NOVEL KINESIN RELATED MOTOR PROTEIN

驱动蛋白和驱动蛋白相关蛋白（KRP）是微管（MT）- 转运细胞内颗粒沿着 因此，MT在细胞器的放置、囊泡的形成、细胞膜的形成、细胞膜的形成和细胞膜的形成中起重要作用。 运输、减数分裂和有丝分裂。这里提出的工作的目的是 是表征一种新的三聚体驱动蛋白相关蛋白， 以下称为KRP，来自海胆（SU）卵， 果蝇胚胎。纯化的KRP，这是第一个 分离自其天然细胞类型，由两种多肽组成 (Mr 85 kd和95 kd），其具有与 一个115 kd的辅助亚基。KRP显示核苷酸敏感的MT 绑定和捆绑活动，并产生力的MT运动 在运动性测定中。我们提出了一个详细的分析 三聚体KRP的分子结构，其“体外”马达 活动及其生物学功能，主要集中在SU 鸡蛋KRP，虽然我们的分析辅助多肽将 还涉及果蝇KRP（其纯化方案需要 待完成）。我们将使用抗SU的单克隆抗体 运动亚单位，并对SU和苍蝇配件亚单位，以 鉴定编码相应多肽的cDNA。这些 探针将被用来学习序列和结构 天然SU KRP中亚基之间的关系。在 此外，MT结合，ATP酶和运动性测定的结果， 细菌表达的马达多肽和天然复合物 将进行比较，以阐明的功能意义， 是三聚体复合物的组分。我们将探索 利用单克隆抗体对85 kd、95 kd 和SU KRP的115 kd亚基用于免疫定位， 海胆卵显微注射分离实验 卵裂球最后，我们将研究苍蝇115 kd的功能， 使用抗体进行细胞学研究， 产生115 kd功能缺陷的突变体。因此我们 将验证我们的假设，即在有丝分裂细胞中，115 kd亚基 可以调节或将马达多肽靶向到力的位点- 代，其中KRP驱动MT之间的相对滑动， 例如在有丝分裂纺锤体的组装或伸长期间。

Kinesin and kinesin-related proteins (KRPs) are microtubule (MT)- based motor proteins that transport intracellular particles along MTs, and thus play important roles in organelle placement, vesicle trafficking, meiosis and mitosis. The aim of the work proposed here is to characterize a novel trimeric kinesin related protein, hereafter referred to as the KRP, from sea urchin (SU) eggs and Drosophila embryos. The purified KRP, which is the first to be isolated from its native cell-type, consists of two polypeptides (Mr 85kd and 95kd) that have a kinesin motor domain complexed with a 115kd accessory subunit. The KRP displays nucleotide sensitive MT binding and bundling activity, and generates force for MT movement in a motility assay. We propose a detailed analysis of the molecular architecture of the trimeric KRP, its "in vitro" motor activities and its biological functions, focusing mainly on the SU egg KRP, although our analysis of the accessory polypeptide will also involve the Drosophila KRP (whose purification protocol needs to be completed). We will use monoclonal antibodies against the SU motor subunits, and against the SU and fly accessory subunits, to identify cDNAS encoding the corresponding polypeptides. These probes will be used to learn the sequences and structural relationships between the subunits in the native SU KRP. In addition, the results of MT binding, ATPase and motility assays on bacterially-expressed motor polypeptides and the native complex will be compared, to illuminate the functional significance of being components of a trimeric complex. We will probe the biological functions of the complex using mAbs to the 85kd, 95kd and 115kd subunits of the SU KRP for immunolocalization and microinjection experiments on dividing sea urchin eggs and blastomeres. Finally, we will study the function of the fly 115kd homologue using antibodies for cytologicaI studies and by generating mutants that are defective in 115kd function. Thus we will test our hypothesis that, in mitotic cells, the 115kd subunit may regulate or target the motor polypeptides to the site of force- generation, where the KRP drives relative sliding between MTs, for example during assembly or elongation of the mitotic spindle.