Identification of a twin‐arginine leader‐binding protein

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Abstract
The transport and targeting of a number of periplasmic proteins is carried out by the Sec‐independent Mtt (also referred to as Tat) protein translocase. Proteins using this translocase have a distinct twin‐arginine‐containing leader. We hypothesized that specific leader‐binding proteins exist to escort proteins to the translocase complex. A fusion was constructed with the twin‐arginine leader from dimethyl sulphoxide (DMSO) reductase, subunit DmsA, to the N‐terminus of glutathione‐S‐transferase. This leader fusion was bound to a glutathione affinity column through which an Escherichia coli anaerobic cell‐free extract was passed. Proteins that bound to the leader were then separated and identified by N‐terminal sequencing, which identified DnaK and a protein originating from the uncharacterized reading frame ynfI . This gene has been designated dmsD based on the findings presented in this paper. DmsD was purified as a His6 fusion and was shown to interact with preprotein forms of DmsA and TorA (trimethyl amine N‐oxide reductase). A strain carrying a dmsD knock‐out mutation showed a loss of anaerobic growth on glycerol–DMSO medium and reduced growth on glycerol–fumarate medium. This work suggests that DmsD is a twin‐arginine leader‐binding protein.