Translocation can drive the unfolding of a preprotein domain.

Precursor proteins are believed to have secondary and tertiary structure prior to translocation across the Escherichia coli plasma membrane. We now find that preprotein unfolding during translocation can be driven by the translocation event itself. At certain stages, translocation and unfolding can occur without exogenous energy input. To examine this unfolding reaction, we have prepared proOmpA-Dhfr, a fusion protein of the well studied cytosolic enzyme dihydrofolate reductase (Dhfr) connected to the C-terminus of proOmpA, the precursor form of outer membrane protein A. At an intermediate stage of its in vitro translocation, the N-terminal proOmpA domain has crossed the membrane while the folded Dhfr portion, stabilized by its ligands NADPH and methotrexate, has not. When the ligands are removed from this intermediate, translocation occurs by a two-step process. First, 20-30 amino acid residues of the fusion protein translocate concomitant with unfolding of the Dhfr domain. This reaction requires neither ATP, delta mu H+ nor the SecA subunit of translocase. Strikingly, this translocation accelerates the net unfolding of the Dhfr domain. In a second step, SecA and ATP hydrolysis drive the rapid completion of translocation. Thus energy derived from translocation can drive the unfolding of a substantial protein domain.