Elucidation of the mechanism of export of protein in prokaryotic systems will provide information relevant to a phenomenon central to many biological process such as secretion and biogenesis of organelles. Furthermore, the recently developed recombinant DNA technology has made possible the production of medically important eukaryotic proteins in bacteria. Such production can be made most efficient if we understand the details of export in bacteria. Data derived from studies on export is currently organized in terms of either the signal hypothesis or the trigger hypothesis. We propose experiments designed to distinguish between the major features of these hypotheses. We shall determine at what point in synthesis nascent chains initially bind to the membrane, when and by what mechanism they are translocated and proteolytically processed and further characterize the involvement of proton motive force in export. We shall initiate investigations of export in a gram positive bacterium and compare the findings with what is known about export in gram negative bacteria. The proposed research involves techniques of molecular biology and biochemistry such as in vivo pulse-labeling, in vitro protein synthesis, membrane isolation and fractionation and analysis of proteolytic digests by high performance liquid chromatography. The work proposed should result in significant progress toward the understanding of the specific passage of protein through biological membranes.

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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Washington State University
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Crane, Jennine M; Randall, Linda L (2017) The Sec System: Protein Export in Escherichia coli. EcoSal Plus 7:
Suo, Yuying; Hardy, Simon J S; Randall, Linda L (2015) The basis of asymmetry in the SecA:SecB complex. J Mol Biol 427:887-900
Chada, Nagaraju; Sigdel, Krishna P; Gari, Raghavendar Reddy Sanganna et al. (2015) Glass is a Viable Substrate for Precision Force Microscopy of Membrane Proteins. Sci Rep 5:12550
Mao, Chunfeng; Cheadle, Carl E; Hardy, Simon J S et al. (2013) Stoichiometry of SecYEG in the active translocase of Escherichia coli varies with precursor species. Proc Natl Acad Sci U S A 110:11815-20
Sanganna Gari, Raghavendar Reddy; Frey, Nathan C; Mao, Chunfeng et al. (2013) Dynamic structure of the translocon SecYEG in membrane: direct single molecule observations. J Biol Chem 288:16848-54
Suo, Yuying; Hardy, Simon J S; Randall, Linda L (2011) Orientation of SecA and SecB in complex, derived from disulfide cross-linking. J Bacteriol 193:190-6
Crane, Jennine M; Lilly, Angela A; Randall, Linda L (2010) Characterization of interactions between proteins using site-directed spin labeling and electron paramagnetic resonance spectroscopy. Methods Mol Biol 619:173-90
Randall, Linda L; Henzl, Michael T (2010) Direct identification of the site of binding on the chaperone SecB for the amino terminus of the translocon motor SecA. Protein Sci 19:1173-9
Mao, Chunfeng; Hardy, Simon J S; Randall, Linda L (2009) Maximal efficiency of coupling between ATP hydrolysis and translocation of polypeptides mediated by SecB requires two protomers of SecA. J Bacteriol 191:978-84
Lilly, Angela A; Crane, Jennine M; Randall, Linda L (2009) Export chaperone SecB uses one surface of interaction for diverse unfolded polypeptide ligands. Protein Sci 18:1860-8

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