Klotz A, Rutberg M, Denoulet P, Wallin M
Department of Zoophysiology, Göteborg University, Göteborg, Sweden.
Cell Motil Cytoskeleton. 1999 Dec;44(4):263-73. doi: 10.1002/(SICI)1097-0169(199912)44:4<263::AID-CM4>3.0.CO;2-V.
In higher organisms, there is a large variety of tubulin isoforms, due to multiple tubulin genes and extensive post-translational modification. The properties of microtubules may be modulated by their tubulin isoform composition. Polyglutamylation is a post-translational modification that is thought to influence binding of both structural microtubule associated proteins (MAPs) and mechano-chemical motors to tubulin. The present study investigates the role of tubulin polyglutamylation in a vesicle transporting system, cod (Gadus morhua) melanophores. We did this by microinjecting an antibody against polyglutamylated tubulin into these cells. To put our results into perspective, and to be able to judge their universal application, we characterized cod tubulin polyglutamylation by Western blotting technique, and compared it to what is known from mammals. We found high levels of polyglutamylation in tissues and cell types whose functions are highly dependent on interactions between microtubules and motor proteins. Microinjection of the anti-polyglutamylation antibody GT335 into cultured melanophores interfered with pigment granule dispersion, while dynein-dependent aggregation was unaffected. Additional experiments showed that GT335-injected cells were able to aggregate pigment even when actin filaments were depolymerized, indicating that the maintained ability of pigment aggregation in these cells was indeed microtubule-based and did not depend upon actin filaments. The results indicate that dynein and the kinesin-like dispersing motor protein in cod melanophores bind to tubulin on slightly different sites, and perhaps depend differentially on polyglutamylation for their interaction with microtubules. The binding site of the dispersing motor may bind directly to the polyglutamate chain, or more closely than dynein.
在高等生物中，由于存在多个微管蛋白基因以及广泛的翻译后修饰，因而有多种微管蛋白亚型。微管的性质可能会受到其微管蛋白亚型组成的调节。多聚谷氨酰胺化是一种翻译后修饰，被认为会影响结构微管相关蛋白（MAPs）和机械化学马达蛋白与微管蛋白的结合。本研究调查了微管蛋白多聚谷氨酰胺化在囊泡运输系统——鳕鱼（Gadus morhua）黑素细胞中的作用。我们通过向这些细胞显微注射抗多聚谷氨酰胺化微管蛋白抗体来进行此项研究。为了正确看待我们的结果，并能够判断其普遍适用性，我们通过蛋白质免疫印迹技术对鳕鱼微管蛋白多聚谷氨酰胺化进行了表征，并将其与哺乳动物的已知情况进行了比较。我们发现在功能高度依赖于微管与马达蛋白相互作用的组织和细胞类型中，多聚谷氨酰胺化水平很高。向培养的黑素细胞显微注射抗多聚谷氨酰胺化抗体GT335会干扰色素颗粒的分散，而动力蛋白依赖性聚集则不受影响。额外的实验表明，即使肌动蛋白丝解聚，注射了GT335的细胞仍能够聚集色素，这表明这些细胞中色素聚集能力的维持确实基于微管，且不依赖于肌动蛋白丝。结果表明，鳕鱼黑素细胞中的动力蛋白和类驱动蛋白分散马达蛋白与微管蛋白的结合位点略有不同，并且它们与微管相互作用时对多聚谷氨酰胺化的依赖性可能也不同。分散马达蛋白的结合位点可能直接与多聚谷氨酸链结合，或者比动力蛋白结合得更紧密。
