Figure 3.

Overexpression of α1 alters the composition of the CP in pba3Δ cells. (a) Cell extracts of WT or pba3Δ yeast expressing empty vector (EV) or α1 from a high-copy plasmid were separated by non-denaturing PAGE and immunoblotted with antibodies against the α4 subunit (left) and the RP base subunit Rpt1 (right). The positions of doubly-capped CP (RP₂CP), singly-capped CP (RP₁CP), RP, and the RP base are shown. Shown to the right is quantification of α4 levels in fully assembled proteasomes (RP₂CP) normalized to the corresponding RP₂CP band in the Rpt1 blot (n = 8; error bars = s.e.m.; ns = not significant, p = 0.9932). **, CP assembly intermediates. Full-length blot is presented in Supplementary Fig. S8. (b) Workflow of SILAC analysis with resulting peptide ion intensity ratios of CP subunits. Cells lacking PBA3 and harboring the indicated high-copy plasmids were metabolically labeled as described in Materials and Methods and lysed. Equal amounts of protein were mixed before immunoaffinity purification of the CP for LC-MS/MS analysis. The average peptide ion intensity ratios (α1: empty vector) for each CP subunit are shown, with the α4 subunit highlighted in red. Error bars indicate the s.d. of the intensity ratios of the CP subunits. EV, empty vector. Full-length gel is presented in Supplementary Fig. S8.