Raft partitioning and dynamic behavior of human placental alkaline phosphatase in giant unilamellar vesicles.
Much attention has recently been drawn to the hypothesis that cellular membranes organize in functionalized platforms called rafts, enriched in sphingolipids and cholesterol. The notion that glycosylphosphatidylinositol (GPI)-anchored proteins are strongly associated with rafts is based on their insolubility in nonionic detergents. However, detergent-based methodologies for identifying raft association are indirect and potentially prone to artifacts. On the other hand, rafts have proven to be difficult to visualize and investigate in living cells. A number of studies have demonstrated that model membranes provide a valuable tool for elucidating some of the raft properties. Here, we present a model membrane system based on domain-forming giant unilamellar vesicles (GUVs), in which the GPI-anchored protein, human placental alkaline phosphatase (PLAP), has been functionally reconstituted. Raft morphology, protein raft partitioning, and dynamic behavior have been characterized by fluorescence confocal microscopy and fluorescence correlation spectroscopy (FCS). Approximately 20-30% of PLAP associate with sphingomyelin-enriched domains. The affinity of PLAP for the liquid-ordered (l(o)) phase is compared to that of a nonraft protein, bacteriorhodopsin. Next, detergent extraction was carried out on PLAP-containing GUVs as a function of temperature, to relate the lipid and protein organization in distinct phases of the GUVs to the composition of detergent resistant membranes (DRMs). Finally, antibody-mediated cross-linking of PLAP induces a shift of its partition coefficient in favor of the l(o) phase.
Kahya
Nicoletta
N
Max Planck Institute of Molecular Cell Biology and Genetics, Dresden University of Technology, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
Brown
Deborah A
DA
Schwille
Petra
P
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D000818:Animals; D001666:Binding Sites, Antibody; D002784:Cholesterol / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D003902:Detergents; D058851:GPI-Linked Proteins; D006801:Humans; D007106:Immune Sera / Q000737:chemistry; D007527:Isoenzymes / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D008081:Liposomes; D021962:Membrane Microdomains / Q000737:chemistry*; D008567:Membranes, Artificial; D018613:Microscopy, Confocal; D017830:Octoxynol / Q000737:chemistry; D010713:Phosphatidylcholines / Q000737:chemistry; D010920:Placenta / Q000201:enzymology*; D011817:Rabbits; D013109:Sphingomyelins / Q000737:chemistry; D013696:Temperature; D013816:Thermodynamics
D003432:Cross-Linking Reagents; D003902:Detergents; D058851:GPI-Linked Proteins; D007106:Immune Sera; D007527:Isoenzymes; D008081:Liposomes; D008567:Membranes, Artificial; D010713:Phosphatidylcholines; D013109:Sphingomyelins; C033173:N-stearoylsphingomyelin; D017830:Octoxynol; D002784:Cholesterol; D000469:Alkaline Phosphatase; C019839:alkaline phosphatase, placental; C017251:1,2-oleoylphosphatidylcholine