Drs2p-dependent formation of exocytic clathrin-coated vesicles in vivo.
The small GTP binding protein ARF has been implicated in budding clathrin-coated vesicles (CCVs) from Golgi and endosomal membranes. An arf1 synthetic lethal screen identified DRS2/SWA3 along with a clathrin heavy-chain conditional allele (chc1-5/swa5-1) and SWA2, encoding the yeast auxilin-like protein involved in uncoating CCVs. Drs2p/Swa3p is a P-type ATPase and a potential aminophospholipid translocase that localizes to the trans-Golgi network (TGN) in yeast. Genetic and phenotypic analyses of drs2Delta mutants suggested that Drs2p was required for clathrin function. To address a potential role for Drs2p in CCV formation from the TGN in vivo, we have performed epistasis analyses between drs2 and mutations that cause accumulation of distinct populations of post-Golgi vesicles. We find that Drs2p is required to form a specific class of secretory vesicles that accumulate when the actin cytoskeleton is disrupted. Accumulation of these vesicles also requires clathrin and is perturbed by mutation of AP-1, but not AP-2, AP-3, or GGA adaptins. Most of the accumulated vesicles are uncoated; however, clathrin coats can be partially stabilized on these vesicles by deletion of SWA2. These data provide the first in vivo evidence for an integral membrane protein requirement in forming CCVs.
Gall
Walter E
WE
Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235-1634, USA.
Geething
Nathan C
NC
Hua
Zhaolin
Z
Ingram
Michael F
MF
Liu
Ke
K
Chen
Sophie I
SI
Graham
Todd R
TR
D033961:Adaptor Protein Complex 1 / Q000235:genetics / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D033922:Clathrin Heavy Chains / Q000235:genetics / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D005089:Exocytosis; D017353:Gene Deletion; D005800:Genes, Fungal; D008854:Microscopy, Electron; D009154:Mutation; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D033921:Vesicular Transport Proteins
D033961:Adaptor Protein Complex 1; D002352:Carrier Proteins; C104014:DRS2 protein, S cerevisiae; D010750:Phosphoproteins; C419865:SWA2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D033922:Clathrin Heavy Chains; D000252:Calcium-Transporting ATPases