Smotrys Jessica E, Schoenfish Marissa J, Stutz Monica A, Linder Maurine E
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
J Cell Biol. 2005 Sep 26;170(7):1091-9. doi: 10.1083/jcb.200507048.
Palmitoylation of the vacuolar membrane protein Vac8p is essential for vacuole fusion in yeast (Veit, M., R. Laage, L. Dietrich, L. Wang, and C. Ungermann. 2001. EMBO J. 20:3145-3155; Wang, Y.X., E.J. Kauffman, J.E. Duex, and L.S. Weisman. 2001. J. Biol. Chem. 276:35133-35140). Proteins that contain an Asp-His-His-Cys (DHHC)-cysteine rich domain (CRD) are emerging as a family of protein acyltransferases, and are therefore candidates for mediators of Vac8p palmitoylation. Here we demonstrate that the DHHC-CRD proteins Pfa3p (protein fatty acyltransferase 3, encoded by YNL326c) and Swf1p are important for vacuole fusion. Cells lacking Pfa3p had fragmented vacuoles when stressed, and cells lacking both Pfa3p and Swf1p had fragmented vacuoles under normal growth conditions. Pfa3p promoted Vac8p membrane association and palmitoylation in vivo and partially purified Pfa3p palmitoylated Vac8p in vitro, establishing Vac8p as a substrate for palmitoylation by Pfa3p. Vac8p is the first N-myristoylated, palmitoylated protein identified as a substrate for a DHHC-CRD protein.
液泡膜蛋白Vac8p的棕榈酰化对于酵母中的液泡融合至关重要（Veit, M., R. Laage, L. Dietrich, L. Wang, and C. Ungermann. 2001. 《欧洲分子生物学组织杂志》20:3145 - 3155; Wang, Y.X., E.J. Kauffman, J.E. Duex, and L.S. Weisman. 2001. 《生物化学杂志》276:35133 - 35140）。含有天冬氨酸 - 组氨酸 - 组氨酸 - 半胱氨酸（DHHC） - 富含半胱氨酸结构域（CRD）的蛋白质正逐渐成为一类蛋白质酰基转移酶家族，因此是Vac8p棕榈酰化的介导因子候选者。在这里，我们证明了DHHC - CRD蛋白Pfa3p（由YNL326c编码的蛋白质脂肪酰基转移酶3）和Swf1p对液泡融合很重要。缺乏Pfa3p的细胞在应激时液泡会碎片化，而同时缺乏Pfa3p和Swf1p的细胞在正常生长条件下液泡就会碎片化。Pfa3p在体内促进Vac8p与膜的结合及棕榈酰化，并且部分纯化的Pfa3p在体外能使Vac8p发生棕榈酰化，这确定了Vac8p是Pfa3p进行棕榈酰化的底物。Vac8p是首个被鉴定为DHHC - CRD蛋白底物的N - 肉豆蔻酰化、棕榈酰化蛋白。
