The multivesicular body (MVB) pathway is an important biological process that functions in a number of different physiological and pathological settings in the eukaryotic cells, including down-regulation of cell surface receptor signaling, enveloped virus budding and abscission during cytokinesis. The AAA ATPase Vps4 plays a pivotal role in this pathway by catalyzing structural remodeling of the membrane-associated Endosomal Sorting Complexes Required for Transport (ESCRT) machinery. This activity leads to the recycling of the ESCRT machinery and may also be linked to membrane fission. The active form of Vps4 is a double-ring dodecameric structure in the presence of ATP. The crystal structure of S. cerevisiae Vps4 shows the protein contains a canonical AAA domain fold and undergoes conformational change upon nucleotide binding, providing a possible mechanism of oligomerization. Positive regulators Vta1, Vps60 and Did2 and negative regulator Ist1 further modulate the activity of Vps4 in the cell. Structural and biochemical analyses of these proteins and their interactions with Vps4 provide further insights into their mechanisms of action. Vta1 is organized into two well-folded domains connected by a flexible linker region. The dimeric C-terminal domain is responsible for Vps4 binding. It stimulates the ATPase activity of Vps4 by stabilizing the double-ring structure. The N-terminal domain contains two tandem MIT domains previously seen in the N-terminal ESCRT-III-binding domain of Vps4. The second MIT domain contains Vps60 and Did2 binding activity, and these interactions further stimulate the Vps4 activity in a Vta1-dependent manner. Ist1 contains a well-folded N-terminal domain and a C-terminal MIT-interacting motif. The crystal structure of the N-terminal domain of Ist1 reveals it resembles the fold of an ESCRT-III protein, suggesting that Ist1 might inhibit Vps4 activity by acting as a substrate mimic. Ist1 also interacts with Did2 via a novel binding mechanism and this interaction is important for both yeast vacuolar sorting and mammalian cytokinesis. Taken together, these results enable us to better understand the molecular mechanism of Vps4 function and its regulation in the MVB pathway
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