Sphingomyelin chain length influences the distribution of GPI-anchored proteins in rafts in supported lipid bilayers

Abstract

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.

Structural determinants of protein partitioning into ordered membrane domains and lipid rafts

“…GPI-anchor Thy-1 exoplasmic Thy-1 is associated at ~80% with the raft phase of GPMVs (Baumgart, Hammond et al 2007) GFP-GPI exoplasmic GPI-GFP Kp,raft = 1.5 -2 (Goswami, Gowrishankar et al 2008, Sengupta, Hammond et al 2008, Johnson, Stinson et al 2010, Zhou, Maxwell et al 2013 Acetylcholi ne esterase (ACE) exoplasmic In SPB composed of DOPC/brain SM/cholesterol (1:1:1) 41% of GPI-ACE partitioned into the Lo phase (Garner, Smith et al 2007) Sterol anchor Hedgehog (Hh) transmembrane - (Mao, Diehl et al 2009, Shi, Lv et al 2013 Transmembrane domain primary structure CD40, CD44, CD154 transmembrane CD154 DRM association diminished by 40% when TMD was mutated (Perschl, Lesley et al 1995, Bock and Gulbins 2003, Benslimane, Hassan et al 2012 HA transmembrane Mutation in the middle of TMD reduced DRM association from 38 to 2.2% (Scheiffele, Roth et al 1997, Lin, Naim et al 1998 Transmembrane LAT transmembrane Decreasing TMD length by 6 residues (~ 1/ (Diaz-Rohrer, Levental et al…”

GIPC: Glycosyl Inositol Phospho Ceramides, the major sphingolipids on earth

“…This function of FA coupling the 2 leaflets is unclear, it could explain the limited diffusion of proteins in the PM, or thermal adaptation. 21,22 For example, the role of interdigidation of phosphatidylserine with long saturated acyl chains has been recently shown to be required for actin-based membrane nanoclustering. 23 The role of FA interdigitation in plant biology clearly deserves to be fully studied.…”

Cilastatin Attenuates Cisplatin-Induced Proximal Tubular Cell Damage

“…Cilastatin is a molecule specifically designed to inhibit brush border-sorted DHP-I (Parkin et al, 2001;Garner et al, 2007). It is even probable that DHP-I is primarily located in apical cholesterol rafts (Pang et al, 2004) near the Fas site (Dimanche-Boitrel et al, 2005).…”