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 freezefracture particles were analyzed. To characterize the freezefracture particles, we compared the particle cross-sectional area of proteins with ␣-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 nm 2 . 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.
The Na ؉ /galactose cotransporter (vSGLT) of Vibrio parahaemolyticus, tagged with C-terminal hexahistidine, has been purified to apparent homogeneity by Ni 2؉ affinity chromatography and gel filtration. Resequencing the vSGLT gene identified an important correction: the N terminus constitutes an additional 13 functionally essential residues. The mass of His-tagged vSGLT expressed under its native promoter, as determined by electrospray ionization-mass spectrometry (ESI-MS), verifies these 13 residues in wild-type vSGLT. A fusion protein of vSGLT and green fluorescent protein, comprising a mass of over 90 kDa, was also successfully analyzed by ESI-MS. Reconstitution of purified vSGLT yields proteoliposomes active in Na ؉ -dependent galactose uptake, with sugar preferences (galactose > glucose > fucose) reflecting those of wild-type vSGLT in vivo. Substrates are transported with apparent 1:1 stoichiometry and apparent K m values of 129 mM (Na ؉ ) and 158 M (galactose). Freeze-fracture electron microscopy of functional proteoliposomes shows intramembrane particles of a size consistent with vSGLT existing as a monomer. We conclude that vSGLT is a suitable model for the study of sugar cotransporter mechanisms and structure, with potential applicability to the larger SGLT family of important sodium:solute cotransporters. It is further demonstrated that ESI-MS is a powerful tool for the study of proteomics of membrane transporters.
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