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A study of an ubiquitylation-dependent protein sorting regulated by SKD1, an AAA-ATPAse.

一项由 SKD1（一种 AAA-ATPAse）调节的泛素化依赖性蛋白质分选的研究。

基本信息

批准号：
16590050
负责人：
FUJITA Hideaki
金额：
$ 2.3万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16590050/
关键词：
Membrane Traffic lysosome endosome ubiquitin multivesicular body proteolysis AAA-ATPase プロテオミクス

项目摘要

Two major objectives of this study are to characterize the molecular basis of ubiquitin dependent protein sorting at endosomes and to learn the function of SKD1 in this process. SKD1 is a member of an AAA-ATPase family and a mammalian class E Vps. Previously we have reported that the expression of a dominant negative SKD1 (E235Q) caused a defect in a membrane traffic through endosomes and an accumulation of a late endosome-lysosome hybrid organelle. Recently, we found that an expression of SKD1 (E235Q), but not wild type SKD1, significantly altered the distribution of the ubiquitylated proteins to the EQ compartments, which are the aberrant structure of endosomes and lysosomes. We speculate that SKD1 regulate the removal of ubiquitin from the endosomal recycling receptors, thus SKD1 (E235Q) induces the accumulation of ubiquitylated proteins presumably due to the defect in the deubiquitylation process. Over twenty membrane-associated proteins potentially ubiquitylated in the cells expre … More ssing SKD1 (E235Q) were identified by a proteomics analysis. Not only the PM proteins but also endosomal recycling receptor and Golgi/TGN membrnae proteins were specifically ubiquitylated and/or redistributed to the EQ compartments. If the ubiquitylation was impaired, the accumulation of them in the EQ compartments was resumed. The ubiquitylation-dependent protein sorting of them should be relevant to their physiological functions. In order to define the functional role of SKD1 and SBPs in protein sorting at endosomes, we have expressed cDNAs encoding SBPs and their deletion mutants and examine the morphology of several endocytic compartments, early and late endosomes and lysosomes. We found that SKD1, possibly together with SBP1, which is known to bind to lyst (lysosomal trafficking regulator), recruit cytosolic lyst protein to endosomal membrane in an ATP-binding dependent manner. We also found that SBP3, a mammalian homologue of Vps2p, one of ESCRT-III subunits, directly linked SKD1 to ESCRT-III. Less

本研究的两个主要目标是表征内体泛素依赖性蛋白质分选的分子基础，并了解 SKD1 在此过程中的功能。 SKD1 是 AAA-ATPase 家族的成员，也是哺乳动物 E 类 Vps。之前我们报道过显性失活 SKD1 (E235Q) 的表达导致通过内体的膜运输缺陷以及晚期内体-溶酶体混合细胞器的积累。最近，我们发现 SKD1 (E235Q) 的表达（而非野生型 SKD1）显着改变了泛素化蛋白在 EQ 区室中的分布，EQ 区室是内体和溶酶体的异常结构。我们推测 SKD1 调节内体回收受体中泛素的去除，因此 SKD1 (E235Q) 可能由于去泛素化过程的缺陷而诱导泛素化蛋白的积累。通过蛋白质组学分析，鉴定出 20 多种可能在表达 SKD1 (E235Q) 的细胞中泛素化的膜相关蛋白。不仅 PM 蛋白，而且内体再循环受体和高尔基体/TGN 膜蛋白也被特异性泛素化和/或重新分配到 EQ 区室。如果泛素化受损，它们在 EQ 区室中的积累就会恢复。它们的泛素化依赖性蛋白质分选应该与其生理功能相关。为了确定 SKD1 和 SBP 在内体蛋白质分选中的功能作用，我们表达了编码 SBP 及其缺失突变体的 cDNA，并检查了几个内吞区室、早期和晚期内体和溶酶体的形态。我们发现 SKD1 可能与已知与 lyst（溶酶体运输调节因子）结合的 SBP1 一起，以 ATP 结合依赖性方式将胞质裂解蛋白招募至内体膜。我们还发现 SBP3（ESCRT-III 亚基之一 Vps2p 的哺乳动物同源物）直接将 SKD1 与 ESCRT-III 连接。较少的