Chapter 3 Analysis of Membrane Protein Topology Using Alkaline Phosphatase and β-Galactosidase Gene Fusions

Abstract

This Chapter describes a simple genetic method for identifying the disposition of different parts of a polypeptide chain relative to the membrane, the topology of the membrane protein. This approach is based on the finding that the specific activities of certain enzymes (sensor enzymes), when fused to a membrane protein, reflect the subcellular disposition of the membrane protein fusion site. The chapter describes methods in Escherichia coli for using alkaline phosphatase (the PhoA product, normally a periplasmic protein) and β -galactosidase (the LacZ product, normally a cytoplasmic protein) fusions to analyze topologies of cytoplasmic membrane proteins. The rationale for using gene fusions for the study of membrane protein topology is described in the chapter. The subcellular location of alkaline phosphatase or β -galactosidase attached to a membrane protein generally corresponds to the normal location of the junction site in the unfused membrane protein. The combined use of alkaline phosphatase and β -galactosidase fusions thus provides high enzyme activity signals for both periplasmic and cytoplasmic sites in cytoplasmic membrane proteins. It is also possible to interconvert alkaline phosphatase and β -galactosidase fusions to compare the activities of the two enzymes fused at a single site.