SecA alone can Promote Protein Translocation and Ion-Channel Activity: SecYEG Increases Efficiency and Specificity

Abstract

SecA is an essential component of Sec translocase through which unfolded preproteins cross bacterial cytoplasmic membranes. The current prevailing view depicts SecYEG as the essential core of the protein-conducting channel, while SecA is the peripheral subunit that functions as an ATPase to drive the peptide chain through the SecYEG channel. In a major deviation, we now find that SecA with liposomes alone can promote protein translocation, and can elicit ion-channel activity in the oocyte whole cell recordings and in patch clamp single channel recordings. We previously reported that SecA forms ring-like pore structures upon binding with anionic phospholipids, providing the biophysical basis of SecA-liposomes channel activities. Further studies show that SecA-liposomes are less efficient and lose signal peptide recognition specificity, resembling PrlA suppressor mutants. Addition of purified SecYEG in the SecA-liposomes system restores the efficiency and the signal peptide specificity in ion-channel activity and in translocation of proOmpA and ProPhoA. Our data indicate that there is a protein-conducting channel in which SecA plays an important structural role as the core of the channel. Using N-terminal SecA deletion truncated domains of 901 residues SecA and liposomes in the oocytes recordings, we identify two critical SecA domains for the formation of pore channel activity: with phospholipids alone, and for higher activity with SecYEG. The N-terminal region located on amino acid residues #668-828 is required for interaction with liposomes, and #640-649 for interaction with SecYEG, to form functional channels. In a domain reconstitution system, two fragments, N-SecA1-640 and C-SecA610-901 together interact with liposomes to promote channel activity in oocytes. Our findings establish that SecA forms channel with liposomes, and SecA domains can be identified for intramolecular interaction and with SecYEG for electrophysiological channel activity.