Figure 6.

Yeast mutagenesis experiments. (a) Drop test of mutant yeast strains. Indicated cell numbers were spotted on YPD plates and incubated for 1–3 days either at 30 or 37 °C. Growth of the yeast β2 (pup1) G170A-ProA-H₇ mutant is retarded compared to the respective wild type strains and compared to the β2 loss of function mutant T1A. (b) Overexpression of mutant yeast β2-subunits. Wild type yeast (WCG4a) was transformed by either the empty 2 µ plasmid pRS425 or variants thereof encoding wild type PUP1 or mutant pup1 genes. Yeasts were streaked on CM Leu⁻ plates (to select for the plasmid pRS425) and grown at 30 °C. (c) Western blot of equal amounts of β2 (pup1) G170A-ProA-H₇ and β2 (PUP1)-ProA-H₇ cell lysates separated by SDS-PAGE (upper panel) or native PAGE (lower panel). Anti-His immunoblotting of denatured lysates reveals that the mutant accumulates immature β2 (pro-β2), while in wild type cells the processed version of β2 is more abundant. In agreement, probing native lysates with an anti-β7 antibody visualises the accumulation of half 20S proteasomes in the mutant. Note, β7 is the last proteasome subunit that is incorporated into half proteasome precursors. Full-length western blots are presented in Supplementary Fig. S6.