Reconstitution of periplasmic binding protein-dependent glutamine transport in vesicles

This chapter examines how several bacterial active transport systems require, in addition to one or more membrane-bound components, periplasmic binding proteins. Genetic studies, experiments with intact cells, and investigations using spheroplasts have all established the unique role that these proteins play in the transport process. The chapter describes methods for preparing isolated membrane vesicles capable of binding protein-dependent glutamine transport, and for assaying glutamine transport in such vesicles. Two different kinds of mutants are essential for the study of binding protein-dependent transport in isolated membrane vesicles. First, a strain that overproduces the various components of the system under study is needed, both to provide a ready source of the substrate binding protein, and to serve as a parent for the second type of strain. Second, a derivative of the overproducing strain that has a point mutation in the binding protein gene is needed to serve as a source of membrane vesicles. Glutamine-binding protein is purified from a strain that overproduces the various components of the glutamine transport system.