Behaviour of topological marker proteins targeted to the Tat protein transport pathway.
The Escherichia coli Tat system mediates Sec-independent export of protein precursors bearing twin arginine signal peptides. Formate dehydrogenase-N is a three-subunit membrane-bound enzyme, in which localization of the FdnG subunit to the membrane is Tat dependent. FdnG was found in the periplasmic fraction of a mutant lacking the membrane anchor subunit FdnI, confirming that FdnG is located at the periplasmic face of the cytoplasmic membrane. However, the phenotypes of gene fusions between fdnG and the subcellular reporter genes phoA (encoding alkaline phosphatase) or lacZ (encoding beta-galactosidase) were the opposite of those expected for analogous fusions targeted to the Sec translocase. PhoA fusion experiments have previously been used to argue that the peripheral membrane DmsAB subunits of the Tat-dependent enzyme dimethyl sulphoxide reductase are located at the cytoplasmic face of the inner membrane. Biochemical data are presented that instead show DmsAB to be at the periplasmic side of the membrane. The behaviour of reporter proteins targeted to the Tat system was analysed in more detail. These data suggest that the Tat and Sec pathways differ in their ability to transport heterologous passenger proteins. They also suggest that caution should be observed when using subcellular reporter fusions to determine the topological organization of Tat-dependent membrane protein complexes.
Stanley
Nicola R
NR
Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.
Sargent
Frank
F
Buchanan
Grant
G
Shi
Jiarong
J
Stewart
Valley
V
Palmer
Tracy
T
Berks
Ben C
BC
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D019677:Artificial Gene Fusion; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D015415:Biomarkers; D002462:Cell Membrane / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005560:Formate Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D005561:Formates / Q000378:metabolism; D017930:Genes, Reporter; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D010455:Peptides; D019897:Periplasm / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D015415:Biomarkers; D029968:Escherichia coli Proteins; D005561:Formates; D007506:Iron-Sulfur Proteins; D026901:Membrane Transport Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; C408585:twin-arginine translocase complex, E coli; C030544:formic acid; D010088:Oxidoreductases; D005560:Formate Dehydrogenases; C045664:dimethyl sulfoxide reductase; D015500:Chloramphenicol O-Acetyltransferase; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase; D001618:beta-Lactamases