Dual mechanisms specify Doa4-mediated deubiquitination at multivesicular bodies.
Doa4 is a ubiquitin-specific protease in Saccharomyces cerevisiae that deubiquitinates integral membrane proteins sorted into the lumenal vesicles of late-endosomal multivesicular bodies (MVBs). We show that the non-catalytic N terminus of Doa4 mediates its recruitment to endosomes through its association with Bro1, which is one of several highly conserved class E Vps proteins that comprise the core MVB sorting machinery. In turn, Bro1 directly stimulates deubiquitination by interacting with a YPxL motif in the catalytic domain of Doa4. Mutations in either Doa4 or Bro1 that disrupt catalytic activation of Doa4 impair deubiquitination and sorting of MVB cargo proteins and lead to the formation of lumenal MVB vesicles that are predominantly small compared with the vesicles seen in wild-type cells. Thus, by recruiting Doa4 to late endosomes and stimulating its catalytic activity, Bro1 fulfills a novel dual role in coordinating deubiquitination in the MVB pathway.
Richter
Caleb
C
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA.
West
Matthew
M
Odorizzi
Greg
G
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D015153:Blotting, Western; D020134:Catalytic Domain; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport; D011992:Endosomes / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D047468:Immunoprecipitation; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D014057:Tomography, X-Ray Computed; D022161:Transport Vesicles / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*
C084434:DOA4 protein, S cerevisiae; D056827:Endosomal Sorting Complexes Required for Transport; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D033921:Vesicular Transport Proteins; D010450:Endopeptidases; D043222:Ubiquitin Thiolesterase