FIGURE 5:

Der1 function and degradation rate is modulated by its N-terminal sequence. (A) A C-terminally HA-tagged WT Der1 that begins with the sequence Met-Asp (MD) is degraded slowly in NAT3 cells but is destabilized ∼2- to 2.5-fold in a nat3Δ strain. A mutant Der1 (MK) with a Lys at the second position is strongly destabilized (the anti-HA blot required prolonged exposure compared with the others), whereas the ML mutant is degraded at the same slow rate as WT Der1 (bottom). Der1 amounts for each time point in the plot were normalized to PGK levels. (B) The ML-Der1-HA protein is functional in ERAD-L even when not acetylated (mak3Δ cells). A low-copy plasmid expressing ML-Der1-HA from the DER1 promoter was transformed into cells of the indicated genotypes. PGK, loading control.