Ca2+-dependent binding and activation of dormant ezrin by dimeric S100P

S100 proteins are EF hand type Ca2+ binding proteins thought to function in stimulus-response coupling by binding to and thereby regulating cellular targets in a Ca2+-dependent manner. To isolate such target(s) of the S100P protein we devised an affinity chromatography approach that selects for S100 protein ligands requiring the biologically active S100 dimer for interaction. Hereby we identify ezrin, a membrane/F-actin cross-linking protein, as a dimer-specific S100P ligand. S100P-ezrin complex formation is Ca2+ dependent and most likely occurs within cells because both proteins colocalize at the plasma membrane after growth factor or Ca2+ ionophore stimulation. The S100P binding site is located in the N-terminal domain of ezrin and is accessible for interaction in dormant ezrin, in which binding sites for F-actin and transmembrane proteins are masked through an association between the N- and C-terminal domains. Interestingly, S100P binding unmasks the F-actin binding site, thereby at least partially activating the ezrin molecule. This identifies S100P as a novel activator of ezrin and indicates that activation of ezrin's cross-linking function can occur directly in response to Ca2+ transients.     

Membrane composition affects the reversibility of annexin A2t binding to solid supported membranes: a QCM study

By means of the quartz crystal microbalance (QCM) technique, we investigated the interaction of porcine heterotetrametric annexin A2t with solid supported lipid membranes. Dissociation and rate constants of annexin A2t binding to various lipid mixtures were determined as a function of Ca2+ concentrations in solution. In contrast to what has been observed for annexin A1, the binding affinity and kinetics of annexin A2t binding are not influenced by cholesterol. In the experimental setup chosen, the annexin A2t binding is strictly Ca2+-dependent and only affected by the amount of phosphatidylserine (PS) in the membrane and the Ca2+ concentration in solution. By Ca2+-titration experiments at constant annexin A2t concentration, we investigated the reversibility of annexin A2t adsorption and desorption. Surprisingly, Ca2+-titration curves display a significant hysteresis. Protein desorption curves starting from annexin A2t bound to the membrane at 1 mM CaCl2 exhibit high cooperativity with half-maximum Ca2+ concentrations in the submicromolar range. However, protein adsorption curves starting from an EGTA-containing solution with soluble annexin A2t always show two inflection points upon addition of Ca2+ ions. These two inflection points may be indicative of two protein populations differently bound to the solid-supported membrane. The ratio of these two annexin A2t populations depends on the amount of PS molecules and cholesterol in the membrane as well as on the Ca2+ concentration. We propose a model discussing the results obtained in terms of two binding sites differing in their affinity due to lipid rearrangement.     

Identification of hydrophobic amino acid residues involved in the formation of S100P homodimers in vivo

S100 proteins are small dimeric members of the EF-hand superfamily of Ca(2+) binding proteins thought to participate in mediating intracellular Ca(2+) signals by binding to and thereby regulating target proteins in a Ca(2+)-dependent manner. As dimer formation is crucial to S100 function, we applied a yeast two-hybrid approach in analyzing in vivo molecular aspects of S100 dimerization. We chose S100P, a member of the S100 family highly expressed in placenta, for detailed analysis and showed that S100P monomers strongly interact with one another but not with other S100 polypeptides, indicating that homodimer formation is obligatory for S100P. Analysis of the interaction of site-specific S100P mutants with the wild-type polypeptide or with other S100P mutant chains identifies conserved hydrophobic amino acid residues involved in mediating dimerization in vivo. Of these residues, F-15 is crucially important as a mutation to alanine abolishes dimerization even when the F15A S100P mutant polypeptide is allowed to interact with a wild-type chain. On the other hand, I-11, I-12, or F-89 need to be replaced by a less hydrophopic residue in both subunits for there to be a similar extent of interfere with dimerization. This proves that hydrophobic residues implicated through structural studies in S100 dimerization are involved in the dimer interaction in vivo and argues for a hierarchy of hydrophobic contacts stabilizing the dimer and thereby regulating S100 function.     

Phosphatidylserine membrane domain clustering induced by annexin A2/S100A10 heterotetramer

By means of scanning force and fluorescence microscopy of artificial membranes immobilized on mica surfaces, the lateral organization of the annexin A2/S100A10 heterotetramer (annexin A2t) and its influence on the lateral organization of the lipids within the membrane have been elucidated. Planar lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) were prepared on atomically flat mica surfaces by the spreading of unilamellar vesicles. Fluorescence images of fluorescently labeled annexin A2t and scanning force microscopy images of nonlabeled protein bound to POPC/POPS bilayers show the formation of micrometer-sized lateral protein domains in the presence of 1 mM CaCl2. By means of scanning force microscopy, not only protein domains became discernible but also small membrane domains, which were attributed to POPS-enriched areas. A depletion of these POPS domains was observed in the vicinity of annexin A2t protein domains. These results indicate that annexin A2t is a peripheral membrane-binding complex capable of inducing lipid segregation.     

Retinosomes: new insights into intracellular managing of hydrophobic substances in lipid bodies

Lipid bodies form autonomous intracellular structures in many model cells and in some cells of specific tissue origin. They contain hydrophobic substances, a set of structural proteins such as perilipin or adipose differentiation-related protein, enzymes implicated in lipid metabolism, and proteins that participate in signaling and membrane trafficking. Retinosomes, particles reminiscent of lipid bodies, have been identified in retinal pigment epithelium as distinct structures compartmentalizing a metabolic intermediate involved in regeneration of the visual chromophore. These observations suggest that lipid bodies, including retinosomes, carry out specific functions that go beyond those of mere lipid storage organelles.     

Annexin II is required for apical transport in polarized epithelial cells

The sorting of apical proteins comprises an initial recognition step in the trans Golgi network and a final partitioning of the apical pool of proteins into at least two different types of vesicular carriers. One criteria of these carriers is the association or non-association of the protein content with lipid rafts. We have previously characterized a population containing the raft-associated sucrase-isomaltase-carrying vesicles (SAVs) and another one, the non-raft-associated lactase-phlorizin hydrolase-carrrying vesicles (LAVs) that are targeted separately to the apical membrane. Here, we demonstrate biochemically and by employing confocal laser microscopy that the annexin II-S100A10 complex is a component of SAVs and is absent from LAVs. The unequivocal role of annexin II in the apical targeting of SI is clearly demonstrated when down-regulation of this protein by annexin II-specific small interfering RNA drastically decreases the apical delivery of SI in the epithelial cell line Madin-Darby canine kidney. The annexin II-S100A10 complex plays therefore a crucial role in routing SAVs to the apical membrane of epithelial cells.     

Cooperative binding promotes demand-driven recruitment of AnxA8 to cholesterol-containing membranes

The functionality of cellular membranes is critically determined by their lipid composition. Within the endolysosomal system, cholesterol is mainly found in more peripheral compartments. In contrast, cholesterol levels are low in late endosomes/lysosomes (LEL), and the occurrence of enlarged pools of this lipid is commonly linked to endolysosomal dysfunction. Here, we show that Annexin A8 (AnxA8), a member of the annexin family of Ca^2 +-dependent membrane-binding proteins, participates in the endosomal regulation of cholesterol homeostasis. Depletion of AnxA8 caused accumulation of cholesterol in LEL, and pharmacological inhibition of the LEL cholesterol export recruited AnxA8 to the cholesterol-laden LEL. Biophysical analysis revealed that cholesterol enhanced the Ca^2 +-dependent affinity of AnxA8 to lipid bilayers, and induced positive cooperativity of membrane binding. Our findings identify AnxA8 as a regulator of LEL cholesterol balance and point to altered membrane binding cooperativity induced by aberrant lipid composition in the target membrane as a means to control the demand-driven recruitment of this cytosolic regulatory protein.