A conserved late endosome-targeting signal required for Doa4 deubiquitylating enzyme function

J Cell Biol. 2006 Dec 4;175(5):825-35. doi: 10.1083/jcb.200605134.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Conserved Sequence
  • Endopeptidases / genetics
  • Endopeptidases / metabolism
  • Endopeptidases / physiology*
  • Endosomal Sorting Complexes Required for Transport
  • Endosomes / enzymology
  • Endosomes / metabolism*
  • Intracellular Membranes / metabolism
  • Molecular Sequence Data
  • Phenotype
  • Protein Sorting Signals*
  • Protein Structure, Tertiary
  • Protein Transport
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Saccharomyces cerevisiae Proteins / physiology*
  • Sequence Homology, Amino Acid
  • Ubiquitin / metabolism*
  • Ubiquitin Thiolesterase
  • Vesicular Transport Proteins / metabolism

Substances

  • Bro1 protein, S cerevisiae
  • DOA4 protein, S cerevisiae
  • Endosomal Sorting Complexes Required for Transport
  • Protein Sorting Signals
  • Saccharomyces cerevisiae Proteins
  • Ubiquitin
  • Vesicular Transport Proteins
  • Endopeptidases
  • Ubiquitin Thiolesterase