The large first periplasmic loop of SecD and SecF plays an important role in SecDF functioning

Abstract

A remarkable feature of proteins of the SecD and SecF family involved in protein translocation is that they possess a very large first periplasmic domain. Here we report that this large first periplasmic domain is not required for the SecD-SecF interaction but that it is important for catalyzing protein translocation.

FIG. 1.

Schematic representation of different P1 deletions in SecD and SecF and their ability to complement growth of the SecDF-YajC depletion strain JP325. Regions that are conserved are indicated by black boxes. To test the functionality of the deletion, strain JP325 (P_BAD::yajC-secDF) was transformed with plasmids pET545 (yajC-secDF), pNN230 (yajC-secDΔ325-341F), pNN231 (yajC-secDΔ199-279F), pNN232 (yajC-secDΔ33-151F), pNN233 (yajC-secDΔ210-419F), pNN235 (yajC-secDFΔ74-101), and analyzed to restore growth on LB-ampicillin (100 μg/ml) plates containing 0.5% glucose (with or without 300 μM IPTG) at different temperatures (37°C, 30°C, and 22°C). ++, growth; +/−, very poor growth; −, no growth; Cs, growth is cold sensitive (normal growth at 37°C and no growth at 30°C or lower).

FIG. 2.

Expression of P1 SecD/F deletion mutants in cells from which chromosomally encoded YajC-SecDF was depleted. IMVs were prepared from strain JP325 containing the different P1 deletion plasmids grown for six generations on LB medium containing 0.5% glucose. Expression of the plasmid-encoded gene products has been induced with 300 μM IPTG. IMVs were analyzed by SDS-PAGE and CBB staining (A) and by immunostaining using antibodies against SecG (B). (C) After solubilization of the IMVs with dodecylmaltoside, SecF was purified with Ni-NTA beads, and the copurification of SecD was analyzed by SDS-PAGE and CBB staining. (D) The presence of YajC in the Ni-NTA-purified material was analyzed by SDS-PAGE and silver staining. Positions of mutant SecD and SecF proteins are indicated with asterisks and circles, respectively. wt, wild type.

FIG. 3.

SecDF-YajC-depleted IMVs have a kinetic translocation defect. (A) proOmpA translocation into IMVs derived from wild-type and SecDF-YajC-depleted cells (37°C). (B) Quantification results as shown in panel A. (C) proOmpA translocation into IMVs derived from wild-type and SecDF-YajC-depleted cells in the absence and presence of a PMF. IMVs containing P1 SecD/F deletion mutants are defective in protein translocation. (D and E) Initial rates of proOmpA translocation into IMVs containing different P1 SecD/F deletion mutants were analyzed at 37°C and 30°C. Translocation of proOmpA-fluorescein (4 μg/ml) into IMVs containing the different mutant SecDF-YajC complexes (200 μg/ml) were performed in 50 mM HEPES-KOH, pH 7.9, 50 mM KCl, 5 mM MgCl₂, 0.2 mg/ml bovine serum albumin, 2 mM dithiothreitol with SecA (10 μg/ml), SecB (35 μg/ml), 10 mM phosphocreatine, 50 μg/ml creatine kinase, 5 mM succinate, and 10 mM NADH. After the mixture was warmed for 2 min at 37°C, the reaction was initiated by the addition of ATP (2 mM final concentration). After the indicated period (A) or 10 (37°C [D])/15 (30°C [E]) minutes, samples were treated with proteinase K (0.1 mg/ml) for 30 min on ice. Translocation was assayed as described in reference . (B) To dissipate the PMF, nigericin and valinomycin (2 μM final concentration) were added to the translocation mixture. (D and E) wt, wild type.