Monomeric human red cell glucose transporter (Glut1) in non-ionic detergent solution and a semi-elliptical torus model for detergent binding to membrane proteins.
The self-association state of the human red cell glucose transporter (Glut1) in octaethylene glycol n-dodecyl ether (C12E8) and n-octyl beta-D-glucopyranoside (OG) solution was analyzed in the presence of reductant by gel filtration with light-scattering, refractivity and absorbance detection, and by ultracentrifugation. The C12E8-Glut1 complex was essentially monomeric, whereas OG-Glut1 also formed dimers and larger oligomers. C12E8-Glut1 retained substantial glucose transport activity even after depletion of endogenous lipids by gel filtration, as shown by reconstitution and transport measurements. Removal of endogenous lipids from OG-Glut1 abolished the activity unless phosphatidylcholine was included in the eluent. The binding of C12E8 and OG to Glut1 was determined by gel filtration with refractivity and absorbance detection or with radioactive tracer to be 1.86 +/- 0.07 and 1.84 +/- 0.09 g/g polypeptide, respectively. A structural model was proposed in which non-ionic detergent forms a semi-elliptical torus (SET) surrounding the transmembrane protein. The torus thickness was assumed to be equal to the radius (short half-axis) of a spherical (oblate ellipsoidal) free detergent micelle and the polar head groups of the detergent molecules were predicted to be situated just outside the hydrophobic surface of the protein. The experimental detergent binding values and those obtained from the SET model together confirmed that Glut1 was monomeric in C12E8 solution and provided constraints on the shape and size of the hydrophobic transmembrane region of Glut1 in alpha-helical and beta-barrel topology models.
Haneskog
L
L
Department of Biochemistry, Uppsala University, Sweden.
Andersson
L
L
Brekkan
E
E
Englund
A K
AK
Kameyama
K
K
Liljas
L
L
Greijer
E
E
Fischbarg
J
J
Lundahl
P
P
D001692:Biological Transport; D002850:Chromatography, Gel; D003902:Detergents / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D051272:Glucose Transporter Type 1; D005960:Glucosides / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances; D008433:Mathematics; D008565:Membrane Proteins / Q000097:blood*; D008956:Models, Chemical; D009004:Monosaccharide Transport Proteins / Q000097:blood* / Q000737:chemistry; D011092:Polyethylene Glycols / Q000378:metabolism; D017433:Protein Structure, Secondary; D012996:Solutions; D014461:Ultracentrifugation
D003902:Detergents; D051272:Glucose Transporter Type 1; D005960:Glucosides; D046911:Macromolecular Substances; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; C495677:SLC2A1 protein, human; D012996:Solutions; C018619:octyl-beta-D-glucoside; C017272:dodecyloctaethyleneglycol monoether; D011092:Polyethylene Glycols