A dominant negative form of the AAA ATPase SKD1/VPS4 impairs membrane trafficking out of endosomal/lysosomal compartments: class Evpsphenotype in mammalian cells

Abstract

SKD1 is a member of the family of ATPases associated with cellular activities whose yeast homologue Vps4p has been implicated in endosomal/vacuolar membrane transports. When a mutant of SKD1 that lacks ATPase activity [SKD1(E235Q)] was overexpressed in mammalian cells, it induced a dominant negative phenotype characterized by aberrant endosomal structures (denoted as E235Q compartments). Expression of SKD1(E235Q) caused an accumulation of basolateral recycling receptors, such as asialoglycoprotein receptor and low-density lipoprotein in polarized hepatocytes and Madin-Darby canine kidney cells, respectively, in E235Q compartments. In addition, SKD1(E235Q) also abrogated, via endosomes, transport to the trans-Golgi network, as indicated by an accumulation of TGN38 in E235Q compartments. Three lines of evidence further demonstrated that SKD1 participates in the membrane transport from early endosomes to late endosomes/lysosomes: (1) a redistribution of a late endosomal and lysosomal membrane protein endolyn in E235Q compartments; (2) an inhibition of epidermal growth factor receptor degradation, due to an accumulation of the receptors in E235Q compartments; and (3) a mis-sorting of and defect in the proteolytic processing of newly synthesized cathepsin D. An intriguing finding was that the expression of SKD1(E235Q) caused the number of lysosomes to decrease (to one-sixth of control numbers) but their size to increase (2.4-fold larger in diameter than control lysosomes). Indeed, an ultrastructural analysis revealed that the expression of SKD1(E235Q) causes an accumulation of hybrid organelles formed by direct fusion between late endosomes and lysosomes. We conclude that SKD1 regulates multiple steps of membrane transport out of early endosomes and the reformation of lysosomes from a hybrid organelle.

Mammalian class E Vps proteins, SBP1 and mVps2/CHMP2A, interact with and regulate the function of an AAA-ATPase SKD1/Vps4B

“…Indeed, SPR analysis revealed a stable complex of SBP1-SKD1 in vitro. Previously, we reported that E235Q compartments are derived from multiple endogenous membranes, early and late endosomes, and lysosomes (Fujita et al, 2003). GFP-SKD1(E235Q) co-localizes with several endosomal/lysosomal markers but the extent of the co-localization varies.…”

“…This result clearly demonstrates that SKD1 and rab5 regulate endosomal membrane transport independently. Interestingly, despite the distinct molecular mechanisms of SKD1(E235Q) and rab5QL, the resultant morphological alterations on late endosomes and lysosomes are quite similar; both induce the enlargement (Fujita et al, 2003;Rosenfeld et al, 2001). Based on our results and those of others, we assume that the biogenesis of lysosomes is highly ascribed to the membrane traffic at early endosomes.…”

“…Rabbit polyclonal antibodies against SKD1 has been described previously (Fujita et al, 2003). Alexa488-and Alexa594-labeled secondary antibodies were purchased from Molecular Probes (Eugen, OR, USA).…”

“…Plasmid construction SKD1 Plasmids encoding GST-SKD1, GFP-SKD1 and GFP-SKD1(E235Q) were described previously (Fujita et al, 2003). cDNAs encoding the wild-type SKD1 and mutant E235Q were excited from GFP fusion constructs and subcloned into a pcDNA3.1(-) vector.…”

“…We have previously shown that overexpression of SKD1(E235Q), the ATPase-deficient form of SKD1 (suppressor of potassium transport growth defect 1), led to perturbation of various membrane transports via endosomes (Fujita et al, 2003). For instance, recycling receptors are accumulated in the E235Q compartments, the aberrant endosomes induced by SKD1(E235Q).…”