The Rat Central Nervous System Expresses Alzheimer's Amyloid Precursor Protein APP695, but Not APP677 (L-APP Form)

Abstract: A novel splicing form of βA4 amyloid precursor protein (APP) lacking exon 15, corresponding to 18 residues, was first reported in leukocytes and then in ubiquitous organs. To determine which APP molecules (APP₆₉₅, APP₇₅₁, or APP₇₇₀) either with (N-APP) or without (L-APP; leukocytederived APP) exon 15 were expressed in various organs, we investigated the alternative splicing at exon 15 in the rat brain, kidney, heart, and testis by a PCR analysis of reverse-transcribed RNA and Southern blot analysis. Regarding APP₆₉₅ without exons 7 and 8, L-APP was either seldom or never expressed in the brain, whereas both N- and L-APP were expressed in other organs. On the other hand, regarding APP_751/770 containing exon 7, which codes for the Kunitz-type serine protease inhibitor domain, both N- and L-APP were expressed in all the organs examined, including the brain. These results suggest that a particular alternative regulation system related to exon 15 might be present in only APP₆₉₅ of the brain and influence the proteolytic processing of APP.     

A 127-kDa Protein (UV-DDB) Binds to the Cytoplasmic Domain of the Alzheimer's Amyloid Precursor Protein

Abstract : Alzheimer amyloid precursor protein (APP) is an integral membrane protein with a short cytoplasmic domain of 47 amino acids. It is hoped that identification of proteins that interact with the cytoplasmic domain will provide new insights into the physiological function of APP and, in turn, into the pathogenesis of Alzheimer's disease. To identify proteins that interact with the cytoplasmic domain of APP, we employed affinity chromatography using an immobilized synthetic peptide corresponding to residues 645-694 of APP₆₉₅ and identified a protein of ~130 kDa in rat brain cytosol. Amino acid sequencing of the protein revealed the protein to be a rat homologue of monkey UV-DDB (UV-damaged DNA-binding protein, calculated molecular mass of 127 kDa). UV-DDB/p127 co-immunoprecipitated with APP using an anti-APP antibody from PC12 cell lysates. APP also co-immunoprecipitated with UV-DDB/p127 using an anti-UV-DDB/p127 antibody. These results indicate that UV-DDB/p127, which is present in the cytosolic fraction, forms a complex with APP through its cytoplasmic domain. In vitro binding experiments using a glutathione S -transferase-APP cytoplasmic domain fusion protein and several mutants indicated that the YENPTY motif within the APP cytoplasmic domain, which is important in the internalization of APP and amyloid β protein secretion, may be involved in the interaction between UV-DDB/p127 and APP.     

Intracellular Cyclic AMP Inhibits Constitutive and Phorbol Ester-Stimulated Secretory Cleavage of Amyloid Precursor Protein

Abstract: α-Secretase cleaves the full-length Alzheimer's amyloid precursor protein (APP) within the amyloid β peptide sequence, thus precluding amyloid formation. The resultant soluble truncated APP is constitutively secreted. This nonamyloidogenic processing of APP is increased on stimulation of the phospholipase C/protein kinase C pathway by phorbol esters. Here we used C6 cells transfected with APP₇₅₁ to examine whether the α-secretase cleavage is regulated by the adenylate cyclase signal transduction pathway. Forskolin, an activator of adenylate cyclase, inhibited both the constitutive and phorbol ester-stimulated secretion of nexin II (NXII), the secreted product of the α-secretase cleavage of APP₇₅₁. At 1 µ M , forskolin inhibited secretion of NXII by ∼50% without affecting either the intracellular levels of total APP or the secretion of secretory alkaline phosphatase. In contrast, 1,9-dideoxyforskolin, an inactive analogue of forskolin, did not affect secretion of NXII. These results indicated that forskolin specifically inhibited the α-secretase cleavage of APP₇₅₁. Forskolin treatment increased the intracellular concentration of cyclic AMP (cAMP), suggesting that the forskolin effects on APP cleavage may be mediated by cAMP. In support of this suggestion, both dibutyryl cAMP, a cAMP analogue, and isoproterenol, an activator of adenylate cyclase, also inhibited secretion of NXII. These data indicate that forskolin inhibition of the nonamyloidogenic cleavage of APP is mediated by the second messenger cAMP, which together with the protein kinase C signal transduction pathway modulates the secretory cleavage of APP.     

Mhp493 (P216) is a proteolytically processed, cilium and heparin binding protein of Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae induces respiratory disease in swine by colonizing cilia causing ciliostasis, cilial loss and epithelial cell death. Heparin binds to M. hyopneumoniae cells in a dose-dependent manner and blocks its ability to adhere to porcine cilia. We show here that Mhp493 (P216), a paralogue of the cilium adhesin P97 (Mhp183), is cleaved between amino acids 1040 and 1089 generating surface-accessible, heparin-binding proteins P120 and P85. Antiphosphoserine antibodies recognized P85 in 2-D immunoblotting studies and TiO₂ chromatography of trypsin digests of P85 isolated a single peptide with an m/z of 917.3. A phosphoserine residue in the tryptic peptide ⁹⁰VSELpSFR⁹⁶ (position 94 in P85) was identified by MALDI-MS/MS. Polyhistidine fusion proteins (F1_P216, F2_P216, F3_P216) spanning Mhp493 bound heparin with biologically significant Kd values, and heparin, fucoidan and mucin inhibited this interaction. Latex beads coated with F1_P216, F2_P216 and F3_P216 adhered to and entered porcine kidney epithelial-like (PK15) cell monolayers. Microtitre plate-based assays showed that sequences within P120 and P85 bind to porcine cilia and are recognized by serum antibodies elicited during infection by M. hyopneumoniae . Mhp493 contributes significantly to the surface architecture of M. hyopneumoniae and is the first cilium adhesin to be described that lacks an R1 cilium-binding domain.     

Secreted Form of Amyloid β/A4 Protein Precursor (APP) Binds to Two Distinct APP Binding Sites on Rat B103 Neuron-Like Cells Through Two Different Domains, but Only One Site Is Involved in Neuritotropic Activity

Abstract: Recent studies have identified the Alzheimer's disease amyloid β/A4 protein precursor (APP) as a trophic and/or tropic protein on several types of cells, including fibroblasts, primary culture neurons, PC12 cells, and B103 neuron-like cells. Many trophic proteins bind heparin, and it is believed that the heparin-binding domain is crucial for the trophic activity of these proteins. APP also binds heparin. The current studies were undertaken to examine the hypothesis that the neuritotropic activity of APP requires heparin binding. It was found that APP produced in E. coli bound B103 cells through detergent-extractable molecules. Approximately 50% of the binding sites were heparinase-sensitive, and heparin and heparan sulfate competed for APP binding to these sites. The heparinase-insensitive sites were recognized by a stretch of 17 amino acids of APP (residues 319–335) that contains the neuritotropic activity of APP. A mutant APP with a deletion at this site was capable of binding to the heparinase-sensitive sites, although this molecule was not neuritotropic to B103 neuron-like cells. Therefore, the neuritotropic site and the heparin-binding site are distinct in APP, and the neuritotropic effect of APP is produced through its binding to detergent-extractable and heparinase-insensitive sites.     

Phosphoinositide recognition domains

Domains or modules known to bind phosphoinositides have increased dramatically in number over the past few years, and are found in proteins involved in intracellular trafficking, cellular signaling, and cytoskeletal remodeling. Analysis of lipid binding by these domains and its structural basis has provided significant insight into the mechanism of membrane recruitment by the different cellular phosphoinositides. Domains that target only the rare (3-phosphorylated) phosphoinositides must bind with very high affinity, and with exquisite specificity. This is achieved solely by headgroup interactions in the case of certain pleckstrin homology (PH) domains [which bind PtdIns(3,4,5)P₃ and/or PtdIns(3,4)P₂], but requires an additional membrane-insertion and/or oligomerization component in the case of the PtdIns(3)P-targeting phox homology (PX) and FYVE domains. Domains that target PtdIns(4,5)P₂, which is more abundant by some 25-fold, do not require the same stringent affinity and specificity characteristics, and tend to be more diverse in structure. The mode of phosphoinositide binding by different domains also appears to reflect their distinct functions. For example, pleckstrin homology domains that serve as simple targeting domains recognize only phosphoinositide headgroups. By contrast, certain other domains, notably the epsin ENTH domain, appear to promote bilayer curvature by inserting into the membrane upon binding .     

Helicobacter pylori interacts with heparin and heparin-dependent growth factors

Abstract The pathogenic bacterium Helicobacter pylori , which causes active, chronic type B gastritis and peptic ulcer disease, and increases the risk for development of gastric cancer, could tentatively interfere with growth factors and growth factor receptors of importance for the gastroduodenal mucosa, e.g. heparin-binding FGFs (fibroblast growth factors). H. pylori binds FGF with an extremely strong affinity (3.8 × 10⁻¹² M), and also heparan sulfate and heparin with higher affinity ( K _d 9 × 10⁻⁹ M) than FGFs bind to heparin (10⁻⁸–10⁻⁹ M). FGF receptors are also dependent on heparin for their activation. Heparan sulfate binding proteins (HSBP) are exposed on and shed from the surface of H. pylori , which often are localised close to the epithelial stem cells in the gastroduodenal glands. H. pylori could thus efficiently interfere with growth factors and growth factor receptors, tentatively resulting in disturbance of the delicate balance that control the renewal, maintenance and repair of the gastroduodenal mucosa. This mode of action has previously not been considered, but may constitute part of its pathogenic mechanism. Such a dynamic mode of action of H. pylori may explain the reason for that infected victims may either suffer from gastrointestinal symptoms or lack clinical evidence of disease or discomfort.     

Affinity Purification of Proteoglycans that Bind to the Amyloid Protein Precursor of Alzheimer's Disease

Abstract: The binding of the amyloid protein precursor (APP) to heparan sulfate proteoglycans has been shown to stimulate the neurite-promoting activity of APP. In this study, proteoglycans that bind with high affinity to APP were characterized. Conditioned medium from cultures of postnatal day 3 mouse brain cells was applied to an affinity column containing a peptide homologous to a heparin-binding domain of APP. A fraction 17-fold enriched in proteoglycans was recovered by elution with a salt gradient. APP bound saturably and with high affinity to the affinity-purified proteoglycan fraction. Scatchard analysis of the binding showed that APP bound to high- and low-affinity sites with equilibrium dissociation constants of 1.4 × 10⁻¹¹ and 6.5 × 10⁻¹⁰ M , respectively. APP, in conjunction with the affinity-purified proteoglycan fraction, promoted neurite outgrowth. The affinity-purified proteoglycan fraction contained a heparan sulfate proteoglycan and a chondroitin sulfate proteoglycan. Digestion of the affinity-purified fraction with heparitinase I revealed a core protein of 63–69-kDa molecular mass, whereas digestion with chondroitinase ABC revealed a core protein of 100–110 kDa. The results suggest that expression of specific APP-binding proteoglycans may be an important step in the regulation of the neurite outgrowth-promoting activity of APP.     

Solution conformation and heparin-induced dimerization of the full-length extracellular domain of the human amyloid precursor protein

Proteolytic cleavage of the amyloid precursor protein (APP) by beta and gamma-secretases gives rise to the beta-amyloid peptide, considered to be a causal factor in Alzheimer's disease. Conversely, the soluble extracellular domain of APP (sAPPalpha), released upon its cleavage by alpha-secretase, plays a number of important physiological functions. Several APP fragments have been structurally characterized at atomic resolution, but the structures of intact APP and of full-length sAPPalpha have not been determined. Here, ab initio reconstruction of molecular models from high-resolution solution X-ray scattering (SAXS) data for the two main isoforms of sAPPalpha (sAPPalpha(695) and sAPPalpha(770)) provided models of sufficiently high resolution to identify distinct structural domains of APP. The fragments for which structures are known at atomic resolution were fitted within the solution models of full-length sAPPalpha, allowing localization of important functional sites (i.e. glycosylation, protease inhibitory and heparin-binding sites). Furthermore, combined results from SAXS, analytical ultracentrifugation (AUC) and size-exclusion chromatography (SEC) analysis indicate that both sAPPalpha isoforms are monomeric in solution. On the other hand, SEC, bis-ANS fluorescence, AUC and SAXS measurements showed that sAPPalpha forms a 2:1 complex with heparin. A conformational model for the sAPPalpha:heparin complex was also derived from the SAXS data. Possible implications of such complex formation for the physiological dimerization of APP and biological signaling are discussed in terms of the structural models proposed.     

Structural Characterization of the E2 Domain of APL-1, a Caenorhabditis elegans Homolog of Human Amyloid Precursor Protein, and Its Heparin Binding Site

The amyloid β-peptide deposit found in the brain tissue of patients with Alzheimer disease is derived from a large heparin-binding protein precursor APP. The biological function of APP and its homologs is not precisely known. Here we report the x-ray structure of the E2 domain of APL-1, an APP homolog in Caenorhabditis elegans, and compare it to the human APP structure. We also describe the structure of APL-1 E2 in complex with sucrose octasulfate, a highly negatively charged disaccharide, which reveals an unexpected binding pocket between the two halves of E2. Based on the crystal structure, we are able to map, using site-directed mutagenesis, a surface groove on E2 to which heparin may bind. Our biochemical data also indicate that the affinity of E2 for heparin is influenced by pH: at pH 5, the binding appears to be much stronger than that at neutral pH. This property is likely caused by histidine residues in the vicinity of the mapped heparin binding site and could be important for the proposed adhesive function of APL-1.     

The Role of Vitamin B12 Binding In the Uptake of the Vitamin By Euglena Gracilis

The uptake of [⁵⁷Co]B₁₃ (cyanocobalamin) by Euglena gracilis strain Z (ATCC 12716) occurred in 2 distinct phases-an initial rapid phase followed by a slower secondary phase. This secondary phase appeared after the saturation of the binding sites involved in the initial rapid phase and was energy-dependent and completely inhibited by 2,4-dinitrophenot, KCN and sodium azide. the subcellular localization of labeled cyanocobalamin taken up by the cell was mostly contained in the chloroplast fraction. the time course and the saturation kinetics of B₁₂ uptake by purified chloroplast fraction indicated that this fraction and the intact cell had a similar affinity for the vitamin B₁₂. This suggested that the chloroplasts contained the binding sites for vitamin ₁₂ and might regulate the uptake process in the intact cell. the kinetic properties of the overall ₁₂ uptake mechanism suggested that the initial phase represent the binding of vitamin ₁₂ to the available sites on the chloroplast. the secondary phase may represent the de novo synthesis of new binding sites.     

Two different heparin-binding domains in the triple-helical domain of ColQ,the collagen tail subunit of synaptic acetylcholinesterase

ColQ, the collagen tail subunit of asymmetric acetylcholinesterase, is responsible for anchoring the enzyme at the vertebrate synaptic basal lamina by interacting with heparan sulfate proteoglycans. To get insights about this function, the interaction of ColQ with heparin was analyzed. For this, heparin affinity chromatography of the complete oligomeric enzyme carrying different mutations in ColQ was performed. Results demonstrate that only the two predicted heparin-binding domains present in the collagen domain of ColQ are responsible for heparin interaction. Despite their similarity in basic charge distribution, each heparin-binding domain had different affinity for heparin. This difference is not solely determined by the number or nature of the basic residues conforming each site, but rather depends critically on local structural features of the triple helix, which can be influenced even by distant regions within ColQ. Thus, ColQ possesses two heparin-binding domains with different properties that may have non-redundant functions. We hypothesize that these binding sites coordinate acetylcholinesterase positioning within the organized architecture of the neuromuscular junction basal lamina.     

Characterization of the high affinity heparin binding site of the Alzheimer's disease βA4 amyloid precursor protein (APP) and its enhancement by zinc(II)

The Alzheimer's disease βA4 amyloid precursor protein (APP) has been shown to be involved in a diverse set of biological protein precursor-like proteins (APLP1 and APLP2) belong to a superfamily of proteins that are probably functionally related. In order to characterize the cell adhesion properties of APP the brain specific isoform APP₆₉₅ was purified and used to assess the binding to herparin, a structural and functional analogue of the glycosaminoglycan heparan sulfate. We show that APP binds in a time dependent and saturable manner to heparin. The salt concentration of 620 mM at which APP elutes from heparin Sepharose is greater than physiological. Tha apparent equilibrium constant for dissociation was determined to be 300 pM for APP binding to heparin Sepharose. A high affinity heparin binding site was identified within a region conversed in rodent and human APP, APLP1 and APLP2. This binding site was located between residues 316-337 of APP₆₉₅ which is within the carbohydrate domain of APP. We also demonstrate an interaction between this heparin binding site and the zinc(II) binding site which is conserved in all members of the APP superfamily. We show by using an automated surface plasmon resonance biosensor (BIAcore, Pharmacia) that the affinity for heparin is increased two- to four-fold in the presence of micromolar zinc(II). The identification of zinc-enhanced binding of APP to heparin sulfate side chains of proteoglycans offers a molecular link between zinc(II), as a putative environmental toxin for Alzheimer's disease, and aggregation of amyloid βA4 protein.     

Acetylcholinesterase Is Increased in the Brains of Transgenic Mice Expressing the C-Terminal Fragment (CT100) of the β-Amyloid Protein Precursor of Alzheimer's Disease

Abstract: Acetylcholinesterase (AChE) expression is markedly affected in Alzheimer's disease (AD). AChE activity is lower in most regions of the AD brain, but it is increased within and around amyloid plaques. We have previously shown that AChE expression in P19 cells is increased by the amyloid β protein (Aβ). The aim of this study was to investigate AChE expression using a transgenic mouse model of Aβ overproduction. The β-actin promoter was used to drive expression of a transgene encoding the 100-amino acid C-terminal fragment of the human amyloid precursor protein (APP CT100). Analysis of extracts from transgenic mice revealed that the human sequences of full-length human APP CT100 and Aβ were overexpressed in the brain. Levels of salt-extractable AChE isoforms were increased in the brains of APP CT100 mice. There was also an increase in amphiphilic monomeric form (G^A₁) of AChE in the APP CT100 mice, whereas other isoforms were not changed. An increase in the proportion of G^A₁ AChE was also detected in samples of frontal cortex from AD patients. Analysis of AChE by lectin binding revealed differences in the glycosylation pattern in APP CT100 mice similar to those observed in frontal cortex samples from AD. The results are consistent with the possibility that changes in AChE isoform levels and glycosylation patterns in the AD brain may be a direct consequence of altered APP metabolism.     

In Vitro Phosphorylation of the Cytoplasmic Domain of the Amyloid Precursor Protein by Glycogen Synthase Kinase-3β

Abstract: The two pathological lesions found in the brains of Alzheimer's disease patients, neurofibrillary tangles and neuritic plaques, are likely to be formed through a common pathway. Neurofibrillary tangles are intracellular aggregates of paired helical filaments, the main component of which is hyperphosphorylated forms of the microtubule-associated protein τ. Extracellular neuritic plaques and diffuse and vascular amyloid deposits are aggregates of β-amyloid protein, a 4-kDa protein derived from the amyloid precursor protein (APP). Using conditions in vitro under which two proline-directed protein kinases, glycogen synthase kinase-3β (GSK-3β) and mitogen-activated protein kinase (MAPK), were able to hyperphosphorylate τ, GSK-3β but not MAPK phosphorylated recombinant APP_cyt. The sole site of phosphorylation in APP_cyt by GSK-3β was determined by phosphoamino acid analysis and phosphorylation of APP_cyt mutant peptides to be Thr⁷⁴³ (numbering as for APP₇₇₀). This site was confirmed by endoproteinase Glu-C digestion of APP_cyt and peptide sequencing. The ability of GSK-3β to phosphorylate APP_cyt and τ provides a putative link between the two lesions and indicates a critical role of GSK-3β in the pathogenesis of Alzheimer's disease.     

Binding of heparin/heparan sulfate to fibroblast growth factor receptor 4

Fibroblast growth factors (FGFs) are heparin-binding polypeptides that affect the growth, differentiation, and migration of many cell types. FGFs signal by binding and activating cell surface FGF receptors (FGFRs) with intracellular tyrosine kinase domains. The signaling involves ligand-induced receptor dimerization and autophosphorylation, followed by downstream transfer of the signal. The sulfated glycosaminoglycans heparin and heparan sulfate bind both FGFs and FGFRs and enhance FGF signaling by mediating complex formation between the growth factor and receptor components. Whereas the heparin/heparan sulfate structures involved in FGF binding have been studied in some detail, little information has been available on saccharide structures mediating binding to FGFRs. We have performed structural characterization of heparin/heparan sulfate oligosaccharides with affinity toward FGFR4. The binding of heparin oligosaccharides to FGFR4 increased with increasing fragment length, the minimal binding domains being contained within eight monosaccharide units. The FGFR4-binding saccharide domains contained both 2-O-sulfated iduronic acid and 6-O-sulfated N-sulfoglucosamine residues, as shown by experiments with selectively desulfated heparin, compositional disaccharide analysis, and a novel exoenzyme-based sequence analysis of heparan sulfate oligosaccharides. Structurally distinct heparan sulfate octasaccharides differed in binding to FGFR4. Sequence analysis suggested that the affinity of the interaction depended on the number of 6-O-sulfate groups but not on their precise location.     

Key role of teichoic acids on aflatoxin B1 binding by probiotic bacteria

Aims:  To assess the ability of five probiotic bacteria to bind aflatoxin B₁ and to determine the key role of teichoic acids in the binding mechanism.
Methods and Results:  The strains were incubated in aqueous solutions containing aflatoxin B₁ (AFB₁). The amount of free toxin was quantified by HPLC. Stability of the bacteria–aflatoxin complex was evaluated by repeated washes with buffer. In order to understand the binding process, protoplasts, spheroplasts and cell wall components of two strains were analysed to assess their capacity to bind AFB₁. Additionally, the role of teichoic acids in the AFB₁ binding process was assessed. Lactobacillus reuteri strain NRRL14171 and Lactobacillus casei strain Shirota were the most efficient strains for binding AFB₁. The stability of the AFB₁–bacteria complex appears to be related to the binding ability of a particular strain; AFB₁ binding was also pH-dependent. Our results suggest that teichoic acids could be responsible for this ability.
Conclusions:  Our results provide information concerning AFB₁ binding by previously untested strains, leading to enhanced understanding of the mechanism by which probiotic bacteria bind AFB₁.
Significance and Impact of the Study:  Our results support the suggestion that some probiotic bacteria could prevent absorption of aflatoxin from the gastrointestinal tract.     

Kinetic and proteolytic identification of heat-induced conformational changes in the urea herbicide binding site of isolated Phaseolus vulgaris chloroplast thylakoids

Kinetic parameters of 3-(3, 4-dichlorophenyl)-1, 1-dimethyl urea (DCMU)-induced inhibition of electron transport in chloroplast thylakoids isolated from Phaseolus vulgaris L. cv. Oregon 1604 were determined from analysis of a convergent, parallel electrical circuit. Through this analogue, the apparent affinity of the purported binding site for DCMU (K₁) and the relative amount of DCMU-insensitive electron transport (v^max₁/v_o) were obtained using a reiterative non-linear least squares curve-fitting procedure. Exposure of thylakoids to heat caused a gradual increase in K₁ (or decrease in the affinity of the thylakoid for DCMU) with an apparent activation energy of 134 kJ mol⁻¹. Tryptic susceptibility of a protein region regulating K₁ also decreased gradually with exposure to 45°C, suggesting that the heat-induced increase in K₁ might be due to a protein conformational change. On the other hand, thylakoid exposure to 45°C resulted in a rapid (<5 min) irreversible increase in v^max_I/v_o, which was also the apparent result of a conformational change in a region of the protein which regulates this function. These results are suggestive of the existence of differential thermal sensitivities of proteins within the thylakoids and, perhaps, of different regions within a single membrane protein.     

A Conserved, Lipid-Mediated Sorting Mechanism of Yeast Ist2 and Mammalian STIM Proteins to the Peripheral ER

Sorting of yeast Ist2 to the plasma membrane (PM) or the cortical endoplasmic reticulum (ER) requires a cortical sorting signal (CSS^Ist2) that interacts with lipids including phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂) at the PM. Here, we show that the expression of Ist2 in mammalian cells resulted in a peripheral patch-like localization without any detection of Ist2 at the cell surface. Attached to C-termini of mammalian integral membrane proteins, the CSS^Ist2 targeted these proteins to PM-associated domains of the ER and abolished trafficking via the classical secretory pathway. The interaction of integral membrane proteins with PI(4,5)P₂ at the PM created ER–PM contacts. This process is similar to the regulated coupling of ER domains to the PM via stromal interaction molecule (STIM) proteins during store-operated Ca²⁺ entry (SOCE). The CSS^Ist2 and the C-terminus of the ER-located Ca²⁺ sensor STIM2 were sufficient to bind PI(4,5)P₂ and PI(3,4,5)P₃ at the PM, showing that an evolutionarily conserved mechanism is involved in the sorting of integral membrane proteins to PM-associated domains of the ER. Yeast Ist2 and STIM2 share a common basic and amphipathic signal at their extreme C-termini. STIM1 showed binding preference for liposomes containing PI(4,5)P₂, suggesting a specific contribution of lipids to the recruitment of ER domains to the PM during SOCE.