Our overall goal is to use E.coli as a model system to elucidate the basic mechanisms of protein localization that occur in all living cells. Our long-term objective is (i) to define and characterize the E.coli protein export machinery, (ii) to assign functions to its individual components, (iii) to understand how these functions are interelated to produce an efficient protein export pathway, and (iv) to determine how this pathway is regulated and coordinated with other essential cellular processes. At present, 9 putative export machinery components have been identified, but no specific biochemical function has been assigned to any of these components. The specific objective of these studies is to further define and elucidate the role of the secA gene product in catalyzing and regulating protein export in E.coli. Using a mutational analysis and a SecA-dependent in vitro protein translocation system, we will define the translocation activity(ies) catalyzed by SecA protein, determine which portions of the SecA polypeptide encodes this activity, and identify the export-related components that SecA protein interacts with in fulfilling this function. We will dissect the coregulation of secA with protein export proficiency further using a combination of mutational analysis and in vivo and in vitro assays to characterize which portions of the SecA polypeptide encodes its autogenous translational repressor activity, define the location and structure of the export-responsive translational operator, and elucidate the biochemical mechanism responsible for the coregulation observed. Since the mechanisms of protein localization are of central biological importance at all levels, from the functioning of individual protein molecules to the biogenesis and maintenance of normal and abnormal cell states, and since such mechanisms have been conserved throughout evolution, these studies with E.coli should be of broad significance.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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Wesleyan University
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United States
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Auclair, Sarah M; Oliver, Donald B; Mukerji, Ishita (2013) Defining the solution state dimer structure of Escherichia coli SecA using Forster resonance energy transfer. Biochemistry 52:2388-401
Das, Sanchaita; Grady, Lorry M; Michtavy, Jennifer et al. (2012) The variable subdomain of Escherichia coli SecA functions to regulate SecA ATPase activity and ADP release. J Bacteriol 194:2205-13
Grady, Lorry M; Michtavy, Jennifer; Oliver, Donald B (2012) Characterization of the Escherichia coli SecA signal peptide-binding site. J Bacteriol 194:307-16
Auclair, Sarah M; Moses, Julia P; Musial-Siwek, Monika et al. (2010) Mapping of the signal peptide-binding domain of Escherichia coli SecA using Förster resonance energy transfer. Biochemistry 49:782-92
Das, Sanchaita; Stivison, Elizabeth; Folta-Stogniew, Ewa et al. (2008) Reexamination of the role of the amino terminus of SecA in promoting its dimerization and functional state. J Bacteriol 190:7302-7
Jilaveanu, Lucia B; Oliver, Donald B (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 282:4661-8
Jilaveanu, Lucia B; Oliver, Donald (2006) SecA dimer cross-linked at its subunit interface is functional for protein translocation. J Bacteriol 188:335-8
Jilaveanu, Lucia B; Zito, Christopher R; Oliver, Donald (2005) Dimeric SecA is essential for protein translocation. Proc Natl Acad Sci U S A 102:7511-6
Zito, Christopher R; Antony, Edwin; Hunt, John F et al. (2005) Role of a conserved glutamate residue in the Escherichia coli SecA ATPase mechanism. J Biol Chem 280:14611-9
Zito, Christopher R; Oliver, Donald (2003) Two-stage binding of SecA to the bacterial translocon regulates ribosome-translocon interaction. J Biol Chem 278:40640-6

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