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Conjugation-rescue experiments with two Tetrahymena thermophila mutants (exo-) incapable of exocytosis (SB255, SB258) have been used to dissect regulatory steps in assembly of a functional membrane microdomain, the fusion rosette. "Rescue" refers to the recovery of a secretory activity. Exo- mutants fail to secrete mucus normally (form capsules) when stimulated by the secretagogue alcian blue and are blocked before the assembly of a functional fusion rosette in the cell membrane. Two criteria are used to assay recovery of the wild-type (exo+) phenotype: the conjugant's ability to form capsules when stimulated and the presence of assembled rosettes, which disperse upon stimulation. Conjugation of exo+ X SB258 results in restoration of secretion in 60% of the mutant conjugants and reappearance of assembled rosettes. Secretory capacity is restored in the SB258 cell within one-half hour of firm pair formation. This restoration is not due to new gene expression or continued protein synthesis, since it occurs when SB258 is crossed to a "star" strain (A*), which has defective micronuclei and therefore cannot contribute wild-type genes, and restoration occurs in the presence of cycloheximide during conjugation. Conjugation of exo+ X SB255 reveals a real but inefficient restoration of exocytic capacity in the exo- conjugant and a significant decrease of exocytic capacity in the exo+ conjugant. SB255 X SB258 crosses also show a low but significant rescue of exocytic competence, indicating that different components of the exocytic mechanism are affected in the two mutants. This cross leads to restoration of rosette assembly and function in one of the partners, presumably SB258. These results provide data about some of the steps necessary for rosette assembly and suggest that transferable factors that promote and/or inhibit exocytosis are present in these cells.

Conjugation rescue of an exocytosis-competent membrane microdomain in Tetrahymena thermophila mutants.