Assembly of the translocase motor onto the preprotein-conducting channel.

Bacterial protein secretion is catalysed by the SecYEG protein-conducting channel complexed with the SecA ATPase motor. To gain insight into the SecA-SecYEG interaction we used peptide arrays, thermodynamic quantification, mutagenesis and functional assays. Our data reveal that: (i) SecA binds with low affinity on several, peripheral, exposed SecYEG sites.

Preprotein-controlled catalysis in the helicase motor of SecA.

The cornerstone of the functionality of almost all motor proteins is the regulation of their activity by binding interactions with their respective substrates. In most cases, the underlying mechanism of this regulation remains unknown. Here, we reveal a novel mechanism used by secretory preproteins to control the catalytic cycle of the helicase 'DEAD' motor of SecA, the preprotein translocase ATPase.

Identification of the preprotein binding domain of SecA.

SecA, the preprotein translocase ATPase, has a helicase DEAD motor. To catalyze protein translocation, SecA possesses two additional flexible domains absent from other helicases. Here we demonstrate that one of these "specificity domains" is a preprotein binding domain (PBD).

Purification of a functional mature region from a SecA-dependent preprotein.

Most of the bacterial proteins that are active in extracytoplasmic locations are translocated through the inner membrane by the Sec translocase. Translocase comprises a membrane "pore" and the peripheral ATPase SecA. Where preproteins bind to SecA and how they activate translocation ATPase remains elusive.

Escherichia coli SecA truncated at its termini is functional and dimeric.

Terminal residues in SecA, the dimeric ATPase motor of bacterial preprotein translocase, were proposed to be required for function and dimerization. To test this, we generated truncation mutants of the 901aa long SecA of Escherichia coli. We now show that deletions of carboxy-terminal domain (CTD), the extreme CTD of 70 residues, or of the N-terminal nonapeptide or of both, do not compromise protein translocation or viability.

Helicase Motif III in SecA is essential for coupling preprotein binding to translocation ATPase.

The SecA ATPase is a protein translocase motor and a superfamily 2 (SF2) RNA helicase. The ATPase catalytic core ('DEAD motor') contains the seven conserved SF2 motifs. Here, we demonstrate that Motif III is essential for SecA-mediated protein translocation and viability.