FIGURE 6:

ERAD targeting of SZ* correlates with higher aggregation propensity. (A) Microsomes from wild-type yeast expressing SZ* under the control of the TEF promoter in either a CEN (low) or a 2μ (high) plasmid were treated at the indicated temperature and with 10% DDM. Protein residing in the supernatant (S) and pellet (P) fractions was analyzed after centrifugation at 18,000 × g for 30 min and immunolotting. Sec61 was used as a control. (B) Wild-type yeast lysate containing a soluble version of SZ* that lacks a TMD (“NBD2*”) was treated with 100 mM Na₂CO₃, followed by centrifugation as in A and immunoblotting. (C) ERAD dependence of SZ* at high expression was assayed by a cycloheximide chase at 26°C after treated with the proteasomal inhibitor MG132 or DMSO. Data represent the means ± SE of three independent experiments; *p < 0.05 and **p < 0.005. (D) The cellular localization of SZ* expressed at high level, and the position of the nucleus and ER was determined in both pdr5Δ and pep4Δ pdr5Δ cells by indirect immunofluorescence microscopy in the absence of proteasome inhibition as in Figure 2. Arrowheads denote the ER. Scale bar: 5 μm.