Tang, J.; Khamari, L.; Moon, S.; Walter, N. G.

Valosin-containing protein (VCP/p97) is an essential homohexameric AAA+ ATPase that powers ubiquitin-dependent protein quality control by extraction and unfolding of clients for proteasomal degradation. Heterozygous, autosomal-dominant VCP missense mutants are associated with multisystem proteinopathy (MSP) through unclear molecular mechanisms. We developed a single-molecule pull-down assay to quantify VCP hexamer assembly and subunit exchange dynamics directly in human cell lysate. We show the common MSP-associated VCP variant R155H co-assembles with wild-type subunits to form heterohexamers. Wild-type VCP complexes readily undergo subunit exchange in cell lysates, but this exchange is markedly reduced for purified complexes in buffer. We identify the VCP-interactor ASPL as a selective mediator of monomer exchange that efficiently remodels wild-type, but only modestly exchanges R155H variants within multimers. Single-molecule kinetics analyses reveal ~2-fold faster ASPL association with, and ~4-fold slower dissociation from, wild-type VCP than R155H-VCP. We propose that ASPL-driven monomer exchange remodels VCP molecular machines to sustain proteostasis. The failure of ASPL-driven exchange of MSP variant monomers would be predicted to stabilize mutant VCP in assemblies, revealing a potentially targetable defect.