Complex formation by the mrpABCDEFG gene products, which constitute a principal Na+/H+ antiporter in Bacillus subtilis

Abstract

The Bacillus subtilis Mrp (also referred to as Sha) is a particularly unusual Na(+)/H(+) antiporter encoded by mrpABCDEFG. Using His tagging of Mrp proteins, we showed complex formation by the mrpABCDEFG gene products by pull-down and blue native polyacrylamide gel electrophoresis analyses. This is the first molecular evidence that the Mrp is a multicomponent antiporter in the cation-proton antiporter 3 family.

FIG. 1.

Complementation of mrp-deleted strains by each His-tagged form of Mrp. (A) Cells of B. subtilis strains TY1 (ΔA), TY2 (ΔB), TY3 (ΔC), TY4 (ΔD). TY5 (ΔE), TY6 (ΔF), and TY7 (ΔG) and wild-type strain UOT1285 (WT) were streaked onto an LB plate containing 0.2 M NaCl. (B) Strains SK702 (ΔA/A*-his), KY8 (ΔB/B-his), KY9 (ΔC/C-his), KY10 (ΔD/D-his), KY11 (ΔE/E-his), KY12 (ΔF/F-his), and KY13 (ΔG/G-his) grew on an LB plate containing 1 M NaCl and 1 mM IPTG, but SK701 (ΔA/A-his) did not. (C) Western blots to detect His-tagged MrpA. The membrane samples (20 μg of protein) of TY1 (ΔA), SK701 (ΔA/A-his), and SK702 (ΔA/A*-his) were loaded onto an SDS-12.5% polyacrylamide gel without boiling. His-tagged MrpA was immunodetected using an anti-His tag antibody (arrowhead).

FIG. 2.

Pull-down assays. His-tagged MrpA, MrpB, MrpC, MrpD, MrpE, MrpF, and MrpG were purified using TALON metal affinity resin from the membrane samples of SK702 and KY8 to KY13, respectively. The eluted samples (5 μg of protein) were separated by SDS-PAGE using a 10-to-20%-gradient acrylamide gel, and immunodetected by an anti-His tag antibody (top, arrowheads). Eluted samples of the same amount were also separated by Tricine SDS-PAGE using a 4% stacking-10% running acrylamide gel and immunodetected by an anti-MrpE antibody (bottom, star). The membrane sample of UOT1285 was used as the negative control (NC).

FIG. 3.

BN/SDS-PAGE. The solubilized membrane samples of SK702 and KY8 to KY13 were loaded onto the first-dimension (1D) BN-PAGE gel and then the second-dimension (2D) Tricine SDS-PAGE gel (4% stacking-10% running gel) (top). His-tagged Mrp proteins were probed with an anti-His tag antibody. The range in which the signals are detected in all lanes in the 1D is shown with dotted lines, and the molecular size of the center is estimated to be 410 kDa by comparison with a standard curve of a NativeMark unstained protein standard (Invitrogen). No signals were detected with the membrane sample of UOT1285 (the negative control) in the region between the dotted lines (bottom). The molecular sizes of His-tagged Mrp proteins estimated from the molecular standard (Precision Plus protein standards [Bio-Rad]) in the 2D are indicated to the right of the top panel. The migration pattern of the Precision molecular standard is shown to the left of the bottom panel.