University of Groningen Inhibition of preprotein translocation and reversion of the membrane inserted state of SecA by a carboxyl terminus binding

SecA is the peripheral subunit of the preprotein translocaseof Escherichia coli . SecA consists of two independently folding domains, i.e., the N-domain bearing the high-affinity nucleotide binding site (NBS-I) and the C-domain that harbors the low-affinity NBS-II. ATP induces SecA insertion into the membrane during preprotein translocation. Domain-specific monoclonal antibodies (mAbs) were developed to analyze the functions of the SecA domains in preprotein translocation. The antigen binding sites of the obtained mAbs were confined to five epitopes. One of the mAbs, i.e., mAb 300-1K5, recognizes an epitope in the C-domain in a region that has been implicated in membrane insertion. This mAb, either as IgG or as Fab, completely inhibits in Vitro proOmpA translocation and SecA translocation ATPase activity. It prevents SecA membrane insertion and, more strikingly, reverses membrane insertion and promotes the release of SecA from the membrane. Surface plasmon resonance measurements demonstrate that the mAb recognizes the ADPand the AMP-PNP-bound state of SecA either free in solution or bound at the membrane at the SecYEG protein. It is concluded that the mAb actively reverses a conformation essential for membrane insertion of SecA. The other mAbs directed to various epitopes in the N-domain were found to be without effect, although all bind the native SecA. These results demonstrate that the C-domain plays an important role in the SecA membrane insertion, providing further evidence that this process is needed for preprotein translocation. SecA is the central component of the preprotein translocasein Escherichia coli(for reviews, see ref 1, 2). Preprotein translocaseis a multimeric membrane protein complex that in addition to SecA consists of the integral membrane proteins SecY, SecE, and SecG as stable subunits ( 3-5), and SecD and SecF as accessory proteins ( 6, 7). The dynamic distribution of SecA between the cytosol and the membrane is determined by the amount of integral translocase subunits present in the membrane and by growth conditions (8-11). The heterotrimeric SecYEG complex constitutes a high-affinity membrane binding site for SecA (3, 12), and the SecA bound at these sites exposes a carboxyterminal domain to the periplasmic face of the membrane (8). SecA may also directly bind to the phospholipids at the membrane surface that constitute a binding site with low affinity (13, 14). SecA is required for the productive binding of preproteins to thetranslocase(3, 4). It interacts with the signal sequence and with a part of the mature preprotein ( 15, 13) but also binds SecB, a molecular chaperone that is specific for preprotein translocation ( 3). A key feature of the SecYEG-bound SecA is its ATPase activity that is stimulated by preproteins ( 13). SecA couples the hydrolysis of ATP to the translocation of the preprotein across the membrane. SecA is a large protein of 102 kDa, and not only exists (16) but also functions (17) as a stable homodimer. Each of the protomers comprises two independently folding domains of similar size, which have been termed the amino (N) and the carboxy (C) terminal domains (18). The N-domain contains a high-affinity ( KD,ADP≈ 0.15 μM) nucleotide binding site (NBS-I), 1 whereas the C-domain contains a site that binds nucleotides with low affinity ( KD,ADP ≈ 340μM, NBS-II) (19, 20). Both NBSs are able to bind ATP independentlyin Vitro, whereas mutants in either site inhibit the translocation ATPase activity of SecA completely, indicating that both sites are essential for and act in a concerted manner in the SecA-mediated translocation ( 2022). During translocation, preproteins are pushed across the membrane in steps of about 2 -3 kDa (23). This process is thought to be driven by nucleotide-modulated cycles of membrane inserted SecA ( 24, 25, 14). In the presence of preprotein, the binding of ATP (or the nonhydrolyzable ATP analog AMP-PNP) drives the insertion of SecA into SecAdepleted membranes in a SecYEG-dependent fashion. This gives rise to a number of proteinase resistant SecA fragments † This work was supported by a PIONIER grant of the Netherlands Organization for Scientific Research (N.W.O.). * To whom correspondence should be addressed at the Department of Microbiology, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands (Phone: +50363 21 64; Fax:+50363 21 54; E-mail: A.J.M.DRIESSEN@BIOL.RUG.NL). ‡ Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen. § Department of Internal Medicine, University of Groningen. | Utrecht University. X Abstract published inAdVance ACS Abstracts, July 1, 1997. 1 Abbreviations: AMP-PNP, adenosine 5 ′-(â,γ-imidotriphosphate); BSA, bovine serum albumin; DTT, dithiothreitol; ELISA, enzyme linked immunosorbent assay; Fab, ab fragment from IgG; IMV, inverted inner membrane vesicles; mAb, monoclonal antibody; NBS, nucleotide binding site; TBS, Tris-buffered saline; SPR, surface plasmon resonance. 9159 Biochemistry1997,36, 9159-9168 S0006-2960(97)00344-9 CCC: $14.00 © 1997 American Chemical Society using uniformly 35S-labeled SecA ( 26, 14) or a 30-kDa carboxy-terminal fragment using 125I-SecA (24-26; Figure 1). Based on the observation that ATP hydrolysis stimulates the de-insertion of the 30-kDa 125I-SecA fragment, and that a second ATP molecule has to be hydrolyzed to release the SecA from the membrane, it has been suggested that the deinsertion of SecA is an essential part of the preprotein translocation cycle ( 24, 25). However, in 35S-SecAreconstituted membranes, about 50 -60% of the35S-SecA can be released by unlabeled SecA independent of protein translocation and the remaining SecA seems to be stably inserted and fully functional in preprotein translocation ( 14), suggesting, that the de-insertion of SecA may not to be a prerequisite for preprotein translocation. The mechanistic details, how the nucleotide-induced conformational changes in SecA are coupled to preprotein translocation, are only poorly understood. Differential scanning calorimetry and dynamic light scattering experiments with soluble SecA ( 18) indicate that the AMP-PNPbound state of SecA has a more extended conformation than the ADP-bound state. The ADP-bound state may resemble the membrane de-inserted, surface-bound state of SecA, whereas the more extended AMP-PNP-bound state possibly corresponds to the membrane inserted conformation. Binding of nucleotides to NBS-I changes the interaction between the Nand C-domains ( 18), possibly by closing and opening of a cleft between both domains. To provide further information about the functions of the Cand N-domains, we have raised monoclonal antibodies against specific parts of SecA. One mAb recognizes an epitope that is confined to the 30-kDa carboxyl-terminal region of SecA that has been shown to insert into the membrane (26). This mAb efficiently inhibits in Vitro translocation and blocks the SecA translocation ATPase activity. Inhibition is due to the prevention of SecA membrane insertion and, more strikingly, the reversal of the membrane inserted state of SecA. It is concluded that the mAb inhibits preprotein translocation by interfering with a membrane inserted conformation of SecA. EXPERIMENTAL PROCEDURES Bacterial Strains and Growth Media.Unless indicated otherwise, strains were grown in Luria Bertani (LB) broth or on LB-agar supplemented with 50 μg of ampicillin/mL, 0.5% (w/v) glucose, or 1 mM isopropyl 1-thioâ-D-galactopyranoside, as required. SecYEG overproduction was done in SF100 freshly transformed with pET340 (YEG +), SecA in JM109 and NO2947 containing pMKL18, SecB in NO2947 (Table 1) containing pAK330 ( 27), and proOmpA in E. coli strain MM52 transformed with pTAQpOA (gift of N. Nouwen). Materials. SecA, SecB, and proOmpA were isolated as described ( 28-30, respectively). Purified SecA was labeled with carrier-free125I (Radiochemical Centre, Amersham, U.K.) to a specific activity of about 5× 104 cpm/pmol according to the following procedure: SecA (100 μg) in buffer A (50 mM TrisHCl, pH 8.0, 50 mM KCl, 5 mM MgCl2, and 10% glycerol) was transferred to a reaction vial coated with IODO-GEN Iodination Reagent (Pierce, Rockford, IL). The reaction was started by adding 2 μL of K125I (200 μCi), incubated for 15 min at room temperature, and terminated by transferring the mixture into a new reaction vial containing dithiothreitol (DTT) at a final concentration of 10 mM. Free iodine was removed by chromatography on a PD-10 Sephadex column (Pharmacia LKB Biotech AB, Uppsala, Sweden) which was prewashed with buffer A, containing 1 mM DTT. Inverted inner membrane vesicles (IMV; 31) were prepared from E. coli D10 and SF100 which was transformed either with pET324 (8) as a control or with pET340 (SecYEG +; Table 1). The fusion proteins cro-â-galactosidase -SecA(323-706) andcro-â-galactosidase -SecA(149-323) were purified from strain POP2136 containing the cIts857 repressor harboring the plasmids pET111 and pET135, respectively. Monoclonal antibodies were produced as hybridoma supernatants (MCA development b.v. Groningen, The Netherlands) or as ascites (Eurogentec, Gent, Belgium) and purified by protein G-Sepharose 4 Fast Flow affinity chromatography as recommended by the company (Pharmacia). Fab fragments were obtained using the ImmunoPure Fab preparation kit of Pierce as recommended. AMP-PNP was purified of contaminating ADP as described ( 18). Anti-mouse IgG Fcγ was from Pharmacia (Biosensor AB, Uppsala, Sweden). All chemicals were from Sigma (St. Louis, MO) if not stated otherwise. Construction and Expression of the Fusion Gene lacZsecA.A DNA fragment encoding a truncated SecA molecule was obtained by digestion of pMKL18 encoding wild-type SecA withSalI-PstI or with EcoRV-SalI. The fragments were made blunt by T4-polymerase/exonuclease treatment and cloned in-frame withlacZ in the multiple cloning site of pEX1 and pEX2, respectively. The pEX plasmids contain a synthetic operon ex