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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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Mutation of a conserved ATPase motif causes loss of Drs2p function in vivo. (A) Comparison of five conserved consensus motifs of yeast P-type ATPases and the corresponding sequence in Drs2p (adapted from Catty et al. 1997). The arrow indicates the aspartic acid residue (D) at position 560, which was mutated to glutamic acid (E) or asparagine (N). (B) The drs2Δ strain containing plasmids pDRS2(D560E), pDRS2(D560N), pRS315-DRS2 (wild-type), and the pRS315 empty vector (drs2Δ) were grown at 30° or 20°C for 3 d.
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Figure 9: Mutation of a conserved ATPase motif causes loss of Drs2p function in vivo. (A) Comparison of five conserved consensus motifs of yeast P-type ATPases and the corresponding sequence in Drs2p (adapted from Catty et al. 1997). The arrow indicates the aspartic acid residue (D) at position 560, which was mutated to glutamic acid (E) or asparagine (N). (B) The drs2Δ strain containing plasmids pDRS2(D560E), pDRS2(D560N), pRS315-DRS2 (wild-type), and the pRS315 empty vector (drs2Δ) were grown at 30° or 20°C for 3 d.

Mentions: Drs2p is predicted to be a P-type ATPase based on the presence of five well-conserved ATPase motifs involved in the binding and hydrolysis of ATP (Fig. 9 A). For P-type ATPases, an aspartic acid within the second motif forms an aspartyl-phosphate catalytic intermediate that is essential for ATP hydrolysis (Allen and Green 1976; Fagan and Saier 1994). Sequence alignments between Drs2p and other P-type ATPases predict that aspartic acid 560 (D560) in Drs2p would form the aspartyl-phosphate intermediate (Fig. 9 A). To test whether this amino acid is critical for Drs2p function, we mutated D560 to either an asparagine (D560N) or a glutamic acid (D560E) residue and examined the ability of the mutants to complement the cold-sensitive growth defect of the drs2Δ strain. These mutations would be expected to cause minimal structural changes in Drs2p, but should abolish the presumed ATPase activity of this protein. The drs2Δ strain carrying either the wild-type DRS2 gene (Wild-type), an empty vector (drs2Δ), or two independent isolates of each mutant (D560N and D560E) were tested for growth at 30° and 20°C (Fig. 9 B). As previously reported, the drs2Δ strain failed to grow at 20°C, but grew well at 30°C. None of the strains carrying the D560 point mutations were able to grow at 20°C (Fig. 9 B), even though each of the strains expressed a wild-type level of Drs2p (data not shown). These data support the assignment of Drs2p as a P-type ATPase and suggest that the ATPase activity of Drs2p is essential for its function in vivo.


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)

Mutation of a conserved ATPase motif causes loss of Drs2p function in vivo. (A) Comparison of five conserved consensus motifs of yeast P-type ATPases and the corresponding sequence in Drs2p (adapted from Catty et al. 1997). The arrow indicates the aspartic acid residue (D) at position 560, which was mutated to glutamic acid (E) or asparagine (N). (B) The drs2Δ strain containing plasmids pDRS2(D560E), pDRS2(D560N), pRS315-DRS2 (wild-type), and the pRS315 empty vector (drs2Δ) were grown at 30° or 20°C for 3 d.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2168089&req=5

Figure 9: Mutation of a conserved ATPase motif causes loss of Drs2p function in vivo. (A) Comparison of five conserved consensus motifs of yeast P-type ATPases and the corresponding sequence in Drs2p (adapted from Catty et al. 1997). The arrow indicates the aspartic acid residue (D) at position 560, which was mutated to glutamic acid (E) or asparagine (N). (B) The drs2Δ strain containing plasmids pDRS2(D560E), pDRS2(D560N), pRS315-DRS2 (wild-type), and the pRS315 empty vector (drs2Δ) were grown at 30° or 20°C for 3 d.
Mentions: Drs2p is predicted to be a P-type ATPase based on the presence of five well-conserved ATPase motifs involved in the binding and hydrolysis of ATP (Fig. 9 A). For P-type ATPases, an aspartic acid within the second motif forms an aspartyl-phosphate catalytic intermediate that is essential for ATP hydrolysis (Allen and Green 1976; Fagan and Saier 1994). Sequence alignments between Drs2p and other P-type ATPases predict that aspartic acid 560 (D560) in Drs2p would form the aspartyl-phosphate intermediate (Fig. 9 A). To test whether this amino acid is critical for Drs2p function, we mutated D560 to either an asparagine (D560N) or a glutamic acid (D560E) residue and examined the ability of the mutants to complement the cold-sensitive growth defect of the drs2Δ strain. These mutations would be expected to cause minimal structural changes in Drs2p, but should abolish the presumed ATPase activity of this protein. The drs2Δ strain carrying either the wild-type DRS2 gene (Wild-type), an empty vector (drs2Δ), or two independent isolates of each mutant (D560N and D560E) were tested for growth at 30° and 20°C (Fig. 9 B). As previously reported, the drs2Δ strain failed to grow at 20°C, but grew well at 30°C. None of the strains carrying the D560 point mutations were able to grow at 20°C (Fig. 9 B), even though each of the strains expressed a wild-type level of Drs2p (data not shown). These data support the assignment of Drs2p as a P-type ATPase and suggest that the ATPase activity of Drs2p is essential for its function in vivo.

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