ATPase-defective Mammalian VPS4 Localizes to Aberrant Endosomes and Impairs Cholesterol Trafficking

Abstract

The yeast vacuolar sorting protein Vps4p is an ATPase required for endosomal trafficking that couples membrane association to its ATPase cycle. To investigate the function of mammalian VPS4 in endosomal trafficking, we have transiently expressed wild-type or ATPase-defective human VPS4 (hVPS4) in cultured cells. Wild-type hVPS4 was cytosolic, whereas a substantial fraction of hVPS4 that was unable to either bind or hydrolyze ATP was localized to membranes, including those of specifically induced vacuoles. Vacuoles were exclusively endocytic in origin, and subsets of enlarged vacuoles stained with markers for each stage of the endocytic pathway. Sorting of receptors from the early endosome to the recycling compartment or to the trans-Golgi network was not significantly affected, and no mutant hVPS4 associated with these compartments. However, many hVPS4-induced vacuoles were substantially enriched in cholesterol relative to the endosomal compartments of untransfected cells, indicating that expression of mutant hVPS4 gives rise to a kinetic block in postendosomal cholesterol sorting. The phenotype described here is largely consistent with the defects in vacuolar sorting associated with class E vps mutants in yeast, and a role for mammalian VPS4 is discussed in this context.

Vps4 and the ESCRT-III complex are required for the release of infectious hepatitis C virus particles

“…However, our data clearly show that ESCRT perturbation does not block assembly of intracellular infectious virus. We propose instead, that the ESCRT system is involved in either trafficking of complete nascent HCV particles through (and out of) the cell, or possibly the scission event whereby infectious particles are released from cellular membranes.One explanation for our findings is the observation made several years ago that Vps4DN-expressing cells demonstrated impaired cholesterol trafficking (Bishop & Woodman, 2000), suggesting that defects in the ESCRT system might disrupt normal cellular lipid metabolism. Lipids are known to play a vital role in HCV particle production; a number of HCV proteins are known to associate with lipid droplets, notably core (Boulant et al, 2006) and NS5A (Miyanari et al, 2007), and these organelles play a key, albeit uncharacterized, role in virus production (Miyanari et al, 2007;Huang et al, 2007;Ye, 2007).…”

“…Vps4 dissociates ESCRT complexes from the MVB membrane and allows them to recycle for further rounds of vesicle formation. A DN ATPase-defective mutant of Vps4, in which the active site glutamic acid has been mutated to glutamine, leads to defective MVB sorting and formation of aberrant endosomes in BHK cells (Bishop & Woodman, 2000) and also in Huh7 cells. Production of infectious particles of HIV-1 (Garrus et al, 2001), HSV-1 (Crump et al, 2007), HBV (Lambert et al, 2007) and ASV (Pincetic et al, 2008) is inhibited upon expression of the Vps4DN protein.…”

Regulation of Epidermal Growth Factor Receptor Down-Regulation by UBPY-mediated Deubiquitination at Endosomes

“…SKD1 is a mammalian orthologue of yeast Vps4, an AAA-type ATPase that also participates in the endosomal sorting of ubiquitinated membrane proteins . Overexpression of SKD1 E235Q , a dominant-negative SKD1 mutant lacking ATPase activity, causes the accumulation of endo- somal proteins on morphologically aberrant endosomes (Bishop and Woodman, 2000;Yoshimori et al, 2000). In SKD1 E235Q -overexpressing cells, UBPY also localized to the SKD1 E235Q -positive aberrant endosomes ( Figure 5, D-DЉ, arrowheads).…”

“…UBPY colocalized with overexpressed Hrs on the aberrant endosomes ( Figure 5). Second, yeast Vps4 and its mammalian orthologue SKD1 are AAA-type ATPases dysfunction of which results in the accumulation of endocytosed receptors as well as proteins of the endosomal Ub-sorting machinery on aberrant endosomes (Bishop and Woodman, 2000;Yoshimori et al, 2000;Katzmann et al, 2002). UBPY was also accumulated on aberrant endosomes in cells overexpressing SKD1 E235Q , a dominant-negative SKD1 mutant lacking ATPase activity ( Figure 5).…”

Chloroquine-resistant isoforms of the Plasmodium falciparum chloroquine resistance transporter acidify lysosomal pH in HEK293 cells more than chloroquine-sensitive isoforms

“…Co-expression of a dominant negative tetraspanin mutant, CD63-AEVM [39], did not alter PfCRT targeting to the lysosome (not shown). We noted that the P. falciparum genome sequence [40] contains homologues of human vacuolar protein sorting protein VPS4 [41,42] and human sorting nexin 4 (SNX4) [43]. Co-expression of dominant negative mutants of either of these proteins did not perturb PfCRT sorting to the lysosome.…”