CHARACTERIZATION OF YEAST DYANCTIN COMPLEX

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。动力蛋白是大多数动力蛋白活动所必需的多亚基蛋白质复合体，被认为可以作为货物适配器或增强动力蛋白的运动过程。动力蛋白由两部分组成，含有11种不同的多肽。1)类肌动蛋白Arp1细丝在其带刺末端含有常规肌动蛋白和套膜蛋白，在其尖端含有Arp11、p25、p27和p62。2)臂由p150Glued、Dynamitin和p24/22组成。在酿酒酵母中，已经鉴定了几个dynactin亚基的同源物，它们的基因缺失产生了与dynein零突变相同的表型。另一方面，p24/22、p25、p27和p62的同源物还没有被鉴定，酵母动力蛋白的组成也没有被生化定义。在这里，我们报告了在发芽酵母中发现了一个明显的p24/22同源物，基于以下原因：1)零突变体具有动力蛋白样纺锤体运动的表型。2)预测的多肽具有合适的大小和二级结构。3)空突变体是与kar9&#8710；而不是dyn1&#8710；合成的。4)该蛋白定位于微管+末端和纺锤体极体，如Nip100/p150Glued。5)如已知的dynactin突变体所见，dynein在缺失突变体的微管正端积累dynein。完整的动力蛋白复合体的生化分离正在进行中，这可能会识别酵母动力蛋白的其他亚基。根据我们目前的理解，dynactin是将dynein从正端卸载到皮质所必需的。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Dynactin is a multi-subunit protein complex required for most dynein activities, proposed to serve either as a cargo adaptor or to enhance dynein motor processivity. Dynactin consists of two parts with eleven different polypeptides. 1) The actin-like Arp1 filament has conventional actin and capping protein at its barbed end and Arp11, p25, p27, and p62 at its pointed end. 2) The arm consists of p150Glued, dynamitin, and p24/22. In Saccharomyces cerevisae, homologues of several dynactin subunits have been identified, and deletion of their genes produces phenotypes identical to those of dynein null mutations. On the other hand, homologues for p24/22, p25, p27 and p62 have not been identified, and the composition of yeast dynactin has not been defined biochemically. Here, we report the discovery of an apparent p24/22 homolog in budding yeast, based on the following: 1) The null mutant has a dynein-like phenotype for spindle movement. 2) The predicted polypeptide has an appropriate size and secondary structure. 3) The null mutant is synthetic with kar9∆but not dyn1∆. 4) The protein localizes to microtubule plus ends and the spindle pole body, as seen for Nip100 / p150Glued. 5) Dynein accumulates of dynein at the plus end of microtubules in the null mutant, as seen withknown dynactin mutants. Biochemical fractionations of intact dynactin complex are underway, which may identify other subunits of yeast dynactin. Based on our current understanding, dynactin is needed to offload dynein from the plus ends to the cortex.