Figure 6.

Secretory and integral membrane proteins use the same translocon. pOmpA nascent chains were synthesized in the presence of an oligonucleotide causing a translational stop after 125 amino acids (Behrmann et al., 1998). They were subsequently released from the ribosomes by treatment with 8 M urea (Matlack et al., 1997), resulting in a pOmpA derivative carrying a tRNA at its COOH terminus. The tRNA-tethered OmpA (pOmpA::tRNA) was incubated with INV. After centrifugation through a sucrose cushion, these INV carrying the pOmpA::tRNA translocation intermediates (pOmpA::tRNA-INV) were resuspended and assayed for the translocation of OmpA and the integration of MtlA. As a control (mock-INV) the pOmpA nascent chains were incubated with puromycin to remove the ribosome and the tRNA before urea treatment and incubation with the membranes.