Department of Chemistry and the Purdue Center for Cancer Research, Purdue University, West Lafayette, Indiana 47907, USA.
J Biol Chem. 2010 Apr 30;285(18):13380-7. doi: 10.1074/jbc.M109.061366. Epub 2010 Mar 3.
The isoprenylcysteine carboxyl methyltransferase (Icmt) from Saccharomyces cerevisiae, also designated Ste14p, is a 26-kDa integral membrane protein that contains six transmembrane spanning segments. This protein is localized to the endoplasmic reticulum membrane where it performs the methylation step of the CAAX post-translational processing pathway. Sequence analysis reveals a putative GXXXG dimerization motif located in transmembrane 1 of Ste14p, but it is not known whether Ste14p forms or functions as a dimer or higher order oligomer. We determined that Ste14p predominantly formed a homodimer in the presence of the cross-linking agent, bis-sulfosuccinimidyl suberate. Wild-type untagged Ste14p also co-immunoprecipitated and co-purified with N-terminal-tagged His(10)-myc(3)-Ste14p (His-Ste14p). Furthermore, enzymatically inactive His-Ste14p variants L81F and E213Q both exerted a dominant-negative effect on methyltransferase activity when co-expressed and co-purified with untagged wild-type Ste14p. Together, these data, although indirect, suggest that Ste14p forms and functions as a homodimer or perhaps a higher oligomeric species.
酿酒酵母的异戊烯基半胱氨酸羧基甲基转移酶（Icmt），也称为 Ste14p，是一种 26kDa 的完整膜蛋白，含有六个跨膜区段。该蛋白定位于内质网膜上，在那里执行 CAAX 翻译后加工途径的甲基化步骤。序列分析揭示了 Ste14p 跨膜 1 中存在一个假定的 GXXXG 二聚化基序，但尚不清楚 Ste14p 是否形成二聚体或更高阶寡聚体，以及是否发挥功能。我们确定 Ste14p 在交联剂双琥珀酰亚胺辛二酸酯存在下主要形成同源二聚体。野生型未标记的 Ste14p 还与 N 端标记的 His(10)-myc(3)-Ste14p（His-Ste14p）共免疫沉淀和共纯化。此外，当与未标记的野生型 Ste14p 共表达和共纯化时，酶失活的 His-Ste14p 变体 L81F 和 E213Q 均对甲基转移酶活性表现出显性负效应。这些数据虽然间接，但表明 Ste14p 形成并发挥功能作为同源二聚体或更高阶寡聚体。
