PROTEIN SECRETION IN TETRAHYMENA THERMOPHILA

Abstract

Tetrahymena thermophila contains membrane-limited exocytic organelles termed mucocysts. A series of secretory mutant strains of Tetrahymena have been isolated (Orias et al., 1980), and the ultrastructural correlates of nonrelease in one unique mutant strain, designated SB281, are described. Thin-section electron microscopy (EM) of SB281 reveals that these cells possess no docked or free mature mucocysts. Freeze-fracture EM reveals that a characteristic intramembrane particle (IMP) array termed the rosette, which is present in the plasma membrane (PM) of wildtype (WT; SB210) cells above sites of docked mucocysts, is absent in the PM of WT and SB281 indicate that both strains have similar IMP densities in corresponding leaflets of the PM. Furthermore, a 15 nm IMP size class ('rosette size particles') exists in the PM of SB281, however, the frequency of this size class is significantly reduced in the 'PF' fracture face of SB281 relative to the 'PF' face of WT PM. Preparations highly enriched for discharged mucocysts ('mucus') were isolated and characterized. These preparations maintain a polygonal substructure when examined by EM. SDS PAGE analyses reveal 12 major polypeptide constituents ranging in Mr from 180,000 to 8,000. There are three subsets of 'mucus' proteins which are associated by disulfide bonds as determined by 2-D electrophoresis. All these proteins have acidic apparent isoelectric points. The most prominent 'mucus' constituent has an Mr of 34,000 and was isolated by preparative SDS PAGE for use in the preparation and characterization of a rabbit antiserum. A precipitating antiserum was produced which reacts with a single polypeptide with an Mr of 34,000 in 'mucus' preparations and extracts of WT, but not in extracts of SB281 in immunoblot analyses. Use of this antiserum in indirect immunofluorescence studies demonstrates a punctate localization along the secondary meridians of WT (the major sites of docked mucocysts), but only a low level diffuse fluorescence pattern in the secretory mutant strain SB281. In addition, this mucocyst marker labelled several distinct size classes of vesicles in WT cells during mucocyst regeneration following dibucaine stimulation.