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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 accumulates aberrant Golgi membranes and exhibits a deficiency of clathrin-coated vesicles. (A) Wild-type and drs2Δ cells were grown at 30°C and shifted to 15°C for 1 h, and then lysed and subjected to centrifugation at 21,000 g for 30 min. The 21,000-g supernatant was centrifuged at 100,000 g for 80 min to generate a 100,000-g pellet. This pellet was resuspended and further resolved on a Sephacryl S-1000 column. Samples of every other fraction from fraction 15 to 35 were assayed by immunoblotting for the clathrin heavy chain (Chc1p), a late Golgi protein Mnn1p, and an endosomal t-SNARE Pep12p. (B–D) Membranes in fraction 16 from the drs2Δ sample where Mnn1p was found (B), and fraction 26 from wild-type (C) and drs2Δ (D) samples where Chc1p was enriched were fixed, pelleted, and examined by EM. Bars, 50 nm.
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Figure 6: The drs2Δ mutant accumulates aberrant Golgi membranes and exhibits a deficiency of clathrin-coated vesicles. (A) Wild-type and drs2Δ cells were grown at 30°C and shifted to 15°C for 1 h, and then lysed and subjected to centrifugation at 21,000 g for 30 min. The 21,000-g supernatant was centrifuged at 100,000 g for 80 min to generate a 100,000-g pellet. This pellet was resuspended and further resolved on a Sephacryl S-1000 column. Samples of every other fraction from fraction 15 to 35 were assayed by immunoblotting for the clathrin heavy chain (Chc1p), a late Golgi protein Mnn1p, and an endosomal t-SNARE Pep12p. (B–D) Membranes in fraction 16 from the drs2Δ sample where Mnn1p was found (B), and fraction 26 from wild-type (C) and drs2Δ (D) samples where Chc1p was enriched were fixed, pelleted, and examined by EM. Bars, 50 nm.

Mentions: To further assess clathrin function in the drs2Δ mutants, we asked whether CCVs could be purified from this mutant. CCV preparations were generated from drs2Δ and wild-type cells with or without a 1-h shift to 15°C (see Materials and Methods). Cell lysates were centrifuged at 21,000 g for 30 min to pellet large organellar membranes (e.g., plasma membrane, vacuolar membrane, ER, and mitochondria) and the resulting supernatant was centrifuged at 100,000 g to pellet vesicles. The 100,000-g pellet was then applied to a Sephacryl S-1000 gel filtration column (Mueller and Branton 1984), and the fractions were probed for the clathrin heavy chain by immunoblotting. As shown in Fig. 6 A, the clathrin heavy chain was highly enriched in fractions 23 to 27 for both wild-type and drs2Δ samples from cells shifted to 15°C (Fig. 6 A) or 30°C (Fig. 7 D, and data not shown). In addition, from Coomassie blue–stained gels, we estimated that the recovery of clathrin heavy chain in these fractions was comparable for both strains at both temperatures (data not shown).


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 accumulates aberrant Golgi membranes and exhibits a deficiency of clathrin-coated vesicles. (A) Wild-type and drs2Δ cells were grown at 30°C and shifted to 15°C for 1 h, and then lysed and subjected to centrifugation at 21,000 g for 30 min. The 21,000-g supernatant was centrifuged at 100,000 g for 80 min to generate a 100,000-g pellet. This pellet was resuspended and further resolved on a Sephacryl S-1000 column. Samples of every other fraction from fraction 15 to 35 were assayed by immunoblotting for the clathrin heavy chain (Chc1p), a late Golgi protein Mnn1p, and an endosomal t-SNARE Pep12p. (B–D) Membranes in fraction 16 from the drs2Δ sample where Mnn1p was found (B), and fraction 26 from wild-type (C) and drs2Δ (D) samples where Chc1p was enriched were fixed, pelleted, and examined by EM. Bars, 50 nm.
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Figure 6: The drs2Δ mutant accumulates aberrant Golgi membranes and exhibits a deficiency of clathrin-coated vesicles. (A) Wild-type and drs2Δ cells were grown at 30°C and shifted to 15°C for 1 h, and then lysed and subjected to centrifugation at 21,000 g for 30 min. The 21,000-g supernatant was centrifuged at 100,000 g for 80 min to generate a 100,000-g pellet. This pellet was resuspended and further resolved on a Sephacryl S-1000 column. Samples of every other fraction from fraction 15 to 35 were assayed by immunoblotting for the clathrin heavy chain (Chc1p), a late Golgi protein Mnn1p, and an endosomal t-SNARE Pep12p. (B–D) Membranes in fraction 16 from the drs2Δ sample where Mnn1p was found (B), and fraction 26 from wild-type (C) and drs2Δ (D) samples where Chc1p was enriched were fixed, pelleted, and examined by EM. Bars, 50 nm.
Mentions: To further assess clathrin function in the drs2Δ mutants, we asked whether CCVs could be purified from this mutant. CCV preparations were generated from drs2Δ and wild-type cells with or without a 1-h shift to 15°C (see Materials and Methods). Cell lysates were centrifuged at 21,000 g for 30 min to pellet large organellar membranes (e.g., plasma membrane, vacuolar membrane, ER, and mitochondria) and the resulting supernatant was centrifuged at 100,000 g to pellet vesicles. The 100,000-g pellet was then applied to a Sephacryl S-1000 gel filtration column (Mueller and Branton 1984), and the fractions were probed for the clathrin heavy chain by immunoblotting. As shown in Fig. 6 A, the clathrin heavy chain was highly enriched in fractions 23 to 27 for both wild-type and drs2Δ samples from cells shifted to 15°C (Fig. 6 A) or 30°C (Fig. 7 D, and data not shown). In addition, from Coomassie blue–stained gels, we estimated that the recovery of clathrin heavy chain in these fractions was comparable for both strains at both temperatures (data not shown).

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