Placental Alkaline Phosphatase Is Efficiently Targeted to Rafts in Supported Lipid Bilayers

Abstract

Evidence is growing that biological membranes contain lipid microdomains or "rafts" that may be involved in processes such as cellular signaling and protein trafficking. In this study, we have used atomic force microscopy to examine the behavior of rafts in supported lipid bilayers. We show that bilayers composed of equimolar dioleoylphosphatidylcholine and sphingomyelin spontaneously form rafts, which are detectable as raised features. A comparison of the extents of protrusion of the rafts in monolayers and bilayers indicates that the rafts in the two leaflets of the bilayer coincide. The rafts were observed both in the absence and presence of cholesterol (33 mol %). Cholesterol reduced raft protrusion presumably by increasing the thickness of the non-raft bilayer. PLAP (glycosylphosphatidylinositol-anchored protein placental alkaline phosphatase) was purified and shown to exist as a dimer. Following its incorporation into supported lipid bilayers, PLAP was found to be targeted efficiently to rafts, both in the absence and presence of cholesterol. We suggest that atomic force microscopy provides a powerful tool for the study of raft structure and properties.

Characterizing the interactions between GPI-anchored alkaline phosphatases and membrane domains by AFM

“…These results differ from those recently obtained with PLAP in which, using SLB b s, only one-fourth of the total AP-GPI was found, with significant aggregation, in the l o domains at room temperature [18]. The conclusions of this last paper, in which fluorescence correlation spectroscopy and confocal fluorescence were also used, also differ from the previous one on PLAP [82]. They were however close to those obtained by the same group on GUVs using fluorescence markers [52].…”

“…To summarize, these data strongly suggested that the GPI anchor might contribute to the formation of its most appropriate membrane environment in terms of hydrophobic length and lipid order parameter [40]. Like for BIAP, PLAP has been visualized in the gel SM enriched ordered phase in SLB b s made of DOPC/SM [82]. Notice that the BIAP dimers are first visualized at the periphery of the ordered domains (arrows in b) then found in the center of domains for longer incubations (arrows in c).…”

“…Direct visualization of AP-GPI dimers and aggregates inserted in membrane domains was reported both for samples where the protein was added after the SLBs formation (SLB a s) and for samples in which the protein was inserted during the formation of the liposomes (SLB b s), before the SLBs formation by fusion of the proteincontaining vesicles [18,40,68,82]. Two categories of SLBs under fluid-ordered phase separation have been examined by AFM.…”

“…ternary mixtures containing an unsaturated PC, sphingomyelin, and variable concentrations of cholesterol. For PLAP introduced in the liposomes before fusion with the mica (SLB b s), the protein was visualized practically exclusively (92%) in the SM/Chl enriched l o domains of DOPC/SM/ Chl (1:1:1, mol:mol) [82]. A similar conclusion was reached for BIAP inserted in l o domains of DOPC/SM/ Chl (1:1:0.35, mol:mol) SLB a s [68].…”

Cellular function and molecular structure of ecto-nucleotidases

“…They behave as non-cooperative allosteric enzymes in which the stability and the catalytic properties of each monomer are controlled by the conformation of the second subunit [476]. A dimeric structure has also been suggested after incorporation of the protein into supported lipid bilayers and analysis by atomic force microscopy [477]. Each catalytic site contains three metal ions, two Zn 2+ , and one Mg 2+ that are necessary for catalytic activity.…”