The Oligomeric State of the Active Vps4 AAA ATPase

Abstract

The cellular ESCRT pathway drives membrane constriction toward the cytosol and effects membrane fission during cytokinesis, endosomal sorting, and the release of many enveloped viruses, including HIV. A component of this pathway, the AAA ATPase Vps4, provides energy for pathway progression. Although it is established that Vps4 functions as an oligomer, subunit stoichiometry and other fundamental features of the functional enzyme are unclear. Higher-order oligomers have thus far only been characterized for a Walker B mutant of Vps4 in the presence of ATP. Here, we report that although some mutant Vps4 proteins form dodecameric assemblies, active wild-type S. cerevisiae and S. solfataricus Vps4 enzymes can form hexamers in the presence of ATP and ADP, as assayed by size exclusion chromatography and equilibrium analytical ultracentifugation. The Vta1p activator binds hexameric yeast Vps4p without changing the oligomeric state of Vps4p, implying that the active Vta1p:Vps4p complex also contains a single hexameric ring. Additionally, we report crystal structures of two different archaeal Vps4 homologs, whose structures and lattice interactions suggest a conserved mode of oligomerization. Disruption of the proposed hexamerization interface by mutagenesis abolished the ATPase activity of archaeal Vps4 proteins and blocked Vps4p function in S. cerevisiae. These data challenge the prevailing model that active Vps4 is a double ring dodecamer, and argue that, like other type I AAA ATPases, Vps4 functions as a single ring with six subunits.

A Novel Mechanism of Regulating the ATPase VPS4 by Its Cofactor LIP5 and the Endosomal Sorting Complex Required for Transport (ESCRT)-III Protein CHMP5

“…Vps4 can exist in two quaternary structures, an inactive monomer/dimer and an active high-order oligomer (34). Vps4 oligomerization and its membrane recruitment represent major steps in Vps4 regulation (35), which are mediated by a host of proteins, including its cofactor Vta1 (LIP5) and its substrates the ESCRT-III proteins (36 -39).…”

Vps4 Stimulatory Element of the Cofactor Vta1 Contacts the ATPase Vps4 α7 and α9 to Stimulate ATP Hydrolysis

“…Vps4 ATPase activity is regulated in a number of manners. First, oligomerization is one mechanism regulating Vps4 ATPase activity (34,35,50,51). Second, ESCRT-III interactions with Vps4 enhance ATP hydrolysis by relieving autoinhibition of the AAA domain; this occurs via MIM-MIT interactions and ESCRT-III acidic region interactions with the Vps4 linker region (31,51,52).…”

“…Three residues (located on ␣7 and ␣9) of the small AAA domain were identified as a potential interaction surface: methionine 330, leucine 407, and lysine 411. Models of the Vps4 oligomer (49,50) place these residues on the outer surface of the proposed oligomer where they would be accessible to the Vta1 VSE. Vps4 mutants with these residues altered exhibited concentration-dependent ATPase activity comparable with that of WT Vps4 and were responsive to direct stimulation by ESCRT-III, indicating that these mutants do not globally compromise Vps4 structure and function.…”

Drosophila Vps4 promotes Epidermal growth factor receptor signaling independently of its role in receptor degradation

“…Vps2 also recruits the ATPase associated with a variety of cellular activities (AAA) protein Vps4, the only energy-utilizing ESCRT component (Hanson and Cashikar, 2012;Raiborg and Stenmark, 2009). Active Vps4 forms a hexameric complex that disassembles ESCRT-III, allowing recycling of its components, and also plays an active role in scission of the vesicle neck (Adell et al, 2014;Cashikar et al, 2014;Lata et al, 2008;Monroe et al, 2014;Mueller et al, 2012). In addition to their endocytic functions, ESCRT proteins, including Vps4, are required for cytokinesis, viral budding, protecting viral genomes from degradation, exosome secretion, receptor shedding on microvesicles, assembly of nuclear pore complexes, cholesterol transport and plasma membrane wound repair (Barajas et al, 2014;Choudhuri et al, 2014;Du et al, 2013;de Gassart et al, 2004;Jimenez et al, 2014;Morita, 2012;Nabhan et al, 2012;Tang, 2012;Webster et al, 2014).…”