Chapter 9 Molecular genetic analysis of membrane protein topology

Abstract

This chapter discusses the use of gene fusions to analyze membrane protein topology. Fusions of a gene encoding a protein whose enzymatic activity reflects its subcellular location are generated at multiple sites in the gene for a membrane protein, and the activities of the resulting hybrid proteins are compared to the previously formulated models. The activity of each hybrid protein reveals whether the junction site normally faces toward or away from the cytoplasm. Various molecular-genetic approaches are available for the analysis of membrane protein topology in prokaryotes and eukaryotes. Methods based on the generation and analysis of hybrid proteins are simple and generally reliable, but are subject to several difficulties because of the loss of C-terminal membrane protein sequences in the hybrid proteins generated. These difficulties can be overcome by methods that involve the insertion of easily identifiable target sequences into the membrane protein sequence without loss of C-terminal sequences. However, such methods are somewhat laborious and are subject to technical difficulties of their own because of the requirements that the inserted sequences be accessible to external agents and that they not significantly alter the membrane topologies of the proteins modified.