In vivo evidence for the role of the epsilon subunit as an inhibitor of the proton-translocating ATPase of Escherichia coli

Abstract

The function of the £ subunit of the Escherichia coli proton-translocating ATPase has been examined by using a mutant defective in the uncC gene. Strains with a defective uncC gene show a reduction in both growth yield and growth rate that is more severe than for other unc mutants; this deleterious effect is shown to be a result of the ATPase activity of the F1 complex which is missing the £ subunit. In addition, the £-deficient F1 is bound less tightly to the membrane. These data suggest that, in vivo, the £ subunit is capable of inhibiting the ATPase activity of F1 and also functions in the binding of F1 to Fo. The proton-translocating ATPase of Escherichia coli is a complex multimeric enzyme composed of two structural units, Fo and F1. Three subunits, a, b, and c, form the Fo

SecD and SecF are required for the proton electrochemical gradient stimulation of preprotein translocation.

“…(Pogliano and Beckwith, in press) and either plasmid pCGSHI, in which SecD/F synthesis is directed from their wild-type promoter, or pGAPI, in which SecD/F synthesis is arabinose inducible. Cells were grown in LB containing 0.002% arabinose (+) or 0.2% glucose (- Inner membrane vesicles and permeabilized cells depleted of SecD/F are unable to maintain a full proton electrochemical gradient The IMV's A/ (inside positive) and ApH (inside acidic), components of A/tH+, were first measured by the fluorescence of the potentialand pH-sensitive dyes oxonol VI (Apell and Bersch, 1987) and 9-aminoacridine (Klionsky et al, 1984), respectively. IMVs containing SecD/F displayed a change in fluorescence of oxonol VI of 176 relative units (mg protein)-1 (SD 20; n = 4) upon addition SecD and SecF content were determined from quantitative immunoblot analysis as shown in Figure 1 and described in Materials and methods.…”

“…Cultures were then innoculated in LB with either 2% glucose or 0.002% arabinose and were grown for 10 doublings. Inverted inner membrane vesicles were prepared form EcoliKM9 (unc-::TnlO, relAl, spoTl, metbl;Klionsky et al, 1984),DIO (rnalO, relAl, spoTl, metbl), JP91 and JP135 as described by Chang et al…”

Precursor protein translocation by the Escherichia coli translocase is directed by the protonmotive force.

“…Materials and bacterial strains Inverted membrane vesicles were prepared from E.coli strain KM9 (unc-TnJO, relAl, spoTl, metBl; Klionsky et al, 1984) as described by Chang et al (1978). ProOmpA (Crooke et al, 1988), SecA and SecB protein (Weiss et al, 1988;Lecker et al, 1989), bR (Danon and Stoeckenius, 1974;Driessen et al, 1987) and beefheart CytOx (Yu et al, 1975;Driessen et al, 1985) were purified as described.…”

Non-bilayer lipids are required for efficient protein transport across the plasma membrane of Escherichia coli.

“…The fluorescence after addition of gramicidin was taken as a 100% value. The ApH of inverted inner membrane vesicles was determined by measuring the fluorescence quenching of 9-amino-7-chloro-2-methoxyacridine (ACMA) upon addition of 1 mM ATP (Klionsky et al, 1984). The excitation and emission wavelengths were 420 and 500 nm respectively.…”