Subunit a facilitates aqueous access to a membrane-embedded region of subunit c in Escherichia coli F1F0 ATP synthase

Abstract

Rotary catalysis in F(1)F(0) ATP synthase is powered by proton translocation through the membrane-embedded F(0) sector. Proton binding and release occurs in the middle of the membrane at Asp-61 on transmembrane helix 2 of subunit c. Previously, the reactivity of cysteines substituted into F(0) subunit a revealed two regions of aqueous access, one extending from the periplasm to the middle of the membrane and a second extending from the middle of the membrane to the cytoplasm. To further characterize aqueous accessibility at the subunit a-c interface, we have substituted Cys for residues on the cytoplasmic side of transmembrane helix 2 of subunit c and probed the accessibility to these substituted positions using thiolate-reactive reagents. The Cys substitutions tested were uniformly inhibited by Ag(+) treatment, which suggested widespread aqueous access to this generally hydrophobic region. Sensitivity to N-ethylmaleimide (NEM) and methanethiosulfonate reagents was localized to a membrane-embedded pocket surrounding Asp-61. The cG58C substitution was profoundly inhibited by all the reagents tested, including membrane impermeant methanethiosulfonate reagents. Further studies of the highly reactive cG58C substitution revealed that NEM modification of a single c subunit in the oligomeric c-ring was sufficient to cause complete inhibition. In addition, NEM modification of subunit c was dependent upon the presence of subunit a. The results described here provide further evidence for an aqueous-accessible region at the interface of subunits a and c extending from the middle of the membrane to the cytoplasm.

FIGURE 1.

Inhibition of ATP-driven proton pumping by Ag⁺ and NEM. ATP-driven quenching of ACMA fluorescence by cG58C-inverted membrane vesicles was assayed as described under “Experimental Procedures.” Fluorescence quenching was initiated by the addition of ATP at 20 s and terminated by the addition of nigericin at 100 s. The return to maximum fluorescence reached after the addition of nigericin was used to calculate the relative quenching values given in Table 1 and the inhibition values given in Table 2. Treatment with an inhibitor as described under “Experimental Procedures” is indicated.

FIGURE 2.

Reaction of MTS reagents with a cysteine thiolate in a polypeptide chain. MTSEA is membrane-permeable (35). MTS-methyl (MTSM) and the neutral form of MTS-carboxyethyl (MTSCE) are also expected to be membrane-permeable. MTSES and MTSET are membrane-impermeable (35, 37).

FIGURE 3.

Inhibition of ATP-driven proton pumping by MTS reagents. ATP-driven quenching of ACMA fluorescence was measured as described in Fig. 1. The return to maximum fluorescence reached after the addition of nigericin was used to calculate the inhibition values given in Table 2. A, fluorescence quenching of the cG58C mutant with DMSO, 1 mm MTS-methyl (MTSM), 1 mm MTS-carboxyethyl (MTSCE), and 1 mm MTSEA. B, fluorescence quenching of the cG58C mutant with DMSO, 1 mm MTSES, and 1 mm MTSET. C, inhibition of fluorescence quenching of the cG58C mutant by treatment with various concentrations of MTS-methyl (▴), MTS-carboxyethyl (•), MTSEA (▪), MTSES (○), and MTSET (□).

FIGURE 4.

Stoichiometry of NEM modification of G58C subunit c. A, subunit c was purified from inverted membrane vesicles of the cG58C mutant after treatment with 0.5 mm [¹⁴C]NEM for 15 min. The moles of total purified subunit c were determined using a Lowry assay, and moles of incorporated [¹⁴C]NEM were determined by scintillation counting. The molar ratio determined in two experiments is indicated. B and C, subunit c was purified from untreated membranes or membrane samples treated with 5 mm NEM for 15 min and then subjected to ESI-TOF mass spectrometry. Primary signal peaks correspond to unlabeled G58C subunit c at 8330 Da and NEM-labeled G58C subunit c at 8456 Da. The peak areas in panel C were used to calculate a ratio of modified to total subunit c equal to 0.12.

FIGURE 5.

Labeling of G58C subunit c with [¹⁴C]NEM in the presence and absence of subunit a. Inverted membrane vesicles prepared from strains expressing the cG58C mutant and the cG58C ΔuncB mutant were labeled with 0.5 mm [¹⁴C]NEM, and subunit c was purified as described under “Experimental Procedures.” 20 μg of purified subunit c solubilized in SDS sample buffer were subjected to SDS-PAGE. The gel was stained with Coomassie Brilliant Blue (CBB), sealed in cellophane, and exposed to a phosphor storage screen for 48 h. Relative labeling was quantitated by scintillation counting (black bars) and from the phosphor image (PI, gray bars).

FIGURE 6.

Mapping of reactive residues on structural models of subunit c. A, residues on the cytoplasmic side of cTMH2 are represented in an α-helical wheel model. Red coloring indicates reactivity to Ag⁺ (>90% inhibition) or MTSEA (>80% inhibition). Yellow-colored positions were moderately sensitive, and blue-colored positions were insensitive to MTSEA treatment. Black residues could not be tested. Asp-61 is colored cyan. B, this model of the E. coli c-ring was derived by Meier et al. (12) from the x-ray structure of the c-ring from I. tartaricus using the program WHATIF to substitute E. coli residues. The positions of MTSEA-sensitive residues appear as colored patches on the surface of the ring. Coloring indicates Asp-61 (cyan), residues highly sensitive to MTSEA (red), and residues moderately sensitive to MTSEA (yellow). The areas of residues Phe-53 and Gln-52 exposed to the surface are colored blue. The side chains of residues Val-56 and Val-60 lie behind the surface shown. The surface shown has been calculated using the wild type side chains.