Proteins must be localized to the correct subcellular compartment for proper function. Protein translocation machineries fulfill this essential function by mediating signal-dependent transport of protein molecules across membranes. The importance of these transport processes for cell growth and development is evident from their direct role in a number of disease states including bacterial pathogenicity and protein misfolding diseases such as cystic fibrosis. Altered protein transporter structure or perturbed trafficking is responsible for a number of leukemias and cancers. Protein transport machineries ubiquitous in bacteria but absent from humans are excellent targets for antibiotic development. The goal of the proposed research is to advance our understanding of the molecular basis of protein transport mechanisms in bacteria. The Sec and Tat machineries will be examined using single-turnover stopped-flow fluorescence and single-molecule fluorescence microscopy techniques in order to dissect individual kinetic steps of transport.
The specific aims of the project are: (1) to develop a real-time fluorescence-based kinetic assay for the Escherichia coli Sec machinery; (2) to determine the Sec and Tat transport times via stopped-flow fluorescence; (3) to develop a lipid bilayer system suitable for simultaneous electrical and single-molecule fluorescence measurements; and (4) to determine the transport kinetics of single Sec and Tat substrates via single-molecule fluorescence microscopy. The results will be widely applicable to our understanding of membrane transport mechanisms and will significantly advance the field of single-molecule biophysics.
The Tat machinery is responsible for the transport of numerous bacterial virulencefactors of human health significance; and the absence of a functional Tat system oftenleads to growth defects in the host bacterium. The proposed research seeks to betterunderstand the mechanism of Tat protein transport through various biochemical andfluorescence techniques. The ability of the Tat system to transport fully-folded proteincomplexes suggests that it will eventually be used for the expression of proteintherapeutics.
| Hamsanathan, Shruthi; Anthonymuthu, Tamil S; Bageshwar, Umesh K et al. (2017) A Hinged Signal Peptide Hairpin Enables Tat-Dependent Protein Translocation. Biophys J 113:2650-2668 |
| Bageshwar, Umesh K; VerPlank, Lynn; Baker, Dwight et al. (2016) High Throughput Screen for Escherichia coli Twin Arginine Translocation (Tat) Inhibitors. PLoS One 11:e0149659 |
| Whitaker, Neal; Bageshwar, Umesh; Musser, Siegfried M (2013) Effect of cargo size and shape on the transport efficiency of the bacterial Tat translocase. FEBS Lett 587:912-6 |
| Sun, Changxia; Fu, Guo; Ciziene, Danguole et al. (2013) Choreography of importin-?/CAS complex assembly and disassembly at nuclear pores. Proc Natl Acad Sci U S A 110:E1584-93 |
| Liang, Fu-Cheng; Bageshwar, Umesh K; Musser, Siegfried M (2012) Position-dependent effects of polylysine on Sec protein transport. J Biol Chem 287:12703-14 |
| Whitaker, Neal; Bageshwar, Umesh K; Musser, Siegfried M (2012) Kinetics of precursor interactions with the bacterial Tat translocase detected by real-time FRET. J Biol Chem 287:11252-60 |
| Tu, Li-Chun; Musser, Siegfried M (2011) Single molecule studies of nucleocytoplasmic transport. Biochim Biophys Acta 1813:1607-18 |
| Liang, Fu-Cheng; Bageshwar, Umesh K; Musser, Siegfried M (2009) Bacterial Sec protein transport is rate-limited by precursor length: a single turnover study. Mol Biol Cell 20:4256-66 |
| Bageshwar, Umesh K; Whitaker, Neal; Liang, Fu-Cheng et al. (2009) Interconvertibility of lipid- and translocon-bound forms of the bacterial Tat precursor pre-SufI. Mol Microbiol 74:209-26 |
| Sun, Changxia; Yang, Weidong; Tu, Li-Chun et al. (2008) Single-molecule measurements of importin alpha/cargo complex dissociation at the nuclear pore. Proc Natl Acad Sci U S A 105:8613-8 |
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