Cell-free analysis of tail-anchor protein targeting to membranes

We describe cell-free strategies for analysis of the biogenesis of membrane proteins. Special emphasis is placed on the unique challenges presented by tail-anchor proteins for the analysis of: targeting specificity, binding to membranes such as endoplasmic reticulum, mitochondria and pure lipid membranes, protein topology in the membrane and the dependence of various steps on additional proteins. Tail-anchor proteins such as cytochrome b5, Bcl-2 and Bcl-XL are used to address the suitability of both cell-free techniques based on in vitro transcription translation systems and the use of purified recombinant protein and pure lipid membranes. Together these cell-free systems permit detailed characterization of membrane protein biogenesis.