The Vps4p AAA ATPase regulates membrane association of a Vps protein complex required for normal endosome function.
Vps4p is an AAA-type ATPase required for efficient transport of biosynthetic and endocytic cargo from an endosome to the lysosome-like vacuole of Saccharomyces cerevisiae. Vps4p mutants that do not bind ATP or are defective in ATP hydrolysis were characterized both in vivo and in vitro. The nucleotide-free or ADP-bound form of Vps4p existed as a dimer, whereas in the ATP-locked state, Vps4p dimers assembled into a decameric complex. This suggests that ATP hydrolysis drives a cycle of association and dissociation of Vps4p dimers/decamers. Nucleotide binding also regulated the association of Vps4p with an endosomal compartment in vivo. This membrane association required the N-terminal coiled-coil motif of Vps4p, but deletion of the coiled-coil domain did not affect ATPase activity or oligomeric assembly of the protein. Membrane association of two previously uncharacterized class E Vps proteins, Vps24p and Vps32p/Snf7p, was also affected by mutations in VPS4. Upon inactivation of a temperature-conditional vps4 mutant, Vps24p and Vps32p/Snf7p rapidly accumulated in a large membrane-bound complex. Immunofluorescence indicated that both proteins function with Vps4p at a common endosomal compartment. Together, the data suggest that the Vps4 ATPase catalyzes the release (uncoating) of an endosomal membrane-associated class E protein complex(es) required for normal morphology and sorting activity of the endosome.
Babst
M
M
Division of Cellular Molecular Medicine and Howard Hughes Medical Institute, University of California at San Diego, School of Medicine La Jolla, CA 92093-0668, USA.
Wendland
B
B
Estepa
E J
EJ
Emr
S D
SD
D000251:Adenosine Triphosphatases / Q000378:metabolism / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000378:metabolism; D019281:Dimerization; D056827:Endosomal Sorting Complexes Required for Transport; D011992:Endosomes / Q000201:enzymology / Q000502:physiology*; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D007425:Intracellular Membranes / Q000201:enzymology*; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D009711:Nucleotides / Q000502:physiology; D011108:Polymers / Q000378:metabolism; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D013347:Subcellular Fractions / Q000201:enzymology
D056827:Endosomal Sorting Complexes Required for Transport; D005656:Fungal Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D009711:Nucleotides; D011108:Polymers; C083890:SNF7 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C107528:VPS4 protein, S cerevisiae; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases