Nonpolarized distribution of glycosylphosphatidylinositols in the plasma membrane of polarized Madin-Darby canine kidney cells

Abstract

Glycosylphosphatidylinositols (GPIs) are ubiquitous in eukaryotes and serve to anchor a variety of proteins to the exoplasmic leaflet of cellular membranes. GPIs are synthesized in the endoplasmic reticulum (ER), in excess of the amount needed for protein modification. The fate of the excess GPIs is unknown, but they may be retained in the ER, transported to other membranes, and/or metabolized. In relation to this problem, we were interested in determining whether GPIs were transported to the plasma membrane and whether, like GPI-anchored proteins, their presence was confined to the apical plasma membrane domain in polarized epithelial cells. Polarized Madin-Darby canine kidney epithelial cell monolayers were incubated with [3H]mannose or [3H]ethanolamine to label GPIs and then infected with enveloped viruses. We used influenza virus (flu) and vesicular stomatitis virus (VSV) for these experiments as these viruses are assembled at the cell surface and acquire their envelope lipids from the plasma membrane. Furthermore, flu and VSV bud specifically from the apical and basolateral plasma membrane domains, respectively. Flu and VSV were isolated from the apical and basolateral media, respectively, and subjected to lipid analysis. Radiolabeled GPIs were found in both viruses. Moreover, the membrane concentration of GPIs (i.e. GPI radioactivity normalized to membrane mass) in the two viruses was essentially the same. These observations suggest that (i) non-protein-linked GPIs are located at the plasma membrane; (ii) since GPIs are synthesized in the ER, they must be transported from the ER to the plasma membrane; and (iii) transport of non-protein-linked GPIs is not influenced by the sorting processes that target GPI-anchored proteins exclusively to the apical plasma membrane.