CHARACTERIZATION OF PROTEINS ASSOCIATED WITH A MYO2P CONTAINING RNP

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。Myo 2 p是芽殖酵母中V类肌球蛋白的一员，在极性建立、液泡遗传中的分泌囊泡运输以及核分离、高尔基体和过氧化物酶体遗传中起作用。虽然Myo 2 p参与了许多过程，但只有两个对接蛋白复合物是已知的。Myo 2 p通过-Vac 17 p-Vac 8 p复合物与液泡膜结合，Kar 9 p作为Myo 2 p和纺锤体微管之间的接头以允许核分离。我们有几条证据表明Myo 2 p是RNA-蛋白质复合物（RNP）的一部分。这项合作的目标是鉴定这种复合物中的新型蛋白质，包括Myo 2 p与其RNA货物的连接体。我们通过差速离心实验部分纯化了Myo 2 p RNP复合物。该络合物存在于高速粒料级分（P3）中。 以下实验表明Myo 2 p是RNP的一部分：1。在蔗糖速度梯度上，来自P3分级的Myo 2 p随着RNase的加入而显著改变。Myo 2 p不结合积极翻译的核糖体，放线菌酮治疗的分馏模式没有影响。这项合作的目标是鉴定这种复合物中的新型蛋白质，包括Myo 2 p与其RNA货物的连接体。

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. Myo2p, a member of the class V myosins in budding yeast, functions in polarity establishment, in secretory vesicle trafficking in vacuolar inheritance, and in nuclear segregation, golgi, and peroxisome inheritance. Although Myo2p is involved in many processes, only two docking protein complexes are known. Myo2p binds to the vacuolar membrane through the -Vac17p-Vac8p complex, and Kar9p serves as the linker between Myo2p and spindle microtubule to allow nuclear segregation. We have several lines of evidence that show that Myo2p is part of an RNA-protein complex (RNP). The goal of this collaboration is to identify novel proteins in this complex, including linkers of Myo2p to its RNA cargo. We have partially purified the Myo2p RNP complex via differential centrifugation experiments. This complex is present in a high speed pellet fraction (P3). The following experiments show that Myo2p is part of an RNP: 1. On sucrose velocity gradients, Myo2p from P3 fractionation shifts dramatically with the addition of RNase. Myo2p does not bind to actively translating ribosomes, as cycloheximide treatment has no effect on the fractionation pattern. The goal of this collaboration is to identify novel proteins in this complex, including linkers of Myo2p to its RNA cargo.