EXAMINATION OF THE CONTRIBUTION OF VACUOLAR PROTEASES TO INTRACELLULAR PROTEIN-DEGRADATION IN CHARA-CORALLINA

The contribution of proteases in the central vacuole of Chara corallin a internodal cells to overall cellular protein degradation was examine d. I measured the decrease in the trichloroacetic acid (TCA)-precipita ble radioactivity in the cell for a 6-d chase period after labeling ce llular proteins with [H-3]leucine. The kinetics of [3H]leucine-labeled protein disappearance showed that the half-life of the cellular solub le proteins was 4 to 5 d. This value did not change when cells were tr eated with -trans-epoxysuccinyl-L-leucylamido-3-methyl-butane ethyl es ter, a permeant inhibitor of cysteine proteases. This inhibitor mostly inhibited bovine serum albumin-degrading activity in the vacuole. I a lso measured the release of TCA-soluble radioactivity from the TCA-ins oluble fraction in the cell. This experiment showed that 13% of [H-3]l eucine-labeled cellular proteins were degraded in 1 d. This value agre ed well with the half-life obtained for soluble proteins in the above experiment. This value did not change even when both s-epoxysuccinyl-L -leucylamido-(4-guanidino)butane, a cysteine protease inhibitor, and p epstatin A, an aspartic protease inhibitor, were introduced into the v acuole. With this operation, bovine serum albumin-degrading activity i n the vacuole was almost completely inhibited. These data suggest that the cytoplasmic but not the vacuolar proteases contribute to cellular protein turnover in Chara internodal cells.