PLANT PHYSIOLOGY , Vol 109, Issue 4 1309-1315, Copyright © 1995 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
Examination of the Contribution of Vacuolar Proteases to Intracellular Protein Degradation in Chara corallina
Y. Moriyasu
Department of Biology, Faculty of International Relations, University of Shizuoka, 52-1 Yada, Shizuoka-shi, 422 Japan
The contribution of proteases in the central vacuole of Chara corallina
internodal cells to overall cellular protein degradation was examined. I
measured the decrease in the trichloroacetic acid (TCA)-precipitable
radioactivity in the cell for a 6-d chase period after labeling cellular
proteins with [3H]leucine. The kinetics of [3H]leucine-labeled protein
disappearance showed that the half-life of the cellular soluble proteins
was 4 to 5 d. This value did not change when cells were treated with
(2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methyl-butane ethyl ester, a
permeant inhibitor of cysteine proteases. This inhibitor mostly inhibited
bovine serum albumin-degrading activity in the vacuole. I also measured the
release of TCA-soluble radioactivity from the TCA-insoluble fraction in the
cell. This experiment showed that 13% of [3H]leucine-labeled cellular
proteins were degraded in 1 d. This value agreed well with the half-life
obtained for soluble proteins in the above experiment. This value did not
change even when both
trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, a cysteine protease
inhibitor, and pepstatin A, an aspartic protease inhibitor, were introduced
into the vacuole. With this operation, bovine serum albumin-degrading
activity in 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.
