Genetic analysis of pathway specificity during posttranslational protein translocation across the Escherichia coli plasma membrane

Abstract

In Escherichia coli, the SecB/SecA branch of the Sec pathway and the twin-arginine translocation (Tat) pathway represent two alternative possibilities for posttranslational translocation of proteins across the cytoplasmic membrane. Maintenance of pathway specificity was analyzed using a model precursor consisting of the mature part of the SecB-dependent maltose-binding protein (MalE) fused to the signal peptide of the Tat-dependent TorA protein. The TorA signal peptide selectively and specifically directed MalE into the Tat pathway. The characterization of a spontaneous TorA signal peptide mutant (TorA*), in which the two arginine residues in the c-region had been replaced by one leucine residue, showed that the TorA*-MalE mutant precursor had acquired the ability for efficiently using the SecB/SecA pathway. Despite the lack of the "Sec avoidance signal," the mutant precursor was still capable of using the Tat pathway, provided that the kinetically favored Sec pathway was blocked. These results show that the h-region of the TorA signal peptide is, in principle, sufficiently hydrophobic for Sec-dependent protein translocation, and therefore, the positively charged amino acid residues in the c-region represent a major determinant for Tat pathway specificity. Tat-dependent export of TorA-MalE was significantly slower in the presence of SecB than in its absence, showing that SecB can bind to this precursor despite the presence of the Sec avoidance signal in the c-region of the TorA signal peptide, strongly suggesting that the function of the Sec avoidance signal is not the prevention of SecB binding; rather, it must be exerted at a later step in the Sec pathway.

FIG. 1.

Signal peptides and early mature region of proteins used in this study. (A) The hydrophobic h-regions are underlined, and the signal peptidase cleavage site is indicated by an arrow. The RR residues of the twin-arginine motif are shown in bold and italics; the RR residues of the Sec avoidance signal are shown in bold. The mature regions of the TorA-MalE and TorA*-MalE fusion proteins consist of eight amino acid residues of the mature TorA protein followed by a linker sequence of three amino acid residues (shown in italics) and the mature MalE protein. (B) Nature of the spontaneous mutation that results in a replacement of the two arginine residues in the c-region of the TorA signal peptide by a leucine residue. The gene regions encoding the carboxyl-terminal parts of the h-regions and the c-regions of the wild-type (TorA) and mutant (TorA*) signal peptides are shown. Relevant nucleotide positions and amino acid residues are highlighted in bold.

FIG. 2.

Subcellular localization of MalE polypeptides. Cells of the tat wild-type (wt) strain GSJ100 containing pTorA-MalE and the tat deletion mutant (Δtat) GSJ101 containing pTorA-MalE were fractionated into a combined C/M fraction (lanes C/M) and a P fraction (lanes P) by EDTA-lysozyme spheroplasting as described in Materials and Methods. Samples were subjected to SDS-PAGE and Western blotting using anti-MalE antibodies (lanes 1 to 4) or, as a control, antibodies directed against the cytoplasmic protein TalB (lanes 5 to 8). p, TorA-MalE precursor; m, position of mature form of TorA-MalE in the P fraction; asterisks, positions of TorA-MalE degradation products in the C/M fraction.

FIG. 3.

Processing of TorA-MalE. (A) Processing of TorA-MalE in the tat wild-type (wt) strain GSJ100 containing pTorA-MalE and the tat deletion mutant (Δtat) GSJ101 containing pTorA-MalE in the absence (−) or presence (+) of 3 mM sodium azide was analyzed by pulse-chase labeling and subsequent immunoprecipitation with either anti-MalE serum (upper panel) or anti-OmpA serum (lower panel), SDS-PAGE, and fluorography. Cells were labeled with l-[³⁵S]methionine for 1 min prior to chase with an excess of nonradioactive methionine. Samples were withdrawn at the time points indicated above the respective lanes (e.g., 10", 10 s; 2′, 2 min). p, precursor form of TorA-MalE (upper panel) or OmpA (lower panel); m, mature form of TorA-MalE (upper panel) or OmpA (lower panel); asterisks, positions of TorA-MalE degradation products. (B) The subcellular localization of the MalE gene products observed in the pulse-chase experiments in wild-type (wt) or tat mutant (Δtat) cells was analyzed by fractionating samples withdrawn at 10 s or 30 min after the chase into a combined C/M fraction (lanes C/M) and a P fraction (P). Lanes T show the corresponding samples without fractionation. p, precursor form of TorA-MalE; m, mature form of TorA-MalE; asterisks, positions of TorA-MalE degradation products.

FIG. 4.

Processing of TorA-MalE is slowed down by SecB. Processing of authentic pre-MalE in the secB wild-type (wt) strain MC4100 containing pMalE (lanes 1 to 5) and the secB mutant (secB::Tn5) CK1953 containing pMalE (lanes 6 to 10) is shown. Processing of TorA-MalE in MC4100 (wt) containing pATorA-MalE (lanes 11 to 16) and CK1953 (secB::Tn5) containing pATorA-MalE (lanes 17 to 22) is shown. Cells were labeled with l-[³⁵S]methionine for 1 min prior to chase with an excess of nonradioactive methionine. Samples were withdrawn at the time points indicated above the respective lanes (e.g., 10", 10 s; 1′, 1 min) and subjected to immunoprecipitation with anti-MalE serum, SDS-PAGE, and fluorography. p, precursor form of authentic pre-MalE (lanes 1 to 10) or TorA-MalE (lanes 11 to 22); m, mature form of pre-MalE (lanes 1 to 10) or TorA-MalE (lanes 11 to 22); asterisks, positions of TorA-MalE degradation products.

FIG. 5.

Processing of TorA*-MalE. (A) Processing of TorA*-MalE in the tat wild-type (wt) strain GSJ100 containing pTorA*-MalE and the tat deletion mutant (Δtat) GSJ101 containing pTorA*-MalE in the absence (−) or presence (+) of 3 mM sodium azide was analyzed by pulse-chase labeling and subsequent immunoprecipitation with anti-MalE serum, SDS-PAGE, and fluorography. Cells were labeled with l-[³⁵S]methionine for 1 min prior to chase with an excess of nonradioactive methionine. Samples were withdrawn at the time points indicated above the respective lanes (e.g., 10", 10s; 2′, 2 min). p, precursor form of TorA*-MalE; m, mature form of TorA*-MalE; asterisks, positions of TorA*-MalE degradation products. (B) Processing of TorA*-MalE in the secB wild-type (wt) strain MC41000 containing pATorA*-MalE (lanes 1 to 5) and the secB mutant (secB::Tn5) CK1953 containing pATorA*-MalE (lanes 6 to 10). Cells were labeled with l-[³⁵S]methionine for 1 min prior to chase with an excess of nonradioactive methionine. Samples were withdrawn at the time points indicated above the respective lanes and subjected to immunoprecipitation with anti-MalE serum, SDS-PAGE, and fluorography. p, precursor form of TorA*-MalE; m, mature form of TorA*-MalE.