No abstract

The AAA؉ ATPase VPS4 plays an essential role in multivesicular body biogenesis and is thought to act by disassembling ESCRT-III complexes. VPS4 oligomerization and ATPase activity are promoted by binding to LIP5. LIP5 also binds to the ESCRT-III like protein CHMP5/hVps60, but how this affects its function remains unclear. Here we confirm that LIP5 binds tightly to CHMP5, but also find that it binds well to additional ESCRT-III proteins including CHMP1B, CHMP2A/ hVps2-1, and CHMP3/hVps24 but not CHMP4A/hSnf7-1 or CHMP6/hVps20. LIP5 binds to a different region within CHMP5 than within the other ESCRT-III proteins. In CHMP1B and CHMP2A, its binding site encompasses sequences at the proteins' extreme C-termini that overlap with "MIT interacting motifs" (MIMs) known to bind to VPS4. We find unexpected evidence of a second conserved binding site for VPS4 in CHMP2A and CHMP1B, suggesting that LIP5 and VPS4 may bind simultaneously to these proteins despite the overlap in their primary binding sites. Finally, LIP5 binds preferentially to soluble CHMP5 but instead to polymerized CHMP2A, suggesting that the newly defined interactions between LIP5 and ESCRT-III proteins may be regulated by ESCRT-III conformation. These studies point to a role for direct binding between LIP5 and ESCRT-III proteins that is likely to complement LIP5's previously described ability to regulate VPS4 activity.

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 ESCRT (endosomal sorting complex required for transport) machinery comprises a set of protein complexes that regulate sorting and trafficking into multivesicular bodies en route to the lysosome. The physical mechanism responsible for generating lumenal vesicles in this pathway is unknown. Here we review recent studies suggesting that components of the ESCRT-III complex drive lumenal vesicle formation and consider possible mechanisms for this reaction.

National Heart Lung and Blood Institute grants T32HL007525 and R01HL133113.