EXPRESSION OF MUTANT VERTEBRATE MYOSIN I'S

肌球蛋白I在不同的组织和广泛的组织中广泛表达 系统发育背景。在肠上皮刷状缘，肌球蛋白 I定位于微绒毛中，在那里它连接了 膜和肌动蛋白束。肌球蛋白I也定位于 使迁移细胞的边缘活跃地起皱。我们使用的是 杆状病毒/Sf9系统表达全长鸡刷缘肌球蛋白 I重链(BBMI HC)和钙调蛋白(CaM)，以及一个截头 碎片。脊椎动物的肌球蛋白I具有结构性活性，而肌球蛋白 I来自低等真核生物，如棘阿米巴和网柄金龟子 位于肌球蛋白头部结构域的丝氨酸的磷酸化 活动。脊椎动物与棘阿米巴肌球蛋白I‘s的序列比对 揭示脊椎动物的蛋白质含有带负电荷的氨基酸 在棘阿米巴蛋白具有可磷酸化的位置 丝氨酸。我们打算探索，使用定点突变和 杆状病毒/Sf9系统，该部位的负电荷是否 对肌动蛋白激活的镁ATPase活性和体外运动是必不可少的。

Myosin I's are widely expressed in different tissues and across broad phylogenetic backgrounds. In intestinal epithelial brush borders, myosin I is localized in the microvillus where it bridges gaps between the membrane and the actin bundles. Myosin I is also localized near the actively ruffling edges of migrating cells. We are using the Baculovirus/Sf9 system to express full-length chicken brush border myosin I heavy chain (BBMI HC) along with calmodulin (CaM), and a truncated head fragment. Vertebrate myosin I's are constitutively active whereas myosin I's from low eukaryotes such as Acanthamoeba and Dictyostelium require phosphorylation at a serine located in the myosin head domain for activity. Sequence alignments of vertebrate and Acanthamoeba myosin I's reveal that the vertebrate proteins have a negatively charged amino acid at the position where the Acanthamoeba protein has the phosphorylatable serine. We intend to explore, using site-directed mutagenesis and the Baculovirus/Sf9 system, whether a negative charge at this site is essential for actin-activated MgATPase activity and in vitro motility.