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

本研究的两个主要目标是表征泛素依赖的蛋白质内体分选的分子基础，并了解SKD 1在这一过程中的功能。SKD 1是AAA-ATPase家族的成员，也是哺乳动物E类Vps。以前，我们已经报道，显性负SKD 1（E235 Q）的表达引起的缺陷，通过内体和积累的晚期内体-溶酶体混合细胞器的膜交通。最近，我们发现SKD 1（E235 Q）的表达，而不是野生型SKD 1的表达，显著改变了泛素化蛋白质在EQ隔室中的分布，EQ隔室是内体和溶酶体的异常结构。我们推测SKD 1调节泛素从内体回收受体的去除，因此SKD 1（E235 Q）诱导泛素化蛋白的积累，推测是由于去泛素化过程中的缺陷。20多种膜相关蛋白在细胞中表达， ...更多信息 通过蛋白质组学分析鉴定了SKD 1（E235 Q）。不仅PM蛋白，而且内体再循环受体和高尔基体/TGN膜蛋白被特异性泛素化和/或重新分布到EQ隔室。如果泛素化受损，它们在EQ隔室中的积累就会恢复。它们的泛素化依赖性蛋白质分选与其生理功能有关。为了确定SKD 1和SBPs在内体蛋白分选中的功能作用，我们表达了编码SBPs及其缺失突变体的cDNA，并检查了几个内吞隔室，早期和晚期内体和溶酶体的形态。我们发现，SKD 1，可能与SBP 1，这是已知的结合溶胞（溶酶体运输调节），招募胞质溶胞蛋白的内体膜在ATP结合依赖性的方式。我们还发现，SBP 3，Vps 2 p，ESCRT-III亚基之一，直接连接SKD 1 ESCRT-III的哺乳动物同源物。少

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