Structure and function of a membrane component SecDF that enhances protein export

Abstract

Summary Protein translocation across the bacterial membrane, mediated by the SecYEG translocon and the SecA ATPase1–4, is enhanced by proton-motive force (PMF)5,6 and membrane-integrated SecDF7–9, which associates with SecYEG. Here, we determined the crystal structure of Thermus thermophilus SecDF at 3.3 Å resolution, which revealed a pseudo-symmetrical, 12-helix transmembrane (TM) domain belonging to the RND superfamily and major periplasmic domains (P1 and P4). Higher resolution analysis of the latter suggested that P1, which proved to bind an unfolded protein, undergoes functionally important conformational changes. In vitro analyses identified an ATP-independent step of protein translocation that requires both SecDF and PMF. Electrophysiological analyses revealed that SecDF conducts protons in a pH- and unfolded protein-dependent fashion, in which conserved Asp and Arg residues at the TM SecD/SecF-interface play essential roles in the movements of protons and preproteins. Therefore, we propose that SecDF functions as a membrane-integrated chaperone, powered by PMF, to achieve ATP-independent protein translocation.

Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of marine bacteria

“…1B, lanes 4-7). These results suggest that V.SecDF1 is unable to use the proton gradient and can instead use the Na + gradient to execute its protein export function, whereas V.SecDF2 is independent of Na + and presumably H + -dependent, as has been shown for the E. coli SecDF protein (5). Thus, V.SecDF1 has been optimized to the primary habitat, seawater, of the Vibrio strain whereas V.SecDF2 has retained or gained (27) the proton dependence.…”

“…In addition, efficient operation of protein export in bacteria requires SecDF, a membrane protein complex having 12 transmembrane segments and large periplasmic domains of functional importance (3). SecDF is encoded either by the separate secD and secF genes (4) or as a single polypeptide (5). The crystal structures of Thermus thermophilus SecDF (TSecDF) and structure-instructed functional studies of the Escherichia coli ortholog suggest that SecDF drives polypeptide translocation by capturing a substrate that is emerging from the translocon into the periplasmic space and undergoing proton-motive-force-dependent cycles of conformational changes (5,6).…”

Identification and characterization of a translation arrest motif in VemP by systematic mutational analysis

“…biophysical studies, we previously proposed a model for SecDF-mediated facilitation of protein translocation at a late step in protein export (4,5). Our data strongly suggest that E. coli SecDF pulls a translocating polypeptide chain from the periplasmic side, coupled with proton flow from the periplasmic space to the cytosol through a SecD-SecF transmembrane interface.…”

Stoichiometry of SecYEG in the active translocase of Escherichia coli varies with precursor species

“…At the membrane, SecA binds SecY and acts as a motor providing energy from a cycle of binding and hydrolysis of ATP to move precursors through the channel in SecY. In vivo proton-motive force is also coupled to translocation via SecDF/YajC (5,6). An active translocase consists of the two integral membrane heterotrimeric complexes and the motor SecA ATPase, which is considered a peripheral subunit.…”