Figure 6.
Swe1 in vitro ubiquitination. (A) Swe1 is ubiquitinated in whole cell extracts. 35S-labeled Swe1 was incubated with whole yeast cell extracts at 23°C (lanes 2,3) or on ice (lane 1) in the presence of an ATP-regenerating system and ubiquitin (lanes 1,2) or mutant R48-ubiquitin (lane 3), respectively. The reaction mix was separated by SDS-PAGE and analyzed with a PhosphorImager. (B) Swe1 ubiquitination is defective in cdc34 and met30 mutants. In vitro ubiquitination activity of extracts prepared from cdc34-3 or met30-6 mutants was compared with wild-type extracts. (Lane 5) Complementation experiment in which both mutant extracts were mixed (ratio, 2:1 = cdc34-3:met30-6). Extracts were preincubated at 23°C for 5 min to allow complex formation. 35S-labeled Swe1, ATP-regenerating system and ubiquitin were then added and reactions incubated at 23°C (lanes 2– 6) or on ice (lane 1). No extract was added to the reaction shown in lane 6. Reaction products were analyzed as described in A.