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Results for “Biotin proximity labeling”

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PubMed2000

Rapid colchicine competition-binding scintillation proximity assay using biotin-labeled tubulin.

We have developed a rapid [3H]colchicine competition-binding scintillation proximity assay (SPA) to evaluate antimitotic compounds that bind to the colchicine-binding site on tubulin. The premise of our assay is that compounds will compete with radiolabeled colchicine for the tubulin-binding domain. Biotin-labeled tubulin is incubated first with unlabeled compound and radiolabeled ligand. Streptavidin-labeled SPA beads are added, and the radiolabel associated with tubulin is directly counted with no separation steps. Under our experimental conditions, the dissociation constant of binding (Kd) for colchicine to tubulin was determined to be 1.4 microM, which was consistent with previously reported values. Assay validation was performed by competitively inhibiting [3H]colchicine binding to tubulin with known microtubule inhibitors and comparing their inhibition constants (Ki). Our SPA bead method is a powerful tool since it overcomes the disadvantage of traditional filtration techniques, as there are no separation steps. It is extremely easy to set up, multiple samples can be assayed and supply and labor costs are reduced because of the minimal volume and test reagents used.

Aminophenols↗

PubMed2026

ChromID: A Protocol for Mapping Protein Chromatin Interactions in Living Cells.

Chromatin modifications regulate genome function by recruiting proteins that control transcription, genome organization, and DNA repair. Identifying the proteins associated with specific chromatin modifications is therefore essential for understanding how these regulatory processes operate. Traditional approaches, including chromatin immunoprecipitation and affinity purification coupled to mass spectrometry, have uncovered many chromatin-associated proteins. However, they often rely on crosslinking and chromatin fragmentation, which can disrupt native chromatin architecture and limit the detection of transient interactions. Here, we describe a proximity-labeling protocol for identifying the chromatin-dependent protein interactome associated with specific chromatin marks, termed ChromID. ChromID uses engineered chromatin readers (eCRs) fused to a promiscuous biotin ligase, which labels proteins in the immediate vicinity of the targeted chromatin mark. The protocol includes in vivo biotin labeling, nuclear extract preparation, streptavidin-based enrichment, and tryptic digestion for downstream LC-MS/MS analysis. The protocol has been validated across multiple cell types and chromatin contexts and can be extended to other chromatin-associated proteins, providing a versatile approach to profile chromatin-associated proteomes within their native cellular environment. Key features • Maps proteins associated with different chromatin modifications in living cells using engineered chromatin readers fused to TurboID, BASU, or other promiscuous biotin ligases. • Preserves native chromatin organization and captures transient chromatin-associated interactions that are often lost during conventional affinity purification workflows. • Validated across multiple chromatin contexts, including histone modifications, DNA methylation, transcription factors, RNA polymerase II, and DNA damage-associated chromatin states. • Applicable to diverse cell types and organisms and adaptable to other chromatin-associated proteins, including transcription factors and chromatin regulators.

Biotin proximity labeling↗

PubMed2025

Mapping the FOXA1 Interactome in ER+ Breast Cancer Cells Using Proximity Labeling Reveals Novel Interactions with the Orphan Nuclear Receptor NR2C2.

UNLABELLED: FOXA1 is a pioneer transcription factor essential for chromatin accessibility and transcriptional regulation in hormone-driven cancers. In breast cancer, FOXA1 plays a central role in facilitating nuclear receptor binding, reprogramming enhancer landscapes, and promoting transcriptional changes associated with therapy resistance. Whereas FOXA1's function has been primarily studied in the context of estrogen receptor-α (ER), its broader protein interaction network remains incompletely defined. In this study, we systematically map FOXA1-interacting proteins in ER-positive breast cancer cells using proximity-dependent biotin labeling (miniTurbo) combined with quantitative LC-MS/MS proteomics. We engineered MCF-7 cell lines stably expressing miniTurbo-tagged FOXA1 at either the N-terminus or C-terminus to ensure comprehensive coverage of interaction interfaces. This approach recovered known FOXA1 partners, including AR, MLL3, YAP1, and GATA3, and identified 157 previously unreported FOXA1 interactors. Notably, 42 of these novel partners, including NR2C2, were significantly associated with poor relapse-free survival in patients with ER-positive breast cancer. To demonstrate the utility of this resource, we characterized the FOXA1-NR2C2 interaction in depth. Integrating chromatin immunoprecipitation sequencing and RNA sequencing, we show that FOXA1 and NR2C2 co-occupy a subset of genomic regions and drive co-regulated transcriptional programs involved in tumor progression. Our study reveals an expanded FOXA1 interactome and new insights into its functional network in breast cancer, providing candidate proteins for further exploration as biomarkers or therapeutic targets. IMPLICATIONS: These findings expand the FOXA1 interactome in breast cancer and uncover new candidate proteins with potential as biomarkers and therapeutic targets in hormone-driven tumors.

Humans↗

PubMed2025

Influenza A virus induces PI4P production at the endoplasmic reticulum in an ATG16L1-dependent manner to promote the egress of viral ribonucleoproteins.

The genomic RNAs of influenza A viruses (IAVs) are replicated in the nucleus of infected cells in the form of viral ribonucleoproteins (vRNPs) before being exported to the cytoplasm. The small GTPase RAB11A is involved in the transport of vRNPs to the sites of viral assembly at the plasma membrane, but the molecular mechanisms involved remain largely unknown. Here we show that IAV infection remodels the architecture of the endoplasmic reticulum (ER) sheets, where vRNPs tend to accumulate in the absence of RAB11A. To decipher the interplay between RAB11A, vRNPs, and the ER, we investigated viral-induced perturbations of RAB11A proximity interactome. To this end, we generated cells stably expressing a TurboID-RAB11A fusion protein and performed biotin-based proximity labeling upon viral infection. We found that cellular regulators of phophatidylinositol-4-phosphate (PI4P) homeostasis, including the autophagic and stress response protein ATG16L1, are significantly enriched at the vicinity of RAB11A in infected cells. Infection induces an increase in cellular PI4P levels in an ATG16L1-dependent manner, while ATG16L1 relocalizes to ER membranes upon infection. Depletion of ATG16L1 decreases the co-distribution of vRNPs with PI4P punctae on ER membranes, and reduces the accumulation of vRNPs at the plasma membrane as well as the production of IAV infectious particles. Our data extend to IAVs the notion that viruses can modulate the metabolism and localization of phosphoinositides to control host membrane dynamics and point to the ER as an essential platform for vRNP transport. They provide evidence for a pivotal role of ATG16L1 in regulating the identity of endomembranes and coordinating RAB11A and PI4P-enriched membranes to ensure delivery of vRNPs to the plasma membrane.

Endoplasmic Reticulum↗

PubMed2001

Miniaturization of a hepatitis C virus RNA polymerase assay using a -102 degrees C cooled CCD camera-based imaging system.

Innovations in detection technologies have allowed us to develop a novel assay in 1536-well plate format and assess the advantages of screen miniaturization compared with conventional high-throughput compound screening in 96- or 384-well plates. An HCV RNA polymerase assay has been miniaturized in 1536-well plates by using a new detection technology known as LEADseeker homogeneous imaging system. It uses a -102 degrees C cooled charge-coupled device (CCD) camera and newly designed scintillation proximity microparticles. The miniaturized assay used europium-doped streptavidin-coated yttrium oxide (YO(x)) or polystyrene (PS) microspheres to capture biotin-labeled [(3)H]RNA product transcripts. Beads in proximity to the radioisotope convert the emitted beta(-) particles into photons having wavelengths in the red region of the visible spectrum, optimal for detection by the CCD camera. Because the camera collects light from all wells of the plate simultaneously, 1536-well plates are imaged as rapidly as 384-well plates, on the order of 10 min per plate. The assay has a signal to background of approximately 20-fold, satisfactory for high-throughput robotics screening. The enzyme kinetics and potency of a known inhibitor were similar to those obtained from the conventional assay using scintillation proximity assay (SPA) beads and a scintillation plate counter. Furthermore, the newly developed microbeads (emitting at 610 to 620 nm) are less prone to quenching effects caused by yellow-colored compounds, than conventional SPA beads or scintillation fluid (emitting at 400 to 480 nm region). Thus, the LEADseeker imaging system is a useful new tool for miniaturization of assays for high-throughput screening.

Cold Temperature↗

PubMed1999

Role of Fas (CD95) in tubulointerstitial disease induced by unilateral ureteric obstruction.

Murine renal tubular epithelial cells and interstitial fibroblasts may express both Fas (CD95) death receptor and Fas ligand and are vulnerable to Fas-mediated death in vitro. We therefore hypothesized that an absence of renal Fas may protect resident cells from undergoing apoptosis. We performed unilateral ureteric ligation [producing unilateral ureteral obstruction (UUO)] in 6-wk-old normal control mice and C57Bl6/lpr mice, which express a nonfunctional Fas receptor. Obstructed kidneys were removed at days 3, 7, and 14 (n = 6 per group). Tubular cell apoptosis at day 7 was significantly reduced in lpr mice [21.8 +/- 5.8 vs. 45.7 +/- 7.6 cells/10 high-power fields (hpf), P < 0.02]. Importantly, there was no difference in tubular cell proliferation between normal and lpr mice at any time point studied. Interestingly, double labeling with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and the proximal tubule-specific antibody Fx1A indicated that the absence of Fas reduced distal but not proximal tubular death at day 7. In addition, there was no difference in interstitial cell apoptosis or proliferation, suggesting that Fas does not play a significant role in interstitial cell death. Importantly, inflammatory macrophage infiltration and ultimate collagen I deposition was unchanged in lpr mice. In conclusion, the absence of functional cell surface Fas in UUO provides distal tubular cells with partial protection from apoptosis but does not affect interstitial cell fate in this model of tubulointerstitial injury.

Animals↗

PubMed2025

CRISPR-mediated proximity labeling unveils ABHD14B as a host factor to regulate HBV cccDNA transcriptional activity.

BACKGROUND: The long-term goal of chronic hepatitis B research is a functional cure (HBsAg seroclearance). Although currently used nucleos(t)ide analogs can efficiently inhibit viral replication, they do not reduce viral RNAs or proteins produced from covalently closed circular DNA (cccDNA), and rarely achieve a functional cure. To overcome this situation, revealing the mode of the existence of cccDNA is required, including identifying the interreacting proteins with cccDNA. Here, we aimed to identify novel proteins that interact with cccDNA. METHODS: Using an in vitro HBV infection model and a sequence-specific proximity labelling method consisting of dead Cas9 and biotin identification (BioID2), we comprehensively determined proteins that possibly interact with cccDNA. After identifying the candidate proteins, the HBV RNA transcription levels were examined by knocking out the associated genes. RESULTS: We identified ABHD14B as a protein that interacts with cccDNA and inhibits HBV RNA transcription from cccDNA. ABHD14B decreases the acetylation levels of histone proteins that control the transcription levels of HBV RNA in cccDNA. Moreover, ABHD14B interacts with TFII-I, which binds directly to cccDNA in a sequence-dependent manner. These results suggest that the host protein, ABHD14B, is recruited to cccDNA via the TFII-I protein, inhibiting HBV RNA transcription from cccDNA by deacetylating cccDNA histones. CONCLUSIONS: ABHD14B was newly identified as a suppressor of HBV RNA transcription from cccDNA, which may improve our understanding of the mode of existence of cccDNA, providing a basis for development of a functional cure.

Humans↗

PubMed2003

Interacting domains of the HN and F proteins of newcastle disease virus.

The activation of most paramyxovirus fusion proteins (F proteins) requires not only cleavage of F(0) to F(1) and F(2) but also coexpression of the homologous attachment protein, hemagglutinin-neuraminidase (HN) or hemagglutinin (H). The type specificity requirement for HN or H protein coexpression strongly suggests that an interaction between HN and F proteins is required for fusion, and studies of chimeric HN proteins have implicated the membrane-proximal ectodomain in this interaction. Using biotin-labeled peptides with sequences of the Newcastle disease virus (NDV) F protein heptad repeat 2 (HR2) domain, we detected a specific interaction with amino acids 124 to 152 from the NDV HN protein. Biotin-labeled HR2 peptides bound to glutathione S-transferase (GST) fusion proteins containing these HN protein sequences but not to GST or to GST containing HN protein sequences corresponding to amino acids 49 to 118. To verify the functional significance of the interaction, two point mutations in the HN protein gene, I133L and L140A, were made individually by site-specific mutagenesis to produce two mutant proteins. These mutations inhibited the fusion promotion activities of the proteins without significantly affecting their surface expression, attachment activities, or neuraminidase activities. Furthermore, these changes in the sequence of amino acids 124 to 152 in the GST-HN fusion protein that bound HR2 peptides affected the binding of the peptides. These results are consistent with the hypothesis that HN protein binds to the F protein HR2 domain, an interaction important for the fusion promotion activity of the HN protein.

Animals↗

PubMed2004

Biotin is endogenously expressed in select regions of the rat central nervous system.

The vitamin biotin is an endogenous molecule that acts as an important cofactor for several carboxylases in the citric acid cycle. Disorders of biotin metabolism produce neurological symptoms that range from ataxia to sensory loss, suggesting the presence of biotin in specific functional systems of the CNS. Although biotin has been described in some cells of nonmammalian nervous systems, the distribution of biotin in mammalian CNS is virtually unknown. We report the presence of biotin in select regions of rat CNS, as revealed with a monoclonal antibody directed against biotin and with avidin- and streptavidin-conjugated labels. Detectable levels of biotin were primarily found caudal to the diencephalon, with greatest expression in the cerebellar motor system and several brainstem auditory nuclei. Biotin was found as a somatic label in cerebellar Purkinje cells, in cell bodies and proximal dendrites of cerebellar deep nuclear neurons, and in red nuclear neurons. Biotin was detected in cells of the spiral ganglion, somata and proximal dendrites of cells in the cochlear nuclei, superior olivary nuclei, medial nucleus of the trapezoid body, and nucleus of the lateral lemniscus. Biotin was further found in pontine nuclei and fiber tracts, the substantia nigra pars reticulata, lateral mammillary nucleus, and a small number of hippocampal interneurons. Biotin was detected in glial cells of major tract systems throughout the brain but was most prominent in tracts of the hindbrain. Biotin is thus expressed in select regions of rat CNS with a distribution that correlates to the known clinical sequelae associated with biotin deficiencies.

Animals↗

PubMed2025

Proximity Labeling of Cell Surface Proteins via Cell Surface Remodeling.

Within the complex interplay of proteins, lipids and carbohydrates at the cell surface is the surfaceome, a dense layer of proteins and their posttranslationally modified counterparts that serves as a hub for cell signaling and signal transduction. The surfaceome plays crucial roles in mediating interactions between cells and the extracellular environment, which combined with their availability at the cell surface make it an attractive therapeutic target. Despite its importance, the development of technologies to selectively target cell surface proteins for empirical identification is challenged by their structural complexity. Here, we describe a proximity labeling-based technique to covalently label proteins at the cell surface with a biotin handle, enabling downstream streptavidin-based enrichment and manipulation in a variety of modalities, including fluorescence imaging, western blotting, and mass spectrometry-based proteomics.

Membrane Proteins↗

PubMed2026

Comprehensive proximity proteomics expand the known interactome of the oncoprotein β-catenin.

The oncoprotein β-catenin has critical roles in cell adhesion and cell signalling. β-catenin affects human physiology and pathology through numerous interaction partners, of which many have been discovered by standard affinity purification-based proteomics. However, the interaction landscape of β-catenin remains incompletely understood, highlighting a need for new experimental approaches for the exploration of β-catenin biology. Proximity proteomics, which facilitate the discovery of molecules vicinal to proteins-of-interest by mass spectrometry, have recently emerged as a powerful complementary tool for the study of protein-protein interactions, but have not been applied to β-catenin so far. We investigated the interactome of β-catenin in model cell lines by proximity proteomics using expression constructs with the biotin ligases BioID and TurboID. Mass spectrometry analyses following biotin labelling identified numerous candidate interactors of β-catenin, including many that had not been observed in earlier studies using standard proteomics. Enrichment analyses suggested that proximity proteomics capture proteins associated with the known biological functions of β-catenin, including cell adhesion, Wnt/β-catenin signalling, and transcription regulation. The molecular tools and data generated in this study provide new insights into β-catenin biology and highlight potential new regulators of β-catenin function that warrant further exploration.

beta Catenin↗

PubMed2025

Proximity Proteomics to Profile Ebola Virus Protein Interactome in Its Functional Context.

Proximity labeling-based proteomics (proximity proteomics) has emerged as a popular and versatile approach to illuminate the molecular interactions between viruses and their hosts. In this approach, a proximity labeling enzyme tag is fused to a bait protein and labels neighboring proteins with a chemical handle such as biotin, allowing for downstream affinity purification. Compared to another widely used technique, affinity purification coupled mass spectrometry, proximity proteomics enables the detection of low affinity or transient interactors that might have important functions in the viral life cycle. Further, proximity proteomics can identify interactors of a labile bait protein, of which affinity purification is technically challenging. Here, we describe a proximity proteomic protocol to identify cellular interactors of the Ebola virus polymerase. A similar strategy is readily applicable to elucidate the virus-host interactions for Marburg virus.

Ebolavirus↗

PubMed2001

Development of a time-resolved fluorescence resonance energy transfer assay (cell TR-FRET) for protein detection on intact cells.

An assay named Cell TR-FRET based on time-resolved fluorescence resonance energy transfer, here utilized for detection of receptor proteins on intact cells, is described. In this assay, intact membrane-biotinylated Sf9 cells expressing human interleukin-2Ralpha due to infection with a recombinant baculovirus were prelabeled with a streptavidin-europium (Eu(3+)) chelate, the donor. These prelabeled cells were used in a homogeneous assay by addition of a fluorochrome-labeled anti-hIL-2Ralpha-specific antibody, 7G7B6-Cy5, the acceptor. Binding of 7G7B6-Cy5 to hIL-2Ralpha expressed on the cell surface and europium-labeled streptavidin to surface biotin esters brings the donor and the acceptor in close proximity, allowing transfer of energy from the excited state donor to the acceptor. This energy transfer was specifically inhibited by unlabeled antibody and by free biotin. The described assay constitutes a general method since no specific component of the cell membrane is labeled, thereby allowing a number of binding studies on the cell membrane, including receptor density determinations, to be performed. In addition, due to the rapid fashion in which the Cell TR-FRET assay is accomplished, it can be a valuable method not only for identifying novel membrane-associated proteins, but also for drug screening of large samples in high-throughput format.

Animals↗

PubMed1976

Magnetic resonance studies of the proximity and spatial arrangement of propionyl coenzyme A and pyruvate on a biotin-metalloenzyme, transcarboxylase.

A spin-labeled ester of CoA, R-CoA (3-carboxy-2,2,5,5-tetramethyl-1-pyrolidinyl-1-oxy CoA thioester), has been shown by competition studies using electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) to bind specifically to the propionyl-CoA binding sites of transcarboxylase. Titrations indicate 0.7 +/- 0.2 binding site for R-CoA per enzyme-bound biotin with a dissociation constant of 0.33 +/- 0.12 mM. Propionyl-CoA binds to this site with a 1.3-fold lower disonable agreement with kinetically determined inhibitor constants of CoA and propionyl-CoA and propionyl-CoA (D. B. Northrop (1969), J. Biol. Chem. 244, 5808). The bit of this spin-label on 1/T1 of water protons. The formation of a ternary transcarboxylase-R-CoA-pyruvate complex is suggested by the failure of pyruvate to displace R-CoA from the tight site and is established by the paramagnetic effects of enzyme-bound R-CoA on the relaxation rates of the protons and 13C atoms of enzyme-bound pyruvate. From the paramagnetic effects of R-CoA on the relaxation rates of the methyl protons of pyruvate at 40.5 and 100 MHz, and on the 13C-enriched carbonyl and carboxyl carbon atoms of pyruvate at 25.1 MHz, a correlation time of 7 nsec and distances from the bound nitroxide radical to the methyl protons, the carbonyl, and carboxyl carbon atoms of bound pyruvate of 7.9 +/- 0.7, 10.3 +/- 0.8, and 12.1 +/- 0.9 A, respectively, are calculated. These distances establish the close proximity of the CoA ester and keto acid sites on transcarboxylase. Together with the previously determined distances from the enzyme-bound (Co(II) to the methyl protons and 2 carbon atoms of bound pyruvate and to 12 protons and 3 phosphorus atoms of bound propionyl-CoA, the present distances are used to derive a composite model of the bound substrates in the overall transcarboxylation reaction. In this model the distance from the methyl carbon of pyruvate and the methylene carbon of propionyl-CoA, between which the carboxyl transfer takes place is only approximately 7 A. Depending on the detailed mechanism of the carboxyl transfer, the distance through which the carboxybiotin must migrate is therefore between 0 and 7 A. Hence the major role of the 14-A arm of carboxybiotin is not to permit a large carboxyl migration but, rather to permit carboxybiotin to traverse the gap which occurs at the interface of three subunits and to insinuate itself between the CoA and keto acid sites.

Binding Sites↗

PubMed2002

Somatostatin immunoreactivity in axon terminals in rat nucleus tractus solitarii arising from central nucleus of amygdala: coexistence with GABA and postsynaptic expression of sst2A receptor.

Axon terminals synapsing on neurones in the nucleus tractus solitarii (NTS) that originate from the central nucleus of the amygdala (CeA) have been shown to contain gamma-aminobutyric acid (GABA) immunoreactivity. Here we investigated whether such terminals also contain somatostatin (SOM), a neuropeptide found in axons distributed throughout the NTS and in somata in the CeA, and known to modulate cardiovascular reflexes when microinjected into the NTS. With fluorescence microscopy, SOM immunoreactivity was seen in the varicosities of some axons throughout the NTS that were anterogradely labelled with biotin dextran amine injected into the CeA. Such varicosities were frequently observed in close proximity to dendrites of NTS neurones that were immunoreactive for the SOM receptor sst(2A) subtype, and in many cases also for catecholamine synthesising enzymes. In the caudal, cardioregulatory zone of NTS, SOM immunoreactivity was localised by electron microscopic pre-embedding gold labelling to boutons containing dense-cored and clear pleomorphic vesicles and forming symmetrical synapses, mostly onto dendrites. Additional post-embedding gold labelling for GABA suggested that a subpopulation (29%) of GABAergic terminals sampled in this area of NTS contained SOM. Almost all boutons anterogradely labelled from the amygdala were GABA-immunoreactive (-IR) and 21% of these were SOM-IR. A similar proportion of these boutons (22%) formed synapses onto dendrites containing immunoreactivity for the SOM receptor sst(2A) subtype. These observations provide evidence that some of the GABAergic projection neurones in the CeA that inhibit baroreceptor reflex responses in the NTS in response to fear or emotional stimuli could release SOM, which might modulate the activity of NTS neurones via an action on sst(2A) receptors.

Amygdala↗

PubMed2006

CombiMatrix oligonucleotide arrays: genotyping and gene expression assays employing electrochemical detection.

Electrochemical detection has been developed and assay performances studied for the CombiMatrix oligonucleotide microarray platform that contains 12,544 individually addressable microelectrodes (features) in a semiconductor matrix. The approach is based on the detection of redox active chemistries (such as horseradish peroxidase (HRP) and the associated substrate TMB) proximal to specific microarray electrodes. First, microarray probes are hybridized to biotin-labeled targets, second, the HRP-streptavidin conjugate binds to biotin, and enzymatic oxidation of the electron donor substrate then occurs. The detection current is generated due to electro-reduction of the HRP reaction product, and it is measured with the CombiMatrix ElectraSense Reader. Performance of the ElectraSense platform has been characterized using gene expression and genotyping assays to analyze: (i) signal to concentration dependence, (ii) assay resolution, (iii) coefficients of variation, (CV) and (iv) array-to-array reproducibility and data correlation. The ElectraSense platform was also compared to the standard fluorescent detection, and good consistency was observed between these two different detection techniques. A lower detection limit of 0.75 pM was obtained for ElectraSense as compared to the detection limit of 1.5 pM obtained for fluorescent detection. Thus, the ElectraSense platform has been used to develop nucleic acid assays for highly accurate genotyping of a variety of pathogens including bio-threat agents (such as Bacillus anthracis, Yersinia pestis, and other microorganisms including Escherichia coli, Bacillus subtilis, etc.) and common pathogens of the respiratory tract (e.g. influenza A virus).

Bacteriophage lambda↗

PubMed1996

Site-directed spin labeling and chemical crosslinking demonstrate that helix V is close to helices VII and VIII in the lactose permease of Escherichia coli.

Site-directed chemical cleavage of lactose permease indicates that helix V is in close proximity to helices VII and VIII. To test this conclusion further, permease containing a biotin-acceptor domain and paired Cys residues at positions 148 (helix V) and 228 (helix VII), 148 and 226 (helix VII), or 148 and 275 (helix VIII) was affinity purified and labeled with a sulfhydryl-specific nitroxide spin label. Spin-spin interactions are observed with the 148/228 and 148/275 pairs, indicating close proximity between appropriate faces of helix V and helices VII and VIII. Little or no interaction is evident with the 148/226 pair, in all likelihood because position 226 is on the opposite face of helix VII from position 228. Broadening of the electron paramagnetic resonance spectra in the frozen state was used to estimate distance between the 148/228 and the 148/275 pairs. The nitroxides at positions 148 and 228 or 148 and 275 are within approximately 13-15 A. Finally, Cys residues at positions 148 and 228 are crosslinked by dibromobimane, a bifunctional crosslinker that is approximately 5 A. long, while no crosslinking is detected between Cys residues at positions 148 and 275 or 148 and 226. The results provide strong support for a structure in which helix V is in close proximity to both helices VII and VIII and is oriented in such a fashion that Cys-148 is closer to helix VII.

Amino Acid Sequence↗

PubMed1999

Immunohistochemical localization of multispecific renal organic anion transporter 1 in rat kidney.

Renal proximal convoluted tubules have an important role, i.e., to excrete organic anions, including numerous drugs and endogenous substances. Recently, multispecific organic anion transporter 1 (OAT1) was isolated from rat kidney. In this study, the cellular and subcellular localization of OAT1 in rat kidney was investigated. Kidneys from normal rats were perfused and fixed with periodate-lysine-paraformaldehyde solution and were then processed for immunohistochemical analysis using the labeled streptavidin-biotin method, preembedding horseradish peroxidase method, and immunogold method. Light microscopic examination revealed immunostaining for OAT1 in the middle portion of the proximal tubule (S2 segment), but not in the initial portion of the proximal convoluted tubule, next to the glomerulus. Nephron segments other than the S2 segment and the renal vasculature were not stained with antibody to OAT1. Electron-microscopic observation using a preembedding method revealed that OAT1 was exclusively expressed in the basolateral membrane of S2 segments of proximal tubules. The immunogold method showed no labeling for OAT1 in the cytoplasmic vesicles, suggesting that OAT1 may not move together with organic anions into the cells. These results are consistent with previous physiologic data showing that organic anions, including para-aminohippurate, are taken up by the basolateral Na+-independent organic anion/dicarboxylate exchanger and excreted at S2 segments. In conclusion, OAT1 was localized to the basolateral membrane of S2 segments of proximal tubules in rat kidneys.

Animals↗