Jacot Damien, Frénal Karine, Marq Jean-Baptiste, Sharma Pushkar, Soldati-Favre Dominique
Department of Microbiology & Molecular Medicine, CMU/University of Geneva, Rue Michel-Servet 1, CH-1211, Geneva, 4, Switzerland.
Cell Microbiol. 2014 Oct;16(10):1518-32. doi: 10.1111/cmi.12307. Epub 2014 May 23.
Members of the phylum Apicomplexa possess a highly conserved molecular motor complex anchored in the parasite pellicle and associated with gliding motility, invasion and egress from infected cells. This machinery, called the glideosome, is structured around the acylated gliding-associated protein GAP45 that recruits the motor complex composed of myosin A and two associated myosin light chains (TgMLC1 and TgELC1). This motor is presumably firmly anchored to the inner membrane complex underneath the plasma membrane via an interaction with two integral membrane proteins, GAP50 and GAP40. To determine if the previously mapped phosphorylation sites on TgGAP45 and TgMLC1 have a direct significance for glideosome assembly and function, a series of phospho-mimetic and phospho-null mutants were generated. Neither the overexpression nor the allelic replacement of TgMLC1 with phospho-mutants impacted on glideosome assembly and parasite motility. TgGAP45 phosphorylation mutants were functionally investigated using a complementation strategy in a TgGAP45 inducible knockout background. The loss of interaction with TgGAP50 by one previously reported GAP45-mutant appeared to depend only on the presence of a remaining competing wild type copy of TgGAP45. Accordingly, this mutant displayed no phenotype in complementation experiments. Unexpectedly, GAP45 lacking the region encompassing the cluster of twelve phosphorylation sites did not impact on its dual function in motor recruitment and pellicle integrity. Despite the extensive phosphorylation of TgMLC1 and TgGAP45, this post-translational modification does not appear to be critical for the assembly and function of the glideosome.
顶复门的成员拥有一种高度保守的分子运动复合体，该复合体锚定在寄生虫表膜中，并与滑行运动、侵入和从受感染细胞中逸出有关。这种机制称为滑行体，其结构围绕酰化的滑行相关蛋白GAP45构建，GAP45招募由肌球蛋白A和两条相关的肌球蛋白轻链（TgMLC1和TgELC1）组成的运动复合体。该运动复合体可能通过与两种整合膜蛋白GAP50和GAP40的相互作用，牢固地锚定在质膜下方的内膜复合体上。为了确定先前在TgGAP45和TgMLC1上定位的磷酸化位点是否对滑行体的组装和功能具有直接意义，构建了一系列磷酸模拟和磷酸缺失突变体。用磷酸化突变体过表达或等位基因替换TgMLC1均不影响滑行体的组装和寄生虫运动。在TgGAP45诱导敲除背景下，使用互补策略对TgGAP45磷酸化突变体进行了功能研究。一个先前报道的GAP45突变体与TgGAP50相互作用的丧失似乎仅取决于剩余的竞争性野生型TgGAP45拷贝的存在。因此，该突变体在互补实验中未表现出表型。出乎意料的是，缺少包含十二个磷酸化位点簇区域的GAP45对其在运动复合体招募和表膜完整性方面的双重功能没有影响。尽管TgMLC1和TgGAP45存在广泛的磷酸化，但这种翻译后修饰似乎对滑行体的组装和功能并不关键。
