The Transmembrane Topology of the sn-glycerol-3-phosphate Permease of Escherichia Coli Analysed by phoA and lacZ Protein Fusions

Mol Microbiol. 1988 Sep;2(5):655-63. doi: 10.1111/j.1365-2958.1988.tb00074.x.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkaline Phosphatase
  • Amino Acid Sequence
  • Blotting, Western
  • Enzyme Stability
  • Escherichia coli / ultrastructure*
  • Fluorescent Antibody Technique
  • Membrane Proteins / ultrastructure*
  • Membrane Transport Proteins* / metabolism
  • Molecular Sequence Data
  • Peptide Fragments / metabolism
  • Plasmids
  • Protein Sorting Signals / metabolism
  • Recombinant Fusion Proteins
  • Restriction Mapping
  • beta-Galactosidase

Substances

  • GlpT protein, E coli
  • Membrane Proteins
  • Membrane Transport Proteins
  • Peptide Fragments
  • Protein Sorting Signals
  • Recombinant Fusion Proteins
  • Alkaline Phosphatase
  • beta-Galactosidase