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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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drs2Δ is synthetically lethal with arf1Δ, chc1, and pan1 alleles. (A) Genetic analyses between drs2Δ and mutations that perturb the secretory pathway. Strains carrying the indicated mutations (see Materials and Methods) were crossed with a drs2Δ mutant (6210 drs2Δ or PRY6222) to generate diploids. After tetrad analyses of the progeny, the viable double mutants were streaked at 20°, 26.5°, and 37°C to compare the growth relative to parental strains carrying single mutations. *Double mutants of the alleles and drs2 that were able to grow at 20°C, where single drs2 mutants could not. †Double mutants that grew more slowly than either single mutant at 26.5°C. (B) Tetrad analysis of progeny derived from crossing PRY6222 (drs2Δ) with 6210 chc1-5 (chc1-5). Spores that failed to grow were predicted to be drs2Δ chc1-5 double mutants.
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Figure 1: drs2Δ is synthetically lethal with arf1Δ, chc1, and pan1 alleles. (A) Genetic analyses between drs2Δ and mutations that perturb the secretory pathway. Strains carrying the indicated mutations (see Materials and Methods) were crossed with a drs2Δ mutant (6210 drs2Δ or PRY6222) to generate diploids. After tetrad analyses of the progeny, the viable double mutants were streaked at 20°, 26.5°, and 37°C to compare the growth relative to parental strains carrying single mutations. *Double mutants of the alleles and drs2 that were able to grow at 20°C, where single drs2 mutants could not. †Double mutants that grew more slowly than either single mutant at 26.5°C. (B) Tetrad analysis of progeny derived from crossing PRY6222 (drs2Δ) with 6210 chc1-5 (chc1-5). Spores that failed to grow were predicted to be drs2Δ chc1-5 double mutants.

Mentions: A drs2 strain was generated by replacing most of the DRS2 coding sequences with TRP1. Consistent with the phenotype previously reported for drs2Δ strains (Ripmaster et al. 1993) and that exhibited by the swa3 mutants (Chen and Graham 1998), the drs2Δ mutant was unable to grow at 20°C or below, but grew well at temperatures above 23°C. Linkage analysis indicated that SWA3 is allelic to DRS2, and synthetic lethality between arf1Δ and drs2Δ was confirmed by crossing the single mutants and characterizing the progeny by tetrad analysis (Fig. 1 A and data not shown). This genetic interaction suggested that Drs2p may be involved in an ARF-dependent vesicle-mediated protein transport event(s).


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)

drs2Δ is synthetically lethal with arf1Δ, chc1, and pan1 alleles. (A) Genetic analyses between drs2Δ and mutations that perturb the secretory pathway. Strains carrying the indicated mutations (see Materials and Methods) were crossed with a drs2Δ mutant (6210 drs2Δ or PRY6222) to generate diploids. After tetrad analyses of the progeny, the viable double mutants were streaked at 20°, 26.5°, and 37°C to compare the growth relative to parental strains carrying single mutations. *Double mutants of the alleles and drs2 that were able to grow at 20°C, where single drs2 mutants could not. †Double mutants that grew more slowly than either single mutant at 26.5°C. (B) Tetrad analysis of progeny derived from crossing PRY6222 (drs2Δ) with 6210 chc1-5 (chc1-5). Spores that failed to grow were predicted to be drs2Δ chc1-5 double mutants.
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Related In: Results  -  Collection

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Figure 1: drs2Δ is synthetically lethal with arf1Δ, chc1, and pan1 alleles. (A) Genetic analyses between drs2Δ and mutations that perturb the secretory pathway. Strains carrying the indicated mutations (see Materials and Methods) were crossed with a drs2Δ mutant (6210 drs2Δ or PRY6222) to generate diploids. After tetrad analyses of the progeny, the viable double mutants were streaked at 20°, 26.5°, and 37°C to compare the growth relative to parental strains carrying single mutations. *Double mutants of the alleles and drs2 that were able to grow at 20°C, where single drs2 mutants could not. †Double mutants that grew more slowly than either single mutant at 26.5°C. (B) Tetrad analysis of progeny derived from crossing PRY6222 (drs2Δ) with 6210 chc1-5 (chc1-5). Spores that failed to grow were predicted to be drs2Δ chc1-5 double mutants.
Mentions: A drs2 strain was generated by replacing most of the DRS2 coding sequences with TRP1. Consistent with the phenotype previously reported for drs2Δ strains (Ripmaster et al. 1993) and that exhibited by the swa3 mutants (Chen and Graham 1998), the drs2Δ mutant was unable to grow at 20°C or below, but grew well at temperatures above 23°C. Linkage analysis indicated that SWA3 is allelic to DRS2, and synthetic lethality between arf1Δ and drs2Δ was confirmed by crossing the single mutants and characterizing the progeny by tetrad analysis (Fig. 1 A and data not shown). This genetic interaction suggested that Drs2p may be involved in an ARF-dependent vesicle-mediated protein transport event(s).

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