Hettmann C, Herm A, Geiter A, Frank B, Schwarz E, Soldati T, Soldati D
Zentrum für Molekulare Biologie, Universität Heidelberg, D-69120 Heidelberg, Germany.
Mol Biol Cell. 2000 Apr;11(4):1385-400. doi: 10.1091/mbc.11.4.1385.
Obligate intracellular parasites of the phylum Apicomplexa exhibit gliding motility, a unique form of substrate-dependent locomotion essential for host cell invasion and shown to involve the parasite actin cytoskeleton and myosin motor(s). Toxoplasma gondii has been shown to express three class XIV myosins, TgM-A, -B, and -C. We identified an additional such myosin, TgM-D, and completed the sequences of a related Plasmodium falciparum myosin, PfM-A. Despite divergent structural features, TgM-A purified from parasites bound actin in an ATP-dependent manner. Isoform-specific antibodies revealed that TgM-A and recombinant mycTgM-A were localized right beneath the plasma membrane, and subcellular fractionation indicated a tight membrane association. Recombinant TgM-D also had a peripheral although not as sharply defined localization. Truncation of their respective tail domains abolished peripheral localization and tight membrane association. Conversely, fusion of the tails to green fluorescent protein (GFP) was sufficient to confer plasma membrane localization and sedimentability. The peripheral localization of TgM-A and of the GFP-tail fusion did not depend on an intact F-actin cytoskeleton, and the GFP chimera did not localize to the plasma membrane of HeLa cells. Finally, we showed that the specific localization determinants were in the very C terminus of the TgM-A tail, and site-directed mutagenesis revealed two essential arginine residues. We discuss the evidence for a proteinaceous plasma membrane receptor and the implications for the invasion process.
顶复门专性细胞内寄生虫表现出滑行运动，这是一种独特的依赖底物的运动形式，对宿主细胞入侵至关重要，且已证明其涉及寄生虫肌动蛋白细胞骨架和肌球蛋白马达。已证明弓形虫表达三种XIV类肌球蛋白，即TgM-A、-B和-C。我们鉴定出另一种此类肌球蛋白TgM-D，并完成了相关恶性疟原虫肌球蛋白PfM-A的序列测定。尽管结构特征不同，但从寄生虫中纯化的TgM-A以ATP依赖的方式结合肌动蛋白。同工型特异性抗体显示，TgM-A和重组mycTgM-A定位于质膜正下方，亚细胞分级分离表明其与膜紧密结合。重组TgM-D也定位于外周，尽管定位不如前者明确。截断它们各自的尾部结构域消除了外周定位和紧密的膜结合。相反，将尾部与绿色荧光蛋白（GFP）融合足以赋予质膜定位和沉降能力。TgM-A和GFP-尾部融合蛋白的外周定位不依赖于完整的F-肌动蛋白细胞骨架，且GFP嵌合体不定位于HeLa细胞质膜。最后，我们表明特异性定位决定因素位于TgM-A尾部的最末端，定点诱变揭示了两个必需的精氨酸残基。我们讨论了蛋白质质膜受体的证据及其对入侵过程的影响。
