Towns William L, Tauhata Sinji B F, Vaughan Patricia S, Vaughan Kevin T
Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556, USA.
Cell Motil Cytoskeleton. 2009 Feb;66(2):80-9. doi: 10.1002/cm.20327.
Cytoplasmic dynein contributes to the localization and transport of multiple membranous organelles, including late endosomes, lysosomes, and the Golgi complex. It remains unclear which subunits of dynein are directly responsible for linking the dynein complex to these organelles, however the intermediate chain (IC), light intermediate chain (LIC) and light chain (LC) subunits are each thought to be important. Based on previous mapping of a dynein IC phosphorylation site (S84), we measured the impact of transfected ICs on dynein-driven organelle transport (Vaughan et al.,2001). Wild-type and S84A constructs disrupted organelle transport, whereas the S84D construct induced no defects. In this study we investigated the mechanisms of transfection-induced disruption of organelle transport. Transfected ICs did not: (1) disrupt the dynein holoenzyme, (2) incorporate into the native dynein complex, (3) dimerize with native dynein ICs or (4) sequester dynein LCs in a phosphorylation-sensitive manner. Consistent with saturation of dynactin as an inhibitory mechanism, truncated ICs containing only the dynactin-binding domain were as effective as full-length IC constructs in disrupting organelle transport, and this effect was influenced by phosphorylation-state. Competition analysis demonstrated that S84D ICs were less capable than dephosphorylated ICs in disrupting the dynein-dynactin interaction. Finally, two-dimensional gel analysis revealed phosphorylation of the wild-type but not S84D ICs, providing an explanation for the incomplete effects of the wild-type ICs. Together these findings suggest that transfected ICs disrupt organelle transport by competing with native dynein for dynactin binding in a phosphorylation-sensitive manner.
细胞质动力蛋白参与多种膜性细胞器的定位和运输，包括晚期内体、溶酶体和高尔基体复合体。然而，目前尚不清楚动力蛋白的哪些亚基直接负责将动力蛋白复合体与这些细胞器连接起来，不过中间链（IC）、轻中间链（LIC）和轻链（LC）亚基各自都被认为很重要。基于先前对动力蛋白IC磷酸化位点（S84）的定位，我们测量了转染的IC对动力蛋白驱动的细胞器运输的影响（Vaughan等人，2001年）。野生型和S84A构建体破坏了细胞器运输，而S84D构建体未诱导出缺陷。在本研究中，我们调查了转染诱导的细胞器运输破坏机制。转染的IC并未：（1）破坏动力蛋白全酶，（2）整合到天然动力蛋白复合体中，（3）与天然动力蛋白IC二聚化，或（4）以磷酸化敏感的方式隔离动力蛋白轻链。与动力肌动蛋白作为抑制机制的饱和现象一致，仅包含动力肌动蛋白结合结构域的截短IC在破坏细胞器运输方面与全长IC构建体一样有效，并且这种效应受磷酸化状态影响。竞争分析表明，S84D IC在破坏动力蛋白-动力肌动蛋白相互作用方面比去磷酸化的IC能力更弱。最后，二维凝胶分析显示野生型IC发生了磷酸化，而S84D IC未发生磷酸化，这为野生型IC的不完全效应提供了解释。这些发现共同表明，转染的IC通过以磷酸化敏感的方式与天然动力蛋白竞争动力肌动蛋白结合来破坏细胞器运输。
