Perrone C A, Yang P, O'Toole E, Sale W S, Porter M E
Department of Cell Biology and Neuroanatomy, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
Mol Biol Cell. 1998 Dec;9(12):3351-65. doi: 10.1091/mbc.9.12.3351.
To identify new loci that are involved in the assembly and targeting of dynein complexes, we have screened a collection of motility mutants that were generated by insertional mutagenesis. One such mutant, 5B10, lacks the inner arm isoform known as the I1 complex. This isoform is located proximal to the first radial spoke in each 96-nm axoneme repeat and is an important target for the regulation of flagellar motility. Complementation tests reveal that 5B10 represents a new I1 locus, IDA7. Biochemical analyses confirm that ida7 axonemes lack at least five I1 complex subunits. Southern blots probed with a clone containing the gene encoding the 140-kDa intermediate chain (IC) indicate that the ida7 mutation is the result of plasmid insertion into the IC140 gene. Transformation with a wild-type copy of the IC140 gene completely rescues the mutant defects. Surprisingly, transformation with a construct of the IC140 gene lacking the first four exons of the coding sequence also rescues the mutant phenotype. These studies indicate that IC140 is essential for assembly of the I1 complex, but unlike other dynein ICs, the N-terminal region is not critical for its activity.
为了鉴定参与动力蛋白复合体组装和靶向的新基因座,我们筛选了一组通过插入诱变产生的运动突变体。其中一个这样的突变体5B10,缺乏一种称为I1复合体的内臂亚型。这种亚型位于每个96纳米轴丝重复序列中第一个径向辐条的近端,是鞭毛运动调节的重要靶点。互补试验表明,5B10代表一个新的I1基因座,即IDA7。生化分析证实,ida7轴丝至少缺少五个I1复合体亚基。用包含编码140 kDa中间链(IC)的基因的克隆进行Southern杂交,表明ida7突变是质粒插入IC140基因的结果。用IC140基因的野生型拷贝进行转化可完全挽救突变体缺陷。令人惊讶的是,用缺少编码序列前四个外显子的IC140基因构建体进行转化也能挽救突变体表型。这些研究表明,IC140对于I1复合体的组装至关重要,但与其他动力蛋白IC不同,其N端区域对其活性并不关键。