Regulation of myosin V interaction with cargo.

这项提议的目标是确定肌动蛋白为基础的马达，肌凝蛋白V是如何附着在它的货物上的。这是肌凝蛋白V功能的一个关键方面，因为肌凝蛋白V在不同的时间将不同的货物运送到不同的地方。因此，肌凝蛋白V对货物的附着和/或肌凝蛋白V运动活性可能受到剧烈调节。已经假设肌球蛋白- v的c端球状尾结构域与蛋白受体相互作用，并且这些受体对肌球蛋白- v货物相互作用的调节至关重要。我们的实验室最近发现，酵母肌球蛋白v， Myo2p，在细胞分裂过程中将液泡（溶酶体）移动到子细胞。有趣的是，Myo2p球形尾部的特定点突变会破坏液泡运动，但不会破坏Myo2p的其他功能。利用这些液泡特异性突变体，发现了一种新的蛋白质Vac17p。一些证据表明，它是Myo2p血管受体的一个组成部分。本研究的总体目标是了解Vac17p的确切功能，扩展Myo2p尾部构象变化在指定货物中起作用的初步观察，并确定调节Myo2p-液泡关联的机制。这些目标将按以下方式实现：1)绘制Vac17p上与Myo2p相互作用所需的位点。2)确定Vac17p水平是否驱动液泡遗传的细胞周期协调。3)确定Myo2p球状尾部的构象变化是否调节了货物特异性。4)确定Myo2p与液泡结合所需的其他受体亚基和调节分子。这些研究应该揭示肌凝蛋白V和它的受体是如何调节将货物在适当的时间运送到正确的地方。这个问题显然与人类健康相关，因为肌球蛋白V是突触小泡、内质网和黑素小体正确定位所必需的。此外，由于肌球蛋白Va突变而患有Griscelli综合征的患者表现出神经和色素沉着缺陷。

The goal of this proposal is to determine how the actin based motor, myosin V attaches to its cargo. This is a key aspect of myosin V function because myosin V moves diverse cargo to unique places at distinct times. Therefore, it is likely that myosin V attachment to cargo and/or myosin V motor activity is acutely regulated. It has been postulated that the C-terminal globular tail domain of myosin-V interacts with protein receptors, and that these receptors are critical for the regulation of myosin V-cargo interactions. Our laboratory recently discovered that the yeast myosin-V, Myo2p, moves the vacuole (lysosome) to the daughter cell during cell division. Interestingly, specific point mutations in the globular tail of Myo2p disrupt vacuole movement but not other Myo2p functions. Using these vacuole-specific mutants, a novel protein, Vac17p, was discovered. Several lines of evidence suggest that it is a component of the vascular receptor for Myo2p. The overall goals of this proposal are to understand the precise function of Vac17p, to extend preliminary observations that conformational changes in the Myo2p tail play a role in specifying cargo, and to identify the mechanisms that regulate Myo2p-vacuole association. These goals will be pursued as follows: 1) Map the sites on Vac17p that are required for its interaction with Myo2p. 2) Determine whether Vac17p levels drive the cell-cycle coordination of vacuole inheritance. 3) Determine whether conformational changes within the Myo2p globular tail regulate cargo specificity. 4) Identify additional receptor subunits and regulatory molecules that are required for Myo2p association with the vacuole. These studies should reveal how regulation of both myosin V and its receptor move cargo to the correct place at the proper time. This question is of clear relevance to human health because myosin V is required for correct positioning of synaptic vesicles, the endoplasmic reticulum, and melanosomes. Moreover, patients with Griscelli's syndrome due to mutations in myosin Va, display both neurological and pigmentation defects.