The role of preassembled cytoplasmic complexes in assembly of flagellar dynein subunits
The role of preassembled cytoplasmic complexes in assembly of flagellar dynein subunits
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DOI：
10.1091/mbc.9.9.2337
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发表时间：
1998-09-01
影响因子：
3.3
通讯作者：
Mitchell, DR
中科院分区：
文献类型：
--
作者：
Fowkes, ME;Mitchell, DR
先前的工作已经揭示了鞭毛前体蛋白质的细胞质池能够有助于新鞭毛的组装，但这些组件如何以及在哪里组装是未知的。我们测试了衣原体外动力蛋白臂亚基的稳定性和组装在野生型细胞和11个外动力蛋白臂组装突变株（oda 1-oda 11）的细胞质提取物，或从这些提取物的免疫沉淀，与5个外排动力蛋白亚基特异性抗体的蛋白质印迹。蛋白质印迹显示，至少有三个oda突变体（oda 6，oda 7和oda 9）改变了一个亚基的水平，而不是突变基因的产物。免疫沉淀显示，含有所有五个测试亚基（三个重链和两个中间链）的大型预组装鞭毛复合物存在于野生型细胞质内。当这些亚基的预组装在oda菌株中进行检查时，我们观察到三种模式：完全共组装（oda 1，3，5，8和10），部分共组装（oda 7和oda 11），以及没有共组装（oda 2，6和9）的四个测试亚基与HC β。我们的数据，加上以前的研究，表明鞭毛外动力蛋白臂预组装成一个完整的M-r类似或等于2 × 10（6）动力蛋白臂，驻留在细胞质前体池运输到鞭毛室之前。
Previous work has revealed a cytoplasmic pool of flagellar precursor proteins capable of contributing to the assembly of new flagella, but how and where these components assemble is unknown. We tested Chlamydomonas outer-dynein arm subunit stability and assembly in the cytoplasm of wild-type cells and 11 outer dynein arm assembly mutant strains (oda1-oda11) by Western blotting of cytoplasmic extracts, or immunoprecipitates from these extracts, with five outer-row dynein subunit-specific antibodies. Western blots reveal that at least three oda mutants (oda6, oda7, and oda9) alter the level of a subunit that is not the mutant gene product. Immunoprecipitation shows that large preassembled flagellar complexes containing all five tested subunits (three heavy chains and two intermediate chains) exist within wild-type cytoplasm. When the preassembly of these subunits was examined in oda strains, we observed three patterns: complete coassembly (oda 1, 3, 5, 8, and 10), partial coassembly (oda7 and oda11), and no coassembly (oda2, 6, and 9) of the four tested subunits with HC beta. Our data, together with previous studies, suggest that flagellar outer-dynein arms preassemble into a complete M-r similar or equal to 2 x 10(6) dynein arm that resides in a cytoplasmic precursor pool before transport into the flagellar compartment.