Crosslinking and Reconstitution Approaches to Study Protein Transport

To identify the translocation components in cells, and to understand how they function in protein transport and membrane insertion, a variety of techniques have been used such as genetics, biochemistry, structural biology and single molecule methods. In particular, site-directed crosslinking between the client proteins and components of the translocation machineries have contributed significantly in the past and will do so in the future. One advantage of this technology is that it can be applied in vivo as well as in vitro and a comparison of the two approaches can be made. Also, the in vivo techniques allow time-dependent protocols which are essential for studying cellular pathways. Protein purification and reconstitution into proteoliposomes are the gold standard for studying membrane-based transport and translocation systems. With these biochemically defined approaches the function of each component in protein transport can be addressed individually with a plethora of biophysical techniques. Recently, the use of nanodiscs for reconstitution has added another extension of this reductionistic approach. Fluorescence based studies, cryo-microscopy and NMR spectroscopy have significantly added to our understanding how proteins move into and across membranes and will do this also in future.