Purification and Electron Microscopic Characterization of the Membrane Subunit (IICBGlc) of the Escherichia coli Glucose Transporter

The glucose transporter of the bacterial phosphotransferase system mediates sugar transport across the cytoplasmic membrane concomitant with sugar phosphorylation. It consists of a cytoplasmic subunit IIAGlc and the transmembrane subunit IICBGlc. IICBGlc was purified to homogeneity by urea/alkali washing of membranes and nickel-chelate affinity chromatography. About 1.5 mg highly pure IICBGlc representing 77% of the total activity present in the membranes was obtained from 8g (wet weight) of cells. IICBGlc was reconstituted into lipid bilayers by temperature-controlled dialysis to yield small 2D crystals and by a rapid detergent-dilution procedure to yield densely packed vesicles. Electron microscopy and digital image processing of the negatively stained 2D crystals revealed a trigonal lattice with a unit cell size of a = b = 14.5 nm. The unit cell morphology exhibited three dimers of IICBGlc surrounding the threefold symmetry center. Single particle analysis of IICBGlc in proteoliposomes obtained by detergent dialysis also showed predominantly dimeric structures.