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Mutational analysis of S1 subunit requirements for folding and assembly into pertussis holotoxin

, : Mutational analysis of S1 subunit requirements for folding and assembly into pertussis holotoxin. Abstracts of the General Meeting of the American Society for Microbiology 101: 109-110

Disulfide bond formation in pertussis toxin was analyzed by Western blot after electrophoresis under either reducing or non-reducing conditions. Folding of S1, the A-subunit, but not the B-subunit, was found to involve a putative chaperone. All of the periplasmic B-subunits can be accounted for in holotoxin. In contrast, more than half of the S1 (28 kDa) was present as a 46-kDa complex that was formed by an intermolecular disulfide bond with a putative 18-kDa chaperone. Mutation of the S1 signal sequence resulted in failure to form the 46-kDa complex suggesting secretion is required for complex formation. The 46-kDa form was membrane-associated, whereas holotoxin was soluble. The 46-kDa complex was found in mutants lacking the B subunits, the Ptl secretion proteins, or the Dsb enzymes suggesting that the 18-kDa partner is not one of these proteins. Furthermore, its presence in Dsb mutants suggests the disulfide bond is formed by a novel mechanism. Under reducing conditions, S1 in the 46-kDa form was rapidly degraded unless the samples were boiled in the presence of protease inhibitors. Folded S1 has one intramolecular disulfide bond formed between Cys-41 and Cys-201. S1 was not detected in Cys-41 mutants, but the absence of Cys-201 did not affect the formation of the 46-kDa complex, suggesting complex formation involves Cys-41 and is essential for stability. Mutants deleted for the C-terminus of S1 (which cannot associate with the B subunit) or mutants lacking the B subunit proteins still produced the S1 complex, but upon reduction no S1 was detected, suggesting association with the 18-kDa partner or the B subunit is required for stability. These results suggest that S1 is stabilized as a disulfide-bound intermediate prior to incorporation into holotoxin.


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