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.
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4 December 2006
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December 04 2006
A conserved late endosome–targeting signal required for Doa4 deubiquitylating enzyme function
Alexander Amerik,
Alexander Amerik
1Department of Pharmacology, University of Connecticut Health Center, Farmington, CT 06030
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Nazia Sindhi,
Nazia Sindhi
1Department of Pharmacology, University of Connecticut Health Center, Farmington, CT 06030
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Mark Hochstrasser
Mark Hochstrasser
2Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520
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Alexander Amerik
1Department of Pharmacology, University of Connecticut Health Center, Farmington, CT 06030
Nazia Sindhi
1Department of Pharmacology, University of Connecticut Health Center, Farmington, CT 06030
Mark Hochstrasser
2Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520
Correspondence to Alexander Amerik: [email protected]; or Mark Hochstrasser: [email protected]
Abbreviations used in this paper: CPS, carboxypeptidase S; DUB, deubiquitylating enzyme; ESCRT, endosomal sorting complex required for transport; HAUSP, herpesvirus-associated ubiquitin-specific protease; IsoT, isopeptidase T; LEL, late endosome localization; MVB, multivesicular body; pCPS, CPS precursor; RHD, rhodanese homology domain; UBP, ubiquitin-specific processing protease; VPS, vacuolar protein sorting.
Received:
May 22 2006
Accepted:
October 31 2006
Online ISSN: 1540-8140
Print ISSN: 0021-9525
The Rockefeller University Press
2006
J Cell Biol (2006) 175 (5): 825–835.
Article history
Received:
May 22 2006
Accepted:
October 31 2006
Citation
Alexander Amerik, Nazia Sindhi, Mark Hochstrasser; A conserved late endosome–targeting signal required for Doa4 deubiquitylating enzyme function . J Cell Biol 4 December 2006; 175 (5): 825–835. doi: https://doi.org/10.1083/jcb.200605134
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