Binding, activation and dissociation of the dimeric SecA ATPase at the dimeric SecYEG translocase

Abstract

The bacterial preprotein translocase is comprised of a membrane-embedded oligomeric SecYEG structure and a cytosolic dimeric SecA ATPase. The associations within SecYEG oligomers and SecA dimers, as well as between these two domains are dynamic and reversible. Here, it is shown that a covalently linked SecYEG dimer forms a functional translocase and a high affinity binding site for monomeric and dimeric SecA in solution. The interaction between these two domains stimulates the SecA ATPase, and nucleotides modulate the affinity and ratio of SecA monomers and dimers bound to the linked SecYEG complex. During the translocation reaction, the SecA monomer remains in stable association with a SecYEG protomer and the translocating preprotein. The nucleotides and translocation-dependent changes of SecA-SecYEG associations and the SecA dimeric state may reflect important facets of the preprotein translocation reaction.

Fig. 1. The covalently linked SecYEG dimer is functional for pre protein translocation. (A) Overproduction of SecYYEG suppresses the SecDFyajC^– cold-sensitive and SecY thermo-sensitive phenotypes. Strain BL325 (BL21 tgt::kan-araC⁺ -P_BAD::yajCsecDF) or CJ107 (Wolfe et al., 1985) were transformed with the isopropyl-β-d-thio galactopyranoside (IPTG)-inducible plasmid pTrc99 or pTrc99 encoding SecYE_HAG (pTrcEYG) or SecYYE_HAG (pTrcEYYG). Transformants were plated on LB/ampicillin (50 µg/ml) contain ing 1 mM IPTG and incubated at the restrictive temperature. (B) Immunostaining of IMV preparations. About 1 µg of IMV proteins prepared from E.coli BL21 overexpressing SecYE_HAG (IMVs-Y) or SecYYE_HAG (IMVs-YY) were analyzed by SDS–PAGE and immunostained with the indicated antibodies. In the presence of SDS, SecY (48.5 kDa) and SecYY (97 kDa) migrate aberrantly. (C and D) Translocase activity of the IMV preparations. Translocation reactions were performed and analyzed as described in Materials and methods.

Fig. 2. Oligomeric behavior of covalently-linked SecYEG dimer and SecA. (A) Immunodetection of SecYEG oligomers in membranes enriched with SecYEG (IMVs-Y) or SecYYE²G² (IMVs-YY). IMVs were solubilized in TSG buffer at the indicated detergent concentration. Protein aliquots (∼0.2 µg) were analyzed by linear gradient BN–PAGE (4–12%) and immunostained. Where indicated, IMVs were incubated with SecA or SecA₉₅ (4 µg; 0.8 µM protomer). The asterisks indicate the probable complex of SecYYE²G² with monomeric and dimeric SecA. (B) SecA, His₆-tagged SecA or His₆-tagged SecA₉₅ in TSG buffer (±SDS) were analyzed on a native gel followed by Coomassie blue staining. Molecular weight markers are BSA and catalase. (C) [³⁵S]SecA (∼20 000 c.p.m., ∼100 ng) was mixed at room temperature with unlabeled SecA to reach the desired concentration. Samples were diluted in TSG buffer containing DDM and analyzed by BN–PAGE (4–10%) and autoradiography. The final SecA and DDM concentrations in each sample are indicated.

Fig. 3. The stabilized SecYEG dimer forms a stable binding site for SecA monomers and dimers. (A) Oligomeric state of the purified SecYEG complexes. [¹²⁵I]SecYEG and [¹²⁵I]SecYYE²G² purified complexes (∼30 000 c.p.m., ∼20 ng, in TSG buffer 0.03% DDM or 0.1% SDS) were analyzed by BN–PAGE (5–13%) and autoradio graphy. (B–D) Complexes between radiolabeled SecYEG and unlabeled SecA. [¹²⁵I]SecYEG and [¹²⁵I]SecYYE²G² purified complexes (∼30 000 c.p.m., 0.7 µM) were incubated (5 min, 22°C) with SecA or SecA₉₅ at the indicated concentration in TSG buffer containing 0.04% DDM. The resulting complexes were separated by BN–PAGE (4–12%) and revealed by autoradiography. (E) Complexes between radiolabeled SecA and unlabeled SecYYE²G². [³⁵S]SecA (∼20 000 c.p.m., 1 µM) was incubated in TSG buffer containing 0.04% DDM with the indicated concentration of the purified SecYYE²G² complex. The resulting complexes were analyzed by BN–PAGE (4–12%) and autoradiography. For M_r reference, a sample of the [¹²⁵I]SecYYE²G² complex incubated with unlabeled SecA is loaded in the left lane of the gel. Calculated molecular weights of the Sec proteins and Sec complexes: SecY (48.5 kDa); SecYY (97 kDa); His-SecE (14 kDa); SecG (11 kDa); His-SecA (103 kDa); His-SecA₉₅ (95 kDa); SecYEG (73.5 kDa); SecY²E²G² (147 kDa); SecY⁴E⁴G⁴ (294 kDa); SecY²E²G²–SecA (250 kDa); SecY²E²G²–SecA² (353 kDa).

Fig. 4. Cross-linking production between SecYYE²G² and SecA. (A) [¹²⁵I]SecYYE²G² (0.1 µM) or [³⁵S]SecA (1 µM) were incubated in CL buffer (5 min, 22°C) with the indicated concentration of unlabeled SecYYE²G² or unlabeled SecA, respectively. After cross-linking by EGS and dissociation of the non-covalent protein complexes by SDS, samples were analyzed by BN–PAGE (4–12%) and autoradiography. (B) [¹²⁵I]SecYYE²G² or unlabeled SecYYE²G² were incubated as in (A) with SecA or SecA₉₅, and cross-links were revealed by autoradiography or immunostaining.

Fig. 5. Nucleotide-dependent SecA–SecYEG associations. (A) Nucleotides do not change the oligomeric state of SecA in solution. [³⁵S]SecA (∼20 000 c.p.m., 1 µM) was incubated (10 min, 22°C) in TSG buffer containing 0.04% DDM with the indicated nucleotides (1 mM). Samples were analyzed by BN–PAGE (4–12%) and autoradiography. (B) Complexes between radiolabeled SecYEG and unlabeled SecA after addition of nucleotides. [¹²⁵I]SecYYE²G² (0.7 µM) was mixed with SecA (1 µM) in TSG buffer (0.04% DDM; ±5 mM MgCl₂). After incubation (5 min, 22°C), nucleotides were added and the incubation was prolonged for 5 min. Samples (lanes 5 and 6) were treated with potato apyrase (grade VII; 20 U/ml) for an additional 2 min. All samples were returned to ice before analysis by BN–PAGE (4–12%) and autoradiography. (C) Cross-link production between SecYYE²G² and SecA after addition of nucleotides. [¹²⁵I]SecYYE²G² (0.1 µM) or [³⁵S]SecA (1 µM) were incubated in CL buffer (±5 mM MgCl₂) with unlabeleled SecA (2 µM) or unlabeled SecYYE²G² (0.5 µM), respectively. After incubation with nucleotides (as described in B), samples were treated by EGS, then dissolved by SDS and analyzed by BN–PAGE (4–12%) and autoradiography.

Fig. 6. Activation of the SecA ATPase by SecYEG. SecA (0.8 µM) was incubated at room temperature with the purified SecYEG or SecYYE²G² complexes in TSG buffer (±0.04% DDM; ±MgCl₂) containing 2 mM of ATP. The Sec concentration refers to that of the SecYEG protomer (SecYYE²G² = 2 × SecYEG). After 10 min incubation at 22°C, the inorganic phosphate produced by the SecA ATPase was measured by the photocolorimetric method of Lanzetta et al. (1979).

Fig. 7. The SecA monomer is engaged with preprotein and the SecYEG channel during translocation. (A) [¹²⁵I]ProOmpA–BPTI (∼100 000 c.p.m.; 4 µg/ml) was incubated (10 min, 37°C) in TL buffer (50 µl final volume) with SecYEG- or SecYYE²G²-proteoliposomes (15 µg/ml), BSA (200 µg/ml), SecA (80 µg/ml; 0.8 µM) and ATP (2 mM) or DTT (2 mM) where indicated. Reactions were terminated by addition of apyrase (20 U/ml). Aliquots of the translocation reactions (1/10 vol.) were treated with proteinase K and analyzed by 12% SDS–PAGE and autoradiography. (B) The remainder of the translocation reaction performed in (A) was solubilized with an equal volume of TSG buffer containing 0.12% DDM. Aliquots were analyzed by BN–PAGE (4–10%) and autoradiography. For M_r reference, [¹²⁵I]SecYYE²G² complexed to SecA was loaded on the same gel (lane 11). (C) As in (B) lanes 5 and 6, but using the truncated SecA₉₅ or wild-type SecA to drive translocation in SecYEG- or SecYYE²G²-proteoliposomes. (D) The translocation reactions described in (A) were supplemented with [¹²⁵I]SecA (∼100 000 c.p.m.; 4 nM). At the end of the incubation (10 min, 37°C), proteoliposomes were solubilized with 300 µl of TSG buffer, 0.1% DDM (10 min, 4°C). After centrifugation (10 min, 100 000 g), the membrane extract was incubated with anti-SecG IgG coupled to protein A–Sepharose beads. Immuno precipitates were washed (3 × 0.3 ml of TSG buffer) and analyzed by 10% SDS–PAGE and autoradiography. Theoretical molecular weight of the SecYEG–SecA–pOA complexes: pOA (37 kDa); SecYEG– pOA (112 kDa); (SecYEG)²–pOA (187 kDa); SecYEG–SecA–pOA (212 kDa); (SecYEG)²–SecA (250 kDa); (SecYEG)²–SecA–pOA (287 kDa); (SecYEG)⁴–pOA (343 kDa).