Activation of Human VPS4A by ESCRT-III Proteins Reveals Ability of Substrates to Relieve Enzyme Autoinhibition

Abstract

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 ϳ50 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 acidtagged 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 liposomeenhanced 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. Multivesicular bodies (MVBs)2 are endosomes that contain intralumenal vesicles generated by invagination from the endosomal membrane. The MVB is an intermediate compartment en route to the lysosome; surface receptors destined for degradation are sorted into intralumenal vesicles and delivered to the lysosome. Impairment in this pathway leads to many cellular problems, including prolonged signaling of receptors normally silenced by internalization into the MVB (reviewed in Refs. 1-5). Four ESCRT (endosomal sorting complexes required for transport) complexes (ESCRT-0, -I, -II, and -III) are responsible for cargo sorting and vesicle formation in the MVB pathway. The ESCRT complexes also play essential roles in topologically similar membrane remodeling events including enveloped virus budding and cell abscission at the end of cytokinesis (6 -8).ESCRT-III differs from the other ESCRT complexes in that it is a loosely defined polymer that assembles when its subunits are recruited to the appropriate membrane and bind to each other (9). There are seven ESCRT-III-related proteins in yeast and 12 in human cells. Human ESCRT-III proteins are referred to either as orthologues of their yeast counterparts or as CHMPs (charged multivesicular body proteins). To standardize our discussion, we will primarily use th...

The Linker Region Plays a Regulatory Role in Assembly and Activity of the Vps4 AAA ATPase

“…To better understand the contributions of the linker region, biochemical characterization was undertaken. The analysis of recombinant Vps4 mutant proteins demonstrated that loss of the linker region caused an increase in maximal ATPase activity, a result that is consistent with the idea that the linker is involved in an auto-inhibition mechanism (21). However, the protein concentration necessary to induce oligomerization was higher for linker-deleted Vps4 than for the wild-type protein.…”

“…However, deleting both the MIT domain and the linker abolished Vps4 hydrolysis activity at the concentrations examined (Vps4(⌬1-116), Fig. 3B), which is in contrast to human VPS4A, where deletion of the corresponding residues (1-100) results in higher ATPase activity when compared with the wildtype protein (21). Our data suggested that the MIT domain and the linker affect both oligomerization and ATPase activity of the assembled Vps4 complex.…”

“…An additional layer of regulation has recently been proposed by studies of human Vps4. The deletion of the N-terminal MIT domain and adjacent linker resulted in increased ATPase activity of Vps4 in vitro, suggesting that these regions of the proteins are part of an auto-inhibitory mechanism that ensures Vps4 activation only when associated with the substrate ESCRT-III (21). In our study, we demonstrate that deletion of just the Vps4 linker region results in an active enzyme that is functional in vivo.…”

“…Substrate binding has been shown to increase the hydrolysis activity of Vps4 (16,21). Therefore, one possible model is that the Vps4 mutants assemble into a conformation that resembles the ESCRT-III bound state of Vps4.…”

Interactions of the Human LIP5 Regulatory Protein with Endosomal Sorting Complexes Required for Transport

“…VPS4 enzymes are recruited to their sites of action, at least in part, by binding to a variety of different and often partially redundant MIM elements that are exposed upon ESCRT-III filament formation (19,(32)(33)(34)(35)(36)(37)(38). In addition to these general ESCRT-III interactions, two binary complexes, CHMP1-IST1 and LIP5-CHMP5, play particularly important roles in VPS4 recruitment and activation.…”