MISSENSE MUTATIONS THAT INACTIVATE ESCHERICHIA-COLI LAC PERMEASE

Although missense mutations that inactivate integral membrane proteins cause a variety of diseases, the mechanisms by which they act are poo rly understood. To establish a model for investigating this issue, we identified 51 missense mutations arising in vivo that inactivate Esche richia coli lac permease, a well-characterized membrane transport prot ein. The mutants were isolated using a genetic screening procedure whi ch eliminates mutations that block expression of the lac permease gene , such as nonsense and frameshift mutations. The majority of the 51 mi ssense mutations caused highly non-conservative changes in membrane-sp anning sequences, such as the introduction of charged residues. Nevert heless, the greatest clustering of substitutions occurred in the two r egions of lac permease thought to be most important for transport func tion. The existence of this clustering indicates that even highly non- conservative substitutions may cause relatively localized structural d efects. Conservative inactivating substitutions were scattered through out lac permease and may affect residues that make contacts required f or normal folding. Two unexpected phenotypes were observed in the coll ection of mutants: about 20% of the substitutions led to cold-sensitiv e lactose utilization, and one substitution made the mutant lac permea se toxic to cells. This relatively unbiased collection of mutants shou ld provide a resource for further studies of how missense mutations in activate membrane proteins in vivo. (C) 1998 Academic Press Limited.