title: Identification of a second amphipathic lipid-packing sensor-like motif that contributes to Gcs1p function in the early endosome-to-TGN pathway.
abstract: ArfGAPs, GTPase-activating proteins for Arf small GTPases, are involved in multiple steps of vesicle formation of various transport pathways. Amphipathic lipid-packing sensor (ALPS) motif was first identified in the C-terminal regions of ArfGAP1 and its yeast homologue Gcs1p as a region that adsorbs preferentially onto highly curved membranes by folding into an amphipathic α-helix (AH). We previously showed that Gcs1p functionally interacted with the phospholipid flippase Cdc50p-Drs2p in the early endosome-to-TGN retrieval pathway. In this study, we performed functional analyses of the C-terminal region of Gcs1p containing ALPS. Hydrophobic cluster analysis suggested that there is another potential AH-forming region downstream of ALPS in Gcs1p. Mutational analysis suggested that the ALPS motif is important for the Gcs1p function in the Golgi-to-ER retrograde pathway, whereas ALPS and the predicted AH region redundantly function in the post-Golgi pathways including the early endosome-to-TGN pathway. Liposome flotation assay indicated that this downstream region preferentially interacted with liposomes of smaller size. The region containing the ALPS motif was also required for the interaction with SNARE proteins including Snc1p and Tlg1p. These results suggest that ALPS and the predicted AH region are involved in the regulation and function of Gcs1p by interacting with membrane phospholipids and vesicle proteins.
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: Zahra
authors.initials: Z
authors.lastname: Zendeh-boodi
authors.forename: Takaharu
authors.initials: T
authors.lastname: Yamamoto
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sakane
authors.forename: Kazuma
authors.initials: K
authors.lastname: Tanaka
mesh_terms: D020816:Amino Acid Motifs; D002462:Cell Membrane / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D008565:Membrane Proteins / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D022161:Transport Vesicles / Q000378:metabolism; D021601:trans-Golgi Network / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; C089147:GCS1 protein, S cerevisiae; D020690:GTPase-Activating Proteins; D008565:Membrane Proteins; D010743:Phospholipids; D029701:Saccharomyces cerevisiae Proteins
keywords: ALPS motif; Arf GAP; endocytic recycling; flippase; yeast