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

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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