FIGURE 1.

Orphan Fas2 is proteolytically susceptible in vitro and its degradation depends on the 26 S proteasome in vivo. A, in vitro trypsin sensitivity assay. Native cell extracts from wild type (FAS1 FAS2) and Δfas1 cells expressing endogenous Fas2 were prepared as described under “Experimental Procedures” and treated with (+) or without (−) trypsin. Samples were taken at the indicated time points, and proteins were precipitated, separated, and analyzed by SDS-PAGE/Western blotting using Fas antibody, which detects Fas1 and Fas2 subunits. Endogenously expressed PGK was used as a control. B, in vivo degradation of orphan Fas2 is dependent on the 26 S proteasome. Cycloheximide-chase experiments of endogenously expressed Fas2 were performed as described under “Experimental Procedures” in the proteasomal mutant cim3-1 Δfas1 and in the respective control strain (CIM3 Δfas1). Samples were taken at the indicated time points and subjected to SDS-PAGE and Western blot analysis. Immunoblots were analyzed with Fas and PGK antibody. PGK served as loading control. The data represent the means of two independent experiments. Error bars indicate the standard deviation of the mean.