Kif5B and Kifc1 interact and are required for motility and fission of early endocytic vesicles in mouse liver
Kif5B and Kifc1 interact and are required for motility and fission of early endocytic vesicles in mouse liver
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DOI：
10.1091/mbc.e06-06-0524
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发表时间：
2007-05-01
影响因子：
3.3
通讯作者：
Wolkoff, Allan W.
中科院分区：
文献类型：
--
作者：
Nath, Sangeeta;Bananis, Eustratios;Wolkoff, Allan W.
从小鼠肝脏制备负载有德克萨斯红脱唾液酸类粘蛋白的早期内吞囊泡。这些囊泡在体外与微管结合，加入ATP后，它们双向移动，经常分裂成两个子囊泡。钒酸盐（动力蛋白抑制剂）对运动没有影响，而5 '-腺苷酰亚氨基二磷酸（驱动蛋白抑制剂）具有高度抑制作用。特异性抗体的研究证实，动力蛋白与这些囊泡无关，Kif 5 B和负端驱动蛋白Kifc 1分别介导了它们的正端和负端运动性。超过90%的囊泡与Kifc 1也包含Kif 5 B，和Kifc 1与抗体的抑制导致增强的正末端定向运动。当Kifc 1或Kif 5 B活性被抗体抑制时，囊泡分裂减少，表明分裂发生需要两个马达活性产生的相反力。在293 T细胞中表达FLAG-Kifc 1后，FLAG抗体对天然Kif 5 B的免疫沉淀表明这两个马达可以相互作用。它们是直接相互作用还是通过潜在的调节蛋白复合物相互作用，需要在未来的研究中阐明。然而，目前的研究表明，这些驱动蛋白的协调活动是必不可少的运动和处理早期内吞囊泡。
Early endocytic vesicles loaded with Texas Red asialoorosomucoid were prepared from mouse liver. These vesicles bound to microtubules in vitro, and upon ATP addition, they moved bidirectionally, frequently undergoing fission into two daughter vesicles. There was no effect of vanadate (inhibitor of dynein) on motility, whereas 5'-adenylylimido-diphosphate (kinesin inhibitor) was highly inhibitory. Studies with specific antibodies confirmed that dynein was not associated with these vesicles and that Kif5B and the minus-end kinesin Kifc1 mediated their plus- and minus-end motility, respectively. More than 90% of vesicles associated with Kifc1 also contained Kif5B, and inhibition of Kifc1 with antibody resulted in enhancement of plus-end-directed motility. There was reduced vesicle fission when either Kifc1 or Kif5B activity was inhibited by antibody, indicating that the opposing forces resulting from activity of both motors are required for fission to occur. Immunoprecipitation of native Kif5B by FLAG antibody after expression of FLAG-Kifc1 in 293T cells indicates that these two motors can interact with each other. Whether they interact directly or through a complex of potential regulatory proteins will need to be clarified in future studies. However, the present study shows that coordinated activity of these kinesins is essential for motility and processing of early endocytic vesicles.