Proteolytic processing of secretory proteins in Paramecium: immunological and biochemical characterization of the precursors of trichocyst matrix proteins.
We used polyclonal serum raised against mature trichocyst matrix proteins to detect their unprocessed precursors, a group of proteins (45-55 kDa) present in the whole-cell extract. These precursor proteins were partially purified from the soluble fraction of wild-type cells by ammonium sulfate precipitation and anion-exchange chromatography. Using monoclonal antibodies against each of four families of mature (processed) matrix proteins, we showed that each family was derived from a separate group of precursors. Our results also suggest that in three of four precursors, those in which the mature proteins consist of disulfide-linked heterodimers, intrachain disulfide bonds form before proteolytic processing. Purified precursors eluted from preparative SDS-gels were used to raise rabbit antiserum, which after preadsorption with mature processed proteins specifically recognized precursors, as judged by ELISA and immunoblots. In cross-sections of developing trichocysts, the anti-precursor serum after preadsorption no longer stained the central, paracrystalline region, but still stained the peripheral as well as the structureless region of the secretory granule. In trichocyst-developing mutants tl (trichless) and ftA (football A), the precursors for all four groups of mature proteins were present but their processing was affected: severely blocked in tl (which has no recognizable crystalline trichocyst matrix), and partially blocked in ftA (which has some abnormal trichocyst matrices with crystalline centers). These observations constitute further evidence that proteolytic processing of precursors occurs in parallel with crystallization.
Shih
S J
SJ
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53706.
Nelson
D L
DL
D000818:Animals; D000911:Antibodies, Monoclonal; D002118:Calcium / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000378:metabolism; D016253:Microscopy, Immunoelectron; D008970:Molecular Weight; D009154:Mutation; D010084:Oxidation-Reduction; D010247:Paramecium / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D011487:Protein Conformation; D011498:Protein Precursors / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000235:genetics
D000911:Antibodies, Monoclonal; D011498:Protein Precursors; D002118:Calcium