Ubiquitin-specific proteases of Saccharomyces cerevisiae. Cloning of UBP2 and UBP3, and functional analysis of the UBP gene family.
In eukaryotes, both natural and engineered ubiquitin (Ub) fusions to itself or other proteins are cleaved by processing proteases after the last (Gly76) residue of ubiquitin. YUH1 and UBP1, the genes for two ubiquitin-specific proteases of the yeast Saccharomyces cerevisiae, have been cloned previously and shown to encode nonhomologous proteins. Using an Escherichia coli-based genetic screen, we have isolated two other yeast genes for ubiquitin-specific proteases, named UBP2 and UBP3. Ubp2 (1,264 residues), Ubp3 (912 residues), and the previously cloned Ubp1 (809 residues) are largely dissimilar except for two short regions containing Cys and His which encompass their putative active sites. Neither of these proteases has sequence similarities to Yuh1. Both Ubp2 and the previously identified Ubp1 cleave in vitro at the C terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their size, poly-Ub being the exception. However, both Ubp1 and Ubp2 are also capable of cleaving poly-Ub when coexpressed with it in E. coli, suggesting that such cleavage is largely cotranslational. Although inactive in E. coli extracts, Ubp3 was active with all of the tested ubiquitin fusions except poly-Ub when coexpressed with them in E. coli. Null yuh1 ubp1 ubp2 ubp3 quadruple mutants are viable and retain the ability to deubiquitinate ubiquitin fusions, indicating the presence of at least one more ubiquitin-specific processing protease in S. cerevisiae.
Baker
R T
RT
Division of Biology, California Institute of Technology, Pasadena 91125.
Tobias
J W
JW
Varshavsky
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular / Q000379:methods; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D010450:Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D005810:Multigene Family; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D012331:RNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D004271:DNA, Fungal; D009838:Oligodeoxyribonucleotides; D012331:RNA, Fungal; D011994:Recombinant Proteins; D010450:Endopeptidases; C059237:ubiquitin-Nalpha-protein hydrolase