Archaeal and bacterial SecD and SecF homologs exhibit striking structural and functional conservation

Abstract

The majority of secretory proteins are translocated into and across hydrophobic membranes via the universally conserved Sec pore. Accessory proteins, including the SecDF-YajC Escherichia coli membrane complex, are required for efficient protein secretion. E. coli SecDF-YajC has been proposed to be involved in the membrane cycling of SecA, the cytoplasmic bacterial translocation ATPase, and in the stabilizing of SecG, a subunit of the Sec pore. While there are no identified archaeal homologs of either SecA or SecG, many archaea possess homologs of SecD and SecF. Here, we present the first study that addresses the function of archaeal SecD and SecF homologs. We show that the SecD and SecF components in the model archaeon Haloferax volcanii form a cytoplasmic membrane complex in the native host. Furthermore, as in E. coli, an H. volcanii deltasecFD mutant strain exhibits both severe cold sensitivity and a Sec-specific protein translocation defect. Taken together, these results demonstrate significant functional conservation among the prokaryotic SecD and SecF homologs despite the distinct composition of their translocation machineries.

FIG. 1.

The SecD•6xHis protein localizes to the H. volcanii cytoplasmic membrane. (A) Predicted topology of H. volcanii SecD and SecF using TMHMM (extracytoplasmic [ext] and cytoplasmic [cyt] sides of the membrane, respectively). (B) Cytoplasmic (cyt) and membrane (mem) fractions prepared from H. volcanii strain RK19.1, expressing SecD•6xHis, and the control strain, WR-Hv-NP15, containing a novobiocin-resistant shuttle vector not expressing this tagged protein, were subjected to Western blot analysis using an anti-pentahistidine antibody. Migrations of molecular mass markers are indicated by arrows (in kilodaltons).

FIG. 2.

In vivo copurification of H. volcanii SecF with SecD•6xHis. Membrane proteins were purified from dodecyl maltoside-solubilized membranes of the SecD•6xHis-expressing strain RK19.1 and the control strain, WR-Hv-NP15, using Ni-NTA affinity chromatography. Eluates were subjected to reducing, “seminative” denaturing conditions. Samples were separated on a 3 to 8% Tris-acetate gradient gel, and the proteins were visualized by Coomassie stain. The identities of the bands indicated were verified by matrix-assisted laser desorption ionization-time of flight analysis. Migrations of molecular mass markers are indicated by arrows (in kilodaltons).

FIG. 3.

Deletion of the secFD operon confers a severe cold-sensitive growth defect. Growth of H. volcanii secFD⁺ and ΔsecFD strains was measured in triplicate by plotting the optical density at 600 nm (OD₆₀₀) against time (in hours). In all three panels, growth of secFD⁺ strains is indicated by solid lines, and that of the ΔsecFD strains is indicated by dashed lines (error bars indicate ±1 standard deviation). (A and B) Growth of secFD⁺ (H98) and ΔsecFD (NH-Hv10) strains at (A) 45°C and (B) 30°C. (C) Growth of secFD⁺ (NH-Hv11) and ΔsecFD (NH-Hv12) strains expressing SecFD•6xHis (on plasmid pRK19.1) at 30°C.

FIG. 4.

The H. volcanii ΔsecFD strain confers a Sec translocation defect. (A) Protein levels of Gly-DM and GlyDTED-DM in cell (c) and culture supernatant (s) fractions from secFD⁺ and ΔsecFD H. volcanii cultures expressing these constructs were determined by Western blot analysis (using anti-Myc antibodies). Predicted positions of precursor (p) and mature (m) reporter proteins are indicated. Standardized equivalents of cell and supernatant fractions were analyzed. Strain H98 was used as a negative control (control). (B) CWG signal sequence (wild-type) and mutated CWG signal sequence (mutant). The residues that are altered in this GlyDTED construct are underlined and colored gray.

FIG. 5.

Deletion of the secFD operon perturbs proteinase IV levels. Levels of chromosomally encoded CWG (∼98 kDa) and plasmid-encoded pIV-Myc (∼35 kDa) in secFD⁺ (NH-Hv43) and ΔsecFD (NH-Hv44) strains were determined by Western blot analysis of cell lysates from H. volcanii cultures. Top and bottom panels correspond to identical lanes of a blot cut and probed separately with anti-CWG and anti-Myc antibodies, respectively. Standardized equivalents of cell lysates were analyzed.

FIG. 6.

The ΔsecFD translocation defect is Sec specific. Protein levels of arabinanase-Myc (Arb) and α-amylase-Myc (Amy) in cell (c) and culture supernatant (s) fractions from secFD⁺ and ΔsecFD H. volcanii cultures expressing these constructs were determined by Western blot analysis (using anti-Myc antibodies). Predicted positions of precursor (p) and mature (m) reporter proteins are indicated. Standardized equivalents of cell and supernatant fractions were analyzed.