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The transmembrane topology of the sn ‐glycerol‐3‐phosphate permease of Escherichia coli analysed by phoA and lacZ protein fusions
Author(s) -
Gött P.,
Boss W.
Publication year - 1988
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1988.tb00074.x
Subject(s) - permease , biology , biochemistry , antiporter , periplasmic space , escherichia coli , membrane topology , lactose permease , transmembrane protein , cytoplasm , membrane transport protein , transmembrane domain , gene , transporter , membrane , receptor
Summary 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 β‐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 β‐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 Tn phoA 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.