ATPase-defective mammalian VPS4 localizes to aberrant endosomes and impairs cholesterol trafficking.
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.
Bishop
N
N
School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom.
Woodman
P
P
D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002451:Cell Compartmentation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism*; D006224:Cricetinae; D056827:Endosomal Sorting Complexes Required for Transport; D011992:Endosomes / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D025262:Vacuolar Proton-Translocating ATPases; D033921:Vesicular Transport Proteins
D056827:Endosomal Sorting Complexes Required for Transport; D005656:Fungal Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C107528:VPS4 protein, S cerevisiae; D033921:Vesicular Transport Proteins; D002784:Cholesterol; D000251:Adenosine Triphosphatases; D025262:Vacuolar Proton-Translocating ATPases; D000074183:ATPases Associated with Diverse Cellular Activities; C490305:VPS4A protein, human; C089217:Vps4b protein, mouse