Biosynthesis and processing of secretory proteins in Tetrahymena thermophila wild type and secretory mutants

Abstract

The secretory products of Tetrahymena consist of a series of acidic polypeptides. Two of these polypeptides (34kD and 70kD) were isolated from released products and polyclonal antibodies were raised against them. We demonstrate that a 57kD polypeptide is a probable precursor to the 34kD secretory polypeptide. We postulate that the precursor accumulates in the mutant SB281 cells because it cannot be cleaved. In mutant cells, the 34kD epitope was localized in two different types of cytoplasmic vesicles: small electron dense vesicles and large electron lucent vacuoles. Immunoblot/immunoprecipitation analyses of homogenates of mutant cells with the anti34kD serum revealed a reactive protein of M{dollar}\sb{lcub}\rm r{rcub}{dollar} 57kD. Further evidence for a precursor relation of the 57kD to the 34kD mucus polypeptide was obtained by the use of drugs that block secretory product processing in wt cells. Results indicate that processing of the precursor to the 34kD polypeptide occurs in an acidic compartment(s) possibly in either the trans Golgi network, or condensing vacuoles or both.;Immunoblot analyses were performed using antibodies to the 70kD mucus polypeptide, to chromogranin A (CgA) and to a broad distribution of its peptide sequences. Anti70kD mucus antibodies recognized CgA from lysates of bovine chromaffin granules. Antibodies against the synthetic peptides of CgA recognized the 70kD mucus protein both from isolated mucus and from the highly purified 70kD polypeptide. The 70kD polypeptide isolated from released secretory products of Tetrahymena is a CgA-like molecule in this cell. Comparing this sequence with CgA and its synthetic peptides showed no sequence homology probably because the 20 amino acid sequence lies beyond the homologous region.;Immobilization antigen H{dollar}\sb3{dollar} was used as a marker to follow the pathways of its biosynthesis and transport to the cell surface. H{dollar}\sb3{dollar} is present on the surface of both mutants in immunolocalization and immunoblotting. Therefore a block at either regulated pathway or constitutive pathway does not affect H{dollar}\sb3{dollar} intracellular transport. We suggest that H{dollar}\sb3{dollar} intracellular transport does not discriminate between the established pathways. (Abstract shortened with permission of author.).