FIG. 9.

Suppression of the small Ub-containing peptides of doa4 cells by mutation in NPI1/RSP5 or overexpression of UbK63R. (A) 27061b (WT), 27064b (npi1), 27081a (npi2), or 33276d (npi1 npi2) cells were grown in YNB plus glucose. Cells were collected in exponential growth phase and used to prepare total protein extracts (NaOH-TCA technique). Aliquots were analyzed by Western immunoblotting using an antibody against Ub. The positions of Ub, di-Ub (Ub2), and tri-Ub (Ub3) are indicated. Small Ub peptides above di-Ub characteristic of doa4 cells are indicated (∗). (B) 27061b (WT) transformed with YEp96 (2 μm TRP1 CUP1-UB), and 27081a (npi2) cells transformed with YEp96, YEp96-UbK29, 48R, or YEp96-UbK63R were grown in YNB with glucose as the carbon source and induced for 2 h with CuSO₄. Cells were collected and used to prepare protein extracts analyzed as in panel A.