Mapping of cystic fibrosis transmembrane conductance regulator membrane topology by glycosylation site insertion.
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.
Chang
X B
XB
Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Hou
Y X
YX
Jensen
T J
TJ
Riordan
J R
JR
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003994:Bucladesine / Q000494:pharmacology; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D018118:Chloride Channels / Q000096:biosynthesis / Q000737:chemistry; D005576:Colforsin / Q000494:pharmacology; D016384:Consensus Sequence; D006224:Cricetinae; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006031:Glycosylation; D006801:Humans; D008051:Lipid Bilayers; D008322:Mammals; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000187:drug effects; D014162:Transfection
C505032:CFTR protein, human; D018118:Chloride Channels; D008051:Lipid Bilayers; D008565:Membrane Proteins; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D003994:Bucladesine; D015056:1-Methyl-3-isobutylxanthine