Title : Mapping of
cystic fibrosis transmembrane conductance regulator membrane topology by glycosylation
site insertion
Abstract :
- Technical difficulties in obtaining three-dimensional structures of intrinsic membrane proteins continues to limit understanding of their function
- However, considerable insight can be gained from their two-dimensional topological arrangement in the lipid bilayer
- Efficient molecular genetic approaches are available to discern the topology of prokaryotic but not of eukaryotic membrane proteins
- The absolute asymmetry of the sidedness of their N-glycosylation was employed here to develop such a method using the cystic fibrosis transmembrane conductance regulator ( CFTR )
- Insertion by in vitro mutagenesis of N-glycosylation consensus sequences (NXS/T) in predicted cytoplasmic and extracytoplasmic loops between hydrophobic sequences capable of traversing the membrane established the membrane topology of CFTR
- This provides the first experimental evaluation of the original topological model of CFTR based solely on hydropathy algorithms and a method which may be generally applicable for the in vivo evaluation of the topology of other mammalian membrane proteins