Identification of a twin-arginine leader-binding protein.
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.
Oresnik
I J
IJ
Structural Biology Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta, T2N 1N4, Canada.
Ladner
C L
CL
Turner
R J
RJ
D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004121:Dimethyl Sulfoxide / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins; D047908:Intracellular Signaling Peptides and Proteins; D007506:Iron-Sulfur Proteins; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism; D021382:Protein Sorting Signals; D021381:Protein Transport; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D002352:Carrier Proteins; C434268:DmsD protein, E coli; D029968:Escherichia coli Proteins; D047908:Intracellular Signaling Peptides and Proteins; D007506:Iron-Sulfur Proteins; D026901:Membrane Transport Proteins; D021382:Protein Sorting Signals; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C408585:twin-arginine translocase complex, E coli; D001120:Arginine; D010088:Oxidoreductases; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase; C045664:dimethyl sulfoxide reductase; D004121:Dimethyl Sulfoxide