A conserved late endosome-targeting signal required for Doa4 deubiquitylating enzyme function.
Enzyme specificity in vivo is often controlled by subcellular localization. Yeast Doa4, a deubiquitylating enzyme (DUB), removes ubiquitin from membrane proteins destined for vacuolar degradation. Doa4 is recruited to the late endosome after ESCRT-III (endosomal sorting complex required for transport III) has assembled there. We show that an N-terminal segment of Doa4 is sufficient for endosome association. This domain bears four conserved elements (boxes A-D). Deletion of the most conserved of these, A or B, prevents Doa4 endosomal localization. These mutants cannot sustain ubiquitin-dependent proteolysis even though neither motif is essential for deubiquitylating activity. Ubiquitin-specific processing protease 5 (Ubp5), the closest paralogue of Doa4, has no functional overlap. Ubp5 concentrates at the bud neck; its N-terminal domain is critical for this. Importantly, substitution of the Ubp5 N-terminal domain with that of Doa4 relocalizes the Ubp5 enzyme to endosomes and provides Doa4 function. This is the first demonstration of a physiologically important DUB subcellular localization signal and provides a striking example of the functional diversification of DUB paralogues by the evolution of alternative spatial signals.
Amerik
Alexander
A
Department of Pharmacology, University of Connecticut Health Center, Farmington, CT 06030, USA. amerik@uchc.edu
Sindhi
Nazia
N
Hochstrasser
Mark
M
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D056827:Endosomal Sorting Complexes Required for Transport; D011992:Endosomes / Q000201:enzymology / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D008969:Molecular Sequence Data; D010641:Phenotype; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D025801:Ubiquitin / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase; D033921:Vesicular Transport Proteins / Q000378:metabolism
C480237:Bro1 protein, S cerevisiae; C084434:DOA4 protein, S cerevisiae; D056827:Endosomal Sorting Complexes Required for Transport; D021382:Protein Sorting Signals; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D033921:Vesicular Transport Proteins; D010450:Endopeptidases; D043222:Ubiquitin Thiolesterase