FIG. 6.

rpt1 is defective in degrading nascent proteins. (A) Wild-type and rpt1 strains were subjected to ATP depletion conditions. In Fig. 5, yeast cells were grown at 30°C, while in the experiments described here, yeast cells were grown at the nonpermissive temperature for rpt1 (37°C for 6 h). Pre1-FLAG was precipitated and the levels of eEF1A in the proteasome were threefold lower in rpt1 than in the wild-type strain. (B) The same immunoblot was incubated with antibodies against βgal, and the level of Met-βgal in the proteasome was determined. (C) The levels of Met-βgal in cell extracts were determined. Note the much faster elimination of Met-βgal in this study (37°C) than in Fig. 5A (30°C). Essentially the same βgal cross-reacting bands were detected in the extract and in the proteasome (compare bands in panels B and C). (D) The levels of Ub cross-reacting material were investigated in wild-type and rpt1 proteasomes. Note that the film exposure for the wild-type samples (lanes 1 to 6) was 10 times longer than for the rpt1 samples (lanes 7 to 12). (E) The interaction between eEF1A and the proteasome was examined in other proteasome mutants (rpt4 and rpt6) following ATP depletion. WT, wild type; IP, immunoprecipitation.