FIGURE 8.

Analysis of a stable mutant, S215A. A, degradation of Ser-215 variants. B, S215A can enter the HRD pathway when Hrd1 is constitutively overexpressed (OE Hrd1) from the strong TDH3 promoter. EV, empty vector control plasmid. C, S215A cannot be regulated when Hrd1 is overexpressed. The stability (A, CHX) and regulated degradation (C, Lova) of each Hmg2-GFP variant were assayed as in Fig. 2 in strains expressing Hrd1 from the strong TDH3 promoter or in strains carrying an empty vector control plasmid. D, Ser-215 variants show reduced ubiquitination. Log-phase cultures were treated with ZA for 10 min (or untreated). Hmg2 proteins were isolated by anti-GFP immunoprecipitation and separated by SDS-PAGE, and then immunoblotting for ubiquitin and GFP was done. E, S215A structure does not change when degradation signal is present. Dynamics of Hmg2 structure were analyzed by an in vitro trypsinolysis assay (22, 25) whereby microsomes (treated with farnesol (FOH) or untreated) were treated with trypsin, and samples were taken at different times. Proteins were resolved by SDS-PAGE, and then immunoblotting was performed for the lumenal Myc epitope present in the Hmg2-GFP variants.