Interaction of membrane palmitoylated protein-1 with model lipid membranes

Abstract

Membrane palmitoylated protein-1 (MPP1) plays an important role in the formation of raft domains in erythroid membranes. We have shown recently that MPP1 interacts with membrane lipids composed of dioleoylphosphatidylcholine (DOPC), sphingomyelin (SM) and cholesterol. Here we further extend this investigation. Our results obtained from FRET assays revealed that MPP1 binds liposomes with high affinity (K D ~ 135 ± 15 nM). Preincubation of MPP1 with cholesterol before its addition to the Langmuir subphase resulted in a dramatic reduction in the membrane insertion/binding of MPP1, indicating the role of direct MPP1/cholesterol complexes in the interaction of MPP1 with membrane lipids. The generalized polarization values of liposomes as well as the constant surface area experiments on monolayers composed of DOPC/SM/Chol indicated a change in the lipid mono-and bilayer properties upon the addition of MPP1. Furthermore, the presence of flotillins did not affect the binding of MPP1 to membrane lipids. Also, MPP1 containing palmitoylation-mimicking mutation (C242F) bound DOPC/SM/Chol mono-and bilayer with an affinity very similar to that obtained for wild-type MPP1. In conclusion, our results suggest that the direct binding of MPP1 with membrane lipids could be involved in the mechanism of membrane association of MPP1 in erythroid cells.

Flotillins: At the Intersection of Protein S-Palmitoylation and Lipid-Mediated Signaling

“…This hydrophobic stretch likely has a substantial input to the membrane association of flotillin-1, since a fragment of flotillin-1 encompassing amino acids 36-179 binds to membranes when expressed in cells while the corresponding fragment of flotillin-2 does not [62]. Accordingly, Langmuir monolayer studies have shown that the penetration of recombinant non-acylated flotillin-1 into a monolayer with a lipid composition mimicking the inner leaflet of the erythrocyte membrane exceeded that of flotillin-2 [77].…”

MPP1-based mechanism of resting state raft organization in the plasma membrane. Is it a general or specialized mechanism in erythroid cells?

“…We believe that flotillin-1 and -2 are important components of these nanoclusters, which was confirmed by the fact that flotillins are found in the DRM residual fraction even after treatment with beta methyl cyclodextrin. Binding of palmitoylated MPP1 to the pre-existing nanocomplexes via flotillins and perhaps via cholesterol-binding regions as was recently documented [75,[84][85][86] induces their fusion and stabilizes them as membrane resting-state rafts, which are larger (~20 nm) in diameter [90], more stable (~1 s), detergent-resistant, and which become functional (see Fig. 3).…”

“…In addition, this interaction was found to be sensitive to the presence of cholesterol in the lipid monolayer, as adding MPP1 to the subphase of lipid monolayers containing cholesterol resulted in a much larger increase in surface area than when MPP1 is injected into the subphase of a lipid monolayer devoid of cholesterol [85]. Moreover, MPP1-bound cholesterol was found to competitively inhibit surface pressure changes of the phospholipid monolayer [86].…”

Spectroscopy of model-membrane liposome-protein systems: complementarity of linear dichroism, circular dichroism, fluorescence and SERS

“…The work also illustrates the value of molecular dynamics simulations to interpret fluorescence data. The same principle was used by Elderdfi and Sikorsk [18] to find the equilibrium dissociation constant (B) Fluorescence of gramicidin A (a linear pentadecapeptide from Bacillus brevis with alternating D and L amino acids of which 4 are tryptophans) in TFE (black), 10% TFE (blue) and 10% TFE in the presence of PC liposomes (red) [12]. value of the binding affinity of membrane palmitoylated protein-1 (MPP1) with DOPC/SM/CH liposomes.…”