Semipermeabilized Cells to Study Procollagen Assembly

This chapter will describe the preparation and use of a semipermeabilized (SP) cell system that reconstitutes the initial stages in the assembly and modification of proteins entering the secretory pathway (1 ). The procedure involves treating cells grown in culture with the detergent digitonin and isolating the cells free from their cytosolic component (2 ). The expression of proteins in an SP cell system allows protein assembly to be studied in an environment that more closely resembles that of the intact cell. As this is an in vitro system, the individual components can be manipulated easily, providing a means by which cellular processes can be studied under a variety of conditions. In addition, membrane-permeable chemical crosslinking reagents can be added in order to facilitate the study of interaction between proteins within the endoplasmic reticulum (ER) lumen (1 ,3 ). Furthermore, as the ER remains morphologically intact, the spatial localization of folding and transport processes within the reticular network may also be examined.