Analysis of protein translocation pathways in bacteria: its genetics, regulation and biochemistry
We are using the simple, well-characterized, and genetically facile bacterium E. coli as a model system to study the molecular details of protein translocation across the plasma membrane. Our major focus is on SecA ATPase, a DEAD motor protein that binds preproteins and the SecYEG channel complex, and which undergoes ATP-driven conformational cycles at the membrane that drive the stepwise translocation of proteins across the membrane. Genetic, biochemical and biophysical approaches are being utilized along with recent X-ray structures of SecA and SecYEG proteins in order to elucidate a number of important questions in this system:
- SecA ATPase enzymology. SecA is a multi-domain protein whose conformational cycling is regulated by binding nucleotide, preproteins, SecYEG, and acidic phospholipids. SecA mutant proteins are being generated and utilized to define the enzymology of this complex ATPase.
- SecA association with preproteins and SecYEG. Multidisciplinary approaches are being utilized to define the structural details of SecA interaction with signal peptides, secretory preproteins, and SecYEG and to elucidate their functional consequences in order to work out the protein translocation cycle.
- SecA dimer function. The nature of the requirement for SecA homo-dimerization in protein translocation is being elucidated.
- SecA regulation. SecA represses its own translation in response to the protein secretion-proficient state of the cell. This novel form of translational regulation, that utilizes the upstream gene, secM, encoding a secreted protein whose translation and transport are coupled, is being elucidated.
An X-ray structure of SecA (top) bound to the SecYEG channel complex (bottom). The two-helix finger portion of SecA (green alpha helical hairpin) that inserts into the mouth of the channel has been proposed to act as a translocation ratchet to drive protein transport through the channel utilizing the ATPase cycle of the SecA DEAD motor (shown in dark and light blue ribbon).