Chemical cross-linking of thioredoxin to hybrid membrane fraction in Escherichia coli.

Thioredoxin was cross-linked to a membrane fraction in vivo using the heterobifunctional photoreactive cross-linking reagent p-azidophenacyl bromide, chosen to couple thioredoxin via its highly reactive thiol. Under mild reaction conditions, a significant amount of thioredoxin (30%) was rapidly cross-linked to the crude membrane fraction. The cross-linking reaction was selective, with thioredoxin purified 15-fold in the cross-linked membrane fraction. Membrane fractionation studies showed that thioredoxin associated with the inner membrane and with a hybrid membrane fraction. This hybrid membrane fraction banded at a density between the inner and outer membranes. This result is consistent with the localization of thioredoxin in association with the bacterial membrane adhesion sites first described by Bayer (Bayer, M. (1968) J. Gen. Microbiol. 53, 395-404). Association of thioredoxin with the membrane adhesion sites defines a structure corresponding to the osmotically sensitive cytoplasmic compartment (Lunn, C. A., and Pigiet, V. (1982) J. Biol. Chem. 257, 11424-11430).