P4-ATPase TAT-1和分子伴侣CHAT-1调控线虫内吞分选及循环的机制研究

发布时间：2018-01-11 05:03

本文关键词：P4-ATPase TAT-1和分子伴侣CHAT-1调控线虫内吞分选及循环的机制研究　出处：《北京协和医学院》2011年博士论文　论文类型：学位论文

【摘要】：在真核细胞中,从细胞膜外吞进的货物首先被送到早期内涵体。早期内涵体可将货物分选,经分选后的货物或进入循环回收通路重新回到细胞膜上,或进入降解通路在溶酶体中被降解,或被送到顺式高尔基网。早期内涵体为多形态结构,主体中心是球状膜泡,大部分膜成分会向外出芽形成很多管状膜泡。即将被循环回收的膜受体和其他蛋白会分选进入管状膜泡区域。管状膜泡会与主体膜泡分离,通过快速循环回收通路直接去和细胞膜融合,或者通过慢速循环回收通路经过循环内涵体再回到细胞膜。内吞分选和循环回收两个过程都需要改变膜的形态从而形成管状膜泡。但是关于管状膜泡形成与维持的机制仍然不清楚。通过筛选磷脂酰丝氨酸(PS)分布的调控因子,我们获得了若干tat-1(transbilayer amphipath transporters)突变体和一个chat-1(chaperone of tat-1)突变体。tat-1编码的是P4类ATP水解酶,在细胞膜上负责转运磷脂分子,如PS和磷脂酰乙醇胺(PE),从而特异地使相关磷脂分子保持在细胞膜内侧。在tat-1突变体中,PS不仅出现在凋亡细胞表面也出现在活细胞表面。chat-1是线虫里的Cdc50家族成员。chat-1 (qx36)突变体和tat-1突变体具有相同的表型。这表明CHAT-1也能参与调控细胞膜上PS的不对称性分布。与酵母、哺乳动物细胞的P4类ATP水解酶和Cdc50蛋白一样,TAT-1和CHAT-1也需要彼此才能从ER运出。在tat-1和chat-1突变体,内吞分选的缺陷导致循环回收通路和降解通路都有障碍。TAT-1和CHAT-1具有相同的表达谱,在多种组织广泛表达。它们不仅共定位于细胞膜上,也能共定位在细胞内膜泡上,包括早期内涵体和循环内涵体。这些膜泡上表面都有PS。TAT-1和CHAT-1功能缺失会造成内涵体膜上PS不对称性分布被打破从而影响与循环回收相关的管状膜泡不能正常形成。另外,我们发现在tat-1和chat-1突变体中,电荷分布和某些蛋白定位也发生了改变。因此,TAT-1和CHAT-1可能是通过维持PS在内涵体膜上的不对称性分布促进管状膜泡的形成从而调控内吞分选和循环通路。此外,TAT-1和CHAT-1还可能通过将PS限制在膜的细胞质侧,调控某些蛋白的膜定位,从而介导膜泡运输。为了寻找与TAT-1并行作用的基因,我们通过正向遗传筛选,获得了5个与tat-1共失活致死的突变体。它们位于不同的染色体上。目前,基因的定位与克隆正在进行中。
[Abstract]:In eukaryotic cells, goods swallowed out of the cell membrane are first delivered to the early intension, which can be sorted and returned to the cell membrane after sorting the goods or entering the recycling pathway. Either enter the degradation pathway is degraded in lysosome, or be sent to the cis Golgi net. The early intension body is multi-morphological structure, the main center is spherical membrane vesicles. The membrane receptors and other proteins that will be recycled will be separated into the tubular vesicle area. The tubular membrane vesicles will be separated from the main membrane vesicles. The cell membrane is fused directly through the rapid recycling pathway. Either through the slow cycle recovery pathway, the membrane is returned to the cell membrane through the circulatory intension. Both endocytosis and recycling need to change the shape of the membrane to form a tubular membrane bubble. However, the formation and maintenance of the tubular membrane vesicle are related to the formation and maintenance of the tubular membrane bubble. The mechanism remains unclear. The factors regulating the distribution of phosphatidyl serine were screened. We have obtained several tat-1(transbilayer amphipath transporters) mutants and a chat-1 (. Chaperone of tat-1) mutant. Tat-1 encodes P4 ATP hydrolase. Responsible for the transport of phospholipid molecules such as PS and phosphatidyl ethanolamine on the cell membrane, which specifically keeps the associated phospholipid molecules inside the membrane. In tat-1 mutants. PS appears not only on the surface of apoptotic cells but also on the surface of living cells. Chat-1 is a member of the Cdc50 family of nematodes. The mutants and tat-1 mutants have the same phenotype, which suggests that CHAT-1 can also play a role in regulating the asymmetric distribution of PS on the cell membrane. The P4 ATP hydrolases of mammalian cells, like Cdc50 proteins, also require each other to be transported from ER. In tat-1 and chat-1 mutants, the defect of endocytosis resulted in the obstruction of both recycling and degradation pathways. TAT-1 and CHAT-1 had the same expression profile. They are widely expressed in a variety of tissues. They are not only located on the cell membrane, but also on the intimal vesicles. The absence of PS.TAT-1 and CHAT-1 functions on the upper surface of these vesicles can cause the asymmetric distribution of PS on the membranes to be broken, thus affecting and recycling. The associated tubular vesicles are not normally formed. We found that in tat-1 and chat-1 mutants, the distribution of charge and the localization of some proteins also changed. TAT-1 and CHAT-1 may regulate endocytosis and circulatory pathway by maintaining the asymmetric distribution of PS on the inner membrane to promote the formation of tubular vesicles. TAT-1 and CHAT-1 may also mediate membrane vesicle transport by limiting PS to the cytoplasmic side of the membrane and regulating the membrane localization of some proteins. In order to search for genes that interact with TAT-1, we obtained five mutants that were inactivated with tat-1 through forward genetic screening. They are located on different chromosomes. Gene mapping and cloning are in progress.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R329

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