Ligand extraction properties of the GM2 activator protein and its interactions with lipid vesicles.
The GM2 activator protein (GM2AP) is an accessory protein required for the enzymatic conversion of GM2 to GM3 by hydrolases in the lysosomal compartments of cells. Here, GM2AP interactions with lipid vesicles are investigated by sucrose-loaded vesicle sedimentation and gel filtration assays, and the effects of pH and lipid composition on membrane binding and lipid extraction are characterized. The sedimentation experiments allow for facile quantification of the percentage of protein in solution and on the bilayer surface, with detailed analysis of the protein:lipid complex that remains in solution. Optimum binding and ligand extraction is found for pH 4.8 where <15% of the protein remains surface associated regardless of the lipid composition. In addition to extracting GM2, we find that GM2AP readily extracts dansyl-headgroup-labeled lipids as well as other phospholipids from vesicles. The ability of GM2AP to extract dansyl-DHPE from vesicles is altered by pH and the specific ligand GM2. Although the unique endosomal lipid, bis(monoacylglycero)phosphate, is not required for ligand extraction, it does enhance the extraction efficiency of GM2 when cholesterol is present in the vesicles.
Ran
Yong
Y
Department of Chemistry, University of Florida, Gainesville, Florida 32611-7200, USA.
Fanucci
Gail E
GE
D002784:Cholesterol / Q000737:chemistry; D002850:Chromatography, Gel; D003619:Dansyl Compounds / Q000737:chemistry; D049289:G(M2) Activator Protein / Q000737:chemistry*; D005732:Gangliosides / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry; D008246:Lysophospholipids / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D050178:Monoglycerides / Q000737:chemistry; D010713:Phosphatidylcholines / Q000737:chemistry; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010715:Phosphatidylglycerols / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013395:Sucrose; D053835:Unilamellar Liposomes / Q000737:chemistry*
D003619:Dansyl Compounds; D049289:G(M2) Activator Protein; D005732:Gangliosides; D008051:Lipid Bilayers; D008081:Liposomes; D008246:Lysophospholipids; D050178:Monoglycerides; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010715:Phosphatidylglycerols; D010743:Phospholipids; D053835:Unilamellar Liposomes; C012786:bis(monoacylglyceryl)phosphate; C025447:sialogangliosides; C007510:1,2-dipalmitoyl-3-phosphatidylethanolamine; D013395:Sucrose; C060037:1-palmitoyl-2-oleoylglycero-3-phosphoglycerol; D002784:Cholesterol; C028694:1-palmitoyl-2-oleoylphosphatidylcholine