Sphingomyelin chain length influences the distribution of GPI-anchored proteins in rafts in supported lipid bilayers.
Glycosyl-phosphatidylinositol (GPI)-anchored proteins are enriched in cholesterol- and sphingolipid-rich lipid rafts within the membrane. Rafts are known to have roles in cellular organization and function, but little is understood about the factors controlling the distribution of proteins in rafts. We have used atomic force microscopy to directly visualize proteins in supported lipid bilayers composed of equimolar sphingomyelin, dioleoyl-sn-glycero-3-phosphocholine and cholesterol. The transmembrane anchored angiotensin converting enzyme (TM-ACE) was excluded from the liquid ordered raft domains. Replacement of the transmembrane and cytoplasmic domains of TM-ACE with a GPI anchor (GPI-ACE) promoted the association of the protein with rafts in the bilayers formed with brain sphingomyelin (mainly C18:0). Association with the rafts did not occur if the shorter chain egg sphingomyelin (mainly C16:0) was used. The distribution of GPI-anchored proteins in supported lipid bilayers was investigated further using membrane dipeptidase (MDP) whose GPI anchor contains distearoyl phosphatidylinositol. MDP was also excluded from rafts when egg sphingomyelin was used but associated with raft domains formed using brain sphingomyelin. The effect of sphingomyelin chain length on the distribution of GPI-anchored proteins in rafts was verified using synthetic palmitoyl or stearoyl sphingomyelin. Both GPI-ACE and MDP only associated with the longer chain stearoyl sphingomyelin rafts. These data obtained using supported lipid bilayers provide the first direct evidence that the nature of the membrane-anchoring domain influences the association of a protein with lipid rafts and that acyl chain length hydrophobic mismatch influences the distribution of GPI-anchored proteins in rafts.
Garner
Ashley E
AE
Proteolysis Research Group, Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, and Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds, UK.
Smith
D Alastair
DA
Hooper
Nigel M
NM
D000818:Animals; D016466:CHO Cells; D002784:Cholesterol / Q000737:chemistry* / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D003902:Detergents / Q000737:chemistry; D004150:Dipeptidases / Q000737:chemistry / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000737:chemistry* / Q000378:metabolism*; D007668:Kidney / Q000737:chemistry / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D008081:Liposomes; D021962:Membrane Microdomains / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D018625:Microscopy, Atomic Force / Q000379:methods; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry / Q000378:metabolism; D010713:Phosphatidylcholines; D012995:Solubility; D013109:Sphingomyelins / Q000737:chemistry* / Q000378:metabolism; D013552:Swine
D003902:Detergents; D017261:Glycosylphosphatidylinositols; D008051:Lipid Bilayers; D008081:Liposomes; D008565:Membrane Proteins; D010713:Phosphatidylcholines; D013109:Sphingomyelins; D002784:Cholesterol; D004150:Dipeptidases; C038937:dipeptidase; D007703:Peptidyl-Dipeptidase A; C017251:1,2-oleoylphosphatidylcholine