Thermal stability of bivalent cation/phosphoinositide domains in model membranes.

As key mediators in a wide array of signaling events, phosphoinositides (PIPs) orchestrate the recruitment of proteins to specific cellular locations at precise moments. This intricate spatiotemporal regulation of protein activity often necessitates the localized enrichment of the corresponding PIP. We investigate the extent and thermal stabilities of phosphatidylinositol-4-phosphate (PI(4)P), phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2 and phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) clusters with calcium and magnesium ions. We observe negligible or minimal clustering of all examined PIPs in the presence of Mg2+ ions. While PI(4)P shows in the presence of Ca2+ no clustering, PI(4,5)P2 forms with Ca2+ strong clusters that exhibit stablity up to at least 80°C. The extent of cluster formation for the interaction of PI(3,4,5)P3 with Ca2+ is less than what was observed for PI(4,5)P2, yet we still observe some clustering up to 80°C. Given that cholesterol has been demonstrated to enhance PIP clustering, we examined whether bivalent cations and cholesterol synergistically promote PIP clustering. We found that the interaction of Mg2+ or Ca2+ with PI(4)P remains extraordinarily weak, even in the presence of cholesterol. In contrast, we observe synergistic interaction of cholesterol and Ca2+ with PI(4,5)P2. Also, in the presence of cholesterol, the interaction of Mg2+ with PI(4,5)P2 remains weak. PI(3,4,5)P3 does not show strong clustering with cholesterol for the experimental conditions of our study and the interaction with Ca2+ and Mg2+ was not influenced by the presence of cholesterol.

Paratore TA ，Schmidt GE ，Ross AH ，Gericke A ... -  《-》

Phosphatidylinositol-4,5-bisphosphate ionization in the presence of cholesterol, calcium or magnesium ions.

Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) is an important signaling lipid and plays a crucial role in a wide variety of cellular processes by interacting with protein targets and localizing proteins at the plasma membrane. These interactions are strongly influenced by the lateral distribution of PI(4,5)P2 as well as its ionization state. The characterization of the PI(4,5)P2 ionization state provides important information about how PI(4,5)P2 interacts with other membrane resident or associated chemical species. In this study we have used solid-state MAS (31)P NMR to investigate the interactions of PI(4,5)P2 with potential cluster promoting agents, divalent cations and cholesterol. Both Ca(2+) and cholesterol were found previously to promote formation of local PI(4,5)P2 clusters in vitro. The NMR approach allows us to probe independently the ionization state of PI(4,5)P2 two phosphomonoester groups. We investigated mixed phosphatidylcholine (PC)/PI(4,5)P2 multilamellar vesicles in the presence of micro and millimolar concentrations of Ca(2+) and Mg(2+). We found that both cations lead to an increased downfield chemical shift of the PI(4,5)P2 phosphomonoester peaks, indicating an increased ionization in the presence of the divalent cations. Ca(2+) has a much larger effect on PI(4,5)P2 as compared to Mg(2+) at similar concentrations. Physiological concentrations of Ca(2+) are significantly lower than those found for Mg(2+) and the comparison of the PI(4,5)P2 ionization in the presence of Ca(2+) and Mg(2+) at physiological concentrations resulted in similar charges of the phosphomonoester groups for both cations. PI(4,5)P2 was also examined with vesicles containing cholesterol since cholesterol has been shown to promote PI(4,5)P2 clustering. In the presence of 40 mol% cholesterol, the PI(4,5)P2 phosphomonoester (31)P NMR peaks shifted slightly downfield, indicating a small increase in charge. Previously published data suggest that PI(4,5)P2 is capable of forming an intra- and intermolecular hydrogen bond network, which leads to a reduction of the charge at the phosphomonoester groups through dissipation of the charge across the bilayer/water interface. We hypothesize that cholesterol participates in this intermolecular hydrogen bond network, resulting in a stabilization of PI(4,5)P2 enriched domains due an increased spacing between the PI(4,5)P2 headgroup. We also examined the cumulative effects of cholesterol combined with the divalent cations, phosphatidylethanolamine (PE), and phosphatidylinositol (PI), separately. The combination of cholesterol and divalent cations results in an additive effect on PI(4,5)P2 ionization, while the effect of cholesterol on PI(4,5)P2 ionization is reduced in the presence of PE or PI.

Graber ZT ，Gericke A ，Kooijman EE 《-》

Cholesterol stabilizes fluid phosphoinositide domains.

Local accumulation of phosphoinositides (PIPs) is an important factor for a broad range of cellular events including membrane trafficking and cell signaling. The negatively charged phosphoinositide headgroups can interact with cations or cationic proteins and this electrostatic interaction has been identified as the main phosphoinositide clustering mechanism. However, an increasing number of reports show that phosphoinositide-mediated signaling events are at least in some cases cholesterol dependent, suggesting other possible contributors to the segregation of phosphoinositides. Using fluorescence microscopy on giant unilamellar vesicles and monolayers at the air/water interface, we present data showing that cholesterol stabilizes fluid phosphoinositide-enriched phases. The interaction with cholesterol is observed for all investigated phosphoinositides (PI(4)P, PI(3,4)P2, PI(3,5)P2, PI(4,5)P2 and PI(3,4,5)P3) as well as phosphatidylinositol. We find that cholesterol is present in the phosphoinositide-enriched phase and that the resulting phase is fluid. Cholesterol derivatives modified at the hydroxyl group (cholestenone, cholesteryl ethyl ether) do not promote formation of phosphoinositide domains, suggesting an instrumental role of the cholesterol hydroxyl group in the observed cholesterol/phosphoinositide interaction. This leads to the hypothesis that cholesterol participates in an intermolecular hydrogen bond network formed among the phosphoinositide lipids. We had previously reported that the intra- and intermolecular hydrogen bond network between the phosphoinositide lipids leads to a reduction of the charge density at the phosphoinositide phosphomonoester groups (Kooijman et al., 2009). We believe that cholesterol acts as a spacer between the phosphoinositide lipids, thereby reducing the electrostatic repulsion, while participating in the hydrogen bond network, leading to its further stabilization. To illustrate the effect of phosphoinositide segregation on protein binding, we show that binding of the tumor suppressor protein PTEN to PI(5)P and PI(4,5)P2 is enhanced in the presence of cholesterol. These results provide new insights into how phosphoinositides mediate important cellular events.

Jiang Z ，Redfern RE ，Isler Y ，Ross AH ，Gericke A ... -  《-》

Differential isolation and identification of PI(3)P and PI(3,5)P2 binding proteins from Arabidopsis thaliana using an agarose-phosphatidylinositol-phosphate affinity chromatography.

A phosphatidylinositol-phosphate affinity chromatographic approach combined with mass spectrometry was used in order to identify novel PI(3)P and PI(3,5)P2 binding proteins from Arabidopsis thaliana suspension cell extracts. Most of the phosphatidylinositol-phosphate interacting candidates identified from this differential screening are characterized by lysine/arginine rich patches. Direct phosphoinositide binding was identified for important membrane trafficking regulators as well as protein quality control proteins such as the ATG18p orthologue involved in autophagosome formation and the lipid Sec14p like transfer protein. A pentatricopeptide repeat (PPR) containing protein was shown to directly bind to PI(3,5)P2 but not to PI(3)P. PIP chromatography performed using extracts obtained from high salt (0.4M and 1M NaCl) pretreated suspensions showed that the association of an S5-1 40S ribosomal protein with both PI(3)P and PI(3,5)P2 was abolished under salt stress whereas salinity stress induced an increase in the phosphoinositide association of the DUF538 domain containing protein SVB, associated with trichome size. Additional interacting candidates were co-purified with the phosphoinositide bound proteins. Binding of the COP9 signalosome, the heat shock proteins, and the identified 26S proteasomal subunits, is suggested as an indirect effect of their interaction with other proteins directly bound to the PI(3)P and the PI(3,5)P2 phosphoinositides. PI(3,5)P2 is of special interest because of its low abundance. Furthermore, no endogenous levels have yet been detected in A. thaliana (although there is evidence for its existence in plants). Therefore the isolation of novel interacting candidates in vitro would be of a particular importance since the future study and localization of the respective endogenous proteins may indicate possible targeted compartments or tissues where PI(3,5)P2 could be enriched and thereafter identified. In addition, PI(3,5)P2 is a phosphoinositide extensively studied in mammalian and yeast systems. However, our knowledge of its role in plants as well as a list of its effectors from plants is very limited.

Oxley D ，Ktistakis N ，Farmaki T 《-》

Membrane Order Is a Key Regulator of Divalent Cation-Induced Clustering of PI(3,5)P(2) and PI(4,5)P(2).

Sarmento MJ ，Coutinho A ，Fedorov A ，Prieto M ，Fernandes F ... -  《-》