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 motor protein that binds preproteins and the translocon component SecYEG, and which undergoes ATP-driven conformational cycles at the membrane that promote the stepwise translocation of proteins across this membrane layer. Genetic, biochemical and biophysical approaches are being utilized along with our recent crystal structure of SecA protein in order to elucidate a number of important questions in this system:
1. SecA ATPase enzymology. SecA is a multi-domain protein whose conformational cycling is regulated by binding nucleotide, preproteins, SecYEG, and acidic phospholipids. Oligonucleotide-directed mutagenesis and biochemical and biophysical analysis of the mutant proteins are being used to define the enzymology of this complex ATPase.
2. SecA association with preproteins and the translocon. Genetic, biochemical, and biophysical techniques are also being used to define the interaction of SecA with preproteins and SecYEG protein, and to map the relevant binding sites.
3. SecA membrane cycling. A cysteine-scanning approach along with in vivo sulfhydryl labeling are being utilized to understand the conformational changes that occur when SecA undergoes its membrane insertion and retraction cycle at the translocon that is coupled to the protein translocation cycle.
4. 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, is being elucidated.
Das, S., Stivison, E., Folta-Stogniew, and Oliver, D. 2008. Re-examination of the role of the amino-terminus of SecA in promoting its dimerization and functional state. J. Bacteriol., 190: in press.
Jilaveanu, L. B. and Oliver, D. 2007. In vivo membrane topology of Escherichia coli SecA ATPase reveals extensive periplasmic exposure of multiple functionally important domains clustering on one face of SecA. J. Biol. Chem. 282:4661-4668
Jilaveanu, L. B. and Oliver, D. 2006. SecA dimer cross-linked at its subunit interface is functional for protein translocation. J. Bacteriol. 188: 335-338.
Jilaveanu, L., Zito, C. and D. Oliver. 2005. Dimeric SecA is essential for protein translocation. Proc. Natl. Acad. Sci. USA. 102: 7511-7516.
Zito*, C.R., Antony*, E.A., Hunt, J.F., Oliver, D.B. and Hingorani, M.M. (2005) "Role of a conserved glutamate reside in the E. coli SecA ATPase mechanism". Journal of Biological Chemistry 280:14611-14619.
(*These authors contributed equally to the research)
Kim, P. D., Banack, T., Lerman, D. M., Tracy, J. C., Camara, J. E., Crooke, E., Oliver, D. and W. Firshein. (2003). Identification of a novel membrane-associated gene product that suppresses the toxicity of a TrfA peptide from plasmid RK2 and its relationship to the DnaA host initiation protein. J. Bacteriol. 185: 1817-1824.
Ding, H., Mukerji, I. and D. Oliver. 2003. Nucleotide and phospholipid-dependent control of PPXD and C-domain association of SecA ATPase. Biochemistry 42: 13468-13475.
Butkus, M., Prundeanu, L. and D. Oliver. 2003. Translocon “Pulling” of Nascent SecM Controls the Duration of Its Translational Pause and Secretion-Responsive secA Regulation. J. Bacteriol. Volume 185: 6719-6722.
Zito, C. R. and D. Oliver. 2003. Two-stage binding of SecA to the bacterial translocon regulates compatibility of Sec and SRP-dependent protein translocation pathways. J. Biol. Chem. 278: 40640-40646.
Ding, H., Hunt, J., Mukerji, I. and D. Oliver. 2003. B. subtilis SecA ATPase exists as an antiparallel dimer in solution. Biochemistry 42: 8729-8738.
Hunt, J. F., Weinhauf, S., Henry, L., Fak, J. J., McNicholas, P., Oliver, D. B. and J. Deisenhofer. 2002. Nucleotide control of interdomain interactions in the Conformational Reaction Cycle of SecA. Science 297:2018-2026.
Sarker, S. and D. Oliver. 2002. Critical regions of secM that control its translation and secretion and promote secretion-specific secA regulation. J. Bacteriol. 184: 2360-2369.
Ding. H., Mukerji, I., and D. Oliver. 2001. Lipid and signal peptide-induced conformational changes within the C-domain of Escherichia coli SecA protein. Biochemistry 40:1835-1843
Weinhauf, S., Hunt, J., Scheuring, J., Henry, L., Fak, J., Oliver, D., and J. Deisenhofer. 2001. Conformational stabilization and crystallization of the SecA translocation ATPase from Bacillus subtilis. Acta Crystal. Section D 57: 559-565.
Schmidt, M., Brosh, R., and D. Oliver. 2001. Escherichia coli SecA helicase activity is not required in vivo for efficient protein translocation or autogenous regulation. Submitted to J. Biol. Chem. 276: 37076-37085.
