Inhibition of Preprotein Translocation and Reversion of the Membrane Inserted State of SecA by a Carboxyl Terminus Binding MAb

Abstract

Inhibition of preprotein translocation and reversion of the membrane inserted state of SecA by a carboxyl terminus binding MAb den Blaauwen, T; de Wit, J.G; Gosker, H.; van der Does, C.; Breukink, E.J.; de Leij, Lou; Driessen, A.J.M. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.

In vivo cross-linking of the SecA and SecY subunits of the Escherichia coli preprotein translocase

“…Wild-type D10 cells were cross-linked with formaldehyde and analyzed by SDS-PAGE and immunoblotting with an oligoclonal antibody (OAb; mixture of monoclonal antibodies) directed against SecA (13). In the presence of 0.1% (wt/vol) of formaldehyde, a specific cross-linked protein complex with a molecular mass of 150 kDa was obtained (Fig.…”

The Sec system

“…The kinetics of 30 kDa formation are rather slow as compared to preprotein translocation [27], which is surprising as multiple cycles of SecA membrane insertion/de-insertion are required to completely translocate the preprotein. The 30kDa SecA fragment is accessible from the cytosolic face of the membrane, as a monoclonal antibody directed against this dornain reverses the membrane-insertion [33]. Thus, at least part of the membrane-inserted 30 kDa domain is accessible from the cvtosolic membrane face.…”

The Low-Affinity ATP Binding Site of theEscherichia coliSecA Dimer Is Localized at the Subunit Interface

“…Moreover, preincubation of SecA with a 10-fold excess (500 µM) of ATPγS, a nonhydrolyzable ATP analogue, prior to UV cross-linking by diN 3 ATP effectively prevented the formation of cross-linked products (lanes 6 and 7), while a 10-fold lower amount of ATPγS (5 µM) does not prevent the cross-linking (lanes 4 and 5). Immunostaining of the samples with an oligoclonal antibody directed against SecA (27) demonstrates that the cross-linked products are indeed derived from SecA ( Figure 4B, lane 3). The migration behavior of the 203 kDa polypeptide band on SDS-PAGE is consistent with a cross-linked product that corresponds to a stable dimer of SecA.…”

“…Hydrolysis of ATP dissociates the preprotein from its SecA-bound state and allows the C-domain to de-insert from the membrane (29). Studies with the soluble form of SecA suggest that the protein in its ADP-liganded state resembles a compact globular conformation with extensive contact between the N-and C-domains, and between the two subunits of SecA (21,27). In the ATP-bound form, the protein adopts a nonglobular, more elongated conformation with little interaction between the two domains (21).…”