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

本研究的两个主要目的是表征内体中泛素依赖蛋白分选的分子基础，并了解SKD1在这一过程中的功能。SKD1是aaa - atp酶家族的成员，也是哺乳动物E Vps类。之前我们曾报道过显性阴性SKD1 （E235Q）的表达导致通过核内体的膜运输缺陷和晚期核内体-溶酶体杂交细胞器的积累。最近，我们发现SKD1 （E235Q）的表达，而不是野生型SKD1的表达，显著改变了泛素化蛋白在EQ区室的分布，EQ区室是内体和溶酶体的异常结构。我们推测SKD1调控了内体再循环受体中泛素的去除，因此SKD1 （E235Q）诱导泛素化蛋白的积累可能是由于去泛素化过程中的缺陷。通过蛋白质组学分析，在表达SKD1 （E235Q）的细胞中发现了20多个泛素化的膜相关蛋白。不仅PM蛋白，内体再循环受体和高尔基/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分选可能与其生理功能有关。为了明确SKD1和sbp在核内体蛋白分选中的功能作用，我们表达了编码sbp及其缺失突变体的cdna，并检查了几个内噬室、早期和晚期核内体和溶酶体的形态。我们发现SKD1可能与已知与溶酶体运输调节因子（lyst）结合的SBP1一起，以atp结合依赖的方式将胞质溶酶蛋白募集到内体膜上。我们还发现，ESCRT-III亚基之一Vps2p的哺乳动物同源物SBP3直接将SKD1与ESCRT-III连接起来。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