AUTOPHAGY IN TOBACCO SUSPENSION-CULTURED CELLS IN RESPONSE TO SUCROSESTARVATION

The response of tobacco (Nicotiana tabacum) suspension-cultured cells (BY-2) to nutrient starvation was investigated. When the cells that we re grown in Murashige-Skoog medium containing 3% (w/v) sucrose were tr ansferred to the same medium without sucrose, 30 to 45% of the intrace llular proteins were degraded in 2 d. An analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that proteins were d egraded nonselectively. With the same treatment, protease activity in the cell, which was measured at pH 5.0 using fluorescein thiocarbamoyl -casein as a substrate, increased 3- to 7-fold after 1 d. When the cys teine protease inhibitor -trans-epoxysuccinyl-L-leucylamido-3-methyl-b utane (10 mu M) was present in the starvation medium, both the protein degradation and the increase in the protease activity were effectivel y inhibited. Light microscopy analysis showed that many small spherica l bodies accumulated in the perinuclear region of the cytosol 8 h afte r the start of the inhibitor treatment. These bodies were shown to be membrane-bound vesicles of 1 to 6 mu m in diameter that contained seve ral particles. Quinacrine stained these vesicles and the central vacuo le; thus, both organelles are acidic compartments. Cytochemical enzyme analysis using 1-naphthylphosphate and beta-glycerophosphate as subst rates showed that these vesicles contained an acid phosphatase(s). We suggest that these vesicles contribute to cellular protein degradation stimulated under sucrose starvation conditions.