In vivo expression of the lacY gene in two segments leads to functional lac permease.
The lacY gene of Escherichia coli was cut into two approximately equal-size fragments with Afl II and subcloned individually or together under separate lac operator/promoters in plasmid pT7-5. Under these conditions, lac permease is expressed in two portions: (i) the N-terminal portion (the N terminus, the first six putative transmembrane helices, and most of putative loop 7) and (ii) the C-terminal portion (the last six putative transmembrane helices and the C terminus). Cells harboring pT7-5 encoding both fragments transport lactose at about 30% the rate of cells expressing intact permease to a comparable steady-state level of accumulation. In contrast, cells expressing either half of the permease independently do not transport lactose. As judged by [35S]methionine labeling and immunoblotting, intact permease is completely absent from the membrane of cells expressing lacY fragments either individually or together. Thus, transport activity must result from an association between independently synthesized pieces of lac permease. When the gene fragments are expressed individually, the N-terminal portion of the permease is observed inconsistently, and the C-terminal portion is not observed. When the gene fragments are expressed together, polypeptides identified as the N- and C-terminal moieties of the permease are found in the membrane. It is concluded that the N- or C-terminal halves of lac permease are proteolyzed when synthesized independently and that association between the two complementing polypeptides leads to a more stable, catalytically active complex.
Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90024-1574.
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D000595:Amino Acid Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D007763:Lac Operon; D007785:Lactose / Q000378:metabolism; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D027981:Symporters
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D046911:Macromolecular Substances; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease; D007785:Lactose