A SecE mutation that modulates SecY-SecE translocase assembly, identified as a specific suppressor of SecY defects

Abstract

The SecY39(Cs) (cold-sensitive) alteration of Arg357 results in a defect of translocation initiation. As a means to dissect the Sec translocation machinery, we isolated mutations that act as suppressors of the secY39 defect. A specific secE mutation, designated secE105, was thus isolated. This mutation proved to be identical with the prlG2 mutation and to suppress a number of cold-sensitive secY mutations. However, other prlG mutations did not effectively suppress the secY defects. Evidence indicates that the Ser105-to-Pro alteration in the C-terminal transmembrane segment of SecE weakens SecY-SecE association. In vitro analyses showed that the SecE(S105P) alteration preferentially stimulates the initial phase of translocation. It is suggested that the S105P alteration affects the SecYEG channel such that it is more prone to open and to accept the translocation initiation domain of a preprotein molecule.

FIG. 1.

Effects of the secE105 mutation on secY39 defects. (A) Effects on protein export. Strains HM541 (secY39 secE105) (lanes 1 to 4), HM545 (secY39 secE⁺) (lanes 5 to 8), and AD202 (secY⁺ secE⁺) (lanes 9 to 12) were grown at 37°C in M9-glycerol-amino acids medium supplemented with maltose (0.4%) until early-log phase (upper panel). A portion of the cultures was then shifted to 20°C for 30 min (lower panel). Cells were pulse-labeled with [³⁵S]methionine for 0.5 min (at 37°C) or 1 min (at 20°C) and chased with unlabeled methionine for the indicated periods. At each time point, a portion of culture was directly treated with TCA (final concentration, 5%), and protein precipitates were solubilized and processed for immunoprecipitation of MBP and OmpA. Their precursor (p) and mature (m) forms were visualized by phosphor image analyzer after SDS-PAGE. (B) Effects of prlG expression on growth of the secY39 mutant. Strain GN31 (secY39) was transformed with either pCM134 (secE⁺), pHM401 (prlG1), pHM402 (prlG2), pHM403 (prlG3), or pTWV228 (vector). The transformant cultures, grown at 37°C in l-ampicillin (50 μg/ml), were subjected to 10-fold serial dilutions (from top to bottom) and spotted onto l-ampicillin agar plates, which were then incubated at 37°C for 16 h or at 20°C for 60 h, as indicated. (C) Effects of prlG expression on protein export. The transformant cells shown above were grown at 37 or 20°C, subjected to pulse-labeling, and processed for immunoprecipitation of MBP, as described in the legend to panel A, but without a chase. The percentage of the mature form of MBP is shown below each lane.

FIG. 2.

Specificity of suppression by secE105. (A) Effects on chromosomal secY mutations. Cold-sensitive secY mutants, as indicated, with or without additional secE105 mutation, were grown first at 37°C until early-log phase and then at 20°C for 30 min. Cells were pulse-labeled with [³⁵S]methionine for 1 min and immediately processed for immunoprecipitation of MBP. Labeled MBP molecules were visualized. p, precursor forms; m, mature forms. (B) Effects on dominant-negative secY mutations on plasmids. Strains HM562 (secE⁺) and HM564 (secE105), each carrying plasmid pSTD343 (lacI^q), were further transformed with a compatible plasmid encoding wild-type or mutant SecY. The mutant forms, SecY(R357E) and SecY Δ(346-357), are known to be dominant negative (21). Cells were grown at 37°C in the presence of 1 mM IPTG, 5 mM cyclic AMP, 50 μg of ampicillin/ml, and 20 μg of chloramphenicol/ml and were pulse-labeled with [³⁵S]methionine for 0.5 min.

FIG. 3.

Mutations secE105 and secY24 are synthetically lethal, with destabilization of the SecY24 protein. (A) The secE105 mutation was introduced by P1 transduction into strain EM100 (secY24 F′ lacI^q) carrying pHMC5A (secY⁺ under the control of the lac promoter) in the presence of IPTG to produce HM696. HM695 was a control strain (secE⁺ secY24/pHMC5A). Cell growth on L agar supplemented with 0.4% glucose (L + Glc) and P agar supplemented with 1 mM IPTG (P + IPTG) was recorded after incubation at 30°C for 18 h. (B) Strains HM696 (secY24 secE105) (see above) and HM695 (its secE⁺ derivative) were grown at 30°C in P medium supplemented with 1 mM IPTG and 50 μg of ampicillin/ml to early-log phase. Cells were then collected, washed, and inoculated into L medium containing 0.4% glucose, followed by further incubation at 30°C. Samples were withdrawn at the indicated time points and subjected to anti-SecY immunoblotting, with sample sizes adjusted to those corresponding to a fixed approximate cell number. Arrowhead indicates SecY. Asterisks show background cross-reacting bands. The intensity of each SecY band, relative to that in lane 4, is shown below each lane.

FIG. 4.

Stability of the SecY-SecE association in DM. Two compatible plasmids, pSTD343 (lacI^q) and pNA3 (secY-His₆-myc), were introduced into strains HM562 (secE⁺) and HM564 (secE105). Membrane fractions were prepared from lac-induced cultures of the plasmid-bearing cells, and portions containing 100 μg of protein were solubilized with 2% DM. Supernatants after centrifugation (at 100,000 × g for 30 min at 4°C) were incubated at the indicated temperature for 30 min and then applied to Ni-NTA spin columns (Qiagen), which were washed with a buffer containing 0.03% DM and eluted with 1 M imidazole in the same buffer. Eluates were subjected to SDS-PAGE and immunoblotting with the antibodies shown on the left.

FIG. 5.

SecY-stabilizing ability of SecE(S105P). Strain HM834 (secE⁺) or HM835 (secE105) was transformed with two compatible plasmids, one overexpressing SecY or its derivative and another overexpressing SecE⁺ (lower panel) or SecE(S105P) (upper panel), respectively. The SecY plasmids used were pCM10 (SecY⁺) (lanes 1 to 5), pHM404 [SecY Δ(346-357)] (lanes 6 to 10), and pHM405 [SecY(R357E)] (lanes 11 to 15). Cells were grown in the presence of 1 mM IPTG and 5 mM cyclic AMP at 37°C and were pulse-labeled with [³⁵S]methionine for 30 s, followed by a chase with unlabeled methionine for the indicated periods. The labeled SecY proteins were immunoprecipitated and visualized by phosphor image analyzer after SDS-PAGE.

FIG. 6.

In vitro characterization of SecE(S105P) effects on pro-OmpA translocation. (A) Effects on different SecY mutational defects. IMVs were prepared from strains HM808 (secY39 secE105) (lanes 1 and 2), GN04 (secY39) (lanes 3 and 4), HM809 (secY205 secE105) (lanes 5 and 6), GN05 (secY205) (lanes 7 and 8), HM810 (secY104 secE105) (lanes 9 and 10), GN09 (secY104) (lanes 11 and 12), and TW156 (secY⁺ secE⁺) (lanes 13 and 14). They were subjected to an in vitro translocation assay using ³⁵S-labeled pro-OmpA and wild-type SecA. The PMF was generated (+) or dissipated (−) by addition of 5 mM succinate or 10 μM carbonycyanide-m-chlorophenyl hydrazone, respectively. Reactions were allowed to proceed for 10 min at 37°C (upper panel) or 20°C (lower panel). Samples were treated with proteinase K and analyzed by SDS-PAGE and phosphorimager exposure. p, precursor forms; m, mature forms. (B) pro-OmpA translocation reaction mediated by the SecE(S105P) single-mutant IMV. IMVs were prepared from strains HM811 (secE105) (lanes 1 to 10) and TW156 (secE⁺) (lanes 11 to 20). They were subjected to a ³⁵S-labeled pro-OmpA translocation assay at 20°C in the presence (+) or absence (−) of the PMF for the indicated lengths of time. Shown are SDS-PAGEprofiles after proteinase K treatment. i, fragments generated from incompletely translocated pro-OmpA. (C) Time courses of full-length translocation. Intensities of the full-length proOmpA product (mature and precursor forms) with a SecE(S105P) IMV (circles) or a Sec⁺ IMV (triangles) are plotted against the reaction time. Open and solid symbols, results in the presence and absence of the PMF, respectively. (D) Time courses of generation of incompletely translocated products. Intensities of the incomplete translocation products (corresponding to the “i” bands in panel B) are plotted against the reaction time. Symbols are the same as in panel C.

FIG. 7.

SecE(S105P) preferentially compensates for the early translocation defect of the SecY39 alteration. (A) Translocation. IMVs prepared from strains GN04 (secY39) (lanes 1 to 5), HM808 (secY39 secE105) (lanes 6 to 10), and TW156 (secY⁺) (lanes 11 to 15) were subjected to a ³⁵S-labeled pro-OmpA translocation reaction at 37°C with wild type SecA and in the absence of the PMF. At the indicated time points, samples were treated with proteinase K and analyzed by SDS-PAGE and phosphorimaging. Positions of precursor (p) and mature (m) forms, as well as those of two intermediate forms (I₂₆ and I₁₆), are shown. (B) Processing. The reactions for which results are shown in panel A were terminated directly with 5% TCA. Precursor and mature forms of OmpA were separated by SDS-PAGE. (C) Quantitative representations of data from panels A and B. Inten-sities of full-length translocation products (circles) and the intermediates I₂₆ (triangles) and I₁₆ (squares) in panel A are plotted against reaction time. Processing efficiencies from panel B are also shown (solid diamonds). Left and right graphs show results with the SecY39-SecE(S105P) IMV and the Sec⁺ IMV, respectively. (D) Further translocation of I₁₆ by a superactive SecA derivative. IMVs from HM808 (secY39 secE105) were subjected to a ³⁵S-labeled pro-OmpA translocation reaction at 37°C with wild-type SecA (final concentration, 10 μg/ml) in the absence of the PMF. After 5 min, either SecA329 or wild-type SecA (final concentration, 20 μg/ml) was added to the reaction mixture, followed by further incubation at 37°C. At the indicated time points, samples were treated with proteinase K followed by SDS-PAGE and phosphorimaging. (E) Quantitative representations of the data from panel D. Symbols are the same as in panel C.