The Chlamydomonas FLA10 gene encodes a novel kinesin-homologous protein.
The Chlamydomonas FLA10 gene encodes a novel kinesin-homologous protein.
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DOI：
10.1083/jcb.126.1.175
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发表时间：
1994-07
影响因子：
7.8
通讯作者：
Hall, J L
中科院分区：
文献类型：
--
作者：
Walther, Z;Vashishtha, M;Hall, J L
衣藻单连锁群上的许多基因影响基体/鞭毛器。其中有五个 FLA 基因，其突变会导致鞭毛组装中的温度敏感缺陷。我们对映射到 fla10 并在功能上拯救 fla10 表型的基因进行了分子分析。核苷酸测序显示该基因编码驱动蛋白同源蛋白 KHP1。 87-kD 预测的 KHP1 蛋白与驱动蛋白重链一样，具有氨基末端运动结构域、中央 α 螺旋柄和基本的球状羧基末端尾部。与其他驱动蛋白超家族成员的比较表明，它与主要在小脑中表达的小鼠基因 KIF3 具有惊人的相似性（运动结构域有 64% 的同一性）。在同步培养中，KHP1 mRNA 在细胞分裂后积累，鞭毛动力蛋白 mRNA 也是如此。去鞭毛后 KHP1 mRNA 水平也增加。多克隆抗体在纯化的鞭毛和轴丝的蛋白质印迹中检测到 KHP1 蛋白。 ATP 处理时该蛋白质部分从轴丝中释放，但 AMP-PNP 处理时则不然。对各种运动突变体的轴丝进行蛋白质印迹分析表明，KHP1 不是径向辐条、动力蛋白臂或中央复合体的组成部分。突变体 fla10-1 轴丝中 KHP1 蛋白的数量显着减少，但在其他两个单连锁突变体 fla9 和 fla11 中未观察到减少。此外，通过用KHP1基因组DNA转化来拯救fla10-1。这些结果表明 KHP1 是 FLA10 的基因产物，并表明这种驱动蛋白相关蛋白在鞭毛组装和维护中具有新的作用。
Many genes on the uni linkage group of Chlamydomonas affect the basal body/flagellar apparatus. Among these are five FLA genes, whose mutations cause temperature-sensitive defects in flagellar assembly. We present the molecular analysis of a gene which maps to fla10 and functionally rescues the fla10 phenotype. Nucleotide sequencing revealed that the gene encodes a kinesin-homologous protein, KHP1. The 87-kD predicted KHP1 protein, like kinesin heavy chain, has an amino- terminal motor domain, a central alpha-helical stalk, and a basic, globular carboxy-terminal tail. Comparison to other kinesin superfamily members indicated striking similarity (64% identity in motor domains) to a mouse gene, KIF3, expressed primarily in cerebellum. In synchronized cultures, the KHP1 mRNA accumulated after cell division, as did flagellar dynein mRNAs. KHP1 mRNA levels also increased following deflagellation. Polyclonal antibodies detected KHP1 protein in Western blots of purified flagella and axonemes. The protein was partially released from axonemes with ATP treatment, but not with AMP- PNP. Western blot analysis of axonemes from various motility mutants suggested that KHP1 is not a component of radial spokes, dynein arms, or the central pair complex. The quantity of KHP1 protein in axonemes of the mutant fla10-1 was markedly reduced, although no reduction was observed in two other uni linkage group mutants, fla9 and fla11. Furthermore, fla10-1 was rescued by transformation with KHP1 genomic DNA. These results indicate that KHP1 is the gene product of FLA10 and suggest a novel role for this kinesin-related protein in flagellar assembly and maintenance.