A role for regulated binding of p150(Glued) to microtubule plus ends in organelle transport.
A role for regulated binding of p150(Glued) to microtubule plus ends in organelle transport.
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DOI：
10.1083/jcb.200201029
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发表时间：
2002-07-22
影响因子：
7.8
通讯作者：
Vaughan, Kevin T
中科院分区：
文献类型：
--
作者：
Vaughan, Patricia S;Miura, Pedro;Henderson, Matthew;Byrne, Belinda;Vaughan, Kevin T
微管相关蛋白的一个亚群，包括细胞质连接蛋白(CLIP)-170、动力蛋白、EB1、大肠腺瘤性息肉病、细胞质动力蛋白、CLASPs和LIS-1，最近被证明可以靶向微管的正端。这种结合特异性的机制和功能尚不清楚，尽管已经提出了促进微管延伸的作用。为了进一步研究dynactin在细胞器运输中的作用，我们通过活细胞成像分析了p150glue。对p150glue的时间推移分析显示其靶向生长微管的正端，需要nh2末端细胞骨架相关蛋白-甘氨酸富结构域，而不需要EB1或CLIP-170。蛋白激酶A的效应物调节微管结合，并提示p150glue磷酸化是正端结合特异性的一个因素。利用磷酸敏感单克隆抗体，我们将p150glue磷酸化位点定位到Ser-19上。磷酸化位点突变体的体内和体外分析表明，p150glue磷酸化介导了与微管的动态结合。为了研究动态结合的功能，我们对gfp - p150glue在高尔基膜的动力蛋白依赖性运输过程中进行了成像。活细胞分析显示，高尔基膜和gfp -p150胶标记的微管在运输之前发生了短暂的相互作用，这表明微管和动力蛋白参与了负端定向细胞器的搜索-捕获机制。
A subset of microtubule-associated proteins, including cytoplasmic linker protein (CLIP)-170, dynactin, EB1, adenomatous polyposis coli, cytoplasmic dynein, CLASPs, and LIS-1, has been shown recently to target to the plus ends of microtubules. The mechanisms and functions of this binding specificity are not understood, although a role in encouraging microtubule elongation has been proposed. To extend previous work on the role of dynactin in organelle transport, we analyzed p150Glued by live-cell imaging. Time-lapse analysis of p150Glued revealed targeting to the plus ends of growing microtubules, requiring the NH2-terminal cytoskeleton-associated protein–glycine rich domain, but not EB1 or CLIP-170. Effectors of protein kinase A modulated microtubule binding and suggested p150Glued phosphorylation as a factor in plus-end binding specificity. Using a phosphosensitive monoclonal antibody, we mapped the site of p150Glued phosphorylation to Ser-19. In vivo and in vitro analysis of phosphorylation site mutants revealed that p150Glued phosphorylation mediates dynamic binding to microtubules. To address the function of dynamic binding, we imaged GFP-p150Glued during the dynein-dependent transport of Golgi membranes. Live-cell analysis revealed a transient interaction between Golgi membranes and GFP-p150Glued–labeled microtubules just prior to transport, implicating microtubules and dynactin in a search–capture mechanism for minus-end–directed organelles.