Figure 5. Acm1 proteolysis does not require ubiquitin acceptor sites on Acm1.

A) YKA247 cells carrying P_GAL1 expression plasmids for 3FLAG-Cdh1, 3HA-Acm1, HA-Acm1-ken, and 3HA-Acm1^K0 in the indicated combinations were grown until mid-exponential phase. 10-fold serial dilutions were spotted on selective media containing either glucose or galactose. Plates were incubated at 30°C for 2–3 days. B) and C) YKA247 cells carrying P_GAL1 expression plasmids for 3HA-Acm1 (pHLP117) or lysine-less 3HA-Acm1^K0 mutant (pHLP330) were grown in YP-raffinose to early exponential phase. Cells were arrested at G1 (panel B) or S phase (panel C). Stability of 3HA-Acm1 and 3HA-Acm1^K0 was monitored over the indicated time period by immunoblotting with anti-HA antibody. G6PDH is a loading control. D) Same as panels B and C, except pdr5Δ cells were used and stability was monitored in the presence and absence of MG-132 as indicated. Longer immunoblot exposures were obtained for detection of ubiquitin (Ub) conjugates. E) cdc15-2 cells carrying centromeric plasmids expressing either wild-type 3HA-Acm1 or 3HA-Acm1^K0 from the natural ACM1 promoter were arrested at the indicated cell cycle stages as described in Materials and Methods. The level of each protein was then compared by anti-HA immunoblotting with G6PD as a loading control.