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. 1990 Sep;172(9):5374–5381. doi: 10.1128/jb.172.9.5374-5381.1990

Reconstitution of an active lactose carrier in vivo by simultaneous synthesis of two complementary protein fragments.

W Wrubel 1, U Stochaj 1, U Sonnewald 1, C Theres 1, R Ehring 1
PMCID: PMC213202  PMID: 2203750

Abstract

Escherichia coli lactose permease mediates the proton-driven translocation of galactosides across the cytoplasmic membrane. To define regions important for membrane insertion as well as for biological function, we constructed plasmids encoding different portions of the lactose carrier. Among several lacY deletions, two were obtained that encoded mutant proteins with complementary amino acid sequences. The truncated polypeptide Y71/1 (amino acid residues 1 to 71) comprises the first two alpha-helices predicted for the intact protein, and polypeptide delta Y4-69 carries an internal deletion of this region. Regulated coexpression of these lacY-DNA segments governed by separate but identical lacOP control regions resulted in functional complementation with the following characteristics. (i) Simultaneous synthesis of both incomplete proteins restored transport activity in transport-negative cells, measured as accumulation of [14C]lactose. (ii) Under complementing conditions, but not in the absence of the smaller N-terminal protein, specific radiolabeling of the larger polypeptide by N-ethylmaleimide was prevented by substrate. (iii) The presence of the complementing N-terminal polypeptide was also required for the detection of the larger C-terminal protein by antibodies directed against the C terminus of lactose permease, indicating a stabilizing effect contributed by the smaller N-terminal fragment. Thus, coexpression of lacY mutant genes encoding two nonoverlapping portions of the lactose carrier resulted in reconstitution of a two-subunit protein in the cytoplasmic membrane exhibiting biological properties of intact lactose permease.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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