title: Interaction of C-terminal loop 13 of sodium-glucose cotransporter SGLT1 with lipid bilayers.
abstract: We have previously shown that C-terminal loop 13 of SGLT1 acts as a major binding domain for the aglucon residues of d-glucose transport inhibitors, phlorizin (Raja, M. M., Tyagi, N. K., and Kinne, R. K. H. (2003) Phlorizin Recognition in a C-terminal Fragment of SGLT1 Studied by Tryptophan Scanning and Affinity Labeling, J. Biol. Chem. 278, 49154-49163) and alkyl glucosides (Raja, M. M., Kipp, H., and Kinne, R. K. H. (2004) C-Terminus Loop 13 of Na(+) Glucose Cotransporter SGLT1 Contains a Binding Site for Alkyl Glucosides, Biochemistry 43, 10944-10951). Topology of this loop with regard to the membrane lipids is hitherto a point of debate. Here we report on in vitro incorporation studies using fluorescence of Trp mutants of loop 13 to determine the position of various parts of the loop with the lipid bilayer. Six single Trp mutants were prepared as described in previous studies (Raja et al., 2003) and subsequently incorporated into DOPC:DOPG (60:40% molar ratio) lipid vesicles. Upon addition of the phospholipids only one mutant, R601W, exhibited no change in the fluorescence intensities, position of maxima, or acrylamide accessibility. Mutants Q581W, E621W, and L630W exhibited the most pronounced blue shifts (3-6 nm) and protection against acrylamide, suggesting a position of these segments within the lipid bilayer. This assumption was confirmed by the result that the fluorescence of only these mutants was quenched by doxyl spin membrane embedded labels in the 5- or 12-positions of the acyl side chain of phospholipids. The other parts of the peptide appear to remain outside of the lipid vesicles. Trp-591 and Trp-611 showed, although to a different extent, increase in fluorescence, blue shift of maxima, and decrease in acrylamide accessibility but no interaction with the spin-labeled phospholipids. This suggests changes in the conformation of the peptide itself. These conformation changes are probably induced by the interaction of an adjacent lysine rich region of the peptide with the negatively charged DOPG, since in the absence of this lipid no incorporation of loop 13 into the bilayer is observed. Trypsin cleavage experiments of loop 13 in proteoliposomes yield a peptide containing amino acid residues 603 to 614, confirming that this part of the loop is accessible at the extravesicular face of the membranes. The studies show that at least in the in vitro system the part of loop 13 essential for the interaction with the transport inhibitors is located extracellularly, making a similar arrangement in the intact SGLT1 probable.
authors.affiliation: Department of Epithelial Cell Physiology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
authors.forename: M Mobeen
authors.initials: MM
authors.lastname: Raja
authors.forename: Rolf K H
authors.initials: RK
authors.lastname: Kinne
mesh_terms: D005453:Fluorescence; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D010713:Phosphatidylcholines / Q000378:metabolism / Q000494:pharmacology; D010715:Phosphatidylglycerols / Q000378:metabolism / Q000494:pharmacology; D017433:Protein Structure, Secondary; D051269:Sodium-Glucose Transporter 1; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D008051:Lipid Bilayers; D008081:Liposomes; D008562:Membrane Glycoproteins; D009004:Monosaccharide Transport Proteins; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D051269:Sodium-Glucose Transporter 1; C051388:1,2-dioleoyl-sn-glycero-3-phosphoglycerol; D014364:Tryptophan; C017251:1,2-oleoylphosphatidylcholine