Purification and electron microscopic characterization of the membrane subunit (IICB(Glc)) 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 IIA(Glc) and the transmembrane subunit IICB(Glc). IICB(Glc) was purified to homogeneity by urea/alkali washing of membranes and nickel-chelate affinity chromatography. About 1.5 mg highly pure IICB(Glc) representing 77% of the total activity present in the membranes was obtained from 8g (wet weight) of cells. IICB(Glc) 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 IICB(Glc) surrounding the threefold symmetry center. Single particle analysis of IICB(Glc) in proteoliposomes obtained by detergent dialysis also showed predominantly dimeric structures.
Zhuang
J
J
M. E. Müller-Institute for Microscopic Structural Biology at the Biocenter, University of Basel, Klingelbergstrasse 70, Basel, CH-4056, Switzerland. zhuang@pyl.unibe.ch
Gutknecht
R
R
Flükiger
K
K
Hasler
L
L
Erni
B
B
Engel
A
A
D002846:Chromatography, Affinity; D019281:Dimerization; D004926:Escherichia coli / Q000737:chemistry* / Q000648:ultrastructure; D007091:Image Processing, Computer-Assisted; D008854:Microscopy, Electron; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000648:ultrastructure*; D010770:Phosphotransferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020836:Protein Structure, Quaternary; D011510:Proteolipids; D012995:Solubility
D009004:Monosaccharide Transport Proteins; D011510:Proteolipids; C020485:proteoliposomes; D010770:Phosphotransferases