EVIDENCE FOR DEFECTS IN MEMBRANE TRAFFIC IN PARAMECIUM SECRETORY MUTANTS UNABLE TO PRODUCE FUNCTIONAL STORAGE GRANULES

The ciliated protozoan Paramecium has a regulated secretory system ame nable to genetic analysis. The secretory storage granules, known as tr ichocysts, enclose a crystalline matrix with a genetically determined shape whose biogenesis involves proteolytic maturation of a family of precursor molecules into a heterogeneous set of small acidic polypepti des that crystallize within the maturing vesicles. We have developed a n original pulse-chase protocol for monoxenic Paramecium cultures usin g radiolabeled bacteria to study the processing of trichocyst matrix p roteins in wild-type and mutant cells. In wild-type cells, proteolytic processing is blocked in the presence of monensin and otherwise rapid ly completed after similar to 20 min of chase, suggesting that the con version occurs in the trans-Golgi and/or in small vesicles soon after sorting to the regulated pathway, probably before crystallization begi ns. In trichless mutant cells, which contain no visible trichocysts, s ecretory proteins are synthesized but not processed and we report cons titutive secretion of the uncleaved precursor molecules. The mutation thus appears to affect sorting to the regulated pathway and should pro ve useful for analysis of the sorting machinery and of the relationshi p between sorting and proteolytic processing of secretory proteins. In mutants bearing misshapen trichocysts with poorly crystallized conten ts (tam33, tam38, stubbyA), the proteolytic processing of the trichocy st matrix proteins appears to be normal, while both pulse-chase and mo rphological data indicate that intracellular transport is perturbed, p robably between ER and Golgi. Precursor molecules are present in the m utant trichocysts but not in wild-type trichocysts and may account for the defective crystallization. Our analysis of these mutants suggests that the temporal coordination of intracellular traffic plays a regul atory role in granule maturation.