Reexamination of the role of the amino terminus of SecA in promoting its dimerization and functional state

Abstract

The SecA nanomotor promotes protein translocation in eubacteria by binding both protein cargo and the protein-conducting channel and by undergoing ATP-driven conformation cycles that drive this process. There are conflicting reports about whether SecA functions as a monomer or dimer during this dynamic process. Here we reexamined the roles of the amino and carboxyl termini of SecA in promoting its dimerization and functional state by examining three secA mutants and the corresponding proteins: SecADelta8 lacking residues 2 to 8, SecADelta11 lacking residues 2 to 11, and SecADelta11/N95 lacking both residues 2 to 11 and the carboxyl-terminal 70 residues. We demonstrated that whether SecADelta11 or SecADelta11/N95 was functional for promoting cell growth depended solely on the vivo level of the protein, which appeared to govern residual dimerization. All three SecA mutant proteins were defective for promoting cell growth unless they were highly overproduced. Cell fractionation revealed that SecADelta11 and SecADelta11/N95 were proficient in membrane association, although the formation of integral membrane SecA was reduced. The presence of a modestly higher level of SecADelta11/N95 in the membrane and the ability of this protein to form dimers, as detected by chemical cross-linking, were consistent with the higher level of secA expression and better growth of the SecADelta11/N95 mutant than of the SecADelta11 mutant. Biochemical studies showed that SecADelta11 and SecADelta11/N95 had identical dimerization defects, while SecADelta8 was intermediate between these proteins and wild-type SecA in terms of dimer formation. Furthermore, both SecADelta11 and SecADelta11/N95 were equally defective in translocation ATPase specific activity. Our studies showed that the nonessential carboxyl-terminal 70 residues of SecA play no role in its dimerization, while increasing the truncation of the amino-terminal region of SecA from 8 to 11 residues results in increased defects in SecA dimerization and poor in vivo function unless the protein is highly overexpressed. They also clarified a number of conflicting previous reports and support the essential nature of the SecA dimer.

FIG. 1.

SecAΔ11/N95 is functional in vivo due to overproduction of SecA protein. BL21.19 containing the indicated plasmids was grown in LB supplemented with appropriate antibiotics at 30°C to an A₆₀₀ of 0.2, and then it was shifted to 42°C for an additional 2 h. The A₆₀₀ of all cultures were adjusted to 1.0 by dilution of LB, and then cells were chilled, harvested by sedimentation at 4°C, and resuspended in sample buffer (2% sodium dodecyl sulfate, 125 mM Tris-HCl [pH 6.8], 5% 2-mercaptoethanol, 15% glycerol, 0.005% bromophenol blue) for analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting with SecA antisera and visualization by enhanced chemiluminescence utilizing SuperSignal West Pico (Pierce) and a Syngene Gelbox system as described previously (17). Prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the protein concentration in each sample was measured utilizing the Bradford assay, and equivalent amounts of total protein were loaded onto the gel. SecA levels were determined using a Syngene Gelbox system; the amount of SecA present in wild-type strain MC4100 lacking any plasmid was arbitrarily defined as 1.0, and the amounts determined are indicated under the lanes.

FIG. 2.

lacO(Con) mutants restore SecAΔ11his function by overproduction. (A) Sequence of the lac operator region with the lacO(Con) suppressor mutations indicated below the sequence. The lacO(Con) mutation at position 12 was present in two independent suppressor mutants, whereas the mutation at position 15 and the mutation at position 17 were each present in a single suppressor mutant. The number below each lacO(Con) mutation was obtained from a previous study (2), and these numbers indicate the corresponding repressor affinities for the mutant operator compared to the wild-type operator, which was defined as 100%. The lac repressor-protected fragment is indicated above the sequence, and the axis of dyad symmetry of the sequence is also indicated. (B) Western blot of BL21.19 containing the indicated plasmids. The analysis was performed like the analysis described in the legend to Fig. 1 except that both SecA and OmpA antisera were used to probe the blot. OmpA was utilized as a control protein to verify that equivalent levels of total protein were loaded on the gel.

FIG. 3.

Subcellular fractionation and cross-linking studies support the in vivo results. BL21.19 strains containing the indicated plasmids were grown and harvested as described in the legend to Fig. 1. Cell pellets were resuspended in 0.02 volume of ice-cold (A) TKMDP (10 mM Tris-Cl [pH 7.5], 50 mM KCl, 10 mM magnesium acetate, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1× protease inhibitor cocktail [Sigma]) or (B) HKM (50 mM HEPES-KOH [pH 7.2], 50 mM KCl, 1 mM magnesium acetate, 1× protease inhibitor cocktail) and broken by two passages at 8,000 lb/in² in a French pressure cell. Unbroken cells were removed by two successive centrifugations at 13,000 × g for 10 min at 4°C, which resulted in the total cleared lysate (Total). (A) For subcellular fractionation, soluble (S300) and membrane (P300) fractions were obtained by centrifugation at 150,000 × g for 25 min at 4°C in a Sorvall Discovery M120 microultracentrifuge. S300 was carefully removed, and P300 was resuspended in the original volume of 0.2 M sodium carbonate (pH 11.5), incubated on ice for 30 min, and resedimented. The carbonate-treated supernatant (P300S) was carefully removed, and the integral membrane pellet (P300P) was resuspended in the original volume of TKMD. (B) For protein cross-linking, the P300 pellet was briefly washed and resuspended in the original volume of HKM. Ten microliters of P300 was subjected to chemical cross-linking with the indicated concentration of EDAC [1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide] in HKM supplemented with 50 mM potassium acetate at room temperature for 15 min in a 40-μl reaction mixture, and then the reaction mixtures were quenched on ice by addition of 3 μl of 1 M Tris-HCl (pH 7.5). All samples were heated to 100°C for 2 min after addition of sample buffer and analyzed by (A) 11.3% or (B) 5.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting and visualization of SecA using appropriate antisera by enhanced chemiluminescence utilizing SuperSignal West Pico (Pierce) and a Syngene Gelbox system.

FIG. 4.

The monomer-dimer equilibrium of SecAΔ11, SecAΔ11/N95, and SecAΔ8 proteins is altered, and these proteins have defects in their SecA-dependent translocation ATPase activities. SecA proteins were overproduced and purified using either His-bind resin (Novagen) or SP Sepharose (Sigma) as previously described (17). (A and B) Light scattering data were collected using a Superose 6 10/30 HR size exclusion chromatography column (GE Healthcare) connected to a high-performance liquid chromatography system (Alliance 2965; Waters Corp.) equipped with an autosampler. The eluate from the size exclusion chromatography column was monitored by using a photodiode array UV/Vis detector (996 PDA; Waters Corp.), a differential refractometer (OPTI-Lab or OPTI-rEx; Wyatt Corp.), and a static, multiangle laser light scattering detector (DAWN-EOS; Wyatt Corp.). The system was equilibrated with a solution containing 10 mM Tris-HCl (pH 7.5), 100 mM or 300 mM KCl, 10 mM magnesium acetate, 1 mM dithiothreitol, and 1× protease inhibitor cocktail at a flow rate of 0.3 ml/min. The weight average molecular mass (MW) for SecA protein was determined for the concentration ranges shown with either (A) 100 mM KCl or (B) 300 mM KCl. Two software packages were used for data collection and analysis; the Millennium software (Waters Corp.) controlled the high-performance liquid chromatograph operation and data collection from the multiwavelength UV/Vis detector, while the ASTRA software (Wyatt Corp.) collected data from the refractive index detector and the light scattering detector and recorded the UV trace at 280 nm sent from the 996 PDA detector. The lines indicate the nonlinear least-square fits of weight average molecular mass determined for the apex of each eluting peak at various concentrations for a monomer-dimer association model; the error bars indicate 5% uncertainties for molecular mass determinations expected from measurement of light scattering. Symbols: filled circles, SecA-his; open squares, SecAΔ8his; filled triangles, SecAΔ11his; open triangles, SecAΔ11/N95. (C) Endogenous, membrane, and translocation ATPase activities of the indicated SecA mutant proteins, determined as described previously (17).