== Plasmids used in this study aLow-copy plasmid. bBracketed numbers indicate encoded amino acids; see Experimental Procedures for details. cHigh-copy plasmid. == Microscopy == Staining with the lipophilic dye FM 4-64 was performed as described (14). also allows cell growth in the absence of the Age2 + Gcs1 pair, but good growth in this situation requires Age1. The ability of Age1 to overcome the need for Age2 + Gcs1 depends on phospholipase D activity that regulates lipid composition. We show by direct assessment of Age1 ArfGAP activity that Age1 is regulated by lipid composition and can provide ArfGAP function for post-Golgi transport. Keywords:Endosomes, G Proteins, Golgi, Membrane Trafficking, Yeast, Arf GTPase-activating Protein, Arf1, Diacylglycerol, Phosphatidylinositol Transfer Protein, Post-Golgi Transport == Introduction == Eukaryotic cells move protein and membrane cargo between the plasma membrane and various organelles by a process termed vesicular transport. Each step in the vesicular-transport process, including vesicle generation, cargo packaging, vesicle targeting, and vesicle fusion, is controlled by a variety of proteins (for review, see Ref.1). Regulated vesicular transport not only ensures the fidelity of cargo delivery but also maintains the structural and functional integrity of membrane organelles. Each stage of vesicular transport relies on a specific set of proteins to form the transport vesicle and direct the vesicle to the target compartment. For example, vesicles involved in retrograde transport from the Golgi to the endoplasmic reticulum require coat protein complex I (COPI) Rabbit Polyclonal to EFNB3 and the small GTPase Arf1 (2,3), whereas transport vesicles originating from thetrans-Golgi network also require the Arf1 GTPase but use the clathrin coat complex (4,5). Arf1 regulates recruitment of coat protein complex I and clathrin coat complexes to the donor membrane, where these coat complexes mediate the generation of transport vesicles by deforming the membrane. Arf regulatory activity depends on a cycle GSK163090 of GTP binding and hydrolysis, which is in turn regulated by two types of proteins, Arf guanine-nucleotide exchange factors and Arf GTPase-activating proteins (ArfGAPs).6Arf guanine-nucleotide exchange factors mediate the exchange of GDP for GTP on Arf to activate Arf and allow coat recruitment (6), whereas ArfGAPs stimulate hydrolysis of Arf-bound GTP to inactivate Arf and allow release of coat proteins from transport vesicles in preparation for fusion of the transport vesicle with a target membrane (7). The budding yeastSaccharomyces cerevisiaecontains ArfGAP proteins that are characterized by a zinc-binding motif (CXXCX16CXXC, where C is cysteine andXis GSK163090 any amino acid) and a nearby invariant arginine residue, which together constitute a catalytic center known as the ArfGAP domain (4). Four yeast proteins, Gcs1, Glo3, Age2, and Age1, have been shown to exert ArfGAP activityin vitro(811), with the Age2 + Gcs1 ArfGAP pair providing essential overlapping function for post-Golgi transport (10). Vesicular transport is also mediated by membrane lipid composition through the effects of lipid-protein interactions on protein function. The membrane lipid phosphatidylinositol is phosphorylated on the inositol ring to produce GSK163090 distinct phospholipid species that, like Arf, are involved in protein recruitment to membranes for vesicle formation. One family of yeast proteins that affects membrane lipid composition is defined by the Sec14 phosphatidylinositol transfer protein that is involved in vesicle formation for post-Golgi transport, presumably by creating a lipid composition permissive for vesicle formation. Indeed, we and others have shown that ArfGAP activity is modulated by lipid environment (12,13) and that increased abundance of the Sec14 homologue GSK163090 Sfh2 enhances Gcs1 activity for post-Golgi transport (14). We have investigated the function of the Age2 + Gcs1 pair for post-Golgi transport by identifying yeast genes that, in increased dosage, alleviate the deleterious effects of impaired ArfGAP function. TheAGE1gene is shown here to alleviate defects resulting from deficient ArfGAP function for post-Golgi transport. TheAGE1gene was initially described as a suppressor of Arf1 temperature sensitivity (SAT1) (15) and was later renamed as encoding an ArfGAP with effector functions (AGE1) (11). Those studies implicated the Age1 protein in Arf1 function because increasedAGE1gene dosage alleviates the temperature sensitivity caused by thearf1-3mutant allele (15). A more direct role for Age1 in Arf1 function was implied by the finding that the Age1 protein has ArfGAP activityin vitro(11). We show here that deletion of theAGE1gene compromises the ability of increasedSFH2gene dosage to alleviate post-Golgi transport defects in cells lacking the Age2 + Gcs1 ArfGAP pair. Moreover, we find that the ability ofAGE1to effectively alleviate post-Golgi transport problems depends upon phospholipase D, an effector of an Sfh2-mediated phosphoinositide metabolic pathway (16). In addition, we display that thein vitroArfGAP activity of Age1 is definitely stimulated by diacylglycerol (DAG), a downstream product of phospholipase D activity. Therefore, our analysis shows the importance of phospholipid rate of metabolism for ArfGAP activity in post-Golgi vesicular transport. == EXPERIMENTAL Methods == == == == == == Candida Strains and Plasmids == Candida strains used in this study are explained inTable 1. Candida.