The transmembrane topology of the sn-glycerol-3-phosphate permease of Escherichia coli analysed by phoA and lacZ protein fusions.
The Escherichia coli glpT gene encodes a transport protein that mediates uptake of sn-glycerol-3-phosphate. This permease is a member of a class of bacterial organophosphate permeases which transport substrates by antiport with inorganic phosphate. The glpT gene product, probably an oligomer of a single polypeptide chain, is thought to span the cytoplasmic membrane several times, as predicted by the hydropathic profile. Protein fusions, in which varying lengths of the amino-terminal end of the permease is attached to alkaline phosphatase (phoA) and to beta-galactosidase (lacZ) were constructed. On the assumption that phoA fusions only exhibit high enzymatic activity when fused to extra-cytoplasmic regions of the target protein, whereas lacZ fusions will only be active when the beta-galactosidase portion is attached to cytoplasmic domains of the target protein, the activities of the fusions were used to test a two-dimensional model for the permease. The model proposes that GlpT contains 12 transmembrane segments divided by a larger cytoplasmic region. Despite some limitation caused by hot-spot sites of transpositions, the TnphoA approach was consistent with the model. In contrast, we feel that the enzymatic activity of lacZ fusions is only a limited parameter for studying the topology of a complex membrane protein.
D000469:Alkaline Phosphatase; D000595:Amino Acid Sequence; D015153:Blotting, Western; D004795:Enzyme Stability; D004926:Escherichia coli / Q000648:ultrastructure*; D005455:Fluorescent Antibody Technique; D008565:Membrane Proteins / Q000648:ultrastructure*; D026901:Membrane Transport Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010957:Plasmids; D021382:Protein Sorting Signals / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D015183:Restriction Mapping; D001616:beta-Galactosidase
C488962:GlpT protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase