pH-dependent domain formation in phosphatidylinositol polyphosphate/phosphatidylcholine mixed vesicles

Abstract

Phosphatidylinositol polyphosphates (PI-PPs) have been shown to mediate a large variety of physiological processes by attracting proteins to specific cellular sites. Such site-specific signaling requires local accumulation of PI-PPs, and in light of the rich headgroup functionality, it is conceivable that hydrogen bond formation between adjacent headgroups is a contributing factor to the formation of PI-PP-enriched domains. To explore the significance of hydrogen bond formation for the mutual interaction of PI-PPs, this study aims to characterize the pH-dependent phase behavior of phosphatidylcholine/phosphatidylinositol bisphosphate and trisphosphate mixed vesicles by differential scanning calorimetry, infrared transmission spectroscopy, and fluorescence resonance energy transfer measurements. For pH values Ͼ 7-7.5, the experiments yielded results consistent with dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylinositol polyphosphate gel phase demixing, whereas for moderately acidic conditions, an enhanced mixing was observed. Similarly, this pH-dependent formation of PI-PPenriched domains was also found for the physiologically important fluid phase. The stability of PI-PP-enriched domains and to some extent the pH dependence of the domain formation was governed by the number as well as the position of the phosphomonoester groups at the inositol ring. ( Fig. 1 ) have been shown to control membrane trafficking events by recruiting proteins to specific cellular sites (1, 2). The temporal control of phosphoinositide-mediated signaling events is facilitated by a broad range of kinases and phosphatases (1, 3), whereas the signaling specificity is rooted in selective protein binding as well as a high degree of phosphoinositide compartmentalization. This compartmentalization is not only evident in the accumulation of distinct phosphoinositide derivatives in particular cell entities [e.g., enrichment of phosphatidylinositol-3-phosphate in early endosomes and phosphatidylinositol-4-phosphate in the Golgi (2, 4-6)] but manifests itself also in the formation of phosphoinositide-enriched domains. Several studies have reported the raft-dependent (7-13) and raft-independent (14-17) accumulation of phosphoinositides in vivo; however, the mechanisms that lead to such phosphoinositide segregation are elusive. For example, the recruitment of certain phosphoinositides to raft domains raises the question of what kind of molecular interactions and physiological conditions aid such targeting, which is generally unfavorable for lipids with unsaturated acyl chains (the typical chain composition of phosphoinositides is stearoyl/arachidonoyl). Simple lipid partitioning models cannot explain phosphoinositide enrichment in rafts, because measurements on monolayer systems failed to show any phosphatidylinositol-4,5-bisphosphate (PI-4,5-P 2 ) accumulation in cholesterol-enriched domains (although such studies have some limitations when compared with bilayer systems) (18).

Surface potential of phosphoinositide membranes: Comparison between theory and experiment

“…Similarly, the total phosphate groups in the inositol ring for PIP 2 have two net negatively charged sites at pH 6.0 or lower, and above pH 8.5, there are four net negatively dissociabled charge sites. More or less similar results were recently shown in other work (Fernandes et al [41]; Redfern and Gericke [42,43]) by using fluorescence techniques. Therefore, the average surface charge density of the surface charge layers, 1 and 2 for these PPIs containing membranes with no cation binding, t , can be easily obtained by knowing the concentration of PPI in PC membrane and the average area per molecule as done in our analysis.…”

Phosphatidylinositol-(4,5)-Bisphosphate Acyl Chains Differentiate Membrane Binding of HIV-1 Gag from That of the Phospholipase Cδ1 Pleckstrin Homology Domain

“…In case the acyl chain sequestration by MA is not involved (as shown in silico by Charlier et al recently [51]), saturated acyl chains may affect Gag membrane binding by altering the orientation of or distance between PI(4,5)P 2 molecules or their headgroups. At pH 7.3, which is the pH of the wheat germ lysate-GUV mixtures, PI(4,5)P 2 self-clusters, according to several in vitro studies (52)(53)(54). Therefore, it is possible that the observed effect of acyl chain saturation on Gag binding to PI(4,5)P 2 may involve clustering (or lack thereof) of this lipid.…”

Absence of clustering of phosphatidylinositol-(4,5)-bisphosphate in fluid phosphatidylcholine

“…In this case, energy transfer efficiencies would decrease not because of the formation of PI(4,5)P 2 clusters but as a result of changes in the lipid/water partition coefficient of the probe, as the PI(4,5)P 2 group becomes fully deprotonated. Redfern and Gericke (12) acknowledge that the extent of membrane incorporation changed considerably for different labeled phosphatidylinositides.…”

“…In a recent study, it was proposed that PI(4,5)P 2 compartmentalization can be achieved simply through hydrogen bonding between PI(4,5)P 2 head groups at or slightly above physiological pH (corresponding to partial or complete deprotonation of the phosphomonoester group) (12), without the contribution of any external agent (cholesterol or proteins). Through fluorescence resonance energy transfer (FRET) experiments in a phosphatidylcholine (PC) matrix in the fluid state, the authors claimed to have detected PI(4,5)P 2 domain formation, and similar behaviors were observed for PI(3,4)P 2 and PI(3,4,5)P 3 as well as for phosphatidylinositol monophosphates in another study (13).…”