title: The Arf activator Gea2p and the P-type ATPase Drs2p interact at the Golgi in Saccharomyces cerevisiae.
abstract: Arf GTPases regulate both the morphological and protein sorting events that are essential for membrane trafficking. Guanine nucleotide exchange factors (GEFs) specific for Arf proteins determine when and where Arf GTPases will be activated in cells. The yeast Gea2p Arf GEF is a member of an evolutionarily conserved family of high molecular mass Arf GEFs that are peripherally associated with membranes. Nothing is known about how these proteins are localized to membranes, and few direct binding partners have been identified. In yeast, Gea2p has been implicated in trafficking through the Golgi apparatus and in maintaining Golgi structure. A major function of the Golgi apparatus is the packaging of cargo into secretory granules or vesicles. This process occurs through a series of membrane transformation events starting with fenestration of a saccular membrane, and subsequent remodeling of the fenestrated membrane into a mesh-like tubular network. Concentration of secretory cargo into nodes of the tubular network leads to enlargement of the nodes, which correspond to forming vesicles/granules, and thinning of the surrounding tubules. The tubules eventually break to release the secretory vesicles/granules into the cytoplasm. This process is highly conserved at the morphological level from yeast to mammalian cells. Drs2p, a multi-span transmembrane domain protein and putative aminophospholipid translocase, is required for the formation of a class of secretory granules/vesicles in yeast. Here we show that Drs2p interacts directly with Gea2p, both in vitro and in vivo. We mapped the domain of interaction of Drs2p to a 20-amino-acid region of the C-terminal cytoplasmic tail of the protein, adjacent to a region essential for Drs2p function. Mutations in Gea2p that abolish interaction with Drs2p are clustered in the C-terminal third of the Sec7 domain, and are important for Gea2p function. We characterize one such mutant that has a thermosensitive phenotype, and show that it has morphological defects along the secretory pathway in the formation of secretory granules/vesicles.
authors.affiliation: Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-5430, USA.
authors.forename: Sophie
authors.initials: S
authors.lastname: Chantalat
authors.forename: Sei-Kyoung
authors.initials: SK
authors.lastname: Park
authors.forename: Zhaolin
authors.initials: Z
authors.lastname: Hua
authors.forename: Ke
authors.initials: K
authors.lastname: Liu
authors.forename: Renée
authors.initials: R
authors.lastname: Gobin
authors.forename: Anne
authors.initials: A
authors.lastname: Peyroche
authors.forename: Alain
authors.initials: A
authors.lastname: Rambourg
authors.forename: Todd R
authors.initials: TR
authors.lastname: Graham
authors.forename: Catherine L
authors.initials: CL
authors.lastname: Jackson
mesh_terms: D000595:Amino Acid Sequence; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques
chemical_list: C104014:DRS2 protein, S cerevisiae; C121632:GEA2 protein, S cerevisiae; D020662:Guanine Nucleotide Exchange Factors; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D000252:Calcium-Transporting ATPases