Figure 6: Sem1 is a ubiquitylation substrate of Rsp5.

Screening of pCD–Sub yeast-fusion library revealed Sem1 as a potential ubiquitylation substrate of Rsp5 in the bacterial selection system. (a) Sem1 ubiquitylation. Bacterial spots, expressing a complete (cDHFR–Sem1, nDHFR–Ub, yeast Ubc4, and Rsp5) or incomplete (ΔE1, ΔE2, ΔUb or ΔE3) ubiquitylation apparatus, were seeded as in Figure 1. Growth of spots was monitored by a timelapse scan using an office scanner (Epson) in 30-min intervals. Scan time postseeding is indicated. (b) Growth curves derived from quantification of the scans using 'Time Series Analyzer' in Fiji. Values are average of eight spots with s.d. bars. (c) Detection of Sem1 ubiquitylation in E. coli. Purified His₆–MBP–Sem1 from E. coli that coexpress ubiquitylation apparatus¹⁷ was resolved on SDS–PAGE and detected by western blot with anti-His-tag antibody. WB, western blot. (d) His₆–Sem1 was expressed from Gal-inducible promoter in wild-type or temperature-sensitive rsp5-1 mutant yeast cells. Cultures grew at 25 °C (permissive conditions). Prior induction temperature was shifted to 37 °C (restrictive temperature) or remained as indicated. His₆–Sem1 was purified under denatured conditions, resolved and detected as in c.