DmsD, a Tat system specific chaperone, interacts with other general chaperones and proteins involved in the molybdenum cofactor biosynthesis Haiming Li, Limei Chang, Jenika M. Howell, and Raymond J. Turner * Department of Biological Sciences, Faculty of Science, University of Calgary, Calgary, Alberta, Canada T2N 1N4 Abstract Many bacterial oxidoreductases depend on the Tat translocase for correct cell localization. Substrates for the Tat translocase possess twin-arginine leaders. System specific chaperones or redox enzyme maturation proteins (REMPs) are a group of proteins implicated in oxidoreductase maturation. DmsD is a REMP discovered in Escherichia coli, which interacts with the twin- arginine leader sequence of DmsA, the catalytic subunit of DMSO reductase. In this study, we identified several potential interacting partners of DmsD by using several in vitro protein–protein interaction screening approaches, including affinity chromatography, co-precipitation, and cross- linking. Candidate hits from these in vitro findings were analyzed by in vivo methods of bacterial two-hybrid (BACTH) and bimolecular fluorescence complementation (BiFC). From these data, DmsD was confirmed to interact with the general molecular chaperones DnaK, DnaJ, GrpE, GroEL, Tig and Ef-Tu. In addition, DmsD was also found to interact with proteins involved in the molybdenum cofactor biosynthesis pathway. Our data suggests that DmsD may play a role as a “node” in escorting its substrate through a cascade of chaperone assisted protein-folding maturation events. Keywords Chaperones; Molybdenum cofactor biosynthesis pathway; DmsD; Protein–protein interactions; Tat 1. Introduction A number of bacterial oxidoreductases depend on the Tat (Twin-arginine translocase) system for correct subcellular localization [1–4]. In bacteria, this system represents a method of protein translocation, which differs from the general Sec system in that folded, cofactor- containing proteins are exported into the periplasm. The Tat translocase is thought to form a pore in the inner membrane, comprised of Tat A, B and C subunits [2,4,5]. The TatBC subunits are believed to be the “receptor complex”, and have been shown to be required for Tat substrate interaction with the membrane [6–9], whereas TatA is thought to form a very large oligomeric ring structure, presumed to be the protein-conducting channel itself [6,10]. * Corresponding author. Department of Biological Sciences, University of Calgary, Calgary, AB Canada T2N 1N4. Tel.: +1 403 220 4308; fax: +1 403 289 9311. turnerr@ucalgary.ca (R.J. Turner). Biochim Biophys Acta. Author manuscript; available in PMC 2012 February 27. Published in final edited form as: Biochim Biophys Acta. 2010 June ; 1804(6): 1301–1309. doi:10.1016/j.bbapap.2010.01.022. CIHR Author Manuscript CIHR Author Manuscript CIHR Author Manuscript