Translocation of specific polypeptides across membranes is essential for all living cells. The pathway of transfer through the membrane provided by the heterotrimeric complex SecYEG in Escherichia coli is highly conserved in all three kingdoms of life, from single cell organisms to mammals. In addition to a pathway through the membrane in almost all cases, whether the process occurs in prokaryotes or eukaryotes, chaperones are involved in the initial stages of binding of precursor polypeptides that are to be transported. The principles underlying the phenomena of capture and movement of polypeptides through membranes are likely to be shared among different systems. Therefore what we learn by studying bacterial export will provide insights into the molecular mechanisms of other export systems. The research plan is designed to elucidate the mechanistic details of the process with emphasis on the dynamics of passage of a precursor polypeptide along the pathway from the chaperone SecB to SecA, the ATPase motor of the system, and on through the SecYEG translocon. The work will increase the depth of understanding of molecular switches including both changes in specific contacts within complexes that are crucial to the movement of the precursor from one binding partner to another as well as conformation changes within SecA that underlie the conversion of energy of binding and hydrolysis of ATP to the mechanical work of translocation through the channel in the membrane. The research strategy makes use of a balance of biophysical and biochemical techniques that complement one another. The experiments proposed range from determinations of binding parameters by titration calorimetry and of hydrodynamic properties of complexes by light scattering and ultracentrifugation to the complete reconstitution into proteoliposomes of the entire translocation system comprising not only the translocon, but also the accessory proteins SecDF/YajC as well as bacterial rhodopsin as a means to generate protonmotive force. Among the innovative techniques are the use of Nanodiscs that allow membrane proteins such as SecYEG to be treated as a soluble particle and the use of single molecule atomic force microscopy. Many questions of interest cannot be readily answered by ensemble methods because interpretation is confounded by the multiple dynamic equilibria that occur along the export pathway. For these studies assessment of complexes by a single molecule technique is essential. An overall advantage of the research plan is that all methods are carried out in solution conditions and protein concentrations that are as close as possible to physiological. The proposal is firmly based on our past research and should move the field nearer to a molecular description of protein localization.

Public Health Relevance

Localization and folding of proteins is an essential process in all living cells and errors in the processes are the basis of many human diseases. The mechanism is highly conserved and thus what we learn by studying export in Escherichia coli will be applicable to the phenomenon in all cells from bacteria to humans.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029798-35
Application #
8641362
Study Section
Special Emphasis Panel (ZRG1-GGG-B (03))
Program Officer
Ainsztein, Alexandra M
Project Start
1981-02-01
Project End
2015-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
35
Fiscal Year
2014
Total Cost
$499,938
Indirect Cost
$167,922
Name
University of Missouri-Columbia
Department
Type
Schools of Medicine
DUNS #
153890272
City
Columbia
State
MO
Country
United States
Zip Code
65211
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
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
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
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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