Kabcenell A K, Goud B, Northup J K, Novick P J
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510.
J Biol Chem. 1990 Jun 5;265(16):9366-72.
The 23.5-kDa Sec4 protein is required for vesicular transport between the Golgi apparatus and the plasma membrane in Saccharomyces cerevisiae. In order to analyze its biochemical properties, we have purified the soluble pool of the wild-type protein from an overproducing yeast strain. At 30 degrees C, Sec4p bound [35S] guanosine 5'-O-(thiotriphosphate) (GTP gamma S) with a rate of 0.18 min-1 in a reaction requiring micromolar concentration of free magnesium ions. The protein had high affinity for guanine nucleotides with Kd values for GTP gamma S and GTP of 3.7 nM and 3.5 nM, respectively, and that for GDP of 77 nM. The dissociation of [3H] GDP from Sec4p occurred with a rate of 0.21 min-1 suggesting that the association of GTP gamma S was the result of exchange for prebound GDP. The release of GTP from Sec4p was slow and correlated with a low inherent GTPase activity of 0.0012 min-1. By analogy with other classes of GTP binding proteins, both the nucleotide exchange and hydrolysis activities of Sec4p may be modulated in vivo to facilitate its role in the regulation of intercompartmental membrane traffic.
23.5-kDa的Sec4蛋白是酿酒酵母中高尔基体与质膜之间囊泡运输所必需的。为了分析其生化特性，我们从一个过量表达的酵母菌株中纯化了野生型蛋白的可溶性部分。在30摄氏度下，Sec4p在需要微摩尔浓度游离镁离子的反应中以0.18 min-1的速率结合[35S]鸟苷5'-O-(硫代三磷酸)(GTPγS)。该蛋白对鸟嘌呤核苷酸具有高亲和力，GTPγS和GTP的Kd值分别为3.7 nM和3.5 nM，GDP的Kd值为77 nM。[3H]GDP从Sec4p上的解离速率为0.21 min-1，这表明GTPγS的结合是预结合GDP交换的结果。GTP从Sec4p上的释放很慢，且与0.0012 min-1的低固有GTPase活性相关。与其他类别的GTP结合蛋白类似，Sec4p的核苷酸交换和水解活性在体内可能受到调节，以促进其在调节隔室间膜运输中的作用。
