Figure 7.

An enzyme closely related to Doa4 encoded by the yeast UBP5 (YER144c) gene. (A) Sequence alignment of Doa4 and Ubp5. The two proteins were aligned with the ClustalW algorithm followed by manual adjustment. Identical residues are boxed in black, and structurally related residues are boxed in gray. The Cys and His boxes are indicated by brackets, and the conserved Cys and His residues in these two motifs are marked by arrowheads. (B) Cleavage of a ubiquitin–protein fusion by Ubp5. Shown is an anti-βgal Western immunoblot analysis of extracts from MC1061 cells harboring a plasmid expressing Ub-M-βgal and either YCplac33 vector or YCplac33-UBP5. (C) Ubiquitin isopeptidase activity of Ubp5 using a Lys48-linked diubiquitin substrate. Substrate was incubated at 30°C in E. coli extracts expressing either GST or GST-Ubp5. Partially purified GST-Doa4 was used as a control.