Post-translational cleavage of mucocyst precursors in Tetrahymena.

Pulse-chase experiments utilizing intact Tetrahymena revealed that at least six proteins (molecular weights, 61,000, 56,000, 51,000, 48,000, 42,000, and 38,000) were unstable and underwent proteolytic cleavage during the first 20 min of the chase period. At least 9 product polypeptides (molecular weights, 45,000, 41,000, 25,000, 21,000, 20,000, 18,000, 17,000, 16,000, and 15,000) appeared during the chase. The proposal that the product polypeptides were actually mucocyst constituents was supported by a variety of observations. First, treatment of whole cells in a complete media with dibucaine caused precursor cleavage and product accumulation with kinetics that were consistent with previous morphologic observations on mucocyst formation. Second, the product polypeptides were enriched in a cell fraction containing just cortical components and amorphous material consistent with aggregated mucus. Third, the labeled product polypeptides in the cortex comigrated with partially purified labeled mucus obtained by dibucaine treatment of whole cells. Fourth, one-dimensional peptide mapping of the 45-kilodalton product confirmed that the post-translationally derived product in whole cell pulse-chase experiments was similar to the purified products in the cortex fraction and in dibucaine-released mucus. Two-dimensional peptide mapping of the 125I-labeled tryptic peptides of three pairs of products in the cortex and mucus further suggested a strong homology. The cleavage of mucocyst precursors was blocked by agents which deplete ATP levels and by N-tosyl-L-phenylalanyl chloromethyl ketone. Preliminary structural relationships were established between some of the precursors and products by one-dimensional peptide mapping. Models for the biogenesis of mucocysts are discussed, and it is proposed that the additional sequence information present in the precursors may be required for the intracellular transport of these proteins or their insertion and assembly within the mucocyst.