The role of annexin A4 in cancer.

Annexin A4 (ANXA4) is a member of the annexin family that binds to both calcium ions and phospholipids. Studies indicate that ANXA4 modulates membrane permeability and membrane trafficking, participates in cellular growth and apoptosis, enhances tumor invasion and promotes anti-tumor drug resistance. The overexpression of ANXA4 has been identified in various clinical epithelial tumors including: lung, gastric, colorectal, pancreatic, gallbladder, breast, renal, ovarian, laryngeal, and prostate cancers. In addition, upregulation and nuclear translocation of ANXA4 have been observed in the progression of colorectal cancer and ovarian serous carcinoma. Knockdown of ANXA4 attenuated migration in ovarian cancer and breast cancer cells. In contrast, knockdown of ANXA4 increased susceptibility to platinum in ovarian cancer and malignant mesothelioma cells. It is conceivable that ANXA4 is an indicator for tumor development, invasion, chemo-resistance, poor outcomes of cancer patients, and may be a potential target for therapeutic intervention.

Role of calcium-binding sites in calcium-dependent membrane association of annexin A4.

Annexin A4 (Anx4) is a cytosolic calcium-binding protein with four repeat domains, each containing one calcium-binding site (CBS). The protein interacts with the phospholipid membrane through the CBS-coordinated calcium ion, although the role of each CBS in the calcium-dependent association is unclear. To determine the role of each CBS, 15 CBS-abolished variants were produced in various combinations by substitution of a calcium-liganding residue on each CBS by Ala. Various mutant combinations produced different influences on calcium-dependent membrane-binding behavior and on the sodium-dependent dissociation of membrane-bound Anx4. Our data suggest the interaction of Anx4 with the lipid membrane consists of strong and weak interactions. CBSs I and IV mediate formation of strong interactions, while CBSs II and III are important for weak interactions. We also suggest Anx4 binds the lipid membrane through CBSs I and IV in the cytoplasmic fluids.

Quantitative analysis of self-association and mobility of annexin A4 at the plasma membrane.

Annexins, found in most eukaryotic species, are cytosolic proteins that are able to bind negatively-charged phospholipids in a calcium-dependent manner. Annexin A4 (AnxA4) has been implicated in diverse cellular processes, including the regulation of exocytosis and ion-transport; however, its precise mechanistic role is not fully understood. AnxA4 has been shown to aggregate on lipid layers upon Ca(2+) binding in vitro, a characteristic that may be critical for its function. We have utilized advanced fluorescence microscopy to discern details on the mobility and self-assembly of AnxA4 after Ca(2+) influx at the plasma membrane in living cells. Total internal reflection microscopy in combination with Förster resonance energy transfer reveals that there is a delay between initial plasma membrane binding and the beginning of self-assembly and this process continues after the cytoplasmic pool has completely relocated. Number-and-brightness analysis suggests that the predominant membrane bound mobile form of the protein is trimeric. There also exists a pool of AnxA4 that forms highly immobile aggregates at the membrane. Fluorescence recovery after photobleaching suggests that the relative proportion of these two forms varies and is correlated with membrane morphology.

Conformational analysis of a genetically encoded FRET biosensor by SAXS.

Genetically encoded FRET (Foerster resonance energy transfer) sensors are exciting tools in modern cell biology. Changes in the conformation of a sensor lead to an altered emission ratio and provide the means to determine both temporal and spatial changes in target molecules, as well as the activity of enzymes. FRET sensors are widely used to follow phosphorylation events and to monitor the effects of elevated calcium levels. Here, we report for the first time, to our knowledge, on the analysis of the conformational changes involved in sensor function at low resolution using a combination of in vitro and in cellulo FRET measurements and small-angle scattering of x rays (SAXS). The large and dynamic structural rearrangements involved in the modification of the calcium- and phosphorylation-sensitive probe CYNEX4 are comprehensively characterized. It is demonstrated that the synergistic use of SAXS and FRET methods allows one to resolve the ambiguities arising due to the rotation of the sensor molecules and the flexibility of the probe.

Crystal structures of sodium-bound annexin A4.

Annexin A4 (Anx4) possesses four repeat domains with one Ca(2+)-binding site (CBS) in each domain. In this study, we resolved two crystal structures of the Na(+)-bound form at high resolution (1.58 and 1.35 A). This is the first report that Anx4 binds the Na(+) ion in CBSs. Electron density maps, valence screening, and atomic absorbance spectrometry confirmed that Anx4 bound the Na(+) ion. One structure (1.58 A) bound the Na(+) ion in CBS I, whereas another structure (1.35 A) bound the Na(+) ion in CBS II and CBS III. We compared the two Na(+)-bound forms by superimposing their C(alpha) traces. The C(alpha) atoms of CBS III largely moved by coordination of the Na(+) ion. In the C(alpha) atoms of CBS I, however, little change resulted from Na(+)-coordination. Only the side chain of Glu71 was moved by Na(+)-coordination in CBS I. These results indicate that Anx4 also binds not only Ca(2+) but also Na(+) ion in the CBS.

Annexin A4 binding to anionic phospholipid vesicles modulated by pH and calcium.

Annexin A4 belongs to a class of Ca(2+)-binding proteins for which different functions in the cell have proposed, e.g. involvement in exocytosis and in the coagulation process. All these functions are related to the ability of the annexins to bind to acidic phospholipids. In this study the interaction of annexin A4 with large unilamellar vesicles (LUV) prepared from phosphatidylserine (PS) or from phosphatidic acid (PA) is investigated at neutral and acidic pH. Annexin A4 strongly binds to either lipid at acidic pH, whereas at neutral pH only weak binding to PA and no binding to PS occurs. Addition of 40 microM Ca(2+) leads to a strong binding to the lipids also at neutral pH. This is caused by the different electric charge of the protein below and above its isoelectric point. Binding of annexin A4 induces dehydration of the vesicle surface. The strength of the effects is much greater at pH 4 than at pH 7.4. At pH 7.4 annexin A4 reduces the Ca(2+)-threshold concentration necessary to induce fusion of PA LUV. The Ca(2+) induced fusion of PS LUV is not affected by annexin A4 at pH 7.4. At pH 4 annexin A4 induces fusion of either vesicles without Ca(2+). Despite the low binding extents at neutral pH annexin A4 induces a Ca(2+) independent leakage of PS- or PA-LUV. The leakage extent is increased at acidic pH. From the data two suggestions are made: (1) At pH 4 annexin A4 (at least partially) penetrates into the bilayer in contrast to the preferred location at the vesicle surface at neutral pH. The conformation of annexin A4 seems to be different at the two conditions. (2) At neutral pH, Annexin A4 seems to be able to bind two PA vesicles simultaneously; however, only one PS vesicle at the same time. This behavior might be related to a recently described double Ca(2+) binding site, which appears to be uniquely suited for PS.

Localization of the N-terminal domain in light-harvesting chlorophyll a/b protein by EPR measurements.

The conformational distribution of the N-terminal domain of the major light-harvesting chlorophyll a/b protein (LHCIIb) has been characterized by electron-electron double resonance yielding distances between spin labels placed in various domains of the protein. Distance distributions involving residue 3 near the N terminus turned out to be bimodal, revealing that this domain, which is involved in regulatory functions such as balancing the energy flow through photosystems (PS) I and II, exists in at least two conformational states. Models of the conformational sub-ensembles were generated on the basis of experimental distance restraints from measurements on LHCIIb monomers and then checked for consistency with the experimental distance distribution between residues 3 in trimers. Only models where residue 3 is located above the core of the protein and extends into the aqueous phase on the stromal side fit the trimer data. In the other state, which consequently is populated only in monomers, the N-terminal domain extends sideways from the protein core. The two conformational states may correspond to two functional states of LHCIIb, namely trimeric LHCIIb associated with PSII in stacked thylakoid membranes and presumably monomeric LHCIIb associated with PSI in nonstacked thylakoids. The switch between these two is known to be triggered by phosphorylation of Thr-6. A similar phosphorylation-induced conformational change of the N-terminal domain has been observed by others in bovine annexin IV which, due to the conformational switch, also loses its membrane-aggregating property.

Annexin A4 reduces water and proton permeability of model membranes but does not alter aquaporin 2-mediated water transport in isolated endosomes.

Annexin A4 (Anx4) belongs to a ubiquitous family of Ca2+-dependent membrane-binding proteins thought to be involved in membrane trafficking and membrane organization within cells. Anx4 localizes to the apical region in epithelia; however, its physiological role is unclear. We show that Anx4 exhibited binding to liposomes (phosphatidylcholine:phosphatidylserine, 1:1) in the presence of Ca2+ and binding was reversible with EDTA. Anx4 binding resulted in liposome aggregation and a reduction in membrane water permeability of 29% (P < 0.001) at 25 degrees C. These effects were not seen in the presence of Ca2+ or Anx4 alone and were reversible with EDTA. Measurements of membrane fluidity made by monitoring fluorescence anisotropy of 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD-HPC) demonstrated that Anx4 binding rigidified the outer leaflet of the bilayer (P < 0.001), thus providing a molecular explanation for the inhibition of water flux. To determine whether Anx4 would produce similar effects on physiological membranes we constructed liposomes which recapitulated the lipid composition of the inner leaflet of the MDCK apical membrane. These membranes exhibited reductions to water permeability upon Anx4 binding (19.5% at 25 degrees C, 31% at 37 degrees C; P < 0.01 and P < 0.001, respectively) and to proton permeability (15% at 25 degrees C, 19.5% at 37 degrees C; P < 0.05). Since our in vitro experiments indicated an effect on membrane permeability, we examined localization of Anx4 in the kidney collecting duct, a region of the nephron responsible for concentrating urine through water reabsorbtion. Anx4 was shown to colocalize apically with aquaporin 2 (AQP2) in collecting duct epithelia. To test for the existence of a functional interaction between Anx4 and AQP2 we isolated AQP2-containing endosomes and exposed them to Anx4/Ca2+. Water flux rates were unchanged, indicating Anx4 does not directly regulate AQP2. We conclude that Anx4 can alter the physical properties of membranes by associating with them and regulate passive membrane permeability to water and protons. These properties represent important new functions for Anx4.

Detection of annexin IV in bovine retinal rods.

The fraction of proteins capable of binding to photoreceptor membranes in a Ca2+-dependent manner was isolated from bovine rod outer segments. One of these proteins with apparent molecular mass of 32 kD (p32) was purified to homogeneity and identified as annexin IV (endonexin) by MALDI-TOF mass-spectrometry. In immunoblot, annexin IV purified from bovine rod outer segments cross-reacted with antibodies against annexin IV from bovine liver. This is the first detection of annexin IV in vertebrate retina.

Phosphorylation mutants elucidate the mechanism of annexin IV-mediated membrane aggregation.

Site-directed mutagenesis, electron microscopy, and X-ray crystallography were used to probe the structural basis of annexin IV-induced membrane aggregation and the inhibition of this property by protein kinase C phosphorylation. Site-directed mutants that either mimic (Thr6Asp, T6D) or prevent (Thr6Ala, T6A) phosphorylation of threonine 6 were produced for these studies and compared with wild-type annexin IV. In vitro assays showed that unmodified wild-type annexin IV and the T6A mutant, but not PKC-phosphorylated wild-type or the T6D mutant, promote vesicle aggregation. Electron crystallographic data of wild-type and T6D annexin IV revealed that, similar to annexin V, the annexin IV proteins form 2D trimer-based ordered arrays on phospholipid monolayers. Cryo-electron microscopic images of junctions formed between lipid vesicles in the presence of wild-type annexin IV indicated a separation distance corresponding to the thickness of two layers of membrane-bound annexin IV. In this orientation, a single layer of WT annexin IV, attached to the outer leaflet of one vesicle, would undergo face-to-face self-association with the annexin layer of a second vesicle. The 2.0-A resolution crystal structure of the T6D mutant showed that the mutation causes release of the N-terminal tail from the protein core. This change would preclude the face-to-face annexin self-association required to aggregate vesicles. The data suggest that reversible complex formation through phosphorylation and dephosphorylation could occur in vivo and play a role in the regulation of vesicle trafficking following changes in physiological states.

Differential lipid specificities of the repeated domains of annexin IV.

The roles of the four domains of annexin IV in binding to phospholipids and glycolipids were assessed by analyzing the binding of a group of mutant annexins IV in which one or more of the four domains was inactivated by replacing a critical amino residue(s) (Asp or Glu) with the neutral residue Ala. The data reveal that individual annexin domains may have characteristic affinities for different lipids. In particular, inactivation of the fourth domain inhibits the binding to phosphatidylserine (PS) and phosphatidylinositol (PI) but not to phosphatidylglycerol (PG), suggesting that this domain specifically can accommodate the larger head groups of PS and PI whereas the other three domains may form more restricted binding pockets. In order to block binding to PG, domain 1, or both domains 2 and 3 must be inactivated in addition to domain 4, suggesting that all four domains may be able to accommodate the headgroup of PG to some extent. Binding to acidic glycolipids (sulfatides) was also sensitive to inactivation of domain 4. However, in the case of sulfatides the nature of the binding reaction is fundamentally different compared with the binding to phospholipids since the interaction with sulfatides was highly sensitive to an increase in ionic strength. The binding to sulfatides may depend therefore on charge-charge interactions whereas the binding to phospholipid may involve a more specific interaction between the lipid headgroup and the protein surface, and/or interaction of the protein with the hydrophobic portion of a lipid bilayer.

Glycosaminoglycan binding properties of annexin IV, V, and VI.

We have previously demonstrated that annexin IV, one of the calcium/phospholipid-binding annexin family proteins, binds to glycosaminoglycans (GAGs) in a calcium-dependent manner (Kojima, K., Yamamoto, K., Irimura, T., Osawa, T., Ogawa, H., and Matsumoto, I. (1996) J. Biol. Chem. 271, 7679-7685). In this study, we investigated the GAG binding specificities of annexins IV, V, and VI by affinity chromatography and solid phase assays. Annexin IV was found to bind in a calcium-dependent manner to all the GAG columns tested. Annexin V bound to heparin and heparan sulfate columns but not to chondroitin sulfate columns. Annexin VI was adsorbed to heparin and heparan sulfate columns in a calcium-independent manner, and to chondroitin sulfate columns in a calcium-dependent manner. An N-terminal half fragment (A6NH) and a C-terminal half fragment (A6CH) of annexin VI, each containing four units, were prepared by digestion with V8 protease and examined for GAG binding activities. A6NH bound to heparin in the presence of calcium but not to chondroitin sulfate C, whereas A6CH bound to heparin calcium-independently and to chondroitin sulfate C calcium-dependently. The results showed that annexin IV, V, and VI have different GAG binding properties. Some annexins have been reported to be detected not only in the cytoplasm but also on the cell surface or in extracellular components. The findings suggest that the some annexins function as recognition elements for GAGs in extracellular space.

Structure of the trigonal crystal form of bovine annexin IV.

The structure of a trigonal crystal form of N-terminally truncated [des-(1-9)] bovine annexin IV, an annexin variant that exhibits the distinctive property of binding both phospholipids and carbohydrates in a Ca2+-dependent manner, has been determined at 3 A (0.3 nm) resolution -space group: R3; cell parameters: a=b=118.560 (8) A and c=82.233 (6) A-. The overall structure of annexin IV, crystallized in the absence of Ca2+ ions, is highly homologous to that of the other known members of the annexin family. The trimeric assembly in the trigonal crystals of annexin IV is quite similar to that found previously in non-isomorphous crystals of human, chicken and rat annexin V and to the subunit arrangement in half of the hexamer of hydra annexin XII. Moreover, it resembles that found in two-dimensional crystals of human annexin V bound to phospholipid monolayers. The propensity of several annexins to generate similar trimeric arrays supports the hypothesis that trimeric complexes of such annexins, including annexin IV, may represent the functional units that interact with membranes.

Interaction of annexins IV and VI with phosphatidylserine in the presence of Ca2+: monolayer and proteolytic study.

Annexins, Ca2+- and phospholipid-binding proteins are known to bind to artificial and biological membranes in a calcium-dependent manner. However, the precise mechanism of the annexin-membrane interactions still remains to be studied in detail. In this paper we describe the results of studies on the interactions of the annexin/Ca complexes with phospholipids, obtained by the Wilhelmy balance method of assessing the surface pressure of a phospholipid monolayer. We show that the annexin IV/Ca as well as annexin VI/Ca complexes significantly reduce the surface pressure of a phosphatidylserine monolayer, when its initial value is close to collapse pressure. The effect is highly specific for monolayers composed of phosphatidylserine and strongly sensitive to pH and ionic strength. The most pronounced changes have been observed at pH 7.0-7.5, at a protein/Ca molar ratio of 1:2 for annexin IV and 1:4 for annexin VI. In the presence of sodium chloride at concentrations exceeding 400mM this effect was almost completely abolished. The obtained results point to the mainly electrostatic character of the annexin/phosphatidylserine interactions. In addition, using large multilamellar lipid vesicles and serine proteases, we demonstrate that annexins, when bound in a ternary complex with phospholipids and calcium ions, are partially protected against proteolysis. Our observation that annexin molecules, complexed with calcium ions, are protected against proteolytic attack in the presence of PS liposomes does not have to be necessarily explained in terms of partial penetration of protein within the membrane bilayer.

A rise in nuclear calcium translocates annexins IV and V to the nuclear envelope.

Following incubation of human fibroblasts with Ca2+ ionophore A23187, we found strong immunofluorescence labelling of the nuclear envelope by annexin IV antibody. Using confocal imaging of cells loaded with Fluo-3, we showed that A23187 generates an intense and sustained rise of Ca2+ in the nucleus. By contrast, stimulation without extracellular Ca2+ produces only a brief rise in nuclear Ca2+ that does not promote annexin IV translocation to the nuclear envelope, and compounds that induce only a transient increase of nuclear Ca2+ do not support translocation of annexin IV. In addition, annexin V was also translocated to the nuclear envelope by A23187, but distribution of annexins I, II, VI and VII is unaffected. In in vitro assays with isolated nuclei, annexin V was also found to bind to the nuclear envelope in a Ca2+-dependent manner. These results demonstrate that the translocation to the nuclear envelope of different types of Ca2+-regulated proteins is directly triggered by a major rise of Ca2+ in the nucleus.

Characterization of carbohydrate-binding protein p33/41: relation with annexin IV, molecular basis of the doublet forms (p33 and p41), and modulation of the carbohydrate binding activity by phospholipids.

A protein, p33/41, expressed in bovine kidney and many other tissues was identified as a lectin which binds to sialoglycoproteins and glycosaminoglycans in a calcium-dependent manner. Partial amino acid sequences of p33/41 are highly homologous to those of calcium/phospholipid-binding annexin protein, annexin IV (endonexin), p33/41 exhibited similar calcium/phospholipid-binding activity (Kojima, K., Ogawa, H., Seno, N., Yamamoto, K., Irimura, T., Osawa, T., and Matsumoto, I. (1993) J. Biol. Chem. 267, 20536-20539). To further characterize p33/41, we cloned the p33/41 cDNA and characterized the recombinant protein encoded by this cDNA. Oligonucleotide probes were synthesized based on partial amino acid sequences of p33/41 and used for screening. A p33/41 cDNA clone was isolated encoding a protein of 319 amino acids with a calculated molecular mass of 35,769 Da. The deduced amino acid sequence was identical to that of bovine annexin IV except for one amino acid substitution. The recombinant protein gave two 33-kDa (p33) and 41-kDa (p41) bands on SDS-polyacrylamide gel electrophoresis under non-reducing conditions, and only one 33-kDa band under reducing conditions, as did the native protein. Mass spectrometric analysis combined with site-directed mutagenesis of each of the four cysteine residues of the recombinant protein revealed that p41 is a dimer of p33 cross-linked at Cys-198 via a disulfide bond. The recombinant protein bound to columns of heparin and fetuin glycopeptides in a calcium dependent manner and to phospholipid vesicles composed of phosphatidylserine (PS)/phosphatidylcholine (PC), phosphatidylethanolamine (PE)/PC or phosphatidylinositol (PI)/PC. Furthermore, concurrent binding assays showed that the binding of the recombinant protein to phospholipid vesicles was not affected by heparin, whereas that to heparin was influenced by the phospholipid composition of the vesicles; the highest binding was observed with vesicles composed of PE/PC. These results suggest that p33/41 binds two types of ligands via different sites and that phospholipids modulate the carbohydrate binding activity of p33/41.

Comparison of the expression of native and mutant bovine annexin IV in Escherichia coli using four different expression systems.

Bovine annexin IV, a Ca(2+)-dependent, membrane-binding protein, was expressed in E. coli using four different prokaryotic expression vector systems. An annexin IV cDNA was mutated in the 5' noncoding region to introduce an NcoI restriction site at the translation initiation site. The coding sequence was then excised and ligated into the expression vectors: pKK233-2 (which uses a hybrid trc promoter), pFOG405 (which uses the alkaline phosphatase promoter and generates a fusion protein with the alkaline phosphatase signal sequence that targets the protein for secretion), pOTSNco12 (which provides temperature-sensitive expression from the lambda phage promoter), and pET11d (which uses the T7lac promoter and a protease-deficient host). Expression of wild type and mutant annexin IV in the various systems was compared. Differences in level of expression, formation of inclusion bodies, and yield of purified protein were observed. The pET11d system was found to be the most effective expression system for annexin IV and various annexin IV mutant constructs, providing the highest yield of functional protein from the soluble fraction of cell lysates. Bovine chromaffin granule binding and aggregating activities of recombinant annexin IV were found to be virtually indistinguishable from those of bovine annexin IV isolated from liver tissue. Truncation constructs containing one, two, or three of the four conserved 70-amino-acid domains of native annexin IV were successfully created and expressed in E. coli, but the recombinant proteins were generally insoluble. pET11d annexin constructs containing point mutations in residues involved in binding calcium produced soluble protein at levels comparable to those of constructs expressing wild type protein.

Combinatorial mutagenesis of the four domains of annexin IV: effects on chromaffin granule binding and aggregating activities.

This study addresses the roles of individual annexin IV domains in calcium-dependent membrane binding and aggregation through an analysis of the activities of mutant annexin IV proteins in which critical residues in one or more domains have been altered. The consensus sequence for high-affinity Ca(2+)-binding pockets obtained from the annexin V crystal structure is GXGT-38 residues-D/E [Huber, R., et al. (1992) J. Mol. Biol. 223, 683-704]. Site-directed mutagenesis was used to change the conserved acidic residues (D/E) in these sequences to alanine residues in each of the four domains of bovine annexin IV, singly or in combinations. Fourteen mutants with one, two, three, or four mutated domains were constructed and expressed in Escherichia coli. Purified recombinant product was evaluated for Ca(2+)-dependent binding to and aggregation of bovine chromaffin granules. Increases in the number of mutated domains resulted in increased Ca2+ requirements for both granule binding and aggregation. Further analysis revealed that mutations in individual domains had preferential effects on the binding or aggregating activities of the protein. For example, mutation of the first or fourth domains had a greater effect on membrane binding than aggregation, while conversely, mutation of the second domain had a more dramatic effect on membrane aggregation. Mutation of the third domain was largely silent in these assays. An additional mutation was made in the fourth domain to substitute a serine for a highly conserved arginine residue (Arg274) present at the C-terminus of the fourth endonexin fold. This mutation increased the calcium requirement for membrane binding 2-fold and for membrane aggregation 3-fold. This mutant protein was found to be an effective inhibitor of membrane aggregation by native annexin IV at intermediate levels of calcium.

Highly polarized expression of carbohydrate-binding protein p33/41 (annexin IV) on the apical plasma membrane of epithelial cells in renal proximal tubules.

p33/41 is a Ca(2+)-dependent carbohydrate-binding protein and is identical to annexin IV, a member of the annexin protein family. The localization of p33/41 in bovine kidney specimens was investigated immunohistochemically by use of specific polyclonal antibodies. The most interesting finding on immunostaining was that p33/41 was highly concentrated in the apical plasma membrane of the epithelial cells in the proximal tubules contrary to the distribution throughout the cytoplasm in the papillary ducts and papilla epithelium. The enrichment of p33/41 in the apical membrane was confirmed by immunoblotting of the brush border membrane fraction prepared from a kidney homogenate. Sequential extraction with EDTA and Triton X-100, and a partition experiment with Triton X-114 revealed that most p33/41 associates with the renal brush border membrane in a Ca(2+)-independent manner and is integrated into the membrane like intrinsic membrane proteins.

Endonexin (annexin IV)-mediated lateral segregation of phosphatidylglycerol in phosphatidylglycerol/phosphatidylcholine membranes.

Endonexin (annexin IV) is a member of the annexin family of homologous proteins that share the ability to bind to pure lipid membranes and to aggregate vesicles in a Ca(2+)-dependent fashion. Endonexin appears to preferentially interact with certain types of lipids such as phosphatidylglycerol (PG) in PG/phosphatidylcholine (PC) mixed lipid membranes. Such preferential binding should result in localization of PG lipids to membrane regions where endonexin is bound. This was tested using a PG derivative containing the fluorophore pyrene, which exhibits fluorescence sensitive to molecular collision frequency. Motional restriction of pyrene-PG upon endonexin-membrane binding was evident from decreased ratios of excimer-to-monomer (E/M) pyrene fluorescence with endonexin binding to 97% PG/3% pyrene-PG vesicles. A maximum decrease of 30% suggests a 30% decrease in the average diffusion constant of pyrene-PG molecules or a 53% decrease assuming that only outer-monolayer lipid molecules interact with endonexin. In vesicles containing 5% and 10% pyrene-PG in PC, segregation of lipids was evident from observed increases in E/M of 14.2 +/- 1.8% and 6.8 +/- 0.1%, respectively, in the presence of endonexin and either 10 mM (5% pyrene-PG) or 2 mM (10% pyrene-PG) free Ca2+. At higher concentrations of Ca2+ (> 10 mM for 5% pyrene-PG and > 2 mM for 10% pyrene-PG), smaller endonexin-dependent increases in E/M are observed as endonexin molecules at high surface densities compete for the limited pool of pyrene-PG. The nature of these interactions of endonexin with mixed lipid systems has implications for the way annexins may modulate membrane structure in cells.