Analysis of the deubiquitinating enzymes of the yeast Saccharomyces cerevisiae.
Attachment of proteins to ubiquitin is reversed by specialized proteases called deubiquitinating enzymes (Dubs), which are also essential for ubiquitin precursor processing. In the genome of Saccharomyces cerevisiae, 17 potential DUB genes can be discerned. We have now constructed strains deleted for each of these genes. Surprisingly, given the essential nature of the ubiquitin system, none of the mutants is lethal or strongly growth defective under standard conditions, although a number have detectable abnormalities. Including results from this study, 14 of the 17 Dubs have now been shown to have ubiquitin-cleaving activity. The most extensively characterized yeast Dub is Doa4, which is required for both ubiquitin homeostasis and proteasome-dependent proteolysis. To help determine what distinguishes Doa4 functionally from other Dubs, we have cloned a DOA4 ortholog from the yeast Kluyveromyces lactis. The K. lactis protein is 42% identical to Doa4, but unexpectedly the K. lactis gene is slightly closer in nucleotide sequence to UBP5, which cannot substitute for DOA4 even in high dosage. The data suggest that the DOA4 locus underwent a duplication after the divergence of K. lactis and S. cerevisiae. This information will facilitate fine-structure analysis of the Doa4 protein to help delineate its key functional elements.
Amerik
A Y
AY
Yale University, Department of Molecular Biophysics and Biochemistry, New Haven, CT 06520, USA.
Li
S J
SJ
Hochstrasser
M
M
D000595:Amino Acid Sequence; D004269:DNA, Bacterial / Q000235:genetics; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D015723:Gene Library; D015151:Immunoblotting; D007716:Kluyveromyces / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D043222:Ubiquitin Thiolesterase; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism*
D004269:DNA, Bacterial; C084434:DOA4 protein, S cerevisiae; D056827:Endosomal Sorting Complexes Required for Transport; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; D010450:Endopeptidases; D043222:Ubiquitin Thiolesterase