Figure 3. The two-helix finger of SecA interacts with a translocating substrate.

a, Translocation substrates (pOA:tRNA) of 206 residues containing cysteines at positions 194 and 201 were incubated with SecA and proteoliposomes containing SecY complex in the absence or presence of ATP. The SecA proteins either lacked a cysteine or contained a cysteine at the tip of the two-helix finger (position 797). SecY lacked a cysteine or contained a cysteine in the pore ring (position 282). After oxidation with Cu²⁺-phenanthroline, the samples were treated with NEM and RNase A, and analyzed by non-reducing SDS-PAGE and autoradiography. The positions of free and crosslinked pOA (pOA, pOAxSecY, pOAxSecA, and pOAxSecYxSecA) are indicated. b, As in a, but the proteoliposomes were sedimented before addition of the oxidant. SecA contained a cysteine at position 797. After solubilization in DDM, 20% of each sample was analyzed directly (lanes 1 and 2), while the remainder was incubated with protein G beads either without antibody or with SecA- or SecY- antibodies (lanes 3-8). c, As in a, but the proteoliposomes containing SecY with a cysteine at position 282 were incubated with a SecA mutant containing a single cysteine at position 797 and pOA:tRNA with a cysteine at position 194 and a second cysteine at the indicated position. The samples were sedimented before addition of the oxidant. d, As in c, but the proteoliposomes containing SecY with a cysteine at position 282 were incubated with pOA:tRNA containing cysteines at positions 194 and 201 and SecA mutants containing single cysteines at the indicated positions.