Translocation of conjugated presecretory proteins possessing an internal non-peptide domain into everted membrane vesicles in Escherichia coli.

Polypeptides comprising 20 amino acid residues (Y2) were covalently bound to the carboxyl terminus of a truncated proOmpA (proOmpA-D72C) through N,N'-bis(3-maleimidopropionyl)-2-hydroxy-1,3-propanediamine (X). The length of the inverted linker domain was 2.8 nm. proOmpA-D72C-X-Y2 thus synthesized was subjected to in vitro translocation into everted membrane vesicles of Escherichia coli. The conjugated protein was translocation-competent in terms of both proteinase K resistance and signal peptide cleavage, when a proton motive force (delta microH+) was imposed. The translocation was ATP-dependent. The proteinase K-treatment resulted in the digestion of SecA, SecE, and SecY in the membrane, suggesting that the proteinase K resistance of the Y2 domain was not due to its interaction with these Sec proteins in the secretory machinery. In the absence of delta microH+, the translocation ceased at the linker domain. Upon the imposition of delta microH+, the linker-Y2 domain underwent translocation, which did not require ATP hydrolysis as in the case of the translocation of the latter portion of usual secretory proteins. The translocation was prevented by anti-Y2 IgG even when delta microH+ was imposed. Another conjugated protein, which possesses a polypeptide comprising 61 amino acid residues after the linker (proOmpA-D72C-X-Lpp'), was synthesized. This compound was also translocated into everted membrane vesicles with cleavage of the signal peptide. These results suggest that substances to be translocated through the secretory machinery need not necessarily be solely held together by polypeptide bonds.