Caveolae Purification and Glycosylphosphatidylinositol-Linked Protein Sorting in Polarized Epithelia

This chapter describes the techniques used for the recombinant expression of glycosylphosphatidylinositol (GPI)-linked proteins in epithelial cell lines and the measurement of cell-surface polarity of endogenous or transfected GPI-linked proteins at steady state and during transport. The chapter also discusses the methods for purification and characterization of caveolae from cultured cells. To study the sorting of endogenous GPI-linked proteins in polarized cells, a series of cell-surface labeling techniques that allow the rapid biochemical determination of the polarity of a given cell-surface antigen is developed. Such labeling techniques depend on the growth of polarized cells on permeable supports that allow for separate access to the apical and basolateral domains. These techniques are then applied to a variety of available intestinal and renal epithelial cell lines, such as the Madin-Darby canine kidney (MDCK), LLC-PK1, Caco-2, and SK-C015 lines, that spontaneously form polarized monolayers in culture. The GPI-linked proteins are detected by their sensitivity to release by treatment with bacterial PI-specific phospholipase C. To measure the polarized sorting of the recombinant proteins during cell-surface transport, additional assays are developed to monitor the cell surface delivery, endocytosis, and transcytosis.