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Jenny Maki
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In eukaryotic and prokaryotic organisms, newly synthesized proteins, which are destined for secretion or membrane integration, are transported across membranes by different translocation pathways. In bacteria one of the major route of preprotein transport is the secretory pathway (Sec pathway), which is composed of the Sec proteins. SecA, a unique protein to bacteria, binds to the signal sequence of preproteins post-translationally and aids in the translocation of these proteins through the translocon (1). Signal sequences share no primary sequence homology but share some common features such as: a charged N-terminal ‘n’ region, a 8 to 20 residue hydrophobic ‘h’ region, and a ‘c’ region of 6 to 10 residues which is polar, uncharged, and includes a signal peptidase motif (2). The exact location of the signal sequence binding pocket on SecA has not been determined. Previous work in our laboratory we discovered that a N-terminal proteolytic fragment of SecA binds to sign al peptides with a higher affinity than intact SecA protein (3). To further define the location of signal peptide binding and to determine how signal sequence recognition occurs, we are using recombinant SecA, a zero-length cross-linker, and LamB signal peptides. Once crosslinked products are obtained, they are subjected to proteolytic cleavage and mass spectrometery to determine which fragment of SecA the signal sequence is binding.
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- Triplett, T.L., Sgrignoli, A.R., Gao, F.-B., Yang, Y.-B., Tai, P.C., Gierasch, L.M. (2001) Journal of Biological Chemistry, 277, 13724-13731.
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