FIG. 6.
Cln2 stabilization and ubiquitination in an rpn3 mutant. (A) Control YE110 (RPN3) cells and rpn3 mutant YE111 (rpn3-4) cells, both with an HA-tagged CLN2 gene fused to the GAL1 promoter, were grown in YEPR medium and arrested in G1 with α-factor. The cultures were then shifted to 37°C for an additional hour and induced to express Cln2. After 45 min, Cln2 induction was terminated by returning the cells to prewarmed pheromone-free glucose-containing medium. Aliquots were taken before (lane −45) and after (lane 0) Cln2 induction and every 20 min after glucose repression. HA-tagged Cln2 and Rpt1 (used as a loading control) were immunodetected with anti-HA and anti-Rpt1 antibodies, respectively (top panel; Cln2-HA3 and Rpt1). CLN2-HA3 mRNAs were detected by Northern blot analysis (middle panel). The bottom panel shows ethidium bromide staining of the corresponding gel as a control for equivalent loading of total RNA in the different lanes. Quantification of HA-tagged Cln2 immunoreactivity at different times as measured by densitometry is shown in the graph. (B) Cln2-ubiquitin conjugates accumulate in the rpn3 mutant. Wild-type (strain YE110), rpn3-4 (strain YE111), and cdc34-2 (strain YE471) cells expressing HA-tagged Cln2 under the control of the GAL1 promoter were arrested in G1 with α-factor and then shifted to 37°C to inactivate Rpn3 and Cdc34. Cln2 expression was induced for 30 min by the addition of galactose. As a control for untagged Cln2, we used rpn3-54 strain YE231 (no tag). HA-tagged Cln2 was immunoprecipitated (IP) with a rabbit serum directed against the HA epitope; half of the immunoprecipitate was probed for ubiquitin conjugates by immunoblotting with a monoclonal antibody to polyubiquitin (top right panel), while the other half was checked for HA-tagged Cln2 by Western blotting (WB) with the anti-HA epitope monoclonal antibody 12CA5 (bottom right panel). Cell lysates used for the immunoprecipitation were separated by SDS-PAGE and analyzed with the antipolyubiquitin [anti-(Ub)n] monoclonal antibody to detect total cellular polyubiquitinated proteins (left panel).