title: Effects of mutations of ABCA1 in the first extracellular domain on subcellular trafficking and ATP binding/hydrolysis.
abstract: ABCA1 mediates release of cellular cholesterol and phospholipid to form high density lipoprotein (HDL). The three different mutants in the first extracellular domain of human ABCA1 associated with Tangier disease, R587W, W590S, and Q597R, were examined for their subcellular localization and function by using ABCA1-GFP fusion protein stably expressed in HEK293 cells. ABCA1-GFP expressed in HEK293 was fully functional for apoA-I-mediated HDL assembly. Immunostaining and confocal microscopic analyses demonstrated that ABCA1-GFP was mainly localized to the plasma membrane (PM) but also substantially in intracellular compartments. All three mutant ABCA1-GFPs showed no or little apoA-I-mediated HDL assembly. R587W and Q597R were associated with impaired processing of oligosaccharide from high mannose type to complex type and failed to be localized to the PM, whereas W590S did not show such dysfunctions. Vanadate-induced nucleotide trapping was examined to elucidate the mechanism for the dysfunction in the W590S mutant. Photoaffinity labeling of W590S with 8-azido-[alpha-(32)P]ATP was stimulated by adding ortho-vanadate in the presence of Mn(2+) as much as in the presence of wild-type ABCA1. These results suggest that the defect of HDL assembly in R587W and Q597R is due to the impaired localization to the PM, whereas W590S has a functional defect other than the initial ATP binding and hydrolysis.
authors.affiliation: Laboratory of Cellular Biochemistry, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan.
authors.forename: Arowu R
authors.initials: AR
authors.lastname: Tanaka
authors.forename: Sumiko
authors.initials: S
authors.lastname: Abe-Dohmae
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Ohnishi
authors.forename: Ryo
authors.initials: R
authors.lastname: Aoki
authors.forename: Gaku
authors.initials: G
authors.lastname: Morinaga
authors.forename: Kei-ichiro
authors.initials: K
authors.lastname: Okuhira
authors.forename: Yuika
authors.initials: Y
authors.lastname: Ikeda
authors.forename: Fumi
authors.initials: F
authors.lastname: Kano
authors.forename: Michinori
authors.initials: M
authors.lastname: Matsuo
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Kioka
authors.forename: Teruo
authors.initials: T
authors.lastname: Amachi
authors.forename: Masayuki
authors.initials: M
authors.lastname: Murata
authors.forename: Shinji
authors.initials: S
authors.lastname: Yokoyama
authors.forename: Kazumitsu
authors.initials: K
authors.lastname: Ueda
mesh_terms: D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D009154:Mutation; D011485:Protein Binding; D021381:Protein Transport; D013347:Subcellular Fractions / Q000378:metabolism*
chemical_list: C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters; D017931:DNA Primers; D000255:Adenosine Triphosphate