Rapid proteasomal degradation of Pex5pC6K variants
Mutant pex5Δ cells or ubp15Δpex5Δ cells were transformed with plasmids expressing Pex5p or indicated variants. Strains were grown on oleic acid containing medium for 16 h. Subsequently cells where shifted to oleic acid containing medium with or without 40 μm MG132 to inhibit the proteasome and grown for additional 4 h. (A) Whole-cell lysates of the strains were prepared and blotted for the presence of Pex5p and mitochondrial porin which served as loading control. (B) Pex5p signal intensities of blots depicted in (A) obtained from samples without (white boxes) and with (black boxes) MG132 treatment was estimated by densitometry. Intensities were normalized to signal from plasmid-encoded wild-type Pex5p expressed in pex5Δ cells or ubp15Δ pex5Δ cells respectively, without MG132 treatment. In contrast with untreated cells, Pex5pC6K and Pex5pC6K/K18R/K24R remained stable when the proteasome was inhibited, indicating that the observed reduced steady-state concentrations are due to a rapid proteasomal degradation. Error bars=S.E.M. with n = 3.