Structural Analysis of Cloned Plasma Membrane Proteins by Freeze-Fracture Electron Microscopy

• Published in: Proceedings of the National Academy of Sciences - PNAS 1998-09, Vol.95 (19), p.11235-11240
• Main Authors: Eskandari, Sepehr, Wright, Ernest M., Kreman, Mike, Starace, Dorine M., Zampighi, Guido A.
• Format: Article
• Language: English
• Subjects: Animals; Aquaporin 1; Aquaporins - ultrastructure; Biological Sciences; Cell Membrane - chemistry; Cell membranes; Cellular biology; Cloning; Connexins; Connexins - ultrastructure; Crystals; Cystic Fibrosis Transmembrane Conductance Regulator - ultrastructure; Eye Proteins - ultrastructure; Freezing; Histograms; Ion Channels - ultrastructure; Membrane Glycoproteins - ultrastructure; Membrane proteins; Membrane Proteins - ultrastructure; Membranes; Microscopy, Electron; Monosaccharide Transport Proteins - ultrastructure; Oocytes; Oocytes - chemistry; Opsins; Particle density; Particle Size; Plasma; Plasma density; Population size; Protein Conformation; Protein Structure, Secondary; Proteins; Recombinant Proteins - ultrastructure; Scientific imaging; Sodium-Glucose Transporter 1; Xenopus laevis
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.95.19.11235

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 α -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.