A potential mucus precursor in Tetrahymena wild type and mutant cells.
By using an antibody to a specific mucus polypeptide (34 kDa) to study whole cell extracts of both a secretory mutant (SB281) and wild type (wt) Tetrahymena, we demonstrate that a 57-kDa polypeptide is a probable precursor to the 34-kDa secretory polypeptide. We postulate that the precursor accumulates in the mutant cells because it cannot be cleaved. This mutant contains no recognizable mature secretory granules (mucocysts). By immunoelectron microscopy, the 34-kDa polypeptide was localized in wt cells specifically to the mature mucocysts and to their released products. Localization in mutant cells occurred in two different types of cytoplasmic vesicles: small electron dense vesicles (0.3-0.5 microns in diameter) and large electron lucent vacuoles (1.2-3.5 microns in diameter). Immunoblot analyses of homogenates of mutant and wt cells with the anti-34-kDa serum revealed a dominant band in the mutant at Mr 57 kDa whereas the wt showed a dominant band only at Mr 34 kDa. Furthermore, the 57-kDa polypeptide is immunoprecipitated with anti-34-kDa serum from the mutant cell. Further evidence for a precursor relation of the 57-kDa polypeptide in mutant cells to the 34-kDa mucus polypeptide of wt cells was obtained by the use of drugs (monensin, chloroquine, NH4Cl) that block secretory product processing in wt cells. Extracts of drug-treated wt cells showed the presence of a 57-kDa cross reacting band even after 18 h of incubation in growth medium whereas untreated control cells contained the 34-kDa mature protein almost exclusively. These results indicate that processing of the precursor to the 34-kDa polypeptide occurs in an acidic compartment(s) possibly in either the trans Golgi network, or condensing vacuoles or both.
Ding
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Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461.
Ron
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Satir
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BH
D000818:Animals; D015153:Blotting, Western; D016253:Microscopy, Immunoelectron; D008970:Molecular Weight; D009093:Mucus / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011233:Precipitin Tests; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D015800:Protozoan Proteins / Q000378:metabolism*; D013768:Tetrahymena / Q000187:drug effects / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure
D010455:Peptides; D011498:Protein Precursors; D015800:Protozoan Proteins