title: Structural analysis of cloned plasma membrane proteins by freeze-fracture electron microscopy.
abstract: We have used freeze-fracture electron microscopy to examine the oligomeric structure and molecular asymmetry of integral plasma membrane proteins. Recombinant plasma membrane proteins were functionally expressed in Xenopus laevis oocytes, and the dimensions of their freeze-fracture particles were analyzed. To characterize the freeze-fracture particles, we compared the particle cross-sectional area of proteins with alpha-helical transmembrane domains (opsin, aquaporin 1, and a connexin) with their area obtained from existing maps calculated from two-dimensional crystals. We show that the cross-sectional area of the freeze-fracture particles corresponds to the area of the transmembrane domain of the protein, and that the protein cross-sectional area varies linearly with the number membrane-spanning helices. On average, each helix occupies 1.40 +/- 0.03 nm2. By using this information, we examined members from three classes of plasma membrane proteins: two ion channels, the cystic fibrosis transmembrane conductance regulator and connexin 50 hemi-channel; a water channel, the major intrinsic protein (the aquaporin 0); and a cotransporter, the Na+/glucose cotransporter. Our results suggest that the cystic fibrosis transmembrane conductance regulator is a dimer containing 25 +/- 2 transmembrane helices, connexin 50 is a hexamer containing 24 +/- 3 helices, the major intrinsic protein is a tetramer containing 24 +/- 3 helices, and the Na+/glucose cotransporter is an asymmetrical monomer containing 15 +/- 2 helices.
authors.affiliation: Department of Physiology, University of California at Los Angeles School of Medicine, Los Angeles, CA 90095-1751, USA. sepehr@physiology.medsch.ucla.edu
authors.forename: S
authors.initials: S
authors.lastname: Eskandari
authors.forename: E M
authors.initials: EM
authors.lastname: Wright
authors.forename: M
authors.initials: M
authors.lastname: Kreman
authors.forename: D M
authors.initials: DM
authors.lastname: Starace
authors.forename: G A
authors.initials: GA
authors.lastname: Zampighi
mesh_terms: D000818:Animals; D051398:Aquaporin 1; D020346:Aquaporins / Q000648:ultrastructure; D002462:Cell Membrane / Q000737:chemistry*; D017630:Connexins / Q000648:ultrastructure; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000648:ultrastructure; D005136:Eye Proteins / Q000648:ultrastructure; D005615:Freezing; D007473:Ion Channels / Q000648:ultrastructure; D008562:Membrane Glycoproteins / Q000648:ultrastructure; D008565:Membrane Proteins / Q000648:ultrastructure*; D008854:Microscopy, Electron; D009004:Monosaccharide Transport Proteins / Q000648:ultrastructure; D009865:Oocytes / Q000737:chemistry; D010316:Particle Size; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000648:ultrastructure; D051269:Sodium-Glucose Transporter 1; D014982:Xenopus laevis
chemical_list: D020346:Aquaporins; D017630:Connexins; D005136:Eye Proteins; D007473:Ion Channels; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D011994:Recombinant Proteins; D051269:Sodium-Glucose Transporter 1; C030328:aquaporin 0; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D051398:Aquaporin 1