Abstract
Fragment A of diphtheria toxin is translocated to the cytosol when the toxin is presented to receptor-positive cells. The toxin binds to cell surface receptors through its B-fragment, and after endocytotic uptake, the low endosomal pH triggers translocation of the A-fragment across the membrane. Translocation can also be induced at the level of the plasma membrane by exposure to low pH medium. Based on the diphtheria toxin crystal structure, we made five double cysteine mutants of the A-fragment, each expected to form an intramolecular disulfide bond. Four of the double cysteine mutants efficiently formed an intramolecular disulfide bridge, and these same mutants showed a strong reduction in their translocating ability. The inhibition of translocation was observed both when the toxin was endocytosed and when translocation was induced by exposing surface-bound toxin to low pH. The data indicate that extensive unfolding of the A-fragment is required for translocation.
Highlights
From the $Department of Biochemistry, Institute for Cancer Research at the Norwegian Radium Hospital,Montebello, 0310 Oslo 3, Norway, the IMolecular Biology Institute and Department of Chemistry and Biochemistry, Universityof California, Los Angeles, California 90024, and the lllepartment of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115
The molecular mecha- disulfide bond could prevent unfolding and inhibit the translonism of the translocation process has been most thoroughly cation process.O n the basis of the crystal structureof the toxin studied in the case of diphtheria toxin. This toxin is secreted (Choe et al, 1992), we have identified paiorfsamino acids that are positioned sufficiently close in space to be likely to form disulfide bonds when mutated to cysteiWnees.constructed five
Formationand Characterization of Mutant Proteins-We constructed five double cysteine mutants of the A-fragment, The mRNA was precipitated with ethanol anddissolved in 10 pol f H,O referred to as CC1 through C C 5 (Fig. lA)
Summary
Different double cysteine mutantsof the A-fragment and studied their abilities both to form intramolecular disulfide bonds and to be translocated to the cytosol. To test ing in parallel a 10-pl aliquot of the lysate in the presence of 5 PM if the double cysteine mutants formed intramolecular disulfide bonds, we compared the migration rates on SDS-PAGE of the In Vitro Reconstitution $Active fragment are mixed together under
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