Translocation can drive the unfolding of a preprotein domain.

Abstract

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.

Kinetic Analysis of the Translocation of Fluorescent Precursor Proteins into Escherichia coli Membrane Vesicles

“…The pore of the active E. coli translocase may also be significantly larger than required for the translocation of an unfolded polypeptide chain. Although large folded proteins such as bovine pancreas trypsin inhibitor and dihydrofolate reductase block translocation (7,26), a disulfide-bridged polypeptide loop up to 18 amino acid residues in proOmpA can still be transported across the membrane provided that a PMF is present (27). Our studies with fluorescently labeled proOmpA indicate that a bulky fluorescent dye such as Texas Red (size of 13-16 Å) is readily translocated.…”

Mapping polypeptide interactions of the SecA ATPase during translocation

“…1B) (17). DHFR folds well in the presence of its substrate analog methotrexate and provides an efficient block for the movement of the C terminus of the substrate through the membrane.…”

Import of cytochrome b2 to the mitochondrial intermembrane space: The tightly folded heme‐binding domain makes import dependent upon matrix ATP

“…Like all protein structures, the heme-binding domain will undergo spontaneous partial unfolding events, which should allow the presequence to penetrate further into the matrix. Normally such oscillations are reversible, but matrix ATP would enable mhsp70 to "capture" the presequence after an inward movement, thereby making unfolding irreversible and leading to vectorial translocation Simon et al, 1992;Arkowitz et al, 1993). It has been suggested that ATP-dependent cytosolic chaperones facilitate translocation by preventing the tight folding of precursor proteins (Deshaies et al, 1988a;Rothman, 1989;Click & Schatz, 1991).…”