Properties of permease dimer, a fusion protein containing two lactose permease molecules from Escherichia coli.
An engineered fusion protein containing two tandem lactose permease molecules (permease dimer) exhibits high transport activity and is used to test the phenomenon of negative dominance. Introduction of the mutation Glu-325-->Cys into either the first or the second half of the dimer results in a 50% decrease in activity, whereas introduction of the mutation into both halves of the dimer abolishes transport. Lactose transport by permease dimer is completely inactivated by N-ethylmaleimide; however, 40-45% activity is retained after N-ethylmaleimide treatment when either the first or the second half of the dimer is replaced with a mutant devoid of cysteine residues. The observations demonstrate that both halves of the fusion protein are equally active and suggest that each half may function independently. To test the possibility that oligomerization between dimers might account for the findings, a permease dimer was constructed that contains two different deletion mutants that complement functionally when expressed as untethered molecules. Because this construct does not catalyze lactose transport to any extent whatsoever, it is unlikely that the two halves of the dimer interact or that there is an oligomeric interaction between dimers. The approach is consistent with the contention that the functional unit of lactose permease is a monomer.
Sahin-Tóth
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Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90024-1662.
Lawrence
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MC
Kaback
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D001483:Base Sequence; D001693:Biological Transport, Active; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins; D007785:Lactose / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship; D027981:Symporters
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; D027981:Symporters; C021499:lactose permease; D007785:Lactose