Molecular Basis of Dynamic Localization of Class-I Myosins

I 类肌球蛋白动态定位的分子基础

• 批准号: 8149555
• 负责人: EDWARD D KORN
• 金额: $ 67.93万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8149555
• 关键词: Molecular; Myosin Type I; base

Last year we established that binding of Acanthamoeba and Dictyostelium class-I myosins to acidic phospholipid vesicles could be attributed to a short sequence of basic and hydrophobic amino acids, in no specific order, in the basic domain of the tail. We also developed a computer search program to identify similar BH regions, i.e. potenital membrane-binding sites, in other proteins, and demonstrated that BH-search correctly identified all previously known, non-structured membrane-binding sites in 16 proteins. BH-search also identified previously unknown membrane-binding sites in multiple proteins, and synthetic peptides and protein fragments containing these sequences were shown to bind to acidic phospholipid vesicles. Dictyostelium provides an excellent model system for testing the application of these results in living cells. Specifically, we are determining the localization of expressed GFP-labeled wild-type and mutated Dictyostelium myosin IB (DMIB) in DMIB-null cells at different stages of growth and development. Current findings show that the BH-region of the DMIB tail, residues 801KKKVLVHTLIRR812, is both necessary and sufficient for binding DMIB to the plasma membrane of vegetative (growing) amoebae. Deletion of the basic region or mutation or substitution of Ala for the hydrophobic amino acids completely blocks membrane binding. When cells are starved, full-length DMIB dissociates from the plasma membrane, becoming uniformly distributed in the cytoplasm, and then localizes with actin at the front of the motile cell. However, expressed tail alone remains tightly bound to the plasma membrane. Thus, the localization of DMIB is dynamically regulated: the BH region in the basic domain of the tail is required and sufficient for binding DMIB to the plasma membrane of non-polarized, vegetative cells, but the motor domain is required for release of DMIB from the plasma membrane and re-localization within the cytoplasm of motile and chemotaxing cells.

去年，我们确定了阿米巴和网骨藻I类肌球蛋白与酸性磷脂囊泡的结合可以归因于尾部碱性结构域中的碱性和疏水性氨基酸的短序列，没有特定的顺序。 我们还开发了一个计算机搜索程序，以确定类似的BH区域，即潜在的膜结合位点，在其他蛋白质，并证明BH搜索正确识别所有以前已知的，非结构化的膜结合位点在16个蛋白质。 BH-搜索还确定了以前未知的膜结合位点在多种蛋白质，合成肽和蛋白质片段含有这些序列被证明可以结合到酸性磷脂囊泡。 Dictyosteoblasts提供了一个很好的模型系统，用于测试这些结果在活细胞中的应用。 具体而言，我们正在确定表达的GFP标记的野生型和突变的网囊藻肌球蛋白IB（DMIB）在DMIB-空细胞在不同的生长和发育阶段的定位。 目前的研究结果表明，BH区的DMIB尾巴，残基801 KKKVLVHTLIRR 812，是必要的和足够的DMIB结合到质膜的营养（生长）阿米巴。 碱性区的缺失或突变或Ala取代疏水氨基酸完全阻断膜结合。 当细胞饥饿时，全长DMIB从质膜解离，变得均匀分布在细胞质中，然后与肌动蛋白一起定位在运动细胞的前部。然而，单独表达的尾巴仍然与质膜紧密结合。 因此，DMIB的定位是动态调节的：尾部的碱性结构域中的BH区是将DMIB结合至非极化的营养细胞的质膜所必需的并且是足够的，但是运动结构域是将DMIB从质膜释放并重新定位在运动细胞和趋化细胞的细胞质内所必需的。