Schmitz Stephan, Grainger Munira, Howell Steven, Calder Lesley J, Gaeb Martina, Pinder Jennifer C, Holder Anthony A, Veigel Claudia
Division of Physical Biochemistry, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
J Mol Biol. 2005 May 27;349(1):113-25. doi: 10.1016/j.jmb.2005.03.056. Epub 2005 Apr 7.
A novel form of actomyosin regulation has recently been proposed in which the polymerisation of new actin filaments regulates apicomplexan parasite motility. Here, we identified actin I in the merozoites of Plasmodium falciparum by mass spectrometry. The only post-translational modification is acetylation of the N terminus (acetyl-Gly-Glu-actin), while methylation of histidine 73, a common modification for actin, is absent. Results obtained with anti-actin antibodies suggest that, in contrast to a previous report, there is no actin-ubiquitin conjugate in merozoites. About half of the extracted monomeric actin polymerised and actin filaments could be sedimented at 500,000g. In contrast, centrifugation at 100,000g, conditions commonly used to sediment filamentous actin, yielded very little F-actin. In a functional characterisation using an in vitro motility assay, actin filaments moved over myosin at a velocity indistinguishable from that of rabbit skeletal actin. Filament length, however, was too short to be resolved by conventional fluorescence microscopy. On electron micrographs an average filament length of approximately 100nm was determined. We also identified by mass spectrometry proteins co-purifying with filamentous actin, which are potential actin-binding proteins. Our results demonstrate differences in actin filament dynamics for an apicomplexan parasite, which could be due to specific properties of the actin and/or actin-regulatory proteins.
最近有人提出了一种新的肌动球蛋白调节形式，即新的肌动蛋白丝的聚合调节顶复门寄生虫的运动。在这里，我们通过质谱法在恶性疟原虫的裂殖子中鉴定出肌动蛋白I。唯一的翻译后修饰是N端乙酰化（乙酰基-甘氨酸-谷氨酸-肌动蛋白），而肌动蛋白常见的组氨酸73甲基化不存在。用抗肌动蛋白抗体获得的结果表明，与之前的一份报告相反，裂殖子中不存在肌动蛋白-泛素共轭物。大约一半提取的单体肌动蛋白发生聚合，并且肌动蛋白丝在500,000g的离心力下可以沉淀。相比之下，在通常用于沉淀丝状肌动蛋白的100,000g离心条件下，产生的F-肌动蛋白非常少。在使用体外运动测定法进行的功能表征中，肌动蛋白丝在肌球蛋白上移动的速度与兔骨骼肌肌动蛋白的速度无法区分。然而，丝的长度太短，无法通过传统荧光显微镜分辨。在电子显微镜照片上，确定平均丝长度约为100nm。我们还通过质谱法鉴定了与丝状肌动蛋白共纯化的蛋白质，这些蛋白质是潜在的肌动蛋白结合蛋白。我们的结果表明顶复门寄生虫的肌动蛋白丝动力学存在差异，这可能是由于肌动蛋白和/或肌动蛋白调节蛋白的特定特性所致。
