Ali N, Duden R, Bembenek M E, Shears S B
Inositol Lipid Section, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
Biochem J. 1995 Aug 15;310 ( Pt 1)(Pt 1):279-84. doi: 10.1042/bj3100279.
Coatomer is an oligomeric complex of coat proteins that regulates vesicular traffic through the Golgi complex and from the Golgi to the endoplasmic reticulum [Pelham (1994) Cell 79, 1125-1127]. We have investigated whether the binding of InsP6 to mammalian coatomer [Fleischer, Xie, Mayrleitner, Shears and Fleischer (1994) J. Biol. Chem. 269, 17826-17832] is conserved in the genetically amenable model Saccharomyces cerevisiae. We have isolated coatomer from S. cerevisiae and found it to bind InsP6 at two apparent classes of binding sites (KD1 = 0.8 +/- 0.2 nM; KD2 = 361 +/- 102 nM). Ligand specificity was studied by displacing 4.5 nM [3H]InsP6 from coatomer with various Ins derivatives. The following IC50 values (nM) were obtained: myo-InsP6 = 6; bis(diphospho)inositol tetrakisphosphate = 6; diphosphoinositol pentakisphosphate = 6; scyllo-InsP6 = 12; Ins(1,3,4,5,6)P5 = 13; Ins(1,2,4,5,6)P5 = 22; Ins(1,3,4,5)P4 = 22; 1-O-(1,2-di-O-octanoyl-sn-glycero-3-phospho)-D-Ins(3,4,5)P3 = 290. Less than 10% of the 3H label was displaced by 1 microM of either Ins(1,4,5)P3 or inositol hexakis-sulphate. A cell-free lysate of S. cerevisiae synthesized diphosphoinositol polyphosphates (PP-InsPn) from InsP6, but our binding data, plus measurements of the relative levels of inositol polyphosphates in intact yeast [Hawkins, Stephens and Piggott (1993) J. Biol. Chem. 268, 3374-3383], indicate that InsP6 is the major physiologically relevant ligand. Thus a reconstituted vesicle trafficking system using coatomer and other functionally related components isolated from yeast should be a useful model for elucidating the functional significance of the binding of InsP6 by coatomer.
包被蛋白复合体是一种由包被蛋白组成的寡聚复合物，它调控着通过高尔基体复合体以及从高尔基体到内质网的囊泡运输[佩勒姆（1994年）《细胞》79卷，1125 - 1127页]。我们研究了肌醇六磷酸（InsP6）与哺乳动物包被蛋白复合体[弗莱施尔、谢、迈尔莱特纳、希尔斯和弗莱施尔（1994年）《生物化学杂志》269卷，17826 - 17832页]的结合在遗传上易于操作的酿酒酵母模型中是否保守。我们从酿酒酵母中分离出了包被蛋白复合体，发现它在两类明显的结合位点上结合InsP6（解离常数KD1 = 0.8 ± 0.2纳摩尔；KD2 = 361 ± 102纳摩尔）。通过用各种肌醇衍生物将4.5纳摩尔[3H]InsP6从包被蛋白复合体上置换下来，研究了配体特异性。得到了以下半数抑制浓度（IC50）值（纳摩尔）：肌醇六磷酸 = 6；双（二磷酸）肌醇四磷酸 = 6；二磷酸肌醇五磷酸 = 6；异肌醇六磷酸 = 12；肌醇（1,3,4,5,6）五磷酸 = 13；肌醇（1,2,4,5,6）五磷酸 = 22；肌醇（1,3,4,5）四磷酸 = 22；1 - O -（1,2 - 二 - O - 辛酰基 - sn - 甘油 - 3 - 磷酸） - D - 肌醇（3,4,5）三磷酸 = 290。1微摩尔的肌醇（1,4,5）三磷酸或肌醇六硫酸酯置换的3H标记不到10%。酿酒酵母的无细胞裂解物从InsP6合成二磷酸肌醇多磷酸（PP - InsPn），但我们的结合数据，加上对完整酵母中肌醇多磷酸相对水平的测量[霍金斯、斯蒂芬斯和皮戈特（1993年）《生物化学杂志》268卷，3374 - 3383页]，表明InsP6是主要的生理相关配体。因此，使用从酵母中分离出的包被蛋白复合体和其他功能相关成分构建的重组囊泡运输系统，应该是阐明包被蛋白复合体结合InsP6功能意义的有用模型。
