title: Interaction of the phospholipid flippase Drs2p with the F-box protein Rcy1p plays an important role in early endosome to trans-Golgi network vesicle transport in yeast.
abstract: Phospholipid composition of biological membranes differs between the cytoplasmic and exoplasmic leaflets. The type 4 P-type ATPases are phospholipid flippases that generate such membrane phospholipid asymmetry. Drs2p, a flippase in budding yeast, is involved in the endocytic recycling pathway. Drs2p is implicated in clathrin-coated vesicle formation, but the underlying mechanisms are not clearly understood. Here we show that the carboxyl-terminal cytoplasmic region of Drs2p directly binds to Rcy1p, an F-box protein that is also required for endocytic recycling. The Drs2p-binding region was mapped to the amino acids 574-778 region of Rcy1p and a mutant Rcy1p lacking this region was defective in endocytic recycling of a v-SNARE Snc1p. We isolated Drs2p point mutants that reduced the interaction with Rcy1p. The mutation sites were clustered within a small region (a.a. 1260-1268) of Drs2p. Although these point mutants did not exhibit clear phenotypes, combination of them resulted in cold-sensitive growth, defects in endocytic recycling of Snc1p and defective localization of Rcy1p to endosomal membranes like the drs2 null mutant. These results suggest that the interaction of Drs2p with Rcy1p plays an important role for Drs2p function in the endocytic recycling pathway.
authors.affiliation: Division of Molecular Interaction, Institute for Genetic Medicine, Hokkaido University Graduate School of Life Science, N15 W7, Kita-ku, Sapporo 060-0815, Japan.
authors.forename: Hisatoshi
authors.initials: H
authors.lastname: Hanamatsu
authors.forename: Konomi
authors.initials: K
authors.lastname: Fujimura-Kamada
authors.forename: Takaharu
authors.initials: T
authors.lastname: Yamamoto
authors.forename: Nobumichi
authors.initials: N
authors.lastname: Furuta
authors.forename: Kazuma
authors.initials: K
authors.lastname: Tanaka
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers; D011992:Endosomes / Q000378:metabolism*; D044783:F-Box Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D017931:DNA Primers; C104014:DRS2 protein, S cerevisiae; D044783:F-Box Proteins; C407703:RCY1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D000252:Calcium-Transporting ATPases
keywords: P4-ATPases; endocytic recycling; flippases; phospholipid asymmetry; yeast