Fig. 4: Ubp6-AA and Rpt1-RK mutants are impaired in the Ubp6 noncatalytic effect.
From: Allosteric control of Ubp6 and the proteasome via a bidirectional switch
a In vitro degradation assay performed with C118A or the C118A I329A L330A triple mutant of Ubp6, proteasome, and HA-Ubn-NCB1 (detected with anti-HA antibody). Ubp6 and Rpn8 are loading controls. b Inhibition of HA-Ubn-NCB1 degradation by Ubp6-UbVME. UbVME was preincubated with Ubp6 variants to covalently modify C118, abolishing deubiquitinating activity. The assay was otherwise as in a. c HA-Ubn-NCB1 degradation by wild-type and mutant proteasomes. d As c but with Ubp6-UbVME added. All in vitro assays have been independently repeated. e In vivo assay of the Ubp6 noncatalytic effect. Plasmids expressing variants of Ubp6 were transformed into either ubp6Δ rpn4Δ or ubp6Δ yeast strains, and colony formation was recorded after incubation at 30 °C for 3–4 days. Loss of noncatalytic activity was seen in ubp6-C118A-AA. f The noncatalytic effect of ubp6-C118A is abrogated by the rpt1-RK mutation.
