The effect of puromycin and cycloheximide on vacuole formation and exocytosis in Tetrahymena pyriformis GL-9.

It has been shown that both puromycin and cycloheximide, at concentrations of 434 and 100 mug/ml respectively, produce a marked inhibition of vacuole formation and exocytosis in Tetrahymena pyriformis GL-9. These effects were analysed in a quantitative manner. At the same time as these inhibitions occurred the incorporation of 1-C14 leucine into trichloroacetic acid precipitable material was inhibited by 90% and 100% respectively over a 40 min period. This inhibition of protein synthesis by cycloheximide occurred almost immediately, whereas the inhibition of vacuole formation and egestion was delayed. The results suggested that the latter processes were dependent upon a continuing supply of proteinaceous material, of which there was only a small store within the cell. Cycloheximide inhibited exocytosis completely -nder the conditions employed (with 100% inhibition of protein synthesis) whereas puromycin (with a 90% inhibition of protein synthesis) only inhibited it by about 50%. This suggested that the amount of newly synthesized protein required for the exocytic egestion process was very small in relation to the total cell requirement for protein synthesis. The entry of both inhibitors into the cell was by means other than vacuole formation. Puromycin appeared to have some effect on vacuole formation which was unconnected with protein synthesis. Microscopic observations of living cells indicated that oral apparatus function and endocytic vacuole formation were probably both affected by the inhibitors. Chloramphenicol, at 200 mug/ml, had little effect on vacuole formation by starved cells with an exposure of an hour. The uptake of 1-C14 leucine from the growth medium was found to be a selective process, giving a concentration of about 2000 times into the cells over a 1 hr period. The results are discussed