GLUT4-containing vesicles are released from membranes by phospholipase D cleavage of a GPI anchor.
We have previously developed a cell-free assay from rat skeletal muscle that displayed in vitro glucose transporter 4 (GLUT4) transfer from large to small membrane structures by the addition of a cytosolic protein fraction. By combining protein fractionation and the in vitro GLUT4 transfer assay, we have purified a glycosylphosphatidylinositol (GPI) phospholipase D (PLD) that induces transfer of GLUT4 from small to large membranes. The in vitro GLUT4 transfer was activated and inhibited by suramin and 1,10-phenanthroline (an activator and an inhibitor of GPI-PLD activity, respectively). Furthermore, upon purification of the GLUT4 transporter protein, the protein displayed an elution profile in which the molecular mass was related to the charge, suggesting the presence or absence of phosphate. Second, by photoaffinity labeling of the purified GLUT4 with 3-(trifluoromethyl)-3-(m-[(125)I]iodopenyl)diazirine, both labeled phosphatidylethanolamine and fatty acids (constituents of a GPI link) were recovered. Third, by using phase transition of Triton X-114, the purified GLUT4 was found to be partly detergent resistant, which is a known characteristic of GPI-linked proteins. Fourth, the purified GLUT4 protein was recognized by an antibody raised specifically against GPI links. In conclusion, GLUT4-containing vesicles may be released from a membrane compartment by action of a GPI-PLD.
Copenhagen Muscle Reseach Centre, Department of Human Physiology, Institute of Exercise and Sports Sciences, University of Copenhagen, 2100 Copenhagen, Denmark.
Søren
S
Kristiansen
Erik A
EA
Richter
D015153:Blotting, Western; D002845:Chromatography / Q000379:methods; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D051275:Glucose Transporter Type 4; D017261:Glycosylphosphatidylinositols / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology*; D006801:Humans; D008566:Membranes / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D018482:Muscle, Skeletal / Q000378:metabolism*; D017830:Octoxynol; D010618:Phenanthrolines / Q000494:pharmacology; D010739:Phospholipase D / Q000494:pharmacology*; D019920:Photoaffinity Labels; D010949:Plasma / Q000502:physiology; D011092:Polyethylene Glycols / Q000494:pharmacology; D013498:Suramin / Q000494:pharmacology
D003902:Detergents; D051275:Glucose Transporter Type 4; D017261:Glycosylphosphatidylinositols; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D010618:Phenanthrolines; D019920:Photoaffinity Labels; C495692:SLC2A4 protein, human; D011092:Polyethylene Glycols; D013498:Suramin; D017830:Octoxynol; C010615:Nonidet P-40; D010739:Phospholipase D; C025205:1,10-phenanthroline