Structural determinant of the vesicle aggregation activity of annexin I.

Some annexins, including annexins I, II, IV, and VII, can promote membrane aggregation. To identify amino acids involved in annexin I-mediated membrane aggregation, we generated truncated mutants of human annexin I lacking various parts of the amino terminus. The in vitro vesicle binding and aggregation activities of these mutants indicated that both the amino-terminal region of annexin I spanning residues 26-29 and the carboxy-terminal core are involved in membrane aggregation. This notion was further supported by the finding that a chimera composed of residues 24-35 of annexin I and the core of annexin V has vesicle aggregation activity that is significantly higher than that of annexin V but lower than that of annexin I. Further site-specific mutations in the amino-terminal region of annexin I indicated that Lys-26 and Lys-29 are essential for its membrane aggregation activity. The comparison of tryptic digest patterns of free and vesicle-bound wild type and K29E mutant suggests that a primary role of Lys-26 and Lys-29 is to induce and stabilize an active conformation of annexin I for vesicle aggregation.     

Annexins V and XII insert into bilayers at mildly acidic pH and form ion channels

The functional hallmark of annexins is the ability to bind to the surface of phospholipid membranes in a reversible, Ca(2+)-dependent manner. We now report that human annexin V and hydra annexin XII reversibly bound to phospholipid vesicles in the absence of Ca(2+) at low pH; half-maximal vesicle association occurred at pH 5.3 and 5. 8, respectively. The following biochemical data support the hypothesis that these annexins insert into bilayers at mildly acidic pH. First, a photoactivatable reagent (3-trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine) which selectively labels proteins exposed to the hydrophobic domain of bilayers reacted with these annexins at pH 5.0 and below but not at neutral pH. Second, in a Triton X-114 partitioning assay, annexins V and XII act as integral membrane proteins at low pH and as hydrophilic proteins at neutral pH; in the presence of phospholipids half-maximal partitioning into detergent occurred at pH approximately 5.0. Finally, annexin V or XII formed single channels in phospholipid bilayers at low pH but not at neutral pH. A model is discussed in which the concentrations of H(+) and Ca(2+) regulate the reversible conversion of three forms of annexins-soluble, peripheral membrane, and transmembrane.     

Annexin I-mediated vesicular aggregation: mechanism and role in human neutrophils.

Whole cytosol isolated from human neutrophils was found to accelerate the Ca(2+)-dependent fusion of phospholipid vesicles with neutrophil plasma membranes as measured by several fluorescence resonance energy transfer lipid dilution assays or by the fate of an encapsulated aqueous soluble fluorophore. The Ca2+ (threshold of 2-10 microM) and protein concentration dependencies for fusion mediated by purified human neutrophil annexin I (lipocortin I), recombinant annexin I and des(1-9)annexin I showed behavior similar to that of whole cytosol. A monoclonal antibody against the N-terminal region of annexin I strongly inhibited the action of isolated annexins as well as whole cytosol, indicating that annexin I is the major activity of this type in whole neutrophil cytosol and that it functions even in this complex mixture of proteins. Residual Ca(2+)-dependent fusion activity in the absence of cytosol or annexin I was not inhibited by several antibodies against annexin I, implicating an as yet unknown protein. Kinetic analysis of liposomal fusion showed that annexin I, as in the case of synexin, accelerates aggregation of vesicles but not the actual fusion event per se. The disposition of annexin I in liposomal aggregates was studied by monitoring binding of the protein with a pyrene-phospholipid and by simultaneously monitoring vesicular aggregation by turbidity. An antibody to the N-terminus of annexin I inhibited vesicular aggregation but not binding, suggesting that initial binding of annexin I is similar to that of annexin V. A relatively small proportion of the bound annexin was involved in intervesicular linkage, and no exchange of bound annexin to subsequently added vesicles was observed. The lack of extensive contact between lipids of aggregated vesicles was supported by a lack of energy transfer between phospholipid probes on separate aggregating vesicles. Covalent linkage of maleimidyl or photoaffinity phospholipid derivatives with annexin I in vesicular aggregates did not allow complete disaggregation of vesicles by EDTA, suggesting that monomers of annexin I can contact two membranes simultaneously at the point of intervesicular linkage. These data are discussed in terms of possible models for the structure of this site.     

Annexins in the Human Neuroblastoma SH-SY5Y: Demonstration of Relocation of Annexins II and V to Membranes in Response to Elevation of Intracellular Calcium by Membrane Depolarisation and by the Calcium Ionophore A23187

Abstract: The human neuroblastoma SH-SY5Y was found to express annexins I, II, IV, V, and VI by western blot analysis. Calcium-dependent membrane-binding proteins were isolated from SH-SY5Y and analysed by 2-dimensional gel electrophoresis. Proteins with M_r and pl values similar to those of annexins I, II, III, IV, V, and VI were observed. The identity of annexins II and V was confirmed by western blotting. The membrane association of annexins II and V was studied in cells that had been stimulated to release noradrenaline by K⁺ depolarisation or by treatment with the ionophore A23187. Annexins II and V were both found to associate with membranes in a manner that was resistant to elution with EGTA and required Triton X-100 for their solubilisation. Homogenisation of cells in calcium-containing buffers also resulted in the formation of EGTA-resistant membrane-associated annexins II and V. The results demonstrate calcium-dependent relocation of annexins II and V to membranes in intact cells and suggest that these annexins bind in a calcium-dependent manner to non-phospholipid components of SH-SY5Y membranes. Examination of cells by immunofluorescence microscopy demonstrated that annexin II was homogeneously associated with the plasma membrane before treatment with ionophore and relocated to discrete patches of staining after treatment. Annexin V was found by immunofluorescence to be present in the cytoplasm and in the nucleus. Stimulation of the cells produced no change in the cytoplasmic staining pattern but resulted in a partial relocation of nuclear annexin V to the periphery of the nucleus. The results argue for a general role for both annexins in calcium signalling at discrete intracellular locations. The results are not consistent with the specific involvement proposed previously for annexin II in membrane fusion at sites of vesicle exocytosis.     

Characterization of a discontinuous epitope on annexin II by site-directed mutagenesis.

Recombinant annexin II mutants were generated to identify amino acids involved in the formation of the discontinuous epitope of the monoclonal antibody H28. Analysis of the various mutant proteins by immunoblotting and enzyme-linked immunosorbent assay revealed that residues Lys27, Arg62, Glu65, and Arg67 are indispensable for H28 reactivity. Residues in equivalent positions are also in close proximity in the recently determined X-ray structure of annexin V, a different member of the same family of Ca2+/lipid-binding proteins. Thus annexins II and V show a similar three-dimensional folding in this region of the molecule. Consequently, the Ca2+ binding sites and the residues phosphorylated by pp60src (Tyr23) and protein kinase C (Ser25) most likely reside on opposite sides of the annexin II molecule.     

Purification of annexin I and annexin II from human placental membranes by high-performance liquid chromatography.

Annexin I and annexin II were extracted from human placental membranes with ethylene glycol bis(beta-amino-ethyl ether)-N,N'-tetraacetic acid (EGTA) and purified by high-performance liquid chromatography by measuring their ability to inhibit phospholipase A2 activity in vitro. Neither protein was capable of binding to a DEAE-5PW HPLC column at neutral pH; however, they were resolved through binding to a Mono S column and passage through size-exclusion HPLC columns. Annexin I and its covalently linked dimer (36 and 66 kDa, respectively, by sodium dodecyl sulfate (SDS)-gel electrophoresis) reacted in one-dimensional immunoblots with monoclonal antibodies to annexin I and calpactin II, and with monoclonal and polyclonal antibodies to lipocortin I, confirming that annexin I, calpactin II, and lipocortin I are the same or closely related proteins. Milligram amounts of monomeric annexin I containing negligible amounts of the cross-linked dimeric annexin I were selectively isolated from placental membranes by using buffers containing the sulfhydryl reagent iodoacetic acid. Milligram amounts of cross-linked annexin I were selectively isolated when placental membranes were initially treated with buffers that did not contain iodoacetic acid and then extracted with Triton X-100, suggesting that sulfhydryl-dependent transglutaminase activity contributes to the selective isolation of this protein. A third phospholipase A2-inhibitory protein (35 kDa by SDS-gel electrophoresis) that reacted in immunoblots with monoclonal antibodies to calpactin I and annexin II, indicating their similar identity, was isolated. The procedure employed allows the rapid purification of annexins I and II in milligram amounts from placental membranes within 2 days.     

Mechanism of annexin I-mediated membrane aggregation

It has been proposed that annexin I has two separate interaction sites that are involved in membrane binding and aggregation, respectively. To better understand the mechanism of annexin I-mediated membrane aggregation, we investigated the properties of the inducible secondary interaction site implicated in membrane aggregation. X-ray specular reflectivity measurements showed that the thickness of annexin I layer bound to the phospholipid monolayer was 31 +/- 2 A, indicating that annexin I binds membranes as a protein monomer or monolayer. Surface plasmon resonance measurements of annexin I, V, and mutants, which allowed evaluation of membrane aggregation activity of annexin I separately from its membrane binding, revealed direct correlation between the relative membrane aggregation activity and the relative affinity of the secondary interaction site for the secondary membrane. The secondary binding was driven primarily by hydrophobic interactions, unlike calcium-mediated electrostatic primary membrane binding. Chemical cross-linking of membrane-bound annexin I showed that a significant degree of lateral association of annexin I molecules precedes its membrane aggregation. Taken together, these results support a hypothetical model of annexin I-mediated membrane aggregation, in which a laterally aggregated monolayer of membrane-bound annexin I directly interacts with a secondary membrane via its induced hydrophobic interaction site.     

Interactions of annexins with membrane phospholipids.

The annexins are proteins that bind to membranes and can aggregate vesicles and modulate fusion rates in a Ca2(+)-dependent manner. In this study, experiments are presented that utilize a pyrene derivative of phosphatidylcholine to examine the Ca2(+)-dependent membrane binding of soluble human annexin V and other annexins. When annexin V and other annexins were bound to liposomes containing 5 mol % acyl chain labeled 3-palmitoyl-2-(1-pyrenedecanoyl)-L-alpha-phosphatidylcholine, a decrease in the excimer-to-monomer fluorescence ratio was observed, indicating that annexin binding may decrease the lateral mobility of membrane phospholipids without inducing phase separation. The observed increases of monomer fluorescence occurred only with annexins and not with other proteins such as parvalbumin or bovine serum albumin. The extent of the increase of monomer fluorescence was dependent on the protein concentration and was completely and rapidly reversible by EDTA. Annexin V binding to phosphatidylserine liposomes was consistent with a binding surface area of 59 phospholipid molecules per protein. Binding required Ca2+ concentrations ranging between approximately 10 and 100 microM, where there was no significant aggregation or fusion of liposomes on the time scale of the experiments. The polycation spermine also displaced bound annexins, suggesting that binding is largely ionic in nature under these conditions.     

Characterizing the binding of annexin V to a lipid bilayer using molecular dynamics simulations

Annexins play critical roles in membrane organization, membrane trafficking and vesicle transport. The family members share the ability to bind to membranes with high affinities, but the interactions between annexins and membranes remain unclear. Here, using long‐time molecular dynamics simulations, we provide detailed information for the binding of an annexin V trimer to a POPC/POPS lipid bilayer. Calcium ions function as bridges between several negatively charged residues of annexin V and the oxygen atoms of lipids. The preferred calcium‐bridges are those formed via the carboxyl oxygen atoms of POPS lipids. H‐bonds and hydrophobic interactions formed by several critical residues have also been observed in the annexin‐membrane interface. The annexin‐membrane binding causes small changes of annexin trimer structures, while has significant effects on lipid bilayer structures. The lipid bilayer shows a bent shape and forms a concave region in the annexin‐membrane interaction interface, which provides an atomic‐level evidence to support the view that annexins could disturb the stability of lipids and bend membranes. This study provides insights into the commonly occurring PS‐dependent and calcium‐dependent binding of proteins to membranes. Proteins 2014; 82:312–322. © 2013 Wiley Periodicals, Inc.     

Formation of tightly bound forms of annexins V and VI in rabbit skeletal muscle membranes isolated in the presence of barium ions

After isolation of rabbit skeletal muscle membranes in the presence of Ba2+ or Ca2+, significant portions of annexin V and VI tightly bind to membranes and become inaccessible for chelating agents. Tightly bound annexin VI is virtually completely solubilized only after treatment with a buffer supplemented both with EGTA and detergent. The portion of tightly bound annexin V cannot be removed even by extraction with buffer containing both EGTA and detergent. In some cases, tightly bound annexin V or VI is detected even in the control (not treated with cations) membranes, thus indicating the possible formation of tight annexin--membrane complexes in situ. The addition of exogenous cations seems to promote only the accumulation of tightly bound annexins within the cell. After temperature-induced phase separation, annexin V and VI bound to the membranes isolated in the presence of Ba2+ or Ca2+ remains mainly in the aqueous phase, similarly to annexins isolated from the control membranes. Neither annexin partitions into the detergent-enriched phase. This indicates the absence of hydrophobicity change in comparison with the standard EGTA-soluble annexins.     

Key role of the N‐terminus of chicken annexin A5 in vesicle aggregation

Annexins are calcium‐dependent phospholipid‐binding proteins involved in calcium signaling and intracellular membrane trafficking among other functions. Vesicle aggregation is a crucial event to make possible the membrane remodeling but this process is energetically unfavorable, and phospholipid membranes do not aggregate and fuse spontaneously. This issue can be circumvented by the presence of different agents such as divalent cations and/or proteins, among them some annexins. Although human annexin A5 lacks the ability to aggregate vesicles, here we demonstrate that its highly similar chicken ortholog induces aggregation of vesicles containing acidic phospholipids even at low protein and/or calcium concentration by establishment of protein dimers. Our experiments show that the ability to aggregate vesicles mainly resides in the N‐terminus as truncation of the N‐terminus of chicken annexin A5 significantly decreases this process and replacement of the N‐terminus of human annexin A5 by that of chicken switches on aggregation; in both cases, there are no changes in the overall protein structure and only minor changes in phospholipid binding. Electrostatic repulsions between negatively charged residues in the concave face of the molecule, mainly in the N‐terminus, seem to be responsible for the impairment of dimer formation in human annexin A5. Taking into account that chicken annexin A5 presents a high sequence and structural similarity with mammalian annexins absent in birds, as annexins A3 and A4, some of the physiological functions exerted by these proteins may be carried out by chicken annexin A5, even those that could require calcium‐dependent membrane aggregation.     

Fluorescent annexin A1 reveals dynamics of ceramide platforms in living cells

Upon its genesis during apoptosis, ceramide promotes gross reorganization of the plasma membrane structure involving clustering of signalling molecules and an amplification of vesicle formation, fusion and trafficking. The annexins are a family of proteins, which in the presence of Ca(2+), bind to membranes containing negatively charged phospholipids. Here, we show that ceramide increases affinity of annexin A1-membrane interaction. In the physiologically relevant range of Ca(2+) concentrations, this leads to an increase in the Ca(2+)sensitivity of annexin A1-membrane interaction. In fixed cells, using a ceramide-specific antibody, we establish a direct interaction of annexin A1 with areas of the plasma membrane enriched in ceramide (ceramide platforms). In living cells, the intracellular dynamics of annexin A1 match those of plasmalemmal ceramide. Among proteins of the annexin family, the interaction with ceramide platforms is restricted to annexin A1 and is conveyed by its unique N-terminal domain. We demonstrate that intracellular Ca(2+)overload occurring at the conditions of cellular stress induces ceramide production. Using fluorescently tagged annexin A1 as a reporter for ceramide platforms and annexin A6 as a non-selective membrane marker, we visualize ceramide platforms for the first time in living cells and provide evidence for a ceramide-driven segregation and internalization of membrane-associated proteins.     

Differential expression of annexins I and II in normal and malignant human mammary epithelial cells

Abstract. Collagen-binding proteins ( CBPs ) of rat mammary tumors are identical to Ca²⁺-binding annexins [49]. We have now isolated a protein of 38 kDa from the human mammary tumor cell line ALAB by collagen type I affinity chromatography as well as by extraction of calcium-binding proteins. The 38-kDa band of both preparations was identified as annexin II (calpactin I) by its reaction with an annexin II-specific monoclonal antibody in Western blot analysis. Annexin I (lipocortin I) was not detectable in these cells. Two other human cell lines, the SV40-transformed cell line SV3 and cell line HBL-100, both established from normal mammary glands, were also positive for annexin II and negative for annexin I.
In vivo expression of annexins was investigated by immunohistological staining of normal and malignant human mammary tissue. The annexin II-specific mAb reacted with normal and tumor parenchyme whereas the annexin I-specific mAb reacted with acini and ductal myoepithelium of the normal mammary gland but showed no reaction with tumor tissue. Immunolocalization studies also showed annexin II expression in both normal and tumor stroma while only tumor stromal cells were found to be reactive with the antibody against annexin I. The differential expression of annexins in normal and malignant human mammary tissue suggests special functions of these proteins in the mammary gland.     

Membrane-bound annexin V isoforms (CaBP33 and CaBP37) and annexin VI in bovine tissues behave like integral membrane proteins.

The distribution of annexin V isoforms (CaBP33 and CaBP37) and of annexin VI in bovine lung, heart, and brain subfractions was investigated with special reference to the fractions of these proteins which are membrane-bound. In addition to EGTA-extractable pools of the above proteins, membranes from lung, heart, and brain contain EGTA-resistant annexins V and VI which can be solubilized with detergents (Triton X-100 or Triton X-114). A strong base like Na2CO3, which is usually effective in extracting membrane proteins, only partially solubilizes the membrane-bound, EGTA-resistant annexins analyzed here. Also, only 50-60% of the Triton X-114-soluble annexins partition in the aqueous phase, the remaining fractions being recovered in the detergent-rich phase. Altogether, these findings suggest that, by an as yet unknown mechanism, following Ca(2+)-dependent association of annexin V isoforms and annexin VI with membranes, substantial fractions of these proteins remain bound to membranes in a Ca(2+)-independent way and behave like integral membrane proteins. These results further support the possibility that the above annexins might play a role in membrane trafficking and/or in the regulation of the structural organization of membranes.     

Amino acid sequence analysis of the annexin super-gene family of proteins.

The annexins are a widespread family of calcium-dependent membrane-binding proteins. No common function has been identified for the family and, until recently, no crystallographic data existed for an annexin. In this paper we draw together 22 available annexin sequences consisting of 88 similar repeat units, and apply the techniques of multiple sequence alignment, pattern matching, secondary structure prediction and conservation analysis to the characterisation of the molecules. The analysis clearly shows that the repeats cluster into four distinct families and that greatest variation occurs within the repeat 3 units. Multiple alignment of the 88 repeats shows amino acids with conserved physicochemical properties at 22 positions, with only Gly at position 23 being absolutely conserved in all repeats. Secondary structure prediction techniques identify five conserved helices in each repeat unit and patterns of conserved hydrophobic amino acids are consistent with one face of a helix packing against the protein core in predicted helices a, c, d, e. Helix b is generally hydrophobic in all repeats, but contains a striking pattern of repeat-specific residue conservation at position 31, with Arg in repeats 4 and Glu in repeats 2, but unconserved amino acids in repeats 1 and 3. This suggests repeats 2 and 4 may interact via a buried saltbridge. The loop between predicted helices a and b of repeat 3 shows features distinct from the equivalent loop in repeats 1, 2 and 4, suggesting an important structural and/or functional role for this region. No compelling evidence emerges from this study for uteroglobin and the annexins sharing similar tertiary structures, or for uteroglobin representing a derivative of a primordial one-repeat structure that underwent duplication to give the present day annexins. The analyses performed in this paper are re-evaluated in the Appendix, in the light of the recently published X-ray structure for human annexin V. The structure confirms most of the predictions and shows the power of techniques for the determination of tertiary structural information from the amino acid sequences of an aligned protein family.     

The association of annexin I with early endosomes is regulated by Ca2+ and requires an intact N-terminal domain.

Annexin I is a member of a multigene family of Ca2+/phospholipid-binding proteins and a major substrate for the epidermal growth factor (EGF) receptor kinase, which has been implicated in membrane-related events along the endocytotic pathway, in particular in the sorting of internalized EGF receptors occurring in the multivesicular body. We analyzed in detail the intracellular distribution of this annexin by cell fractionation and immunoelectron microscopy. These studies used polyclonal as well as a set of species-specific monoclonal antibodies, whose epitopes were mapped to the lateral surface of the molecule next to a region thought to be involved in vesicle aggregation. Unexpectedly, the majority of annexin I was identified on early and not on multivesicular endosomes in a form that required micromolar levels of Ca2+ for the association. The specific cofractionation with early endosomes was also observed in transfected baby hamster kidney cells when the intracellular fate of ectopically expressed porcine annexin I was analyzed by using the species-specific monoclonal antibodies in Western blots of subcellular fractions. Interestingly, a truncation of the N-terminal 26, but not the N-terminal 13 residues of annexin I altered its intracellular distribution, shifting it from fractions containing early to those containing late and multivesicular endosomes. These findings underscore the regulatory importance of the N-terminal domain and provide evidence for an involvement of annexin I in early endocytotic processes.     

Binding of annexin V/placental anticoagulant protein I to platelets. Evidence for phosphatidylserine exposure in the procoagulant response of activated platelets

Proteins of the annexin/lipocortin family act as in vitro anticoagulants by binding to anionic phospholipid vesicles. In this study, we investigated whether annexin V (placental anticoagulant protein I) would bind to human platelets. Annexin V bound to unstimulated platelets in a reversible, calcium-dependent reaction with an apparent Kd of 7 nM and 5000-8000 sites/platelet. Additional binding sites could be induced by several platelet agonists in the following order of effectiveness: A23187 greater than collagen + thrombin greater than collagen greater than thrombin. However, neither ADP nor epinephrine induced additional binding sites. Three other proteins of the annexin family (annexins II, III, and IV) competed for annexin V platelets binding sites with the same relative potencies previously observed for binding to phospholipid vesicles. Phospholipid vesicles containing phosphatidylserine completely inhibited binding of annexin V to platelets. Annexin V completely blocked binding of 125I-factor Xa to thrombin-stimulated platelets. These results support the hypothesis that phosphatidylserine exposure occurs during platelet activation and may be necessary for assembly of the prothrombinase complex on platelet membranes.     

Structural determinants for plant annexin-membrane interactions

The interactions of two plant annexins, annexin 24(Ca32) from Capsicum annuum and annexin Gh1 from Gossypium hirsutum, with phospholipid membranes have been characterized using liposome-based assays and adsorption to monolayers. These two plant annexins show a preference for phosphatidylserine-containing membranes and display a membrane binding behavior with a half-maximum calcium concentration in the sub-millimolar range. Surprisingly, the two plant annexins also display calcium-independent membrane binding at levels of 10-20% at neutral pH. This binding is regulated by three conserved surface-exposed residues on the convex side of the proteins that play a pivotal role in membrane binding. Due to quantitative differences in the membrane binding behavior of N-terminally His-tagged and wild-type annexin 24(Ca32), we conclude that the N-terminal domain of plant annexins plays an important role, reminiscent of the findings in their mammalian counterparts. Experiments elucidating plant annexin-mediated membrane aggregation and fusion, as well as the effect of these proteins on membrane surface hydrophobicity, agree with findings from the membrane binding experiments. Results from electron microscopy reveal elongated rodlike assemblies of plant annexins in the membrane-bound state. It is possible that these structures consist of protein molecules directly interacting with the membrane surface and molecules that are membrane-associated but not in direct contact with the phospholipids. The rodlike structures would also agree with the complex data from intrinsic protein fluorescence. The tubular lipid extensions suggest a role in the membrane cytoskeleton scaffolding or exocytotic processes. Overall, this study demonstrates the importance of subtle changes in an otherwise conserved annexin fold where these two plant annexins possess distinct modalities compared to mammalian and other nonplant annexins.     

Ca(2+)-dependent annexin self-association on membrane surfaces

Annexin self-association was studied with 90 degrees light scattering and resonance energy transfer between fluorescein (donor) and eosin (acceptor) labeled proteins. Synexin (annexin VII), p32 (annexin IV), and p67 (annexin VI) self-associated in a Ca(2+)-dependent manner in solution. However, this activity was quite labile and, especially for p32 and p67, was not consistently observed. When bound to chromaffin granule membranes, the three proteins consistently self-associated and did so at Ca2+ levels (pCa 5.0-4.5) approximately 10-fold lower than required when in solution. Phospholipid vesicles containing phosphatidylserine and phosphatidylethanolamine (1:1 or 1:3) were less effective at supporting annexin polymerization than were those containing phosphatidylserine and phosphatidylcholine (1:0, 1:1, or 1:3). The annexins bound chromaffin granule membranes in a positively cooperative manner under conditions where annexin self-association was observed, and both phenomena were inhibited by trifluoperazine. Ca(2+)-dependent chromaffin granule membrane aggregation, induced by p32 or synexin, was associated with intermembrane annexin polymerization at Ca2+ levels less than pCa 4, but not at higher Ca2+ concentrations, suggesting that annexin self-association may be necessary for membrane contact at low Ca2+ levels but not at higher Ca2+ levels where the protein may bind two membranes as a monomer.     

CTP:phosphocholine cytidylyltransferase binds anionic phospholipid vesicles in a cross-bridging mode

CTP:phosphocholine cytidylyltransferase (CCT) catalyzes the rate-limiting step in phosphatidylcholine (PC) synthesis, and its activity is regulated by reversible association with membranes, mediated by an amphipathic helical domain M. Here we describe a new feature of the CCTalpha isoform, vesicle tethering. We show, using dynamic light scattering and transmission electron microscopy, that dimers of CCTalpha can cross-bridge separate vesicles to promote vesicle aggregation. The vesicles contained either class I activators (anionic phospholipids) or the less potent class II activators, which favor nonlamellar phase formation. CCT increased the apparent hydrodynamic radius and polydispersity of anionic phospholipid vesicles even at low CCT concentrations corresponding to only one or two dimers per vesicle. Electron micrographs of negatively stained phosphatidylglycerol (PG) vesicles confirmed CCT-mediated vesicle aggregation. CCT conjugated to colloidal gold accumulated on the vesicle surfaces and in areas of vesicle-vesicle contact. PG vesicle aggregation required both the membrane-binding domain and the intact CCT dimer, suggesting binding of CCT to apposed membranes via the two M domains situated on opposite sides of the dimerization domain. In contrast to the effects on anionic phospholipid vesicles, CCT did not induce aggregation of PC vesicles containing the class II lipids, oleic acid, diacylglycerol, or phosphatidylethanolamine. The different behavior of the two lipid classes reflected differences in measured binding affinity, with only strongly binding phospholipid vesicles being susceptible to CCT-induced aggregation. Our findings suggest a new model for CCTalpha domain organization and membrane interaction, and a potential involvement of the enzyme in cellular events that implicate close apposition of membranes.