FIGURE 6.

Degradation pathways of Hmg2 and Nsg1. A, steady-state 1mycHmg2 and Nsg1–3HA protein levels in WT and hrd1Δ cells treated with the series of sterol pathway inhibitors. Log phase cultures of WT and hrd1Δ cells were treated with inhibitors for 4 h at 30 °C as in Fig. 2A, lysed, and subjected to SDS-PAGE followed by immunoblotting for 1mycHmg2, Nsg1–3HA, and Pgk1 (load control). The experiment was performed at least three times. B, in vivo ubiquitination of Nsg1 in untreated and terbinafine-treated cells. 20 OD units of wild-type, hrd1Δ, or nsg1Δ cells from log phase cultures were treated with Tb (10 μg/ml) and MG132 (25 μg/ml) or control-treated (ethanol is the control vehicle for Tb, and DMSO is the control vehicle for MG132) for 2 h at 30 °C. Cells were lysed with SUME, and Nsg1–3HA was immunoprecipitated (IP) from clarified lysates with anti-HA affinity matrix. Following SDS-PAGE, ubiquitinated Nsg1–3HA was detected by anti-ubiquitin immunoblotting. The bottom panel shows the amount of Nsg1–3HA pulled down in each sample. The experiment was performed at least three times with similar results. C, degradation of TDH3-Nsg1–3HA occurs in hrd1Δ and doa10Δ mutants. Cells expressing Nsg1–3HA in excess of Hmg2 (by virtue of TDH3 promoter) were grown to log phase, and then the CHX chase assay was performed. At the indicated time points, samples were harvested and lysed, and then SDS-PAGE immunoblotting was performed. Similar experiments were performed at least five times. Lo, lovastatin; Ro, Ro48-8071; Ket, ketoconazole; Fen, fenpropimorph.