Remodeling of Ordered Membrane Domains by GPI-Anchored Intestinal Alkaline Phosphatase

Abstract

Glycosylphosphatidyl-inositol (GPI)-anchored proteins preferentially localize in the most ordered regions of the cell plasma membrane. Acyl and alkyl chain composition of GPI anchors influence the association with the ordered domains. This suggests that, conversely, changes in the fluid and in the ordered domains lipid composition affect the interaction of GPI-anchored proteins with membrane microdomains. Validity of this hypothesis was examined by investigating the spontaneous insertion of the GPI-anchored intestinal alkaline phophatase (BIAP) into the solid (gel) phase domains of preformed supported membranes made of dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC), DOPC/sphingomyelin (DOPC/SM), and palmitoyloleoylphosphatidylcholine/SM (POPC/SM). Atomic force microscopy (AFM) showed that BIAP inserted in the gel phases of the three mixtures. However, changes in the lipid composition of membranes had a marked effect on the protein containing bilayer topography. Moreover, BIAP insertion was associated with a net transfer of phospholipids from the fluid to the gel (DOPC/DPPC) or from the gel to the fluid (POPC/SM) phases. For DOPC/SM bilayers, transfer of lipids was dependent on the homogeneity of the gel SM phase. The data strongly suggest that BIAP interacts with the most ordered lipid species present in the gel phases of phase-separated membranes. They also suggest that GPI-anchored proteins might contribute to the selection of their own microdomain environment.

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

“…For all samples, AFM showed that BIAP spontaneously inserted in the gel, i.e. highly ordered, phase of bilayers [67,68,40]. This spontaneous BIAP insertion into SLBs was in accordance with the observations reported for the insertion of various GPI proteins into biomembranes (see above).…”

“…Finally, differences between fluorescence and AFM data might be related to the pronounced effect of scanning forces on AP-GPI visualization. As recently reported [40], the use of scanning forces higher than 300 pN resulted in the extraction of a large number of BIAP molecules from the ordered domains (Fig. 5).…”

“…It was also affected by the gradient of lipid order at the interface of the fluid and gel phases. 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].…”

“…Direct AFM detection of the AP-GPI presence on SLBs can be obtained upon addition of BIAP in the buffer bathing preformed ordered-fluid phase separated bilayers [40,68]. This is illustrated in Fig.…”

“…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.…”