In eukaryotic cells, newly made proteins are specifically transferred from their sites of synthesis to their sites of function. In most cases, this involves transport into or across at least one membrane. In general, these transport steps involve targeting elements contained within each protein's primary sequence and cellular machinery to recognize and facilitate translocation of the precursor protein. Recent studies have identified homologous protein translocation systems called """"""""Tat"""""""" in the thylakoid membranes of chloroplasts and the cytoplasmic membranes of bacteria and archaea. Tat systems uniquely transport folded proteins using only the transmembrane proton gradient as an energy source. They do this with only three membrane components of machinery and without breaching the permeability of the membrane. The Tat system is essential in plants and some prokaryotes. Importantly, the Tat system is used by at least one human pathogen to deliver virulence factors to its hosts. As Tat components seem absent from mammalian genomes, the Tat system represents a potential target for novel antimicrobial compounds. Our long range goal is to determine the mechanism by which Tat systems translocate proteins. Our recent studies have described steps of the process and identified which of the three known components (cpTatC, Hcf106, and Tha4) participate in each step. Specifically, precursors bind to a 700 kDa receptor complex consisting cpTatC and Hcf106. Precursor binding and the proton gradient trigger assembly of Tha4 to the receptor complex. The precursor is then transported across the membrane and the translocation complex dissociates. Here we propose a series of biochemical studies into the mechanism of translocation. Specifically, purification and crosslinking studies will characterize the receptor complex composition, in situ size, signal peptide binding capabilities and binding site, and changes that it undergoes upon signal binding. A newly developed biochemical complementation assay will investigate the role of Tha4 oligomerization in translocation of folded precursors of varied size. Direct and indirect approaches will test two alternative models for the structure and operation of the translocase. The work proposed here will increase basic knowledge of the mechanisms of cellular machines. It may additionally provide for new strategies to address human microbial diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM046951-13
Application #
6727923
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Shapiro, Bert I
Project Start
1992-02-01
Project End
2008-02-29
Budget Start
2004-03-01
Budget End
2005-02-28
Support Year
13
Fiscal Year
2004
Total Cost
$230,635
Indirect Cost
Name
University of Florida
Department
Miscellaneous
Type
Schools of Earth Sciences/Natur
DUNS #
969663814
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Aldridge, Cassie; Ma, Xianyue; Gerard, Fabien et al. (2014) Substrate-gated docking of pore subunit Tha4 in the TatC cavity initiates Tat translocase assembly. J Cell Biol 205:51-65
Ma, Xianyue; Cline, Kenneth (2013) Mapping the signal peptide binding and oligomer contact sites of the core subunit of the pea twin arginine protein translocase. Plant Cell 25:999-1015
Celedon, Jose M; Cline, Kenneth (2013) Intra-plastid protein trafficking: how plant cells adapted prokaryotic mechanisms to the eukaryotic condition. Biochim Biophys Acta 1833:341-51
Celedon, Jose M; Cline, Kenneth (2012) Stoichiometry for binding and transport by the twin arginine translocation system. J Cell Biol 197:523-34
Aldridge, Cassie; Storm, Amanda; Cline, Kenneth et al. (2012) The chloroplast twin arginine transport (Tat) component, Tha4, undergoes conformational changes leading to Tat protein transport. J Biol Chem 287:34752-63
Skalitzky, Courtney A; Martin, Jonathan R; Harwood, Jessica H et al. (2011) Plastids contain a second sec translocase system with essential functions. Plant Physiol 155:354-69
Rodrigues, Ricardo A O; Silva-Filho, Marcio C; Cline, Kenneth (2011) FtsH2 and FtsH5: two homologous subunits use different integration mechanisms leading to the same thylakoid multimeric complex. Plant J 65:600-9
Colquhoun, Thomas A; Schimmel, Bernardus C J; Kim, Joo Young et al. (2010) A petunia chorismate mutase specialized for the production of floral volatiles. Plant J 61:145-55
Ma, Xianyue; Cline, Kenneth (2010) Multiple precursor proteins bind individual Tat receptor complexes and are collectively transported. EMBO J 29:1477-88
Martin, Jonathan R; Harwood, Jessica H; McCaffery, Michael et al. (2009) Localization and integration of thylakoid protein translocase subunit cpTatC. Plant J 58:831-42

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