Ligand crowding at a nascent signal sequence.

We have systematically analyzed the molecular environment of the signal sequence of a growing secretory protein from Escherichia coli using a stage- and site-specific cross-linking approach. Immediately after emerging from the ribosome, the signal sequence of pOmpA is accessible to Ffh, the protein component of the bacterial signal recognition particle, and to SecA, but it remains attached to the surface of the ribosome via protein L23. These contacts are lost upon further growth of the nascent chain, which brings the signal sequence into sole proximity to the chaperone Trigger factor (TF). In its absence, nascent pOmpA shows extended contacts with L23, and even long chains interact in these conditions proficiently with Ffh. Our results suggest that upon emergence from the ribosome, the signal sequence of an E. coli secretory protein gradually becomes sequestered by TF. Although TF thereby might control the accessibility of pOmpA's signal sequence to Ffh and SecA, it does not influence interaction of pOmpA with SecB.

The entire N-terminal half of TatC is involved in twin-arginine precursor binding.

Translocation of twin-arginine precursor proteins across the cytoplasmic membrane of Escherichia coli requires the three membrane proteins TatA, TatB, and TatC. TatC and TatB were shown to be involved in precursor binding. We have analyzed in vitro a number of single alanine substitutions in tatC that were previously shown to compromise in vivo the function of the Tat translocase. All tatC mutants that were defective in precursor translocation into cytoplasmic membrane vesicles concomitantly interfered with precursor binding not only to TatC but also to TatB. Hence structural changes of TatC that affect precursor targeting simultaneously abolish engagement of the twin-arginine signal sequence with TatB and block the formation of a functional Tat translocase. Since these phenotypes were observed for tatC mutations spread over the first half of TatC, this entire part of the molecule must globally be involved in precursor binding.

Alternate recruitment of signal recognition particle and trigger factor to the signal sequence of a growing nascent polypeptide.

Different from cytoplasmic membrane proteins, presecretory proteins of bacteria usually do not require the signal recognition particle for targeting to the Sec translocon. Nevertheless signal sequences of presecretory proteins have been found in close proximity to signal recognition particle immediately after they have emerged from the ribosome. We show here that at the ribosome, the molecular environment of a signal sequence depends on the nature of downstream sequence elements that can cause an alternate recruitment of signal recognition particle and the ribosome-associated chaperone Trigger factor to a growing nascent chain. While signal recognition particle and Trigger factor might remain bound to the same ribosome, both ligands are clearly able to displace each other from a nascent chain. The data also imply that a signal sequence owes its molecular environment to the fact that it remains closely apposed to the ribosomal exit site during growth of a nascent secretory protein.

Differential interactions between a twin-arginine signal peptide and its translocase in Escherichia coli.

The twin-arginine translocation (Tat) machinery of the Escherichia coli inner membrane is dedicated to the export of proteins harboring a conserved SRRxFLK motif in their signal sequence. TatA, TatB, and TatC are the functionally essential constituents of the Tat machinery, but their precise function is unknown. Using site-specific crosslinking, we have analyzed interactions of the twin-arginine precursor preSufI with the Tat proteins upon targeting to inner membrane vesicles. TatA association is observed only in the presence of a transmembrane H(+) gradient. TatB is found in contact with the entire signal sequence and adjacent parts of mature SufI. Interaction of TatC with preSufI is, however, restricted to a discrete area around the consensus motif. The results reveal a hierarchy in targeting of a Tat substrate such that for the primary interaction, TatC is both necessary and sufficient while a subsequent association with TatB likely mediates transfer from TatC to the actual Tat pore.