Glycosphingolipid-enriched, detergent-insoluble complexes in protein sorting in epithelial cells

Abstract

In simple epithelial cells, the delivery of apical and basolateral proteins to the cell surface is mediated by sorting in the trans-Golgi network and transport via separate vesicular carriers. In order to identify the molecular machinery involved in protein sorting, we have recently studied a detergent-insoluble complex in Madin-Darby canine kidney (MDCK) cells, following CHAPS extraction of exocytic carrier vesicles, specifically including the apical marker protein influenza hemagglutinin (HA). Previously, a Triton X-100 insoluble membrane residue that was enriched in glycosylphosphatidylinositol-anchored (GPI) proteins and glycolipids was characterized and implicated in transport to the apical cell surface [Brown, D., & Rose, J. (1991) Cell 68, 533-544]. In this report, the protein compositions of the CHAPS and Triton complexes have been compared by two-dimensional gel analysis. Only a few major membrane proteins are found in the complexes. The protein compositions are qualitatively similar, but differ quantitatively in the individual components. The CHAPS complex is depleted of GPI-linked proteins and retains a minor fraction of lipids similar in composition to that of the Triton X-100 insoluble complex. We propose that in vivo the complexes form part of a sorting platform that mediates protein segregation and delivery to the apical cell surface.