Attachment of proteins to ubiquitin is reversed by specialized proteases ca
lled deubiquitinating enzymes (Dubs), which are also essential for ubiquiti
n precursor processing. In the genome of Saccharomyces cerevisiae, 17 poten
tial DUE genes can be discerned. We have now constructed strains deleted fo
r each of these genes. Surprisingly, given the essential nature of the ubiq
uitin system, none of the mutants is lethal or strongly growth defective un
der standard conditions, although a number have detectable abnormalities. I
ncluding 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 proteasom
e-dependent proteolysis. To help determine what distinguishes Doa4 function
ally from other Dubs, we have cloned a DOA4 ortholog from the yeast Kluyver
omyces lactis. The K. lactis protein is 42% identical to Doa4, but unexpect
edly 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 a
nd S. cerevisiae. This information will facilitate fine-structure analysis
of the Doa4 protein to help delineate its key functional elements.