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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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Drs2p cofractionates with high-density membranes containing the late Golgi markers, Kex2p and Mnn1p. (A) Specificity of the Drs2p antibodies. Wild-type, drs2Δ, and SEY6210 pRS425-DRS2 (2μ DRS2) cells were grown at 30°C before lysing with glass beads in SDS-urea sample buffer. Total cellular proteins (0.1 OD per lane) were subjected to SDS-PAGE and immunoblotted with affinity-purified antibodies against Drs2p. (B) Wild-type cells grown at 30°C were lysed (lysate, L) and subjected to centrifugation at 21,000 g for 30 min to generate pellet (P21) and supernatant (S21) fractions. The S21 fraction was further centrifuged at 100,000 g for 80 min to generate pellet (P100) and supernatant (S100) fractions. 20 μg of total protein per fraction was immunoblotted for Drs2p and the plasma membrane ATPase, Pma1p. (C) Wild-type cells grown at 30°C were lysed and subjected to differential centrifugation and sucrose gradient fractionation of the P100 fraction. These gradient fractions have been previously used to examine Mnn1p distribution in Figure 6 a of Reynolds et al. 1998. Equal volumes per gradient fraction were assayed by enzyme activity for Kex2p or GDPase and by immunoblotting for Drs2p, Mnn1p, and Pep12p. (D) Wild-type cells were grown at 30°C, lysed, and subjected to differential centrifugation to produce a P100 fraction, which was then subjected to gel filtration chromatography as described in Fig. 6. Samples of every other fraction from fraction 15 to 35 were assayed by immunoblotting for the clathrin heavy chain (Chc1p), the late Golgi protein Mnn1p and Drs2p.
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Figure 7: Drs2p cofractionates with high-density membranes containing the late Golgi markers, Kex2p and Mnn1p. (A) Specificity of the Drs2p antibodies. Wild-type, drs2Δ, and SEY6210 pRS425-DRS2 (2μ DRS2) cells were grown at 30°C before lysing with glass beads in SDS-urea sample buffer. Total cellular proteins (0.1 OD per lane) were subjected to SDS-PAGE and immunoblotted with affinity-purified antibodies against Drs2p. (B) Wild-type cells grown at 30°C were lysed (lysate, L) and subjected to centrifugation at 21,000 g for 30 min to generate pellet (P21) and supernatant (S21) fractions. The S21 fraction was further centrifuged at 100,000 g for 80 min to generate pellet (P100) and supernatant (S100) fractions. 20 μg of total protein per fraction was immunoblotted for Drs2p and the plasma membrane ATPase, Pma1p. (C) Wild-type cells grown at 30°C were lysed and subjected to differential centrifugation and sucrose gradient fractionation of the P100 fraction. These gradient fractions have been previously used to examine Mnn1p distribution in Figure 6 a of Reynolds et al. 1998. Equal volumes per gradient fraction were assayed by enzyme activity for Kex2p or GDPase and by immunoblotting for Drs2p, Mnn1p, and Pep12p. (D) Wild-type cells were grown at 30°C, lysed, and subjected to differential centrifugation to produce a P100 fraction, which was then subjected to gel filtration chromatography as described in Fig. 6. Samples of every other fraction from fraction 15 to 35 were assayed by immunoblotting for the clathrin heavy chain (Chc1p), the late Golgi protein Mnn1p and Drs2p.

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)

Drs2p cofractionates with high-density membranes containing the late Golgi markers, Kex2p and Mnn1p. (A) Specificity of the Drs2p antibodies. Wild-type, drs2Δ, and SEY6210 pRS425-DRS2 (2μ DRS2) cells were grown at 30°C before lysing with glass beads in SDS-urea sample buffer. Total cellular proteins (0.1 OD per lane) were subjected to SDS-PAGE and immunoblotted with affinity-purified antibodies against Drs2p. (B) Wild-type cells grown at 30°C were lysed (lysate, L) and subjected to centrifugation at 21,000 g for 30 min to generate pellet (P21) and supernatant (S21) fractions. The S21 fraction was further centrifuged at 100,000 g for 80 min to generate pellet (P100) and supernatant (S100) fractions. 20 μg of total protein per fraction was immunoblotted for Drs2p and the plasma membrane ATPase, Pma1p. (C) Wild-type cells grown at 30°C were lysed and subjected to differential centrifugation and sucrose gradient fractionation of the P100 fraction. These gradient fractions have been previously used to examine Mnn1p distribution in Figure 6 a of Reynolds et al. 1998. Equal volumes per gradient fraction were assayed by enzyme activity for Kex2p or GDPase and by immunoblotting for Drs2p, Mnn1p, and Pep12p. (D) Wild-type cells were grown at 30°C, lysed, and subjected to differential centrifugation to produce a P100 fraction, which was then subjected to gel filtration chromatography as described in Fig. 6. Samples of every other fraction from fraction 15 to 35 were assayed by immunoblotting for the clathrin heavy chain (Chc1p), the late Golgi protein Mnn1p and Drs2p.
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Figure 7: Drs2p cofractionates with high-density membranes containing the late Golgi markers, Kex2p and Mnn1p. (A) Specificity of the Drs2p antibodies. Wild-type, drs2Δ, and SEY6210 pRS425-DRS2 (2μ DRS2) cells were grown at 30°C before lysing with glass beads in SDS-urea sample buffer. Total cellular proteins (0.1 OD per lane) were subjected to SDS-PAGE and immunoblotted with affinity-purified antibodies against Drs2p. (B) Wild-type cells grown at 30°C were lysed (lysate, L) and subjected to centrifugation at 21,000 g for 30 min to generate pellet (P21) and supernatant (S21) fractions. The S21 fraction was further centrifuged at 100,000 g for 80 min to generate pellet (P100) and supernatant (S100) fractions. 20 μg of total protein per fraction was immunoblotted for Drs2p and the plasma membrane ATPase, Pma1p. (C) Wild-type cells grown at 30°C were lysed and subjected to differential centrifugation and sucrose gradient fractionation of the P100 fraction. These gradient fractions have been previously used to examine Mnn1p distribution in Figure 6 a of Reynolds et al. 1998. Equal volumes per gradient fraction were assayed by enzyme activity for Kex2p or GDPase and by immunoblotting for Drs2p, Mnn1p, and Pep12p. (D) Wild-type cells were grown at 30°C, lysed, and subjected to differential centrifugation to produce a P100 fraction, which was then subjected to gel filtration chromatography as described in Fig. 6. Samples of every other fraction from fraction 15 to 35 were assayed by immunoblotting for the clathrin heavy chain (Chc1p), the late Golgi protein Mnn1p and Drs2p.
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