ENGINEERING TRYPSIN-SENSITIVE SITES IN A MEMBRANE-TRANSPORT PROTEIN

This paper describes a systematic procedure for introducing protease-s ensitive sites into bacterial integral membrane proteins. Such sites s hould make it possible to monitor the subcellular localization of indi vidual domains of a topologically complex protein. Escherichia coli la c permease was used as a model. Site-directed mutagenesis, targeted to a particular periplasmic domain, was used to generate insertion deriv atives containing a lysine residue in different sequence contexts. Ind ividual mutants were then screened for lactose transport activity and efficient cleavage by trypsin. To facilitate this screen, the mutagene sis was carried out using a gene fusion encoding an easily detected, b ifunctional lac permease-galactosidase hybrid. Insertions were identif ied in the fourth and sixth periplasmic domains (P4 and P6) that were efficiently cleaved in both the hybrid protein and in unfused lac perm ease. One of the P6 insertion mutants exhibited lactose transport spec ific activity near that of the wild-type and was shown by sequence ana lysis to be cleaved at the expected site in the inserted sequence, As part of this analysis, we determined the range of cellular concentrati ons of lac permease over which lactose uptake was linear, The activity showed a plateau at a relatively low concentration corresponding to a pproximately five times the wild-type level.