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Functional analyses of dynamin GTPase on early steps of endocytosis

动力 GTP 酶对内吞作用早期步骤的功能分析

基本信息

批准号：
10215202
负责人：
BABA Takeshi
金额：
$ 26.3万
依托单位：
Yamanashi Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2001
项目状态：
已结题

项目摘要

The following results were obtained in the term of current project. 1. Combined morphological and biochemical analyses of dynamin mutant cells : We have analyzed three dominant-negative mutants of dynamin, K44A, S45N, and T65F. All three mutants lacked binding capacity for both GTP and GDP, and biochemically indistinguishable. However, electron microscopic analyses revealed the each mutant showed the accumulation of unique morphological intermediates in transformed cells. These results indicated that these mutations in dynamin may inhibit the endocytosis through unknown dynamin's function not through well-known GTP/GDP-dependent fission steps. 2. Ultrastructural study of lysenin : Lysenin is a sphingomyelin-specific binding protein which induce hemolysis. We have identified the unique hexagonal oligomer on sphingomyelin-containing liposomes. The similar structures were found on the surface of lysenin-treated erythrocytes. These results indicated that lysenin induced hemolysis by pore-forming oligomer on the cell surface. 3. Analyses of in vitro reconstitution of synaptic vesicle formation : We have succeeded in reconstitution of the synaptic vesicle formation by incubating liposomes with brain cytosol, ATP and GTPγS. We then analyzed the effect of various lipid composion of liposomes on the rate of vesicle formation. We found that PIP2 accumulate the formation of vesicles. The accumulation of vesicle formation persisted after rapid degradation of PIP2. These results indicated that PIP2 may recruited various coat proteins to the liposomes required for vesicle budding.

本项目期间取得了以下成果。 1. dynamin突变细胞的形态学和生化联合分析：我们分析了dynamin的三种显性失活突变体，K44A、S45N和T65F。所有三个突变体都缺乏 GTP 和 GDP 的结合能力，并且在生化上无法区分。然而，电子显微镜分析显示，每个突变体都在转化细胞中积累了独特的形态中间体。这些结果表明，动力蛋白中的这些突变可能通过未知的动力蛋白功能而不是通过众所周知的 GTP/GDP 依赖性裂变步骤来抑制内吞作用。 2. lysenin 的超微结构研究： Lysenin 是一种鞘磷脂特异性结合蛋白，可诱导溶血。我们在含鞘磷脂的脂质体上鉴定出了独特的六角寡聚物。在经溶素处理的红细胞表面也发现了类似的结构。这些结果表明，溶血素通过细胞表面上的成孔低聚物诱导溶血。 3.突触小泡形成的体外重建分析：我们通过将脂质体与脑细胞质、ATP和GTPγS一起孵育，成功地重建了突触小泡形成。然后我们分析了脂质体的各种脂质成分对囊泡形成速率的影响。我们发现PIP2积累形成囊泡。 PIP2 快速降解后，囊泡形成的积累持续存在。这些结果表明，PIP2 可能会将各种外壳蛋白招募到囊泡出芽所需的脂质体中。