Drs2p-Dependent Formation of Exocytic Clathrin-Coated Vesicles In Vivo

Abstract

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.

P4-ATPase Requirement for AP-1/Clathrin Function in Protein Transport from the trans-Golgi Network and Early Endosomes

“…These post-Golgi vesicles appear to be equivalent to one of the two classes of exocytic vesicles (the "higher density" class) first described by Harsay and Bretscher (1995). Formation of these exocytic vesicles is only mildly perturbed by deletion of AP-1 subunits (Gall et al, 2002); therefore, they likely differ from AP-1/ CCVs proposed to mediate transport between the TGN and endosomes. Other data linking Drs2p to clathrin function include their colocalization to the TGN, a strong synthetic lethal relationship between drs2, arf1, and chc1 (clathrin heavy chain) alleles, and drs2 mutant phenotypes such as the accumulation of enlarged Golgi cisternae, a deficiency of CCVs, and the mislocalization of TGN resident proteins.…”

“…In addition to the AP-1 and Rcy1 pathways, we previously described a role for Drs2p and clathrin in the formation of one class of post-Golgi exocytic vesicles (Gall et al, 2002). AP-1 mutations partially perturb formation of these exocytic vesicles, although it was not clear at the time whether the small defect observed reflected a primary or secondary consequence of AP-1 disruption.…”

“…The "lighter density" class of exocytic vesicles appears to form normally in drs2⌬ cells, and cargos normally secreted in the dense vesicles, such as invertase, get rerouted into the light exocytic vesicles (Chen et al, 1999;Gall et al, 2002;Hua et al, 2002). In fact, the rate of protein transport from the ER to the Golgi and subsequently to the cell surface in drs2⌬ or drs2⌬ dnf1⌬, or dnf1,2,3⌬ cells is similar to that of WT cells (Chen et al, 1999;Gall et al, 2002;Hua et al, 2002). These data also indicate that COPI and COPII function in the early secretory pathway is unperturbed by loss of Drs2 and Dnf P4-ATPases.…”

“…The proposed phospholipid flippase activity for P4-ATPases is best supported by studies showing a temperature-conditional loss of flippase activity in purified TGN membranes carrying a temperature conditional form of Drs2p (Drs2-ts; Natarajan et al, 2004). Inactivation of Drs2-ts in vivo causes an immediate defect in the budding of dense exocytic vesicles (Gall et al, 2002), and rerouting of Drs2-ts to the plasma membrane, implying a defect in budding AP-1/CCVs from the TGN (this work). Therefore, Drs2p flippase activity appears to drive formation of vesicles from the TGN.…”

“…P4-ATPases also play essential roles in protein transport in the secretory and endocytic pathways (Chen et al, 1999;Hua et al, 2002;Hua and Graham, 2003;Pomorski et al, 2003;Sakane et al, 2006;Furuta et al, 2007). For example, inactivation of Drs2-ts in vivo rapidly blocks formation of a clathrin-dependent class of post-Golgi vesicles carrying exocytic cargo (Gall et al, 2002). These post-Golgi vesicles appear to be equivalent to one of the two classes of exocytic vesicles (the "higher density" class) first described by Harsay and Bretscher (1995).…”