Dietary fatty acids promote lipid droplet diversity through seipin enrichment in an ER subdomain.

Exogenous metabolites from microbial and dietary origins have profound effects on host metabolism. Here, we report that a sub-population of lipid droplets (LDs), which are conserved organelles for fat storage, is defined by metabolite-modulated targeting of the C. elegans seipin ortholog, SEIP-1. Loss of SEIP-1 function reduces the size of a subset of LDs while over-expression of SEIP-1 has the opposite effect. Ultrastructural analysis reveals SEIP-1 enrichment in an endoplasmic reticulum (ER) subdomain, which co-purifies with LDs. Analyses of C. elegans and bacterial genetic mutants indicate a requirement of polyunsaturated fatty acids (PUFAs) and microbial cyclopropane fatty acids (CFAs) for SEIP-1 enrichment, as confirmed by dietary supplementation experiments. In mammalian cells, heterologously expressed SEIP-1 engages nascent lipid droplets and promotes their subsequent expansion in a conserved manner. Our results suggest that microbial and polyunsaturated fatty acids serve unexpected roles in regulating cellular fat storage by promoting LD diversity.

Monitoring Changes in the Oligomeric State of a Candidate Endoplasmic Reticulum (ER) Ceramide Sensor by Single-molecule Photobleaching.

Single-molecule photobleaching has emerged as a powerful non-invasive approach to extract the stoichiometry of multimeric membrane proteins in their native cellular environment. However, this method has mainly been used to determine the subunit composition of ion channels and receptors at the plasma membrane. Here, we applied single-molecule photobleaching to analyze the oligomeric state of an endoplasmic reticulum (ER) resident candidate ceramide sensor protein, SMSr/SAMD8. Co-immunoprecipitation and chemical cross-linking studies previously revealed that the N-terminal sterile alpha motif (or SAM) domain of SMSr drives self-assembly of the protein into oligomers and that SMSr oligomerization is promoted by curcumin, a drug known to perturb ER ceramide and calcium homeostasis. Application of cell spreading surface-active coating materials in combination with total internal reflection fluorescence (TIRF) microscopy allowed us to image GFP-tagged SMSr proteins as single fluorescent spots in the ER of HeLa cells in which expression of endogenous SMSr was abolished. In line with our biochemical analysis, we find that the number of bleaching steps in SMSr-GFP-positive spots displays a substantial drop after removal of the SAM domain. In contrast, treatment of cells with curcumin increased the number of bleaching steps. Our results document the first successful application of single-molecule photobleaching to resolve drug-induced and domain-dependent changes in the oligomeric state of an ER-resident membrane protein, hence establishing a complementary method to unravel the mechanism by which SMSr controls ceramide levels in the ER.

From zero to six double bonds: phospholipid unsaturation and organelle function.

Cellular phospholipids (PLs) differ by the nature of their polar heads as well as by the length and unsaturation level of their fatty acyl chains. We discuss how the ratio between saturated, monounsaturated, and polyunsaturated PLs impacts on the functions of such organelles as the endoplasmic reticulum, synaptic vesicles, and photoreceptor discs. Recent experiments and simulations suggest that polyunsaturated PLs respond differently to mechanical stress, including membrane bending, than monounsaturated PLs owing to their unique conformational plasticity. These findings suggest a rationale for PL acyl chain remodeling by acyltransferases and a molecular explanation for the importance of a balanced fatty acid diet.

Cytochrome P450 system proteins reside in different regions of the endoplasmic reticulum.

Cytochrome P450 (P450) function is dependent on the ability of these enzymes to successfully interact with their redox partners, NADPH-cytochrome P450 reductase (CPR) and cytochrome b5, in the endoplasmic reticulum (ER). Because the ER is heterogeneous in lipid composition, membrane microdomains with different characteristics are formed. Ordered microdomains are more tightly packed, and enriched in saturated fatty acids, sphingomyelin and cholesterol, whereas disordered regions contain higher levels of unsaturated fatty acids. The goal of the present study was to determine whether the P450 system proteins localize to different regions of the ER. The localization of CYP1A2, CYP2B4 and CYP2E1 within the ER was determined by partial membrane solubilization with Brij 98, centrifugation on a discontinuous sucrose gradient and immune blotting of the gradient fractions to identify ordered and disordered microdomains. CYP1A2 resided almost entirely in the ordered regions of the ER with CPR also localized predominantly to this region. CYP2B4 was equally distributed between the ordered and disordered domains. In contrast, CYP2E1 localized to the disordered membrane regions. Removal of cholesterol (an important constituent of ordered domains) led to the relocation of CYP1A2, CYP2B4 and CPR to the disordered regions. Interestingly, CYP1A1 and CYP1A2 localized to different membrane microdomains, despite their high degree of sequence similarity. These data demonstrate that P450 system enzymes are organized in specific membrane regions, and their localization can be affected by depletion of membrane cholesterol. The differential localization of different P450 in specific membrane regions may provide a novel mechanism for modulating P450 function.

Combined biochemical and cytological analysis of membrane trafficking using lectins.

We have tested the application of high-mannose-binding lectins as analytical reagents to identify N-glycans in the early secretory pathway of HeLa cells during subcellular fractionation and cytochemistry. Post-endoplasmic reticulum (ER) pre-Golgi intermediates were separated from the ER on Nycodenz-sucrose gradients, and the glycan composition of each gradient fraction was profiled using lectin blotting. The fractions containing the post-ER pre-Golgi intermediates are found to contain a subset of N-linked α-mannose glycans that bind the lectins Galanthus nivalis agglutinin (GNA), Pisum sativum agglutinin (PSA), and Lens culinaris agglutinin (LCA) but not lectins binding Golgi-modified glycans. Cytochemical analysis demonstrates that high-mannose-containing glycoproteins are predominantly localized to the ER and the early secretory pathway. Indirect immunofluorescence microscopy revealed that GNA colocalizes with the ER marker protein disulfide isomerase (PDI) and the COPI coat protein ß-COP. In situ competition with concanavalin A (ConA), another high-mannose specific lectin, and subsequent GNA lectin histochemistry refined the localization of N-glyans containing nonreducing mannosyl groups, accentuating the GNA vesicular staining. Using GNA and treatments that perturb ER-Golgi transport, we demonstrate that lectins can be used to detect changes in membrane trafficking pathways histochemically. Overall, we find that conjugated plant lectins are effective tools for combinatory biochemical and cytological analysis of membrane trafficking of glycoproteins.

Poxvirus membrane biogenesis: rupture not disruption.

Enveloped viruses acquire their membrane from the host by budding at, or wrapping by, cellular membranes. Transmission electron microscopy (TEM) images, however, suggested that the prototype member of the poxviridae, vaccinia virus (VACV), may create its membrane 'de novo' with free open ends exposed in the cytosol. Within the frame of the German-wide priority programme we re-addressed the biogenesis and origin of the VACV membrane using electron tomography (ET), cryo-EM and lipid analysis of purified VACV using mass spectrometry (MS). This review discussed how our data led to a model of unconventional membrane biogenesis involving membrane rupture and the generation of a single open membrane from open membrane intermediates. Lipid analyses of purified virus by MS suggest an ER origin with a relatively low cholesterol content compared with whole cells, confirming published data. Unlike previous reports using thin-layer chromatography, no depletion of phosphatidylethanolamine was detected. We did detect, however, an enrichment for phosphatidic acid, diacylglycerol and phosphatidylinositol in the virion. Our data are discussed in the light of other pathogens that may requirecellular membrane rupture during their intracellular life cycle.

High-resolution mapping reveals topologically distinct cellular pools of phosphatidylserine.

Phosphatidylserine (PS) plays a central role in cell signaling and in the biosynthesis of other lipids. To date, however, the subcellular distribution and transmembrane topology of this crucial phospholipid remain ill-defined. We transfected cells with a GFP-tagged C2 domain of lactadherin to detect by light and electron microscopy PS exposed on the cytosolic leaflet of the plasmalemma and organellar membranes. Cytoplasmically exposed PS was found to be clustered on the plasma membrane, and to be associated with caveolae, the trans-Golgi network, and endocytic organelles including intraluminal vesicles of multivesicular endosomes. This labeling pattern was compared with the total cellular distribution of PS as visualized using a novel on-section technique. These complementary methods revealed PS in the interior of the ER, Golgi complex, and mitochondria. These results indicate that PS in the lumenal monolayer of the ER and Golgi complex becomes exposed cytosolically at the trans-Golgi network. Transmembrane flipping of PS may contribute to the exit of cargo from the Golgi complex.

Structure-function relations, physiological roles, and evolution of mammalian ER-resident selenoproteins.

Selenium is an essential trace element in mammals. The major biological form of this micronutrient is the amino acid selenocysteine, which is present in the active sites of selenoenzymes. Seven of 25 mammalian selenoproteins have been identified as residents of the endoplasmic reticulum, including the 15-kDa selenoprotein, type 2 iodothyronine deiodinase and selenoproteins K, M, N, S, and T. Most of these proteins are poorly characterized. However, recent studies implicate some of them in quality control of protein folding in the ER, retrotranslocation of misfolded proteins from the ER to the cytosol, metabolism of the thyroid hormone, and regulation of calcium homeostasis. In addition, some of these proteins are involved in regulation of glucose metabolism and inflammation. This review discusses evolution and structure-function relations of the ER-resident selenoproteins and summarizes recent findings on these proteins, which reveal the emerging important role of selenium and selenoproteins in ER function.

Content of endoplasmic reticulum and Golgi complex membranes positively correlates with the proliferative status of brain cells.

Although the molecular and cellular basis of particular events that lead to the biogenesis of membranes in eukaryotic cells has been described in detail, understanding of the intrinsic complexity of the pleiotropic response by which a cell adjusts the overall activity of its endomembrane system to accomplish these requirements is limited. Here we carried out an immunocytochemical and biochemical examination of the content and quality of the endoplasmic reticulum (ER) and Golgi apparatus membranes in two in vivo situations characterized by a phase of active cell proliferation followed by a phase of declination in proliferation (rat brain tissue at early and late developmental stages) or by permanent active proliferation (gliomas and their most malignant manifestation, glioblastomas multiforme). It was found that, in highly proliferative phases of brain development (early embryo brain cells), the content of ER and Golgi apparatus membranes, measured as total lipid phosphorous content, is higher than in adult brain cells. In addition, the concentration of protein markers of ER and Golgi is also higher in early embryo brain cells and in human glioblastoma multiforme cells than in adult rat brain or in nonpathological human brain cells. Results suggest that the amount of endomembranes and the concentration of constituent functional proteins diminish as cells decline in their proliferative activity.

Enhancement of antibody responses to Bacillus anthracis protective antigen domain IV by use of calreticulin as a chimeric molecular adjuvant.

The generation of protective humoral immune responses against the receptor-binding domain (domain IV) of protective antigen [PA(dIV)] of Bacillus anthracis represents a plausible approach against anthrax toxin. In the current study, we have developed a naked DNA vaccine encoding calreticulin (CRT) linked to PA(dIV) of Bacillus anthracis [CRT/PA(dIV)]. We transfected a human embryonic kidney cell line (HEK 293) with CRT/PA(dIV) DNA and performed Western blotting and confocal microscopy analysis. We found that linkage of CRT to PA(dIV) targets PA(dIV) to the endoplasmic reticulum, resulting in secretion of the chimeric CRT/PA(dIV) protein. We then evaluated the ability of CRT/PA(dIV) DNA to generate PA(dIV)-specific antibody responses and protective immunity against lethal anthrax toxin (PA plus lethal factor) challenge. We found that mice immunized with CRT/PA(dIV) DNA were capable of rapidly inducing significantly higher PA(dIV)-specific antibody responses than mice immunized with PA(dIV) DNA alone. Furthermore, we observed that this enhanced antibody response generated by CRT/PA(dIV) DNA was CD4 dependent, since CD4 knockout mice demonstrated a significant reduction in antibody responses. In addition, analysis of the titers and avidity maturation of the induced PA-specific antibodies revealed that vaccination with CRT/PA(dIV) DNA vaccine accelerated the avidity maturation of antibodies to PA(dIV) compared to vaccination with PA(dIV) DNA. Importantly, the enhanced antibody responses correlated to protective immunity against lethal anthrax toxin challenge. Thus, DNA vaccines encoding CRT linked to PA(dIV) may dramatically enhance PA-specific protective antibody responses. Our results have significant clinical applications for biodefense against anthrax toxin.

C-terminal hydrophobic region leads PRSV P3 protein to endoplasmic reticulum.

P3 protein is one of the least characterized potyviral proteins in both functions and sub-cellular localization. In this study, we examined the sub-cellular localization of PRSV P3 and its intermediate, P3-6K1 by expressing their GFP fusion proteins in onion epidermal cells. Our results showed that both P3- and P3-6K1 GFP fusion proteins were localized at the endoplasmic reticulum. Deletion analysis indicated that C-terminal of P3 protein contained localization signal, and a 19 amino acids hydrophobic domain from this region was able to target the GFP fusion protein to endoplasmic reticulum. C-terminal of P3 proteins has been suggested to be involved in both viability and pathogenicity of the potyvirus. Therefore, our result suggests that localization of P3 protein at endoplasmic reticulum is essential for functionality of P3 protein.

Inhibition of recombinant N-type Ca(V) channels by the gamma 2 subunit involves unfolded protein response (UPR)-dependent and UPR-independent mechanisms.

Auxiliary gamma subunits are an important component of high-voltage-activated calcium (Ca(V)) channels, but their precise regulatory role remains to be determined. In the current report, we have used complementary approaches including molecular biology and electrophysiology to investigate the influence of the gamma subunits on neuronal Ca(V) channel activity and expression. We found that coexpression of gamma2 or gamma3 subunits drastically inhibited macroscopic currents through recombinant N-type channels (Ca(V)2.2/beta3/alpha2delta) in HEK-293 cells. Using inhibitors of internalization, we found that removal of functional channels from the plasma membrane is an improbable mechanism of current regulation by gamma. Instead, changes in current amplitude could be attributed to two distinct mechanisms. First, gamma subunit expression altered the voltage dependence of channel activity. Second, gamma subunit expression reduced N-type channel synthesis via activation of the endoplasmic reticulum unfolded protein response. Together, our findings (1) corroborate that neuronal gamma subunits significantly downregulate Ca(V)2.2 channel activity, (2) uncover a role for the gamma2 subunit in Ca(V)2.2 channel expression through early components of the biosynthetic pathway, and (3) suggest that, under certain conditions, channel protein misfolding could be induced by interactions with the gamma subunits, supporting the notion that Ca(V) channels constitute a class of difficult-to-fold proteins.

Proteomic inventory of "anchorless" proteins on the colon adenocarcinoma cell surface.

Surface proteins play important pathophysiological roles in health and disease, and accumulating proteomics-based studies suggest that several "non-membrane" proteins are sorted to the cell surface by unconventional mechanisms. Importantly, these proteins may comprise attractive therapeutic targets and novel disease markers for colon cancer. To perform a proteomics-based inventory of these so-called "anchorless" surface proteins, intact colon adenocarcinoma SW480 cells were labeled with membrane-impermeable biotin after which only soluble biotinylated proteins were isolated and identified by nanoLC-MS/MS. Computer-assisted analysis predicted that only 9 of the 97 identified surface-exposed proteins have predicted secretory signal peptides, whereas 2 other proteins have a putative transmembrane segment. Of the 9 proteins with putative signal peptides, 1 was predicted to be retained at the cell surface by a GPI-anchor, whereas 5 other proteins contained an ER-retention motif (KDEL) that should prevent them from being sorted to the cell surface. The remaining 86 soluble "surface" proteins lack known export signals and the possibility that these proteins are candidate substrates of non-classical transporters or exported by unconventional mechanisms is discussed. Alternatively, the large number of "intracellular" and ER-resident proteins may imply that biotinylation approaches are not only specific for surface proteins, but also biased against a certain subset of non-surface proteins. This underscores the importance of post-proteomic verification of proteomics-based inventories on surface-exposed proteins, which eventually should reveal to which extent non-classical export and retention mechanisms contribute to the sorting of "anchorless" proteins to the surface of colon tumor cells.

Cloning and characterization of an ovine intracellular seven transmembrane receptor for progesterone that mediates calcium mobilization.

Classically, progesterone has been thought to act only through the well-known genomic pathway involving hormone binding to nuclear receptors (nPR) and subsequent modulation of gene expression. However, there is increasing evidence for rapid, nongenomic effects of progesterone in a variety of tissues in mammals, and it seems likely that a membrane PR (mPR) is causing these events. The objective of this study was to isolate and characterize an ovine mPR distinct from the nPR. A cDNA clone was isolated from ovine genomic DNA by PCR. The ovine mPR is a 350-amino acid protein that, based on computer hydrophobicity analysis, possesses seven transmembrane domains and is distinct from the nPR. Message for the ovine mPR was detected in hypothalamus, pituitary, uterus, ovary, and corpus luteum by RT-PCR. In CHO cells that overexpressed a mPR-green fluorescent protein fusion protein, the ovine mPR was localized to the endoplasmic reticulum and not the plasma membrane. Specific binding of 3H-progesterone to membrane fractions was demonstrated in CHO cells that expressed the ovine mPR but not in nontransfected cells. Furthermore, progesterone and 17 alpha-hydroxy-progesterone stimulated intracellular Ca2+ mobilization in CHO cells that expressed ovine mPR in Ca2+-free medium (P < 0.05) but not in CHO cells transfected with empty vector. This rise in intracellular Ca2+ is believed to be from the endoplasmic reticulum as intracellular Ca2+ mobilization is absent when mPR transfected cells are first treated with thapsigargin to deplete Ca2+ stores from the endoplasmic reticulum. Isolation, identification, tissue distribution, cellular localization, steroid binding, and a functional response for a unique intracellular mPR in the sheep are presented.

Expression levels of the putative zinc transporter LIV-1 are associated with a better outcome of breast cancer patients.

We investigated the expression pattern of the breast cancer associated gene LIV-1 on mRNA and protein level in 111 human breast cancer patients by in situ hybridization as well as immunohistochemistry and focused on the unknown potential of LIV-1 expression levels as a prognostic marker. To our knowledge, this is the first study on endogenous LIV-1 protein expression. Results of our study indicate that LIV-1 mRNA and protein expression levels are only weakly correlated, suggesting posttranscriptional regulatory mechanisms. Furthermore, LIV-1 mRNA quantity in combination with a positive ER status seem to represent a better marker than the progesterone receptor status according to the prognostic significance for relapse free survival (RFS). A negative correlation of LIV-1 protein levels with tumor size, grade and stage reflects an association of LIV-1 protein expression with less aggressive tumors. High LIV-1 protein expression seems to be associated with a longer relapse free and overall survival in breast cancer patients with invasive ductal carcinoma. This association, however, seems to be dependent from other prognostic markers. Our data suggest that LIV-1 is a promising candidate for a novel marker for breast cancer patients with better outcome. Furthermore, our study presents a revised cDNA sequence of LIV-1 and demonstrates the localization of endogenous LIV-1 in the endoplasmic reticulum.

The distribution of membrane-bound 14-3-3 proteins in organelle-enriched fractions of germinating lily pollen.

Proteins of the 14-3-3 family show a broad range of activities in plants, depending on their localisation in different cellular compartments. Different organelle membranes of pollen grains and pollen tubes of Lilium longiflorum Thunb. were separated simultaneously using optimised discontinuous sucrose density centrifugation. The obtained organelle-enriched fractions were identified as vacuolar, Golgi, endoplasmic reticulum and plasma membranes, according to their marker enzyme activities, and were assayed for membrane-bound 14-3-3 proteins by immunodetection. 14-3-3 proteins were detected in the cytoplasm as well as in all obtained organelle fractions but were also released into the extracellular medium. In pollen grains, much more plasma membrane-bound 14-3-3 proteins were detected than in the PM-enriched fraction of pollen tubes, whereas the level of Golgi- and ER-associated 14-3-3 proteins was similar in pollen grains and tubes. This shift in the localisation of membrane-associated 14-3-3 proteins is probably correlated with a change in the major function of 14-3-3 proteins, e.g., perhaps changing from initiating pollen grain germination by activation of the PM H +-ATPase to recruitment of membrane proteins via the secretory pathway during tube elongation.

Structure of UDP-glucuronosyltransferases in membranes.

This chapter presents the most recent experimental approaches to the investigation of UDP-glucuronosyltransferase (UGTs) in membranes. The first topic described is the subcellular localization of UGTs with special emphasis on the association of these proteins with the endoplasmic reticulum (ER). Experimental methods include subfractionation of tissue for microsome preparation, evaluation of the purity of the membrane fraction obtained, and measurement of UGT activity in the presence of detergents. Next, the recently demonstrated formation of UGT homo- and heterodimer formation and its functional relevance is discussed and the appropriate methods used to characterize such interactions are given (radiation inactivation, size exclusion chromatography, immunopurification, cross-linking, two-hybrid system). The structural determinants of UGTs in relation to membrane association, residency, and enzymatic activity are the next topic, supplemented by a description of the appropriate methods, including the design and expression of chimeric proteins, membrane insertion, and subcellular localization by immunofluorescence. Also presented is new information on the structure and function of UGTs obtained by molecular modeling, bioinformatics (sequence alignment), and comparison with selected crystallized glycosyltransferases. Finally, we discuss the important, and still not fully developed, issue of UGT active site architecture and organization within the ER. This is addressed from two perspectives: (1) chemical modification of UGT active sites by amino acid-specific probes and (2) photoaffinity labeling of UGTs. The detailed synthesis of a photoaffinity probe for an aglycon-binding site is provided and the use of this probe and direct photoaffinity labeling with retinoids is discussed. The application of proteomics techniques, including proteolytic digestion and protein sequencing by liquid chromatography/tandem mass spectrometry and matrix-assisted laser desorption ionization/time of flight, to the identification of crucial amino acids of the active sites, and subsequent site-directed mutagenesis of identified amino acids, is discussed in detail.

[The effect of alpha-tocopherol in a wide range of concentrations on the structural features of the lipid bilayer regions of endoplasmic reticulum membranes in the mouse liver cells in vitro]. / Vliianie alpha-tokoferola v shirokom spektre kontsentratsii na strukturnye kharakteristiki membran éndoplazmaticheskogo retikuluma kletok pecheni myshei in vitro.

The aim of the present work was to study by ESR-spin-probe technique the effect of the natural antioxidant alpha-tocopherol (alpha-tp) in vitro on the structural parameters (microviscosity, order parameter) of endoplasmic reticulum membranes of the mice liver cells starting from the concentration of 10(-3) mol/l and down to the dilution of 10(-25) mol/l. The stable nitroxyl radicals 16-doxylstearic acid (with the deep localization depth of 20 A) and 5-doxylstearic acid (with the surface localization depth of 8 A) were used as spin probes. It has been shown that alpha-tp causes the increase in microviscosity of the deep lipid bilayer regions and in rigidity of the surface ones at the certain concentrations. The concentration curves obtained have the polymodal shapes being typical of the effects of substances at ultra low doses. Using 16-doxylstearic acid it is detected the increase in the number of thermoinduced structural transitions and appearance of much more high-cooperative ones, as well as the increase in their effective activation energy with the rise of temperature at the supplement of different alpha-tp doses.

Dual subcellular distribution of cytochrome b5 in plant, cauliflower, cells.

Subfractionation studies showed that cytochrome b(5) (cyt b5), which has been considered to be a typical ER protein, was localized in both the endoplasmic reticulum membrane (ER) and the outer membrane of mitochondria in cauliflower (Brassica olracea) cells and was a component of antimycin A-insensitive NADH-cytochrome c reductase system in both membranes. When cDNA for cauliflower cyt b5 was introduced into mammalian (COS-7) and yeast cells as well as into onion cells, the expressed cytochrome was localized both in the ER and mitochondria in those cells. On the other hand, rat and yeast cyt b5s were specifically localized in the ER membranes even in the onion cells. Mutation experiments showed that cauliflower cyt b5 carries information that targets it to the ER and mitochondria within the carboxy-terminal 10 amino acids, as in the case of rat and yeast cyt b5s, and that replacement of basic amino acids in this region of cauliflower cyt b5 with neutral or acidic ones resulted in its distribution only in the ER. Together with the established findings of the importance of basic amino acids in mitochondrial targeting signals, these results suggest that charged amino acids in the carboxy-terminal portion of cyt b5 determine its location in the cell, and that the same mechanism of signal recognition and of protein transport to organelles works in mammalian, plant, and yeast cells.

IRBIT, a novel inositol 1,4,5-trisphosphate (IP3) receptor-binding protein, is released from the IP3 receptor upon IP3 binding to the receptor.

The inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) are IP(3)-gated Ca(2+) channels on intracellular Ca(2+) stores. Herein, we report a novel protein, termed IRBIT (IP(3)R binding protein released with inositol 1,4,5-trisphosphate), which interacts with type 1 IP(3)R (IP(3)R1) and was released upon IP(3) binding to IP(3)R1. IRBIT was purified from a high salt extract of crude rat brain microsomes with IP(3) elution using an affinity column with the huge immobilized N-terminal cytoplasmic region of IP(3)R1 (residues 1-2217). IRBIT, consisting of 530 amino acids, has a domain homologous to S-adenosylhomocysteine hydrolase in the C-terminal and in the N-terminal, a 104 amino acid appendage containing multiple potential phosphorylation sites. In vitro binding experiments showed the N-terminal region of IRBIT to be essential for interaction, and the IRBIT binding region of IP(3)R1 was mapped to the IP(3) binding core. IP(3) dissociated IRBIT from IP(3)R1 with an EC(50) of approximately 0.5 microm, i.e. it was 50 times more potent than other inositol polyphosphates. Moreover, alkaline phosphatase treatment abolished the interaction, suggesting that the interaction was dualistically regulated by IP(3) and phosphorylation. Immunohistochemical studies and co-immunoprecipitation assays showed the relevance of the interaction in a physiological context. These results suggest that IRBIT is released from activated IP(3)R, raising the possibility that IRBIT acts as a signaling molecule downstream from IP(3)R.