Activation of human VPS4A by ESCRT-III proteins reveals ability of substrates to relieve enzyme autoinhibition.
VPS4 proteins are AAA(+) ATPases required to form multivesicular bodies, release viral particles, and complete cytokinesis. They act by disassembling ESCRT-III heteropolymers during or after their proposed function in membrane scission. Here we show that purified human VPS4A is essentially inactive but can be stimulated to hydrolyze ATP by ESCRT-III proteins in a reaction that requires both their previously defined MIT interacting motifs and ∼50 amino acids of the adjacent sequence. Importantly, C-terminal fragments of all ESCRT-III proteins tested, including CHMP2A, CHMP1B, CHMP3, CHMP4A, CHMP6, and CHMP5, activated VPS4A suggesting that it disassembles ESCRT-III heteropolymers by affecting each component protein. VPS4A is thought to act as a ring-shaped cylindrical oligomer like other AAA(+) ATPases, but this has been difficult to directly demonstrate. We found that concentrating His(6)-VPS4A on liposomes containing Ni(2+)-nitrilotriacetic acid-tagged lipid increased ATP hydrolysis, confirming the importance of inter-subunit interactions for activity. We also found that mutating pore loops expected to line the center of a cylindrical oligomer changed the response of VPS4A to ESCRT-III proteins. Based on these data, we propose that ESCRT-III proteins facilitate assembly of functional but transient VPS4A oligomers and interact with sequences inside the pore of the assembled enzyme. Deleting the N-terminal MIT domain and adjacent linker from VPS4A increased both basal and liposome-enhanced ATPase activity, indicating that these elements play a role in autoinhibiting VPS4A until it encounters ESCRT-III proteins. These findings reveal new ways in which VPS4 activity is regulated and specifically directed to ESCRT-III polymers.
Merrill
Samuel A
SA
Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, Missouri 63110, USA.
Hanson
Phyllis I
PI
D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D056827:Endosomal Sorting Complexes Required for Transport / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008081:Liposomes / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D009942:Organometallic Compounds / Q000737:chemistry; D011485:Protein Binding; D055503:Protein Multimerization; D013379:Substrate Specificity; D025262:Vacuolar Proton-Translocating ATPases
D056827:Endosomal Sorting Complexes Required for Transport; D008081:Liposomes; D009942:Organometallic Compounds; C088321:nickel nitrilotriacetic acid; D006639:Histidine; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D025262:Vacuolar Proton-Translocating ATPases; D000074183:ATPases Associated with Diverse Cellular Activities; C490305:VPS4A protein, human; D009571:Nitrilotriacetic Acid
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