. Interestingly, the chc1-5 allele isolated in the arf1Δ synthetic lethal screen also exhibits a partial glycosylation defect and an approximately threefold slower transport kinetics for CPY (Chen and Graham 1998).


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 kinetic defect in CPY transport to the vacuole. Wild-type, drs2Δ, and arf1Δ (all isogenic to SEY6210) strains were labeled for 10 min at 15° or 30°C, and then chased for the times indicated. CPY was recovered from each sample by immunoprecipitation and subjected to SDS-PAGE.
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Related In: Results  -  Collection

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

Figure 4: The drs2Δ mutant exhibits a cold-sensitive kinetic defect in CPY transport to the vacuole. Wild-type, drs2Δ, and arf1Δ (all isogenic to SEY6210) strains were labeled for 10 min at 15° or 30°C, and then chased for the times indicated. CPY was recovered from each sample by immunoprecipitation and subjected to SDS-PAGE.
Mentions: These studies indicated that the endocytic defect observed in drs2Δ cells was attributable to a defect in the endosome-to-vacuole pathway rather than clathrin-dependent endocytosis from the plasma membrane. If so, CPY transport should be affected as well since this protein follows a TGN to endosome to vacuole delivery route. To test this, we examined the transport of CPY to the vacuole in the drs2Δ mutant. CPY is synthesized in the ER as the p1 precursor form and is modified on N-linked oligosaccharides by the Golgi α1,3 mannosyltransferase (Mnn1p) to form the p2 precursor. p2 CPY is sorted from secreted proteins in the TGN and ultimately processed to the mature form in the vacuole (Stevens et al. 1982). Wild-type, drs2Δ, and arf1Δ cells were pulse-labeled and chased at either the permissive or nonpermissive temperature of drs2Δ. Aliquots of cells were removed at the chase times indicated and CPY was recovered by immunoprecipitation. As shown in Fig. 4, drs2Δ cells displayed near wild-type CPY transport kinetics at 30°C, while at 15°C the transport of CPY in drs2Δ mutants was significantly delayed relative to that in the wild-type cells, and was similar to the defect observed in arf1Δ cells at either temperature (approximately threefold slower transport kinetics). The partial glycosylation defect observed in drs2Δ (and arf1Δ cells) prevented the formation of a p2 CPY form that could be resolved from the p1 form in SDS-polyacrylamide gels. Thus, the kinetics of ER-to-Golgi transport could not be assessed for CPY. However, the ER-to-Golgi transport kinetics for α-factor and invertase in drs2Δ cells was found to be nearly wild type at the nonpermissive temperature, as scored by disappearance of the ER core form (Fig. 2, and data not shown). Therefore, it is unlikely that ER-to-Golgi transport for CPY is disturbed in drs2Δ cells. These data are most consistent with the interpretation that protein transport from the late Golgi or endosomes to the vacuole is perturbed by the drs2Δ mutation. Interestingly, the chc1-5 allele isolated in the arf1Δ synthetic lethal screen also exhibits a partial glycosylation defect and an approximately threefold slower transport kinetics for CPY (Chen and Graham 1998).

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