Characterization of beta-galactosidase--lactose-permease chimaeras of Escherichia coli.
Escherichia coli strains have been isolated in which 3, 39 or 805 5'-end codons of lacZ, the gene for the cytoplasmic enzyme beta-galactosidase are fused to codon 9 of lacY, the gene for lactose permease. Lactose-permease-deficient cells, carrying the lacZ-Y fusions on F' lac pro episomes, are phenotypically positive on eosin/methylene blue/lactose or on melibiose plates, demonstrating that the beta-galactosidase--lactose-permease chimaeras transport lactose and melibiose in vivo. The apparent affinity for beta-D-galactopypanosyl 1-thio-beta-D-galactopyranoside (GalSGal) in cells is similar to that of the wild-type gene product. The maximum velocity of active GalSGal transport is reduced in all three fusion strains. Both lactose and p-nitrophenyl alpha-D-galactopyranoside inhibit GalSGal uptake. As demonstrated by immunoblot experiments the chimaeras cross-react with polyclonal antibodies directed against native lactose permease and they are present in the cell envelope fraction of homogenates. Their apparent molecular weights upon electrophoresis in NaDodSO4/polyacrylamide gels correspond to those expected from their respective primary sequences, taking into account the migration properties of wild-type lactose permease. It is proposed that substitution of eight N-terminal lactose permease residues by N-terminal beta-galactosidase residues neither prevents membrane incorporation of permease nor completely impairs the ability to transport galactosides actively. Alternative interpretations of the experimental results are discussed.
D001693:Biological Transport, Active; D055598:Chemical Phenomena; D002621:Chemistry; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005696:Galactosidases / Q000235:genetics* / Q000378:metabolism; D007120:Immunochemistry; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins; D010641:Phenotype; D027981:Symporters
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease; D005696:Galactosidases