MAPPING OF CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR MEMBRANE TOPOLOGY BY GLYCOSYLATION SITE INSERTION

Technical difficulties in obtaining three-dimensional structures of in trinsic membrane proteins continues to limit understanding of their fu nction. However, considerable insight can be gained from their two-dim ensional topological arrangement in the lipid bilayer. Efficient molec ular genetic approaches are available to discern the topology of proka ryotic but not of eukaryotic membrane proteins. The absolute asymmetry of the sidedness of their N-glycosylation was employed here to develo p such a method using the cystic fibrosis transmembrane conductance re gulator (CFTR). Insertion by in vitro mutagenesis of N-glycosylation c onsensus sequences (NXS/T) in predicted cytoplasmic and extracytoplasm ic loops between hydrophobic sequences capable of traversing the membr ane established the membrane topology of CFTR. This provides the first experimental evaluation of the original topological model of CFTR bas ed solely on hydropathy algorithms and a method which may be generally applicable for the in vivo evaluation of the topology of other mammal ian membrane proteins.