title: Doa4 function in ILV budding is restricted through its interaction with the Vps20 subunit of ESCRT-III.
abstract: Assembly of the endosomal sorting complex required for transport (ESCRT)-III executes the formation of intralumenal vesicles (ILVs) at endosomes. Repeated cycles of ESCRT-III function requires disassembly of the complex by Vps4, an ATPase with a microtubule interaction and trafficking (MIT) domain that binds MIT-interacting motifs (MIM1 or MIM2) in ESCRT-III subunits. We identified a putative MIT domain at the N-terminus of Doa4, which is the ubiquitin (Ub) hydrolase in Saccharomyces cerevisiae that deubiquitinates ILV cargo proteins. The Doa4 N-terminus is predicted to have the α-helical structure common to MIT domains, and it binds directly to a MIM1-like sequence in the Vps20 subunit of ESCRT-III. Disrupting this interaction does not prevent endosomal localization of Doa4 but enhances the defect in ILV cargo protein deubiquitination observed in cells lacking Bro1, which is an ESCRT-III effector protein that stimulates Doa4 catalytic activity. Deletion of the BRO1 gene (bro1Δ) blocks ILV budding, but ILV budding was rescued upon disrupting the interaction between Vps20 and Doa4. This rescue in ILV biogenesis requires Doa4 expression but is independent of its Ub hydrolase activity. Thus, binding of Vps20 to the Doa4 N-terminus inhibits a non-catalytic function of Doa4 that promotes ILV formation.
authors: Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA.
authors: Caleb M
authors: CM
authors: Richter
authors: Matthew
authors: M
authors: West
authors: Greg
authors: G
authors: Odorizzi
mesh_terms: D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase / Q000737:chemistry / Q000378:metabolism*
chemical_list: C084434:DOA4 protein, S cerevisiae; D056827:Endosomal Sorting Complexes Required for Transport; D029701:Saccharomyces cerevisiae Proteins; D010450:Endopeptidases; D043222:Ubiquitin Thiolesterase
keywords: Deubiquitination; Multivesicular body; Vesicle budding