Roles of the C-terminal end of SecY in protein translocation and viability of Escherichia coli - PubMed (original) (raw)
Roles of the C-terminal end of SecY in protein translocation and viability of Escherichia coli
Kazuhiko Chiba et al. J Bacteriol. 2002 Apr.
Abstract
SecY, a central component of the membrane-embedded sector of protein translocase, contains six cytosolic domains. Here, we examined the importance of the C-terminal cytosolic region of SecY by systematically shortening the C-terminal end and examining the functional consequences of these mutations in vivo and in vitro. It was indicated that the C-terminal five residues are dispensable without any appreciable functional defects in SecY. Mutants missing the C-terminal six to seven residues were partially compromised, especially at low temperature or in the absence of SecG. In vitro analyses indicated that the initial phase of the translocation reaction, in which the signal sequence region of the preprotein is inserted into the membrane, was affected by the lack of the C-terminal residues. SecA binding was normal, but SecA insertion in response to ATP and a preprotein was impaired. It is suggested that the C-terminal SecY residues are required for SecA-dependent translocation initiation.
Figures
FIG. 1.
Growth and protein export phenotypes of chromosomal secY mutants. (A) Growth at 37 and 20°C. Strains TW156 (secY+), GN5 (secY205), KC5 (secY_Δ_5), KC6 (secY_Δ_6), and KC7 (secY_Δ_7) were cultured at 37°C until mid-log phase. Cells were serially diluted with 0.9% NaCl solution (10-fold dilutions from left to right), and 2 μl of each was spotted on L-agar plates, which were photographed after 12 h at 37°C (left panel) or after 48 h at 20°C (right panel). (B) Protein export phenotypes. Cells were grown at 37°C (upper panel) and then at 20°C for 30 min (lower panel), followed by pulse-labeling with [35S]methionine for 30 s at 37°C or for 1 min at 20°C and chase with unlabeled methionine for 12 s (lanes 1, 4, 7, 10, and 13), 1 min (lanes 2, 5, 8, 11, and 14), and 5 min (lanes 3, 6, 9, 12, and 15). MBP and OmpA were immunoprecipitated and subjected to SDS-PAGE and phosphor imager visualization. (C) Cellular accumulation of the mutant SecY proteins. Whole-cell proteins from a fixed number of cells of mid-log-phase M9 cultures were subjected to SDS-PAGE and anti-SecY immunoblotting.
FIG. 2.
SecG dependence of secY mutants. Strains KC9 (secY+ Δ_secG_::kan), KC10 (secY205 Δ_secG_::kan), KC11 (secY_Δ_5 Δ_secG_::kan), KC12 (secY_Δ_6 Δ_secG_::kan), and KC13 (secY_Δ_7 Δ_secG_::kan), all of which carried p_secG+_ (p_lac_-secG), were grown in peptone-IPTG medium at 37°C until mid-log phase. Then 2 μl of a 100-fold diluted culture was spotted onto peptone-IPTG agar (+) or L-agar (−) and incubated at 37°C for 12 h.
FIG. 3.
In vitro translocation of pro-OmpA into IMVs prepared from secY mutants. IMVs were prepared from strains TW156 (secY+), GN5 (secY205), KC5 (secY_Δ_5), KC6 (secY_Δ_6), and KC7 (secY_Δ_7). They were incubated at 37°C (upper panel) or at 20°C (lower panel) for 5 min in the presence of SecA, SecB, ATP, the ATP regeneration system, and 35S-labeled pro-OmpA. PMF was imposed or dissipated, as indicated. Extents of translocation (solid columns) and signal peptide cleavage (open columns) were assayed. Values represent percentages of radioactivities associated with translocated (solid columns) and processed (open columns) molecules after appropriate corrections for the distribution of methionine residues. The number above each pair of columns indicates the percentage of translocated component in the processed protein.
FIG. 4.
Abilities of mutant IMVs to support insertion of the C-terminal SecA segment. 125I-labeled SecA (2 μg) was bound to 4 M urea-washed IMVs (5 μg of proteins in a final volume of 200 μl) at 0°C for 30 min, and the complexes were isolated by centrifugation. The standard insertion reaction mixture (15) contained the SecA-IMV complex, pro-OmpA, and ATP to measure productive insertion (lanes 1 to 5). To measure the futile mode of insertion, pro-OmpA was omitted and AMP-PNP was included instead of ATP (lanes 6 to 10). Reactions were allowed at 37°C for 20 min, followed by proteinase K treatment. The membrane-protected 30-kDa fragment of SecA was visualized by SDS-PAGE and phosphor imager exposure.
FIG. 5.
Sequence conservation at the C-terminal ends in SecY homologs from some bacterial species. Amino acid sequences of the most C-terminal cytosolic domains were aligned using the ClustalW program. The amino acid numbers of the E. coli protein are shown at the top. Conserved residues are shown in boldface.
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