THE TRANSLOCATION, FOLDING, ASSEMBLY AND REDOX-DEPENDENT DEGRADATION OF SECRETORY AND MEMBRANE-PROTEINS IN SEMI-PERMEABILIZED MAMMALIAN-CELLS

We describe here a semi-permeabilized cell-system which reconstitutes the efficient synthesis, translocation, folding, assembly and degradat ion of membrane and secretory proteins. Cells grown in culture were tr eated with the detergent digitonin which selectively permeabilized the plasma membrane leaving the cellular organelles, such as the endoplas mic reticulum (ER) and trans-Golgi network intact. These permeabilized cells were added to an in vitro translation system, either wheatgerm or reticulocyte lysate, supplemented with RNA coding for either membra ne or secretory proteins. Efficient translocation and modification of proteins by these cells was demonstrated by protease protection, photo crosslinking of nascent chains to components of the translocation appa ratus and by post-translational modifications Such as glycosylation or hydroxylation. A comparison was made between the ability of semi-perm eabilized cells and microsomal vesicles to fold and assemble proteins. The results show that the intact ER within these cells can assemble p roteins much more efficiently than vesicularized ER. Furthermore, the semi-permeabilized cells carried out the redox-dependent degradation o f tissue-type plasminogen activator. This system has all the advantage s of conventional cell-free systems, including speed and, importantly, the ability to manipulate the components of the assay, while retainin g intracellular organelles and, therefore, allowing cellular processes to occur as they would in the intact cell.