title: Proximity of the protein moiety of a GPI-anchored protein to the membrane surface: a FRET study.
abstract: GPI-anchored proteins are ubiquitous on the eukaryotic cell surface, where they are involved in a variety of functions ranging from adhesion to enzymatic catalysis. Indirect evidence suggests that the GPI anchor may hold the protein close to the plasma membrane; however, there is a lack of direct information on the proximity of the protein portion of GPI-anchored proteins to the bilayer surface. The present study uses fluorescence resonance energy transfer (FRET) to address this important problem. The GPI-anchored ectoenzyme placental alkaline phosphatase (PLAP) was purified from a plasma membrane extract of human placental microsomes without the use of butanol. The protein was fluorescently labeled at the N-terminus with 7-(dimethylamino)coumarin-4-acetic acid succinimidyl ester (DMACA-SE) or Oregon Green 488 succinimidyl ester (OG488-SE), and each was reconstituted by detergent dilution into defined lipid bilayer vesicles containing an increasing mole fraction of a fluorescent lipid probe. The fluorescence of the labeled PLAP donors was quenched in a concentration-dependent manner by the lipid acceptors. The energy transfer data were analyzed using an approach that describes FRET between a uniform distribution of donors and acceptors in an infinite plane. The distance of closest approach between the protein moiety of PLAP and the lipid-water interfacial region of the bilayer was estimated to be smaller than 10-14 A. This indicates that the protein portion of PLAP is located very close to the lipid bilayer, possibly resting on the surface. This contact may allow transmission of structural changes from the membrane surface to the protein, which could influence the behavior and catalytic properties of GPI-anchored proteins.
authors.affiliation: Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry, Department of Chemistry and Biochemistry, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
authors.forename: Marty T
authors.initials: MT
authors.lastname: Lehto
authors.forename: Frances J
authors.initials: FJ
authors.lastname: Sharom
mesh_terms: D000469:Alkaline Phosphatase; D002462:Cell Membrane / Q000201:enzymology; D004735:Energy Transfer; D005260:Female; D058851:GPI-Linked Proteins; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D007425:Intracellular Membranes / Q000201:enzymology; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008051:Lipid Bilayers; D008861:Microsomes / Q000201:enzymology; D008958:Models, Molecular; D010713:Phosphatidylcholines / Q000737:chemistry; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
chemical_list: D058851:GPI-Linked Proteins; D017261:Glycosylphosphatidylinositols; D007527:Isoenzymes; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D000469:Alkaline Phosphatase; C019839:alkaline phosphatase, placental