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Results for “Subcellular localization”

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PubMed1994

Subcellular localization of peripheral benzodiazepine receptors on human leukocytes.

BACKGROUND: The peripheral-type benzodiazepine receptor (PBR) was initially identified in many peripheral tissues and in some blood cells. Drugs that bind with high affinity to PBRs have previously been described as having immunomodulating properties. The number of PBRs varies according to the cell population considered. The aim of this study was to study the localization of PBRs in two human leukocyte populations, T4-lymphocytes, and monocytes. EXPERIMENTAL DESIGN: Both cell populations were purified by negative immunoselection in order to keep only the physiologically accessible sites on the viable cells. Mitochondria were quantified by electron microscopy and flow cytometric analysis. Subcellular localization was then studied after PBR photoaffinity labeling using electron microscopic ultrastructural autoradiography. RESULTS: We have shown that monocytes contain twice as many mitochondria as lymphocytes. We have also shown that the global labeling of monocytes by ultrastructural autoradiography is actually higher than that of lymphocytes and the labeling of monocyte mitochondria is higher than that of lymphocyte mitochondria. In addition, the distribution of subcellular labeling indicates that there are different populations of mitochondria in one cell, i.e., labeled and unlabeled, and that the percentage of labeled mitochondria is greater in monocytes. These results are consistent with those obtained in previous binding studies. Finally, over 50% of receptors are localized in cell compartments devoid of visible mitochondria. CONCLUSIONS: The subcellular distribution of the PBR shows that this receptor could have other physiologic functions towards immune cells than a function associated with mitochondria.

Autoradiography↗

PubMed1998

Intrarenal and subcellular localization of rat CLC5.

Dent's disease, an inherited disorder characterized by hypercalciuria, nephrolithiasis, nephrocalcinosis, rickets, low-molecular-weight proteinuria, Fanconi's syndrome, and renal failure, is caused by mutations in the renal chloride channel, CLC5. The normal role of CLC5 is unknown. We have investigated the intrarenal and subcellular localization of CLC5 in rat kidney by in situ hybridization and immunohistochemistry. By in situ hybridization, CLC5 mRNA was detected predominantly in cortical medullary ray and outer medullary tubule epithelial cells. Polyclonal antiserum was generated against a CLC5 fusion protein, affinity purified, and immunoadsorbed against CLC3 and CLC4 to yield a CLC5 isoform-specific antiserum. By immunohistochemistry, CLC5 protein was localized to the intracellular domain of tubular epithelial cells in the S3 segment of the proximal tubule and the medullary thick ascending limb. By subcellular membrane fractionation and flow cytometry, CLC5 expression was found in outer medullary endosomes. These findings are consistent with a model in which CLC5 encodes an endosomal chloride channel that facilitates acidification and trafficking of renal epithelial endosomes.

Animals↗

PubMed2004

The subcellular localization control of integrin linked kinase 1 through its protein-protein interaction with caveolin-1.

Integrin linked kinase 1 (ILK1), a member of the serine/threonine kinases, has been shown to be crucial for the cell survival, differentiation, and Wnt signaling. Firstly, by using a confocal microscopy and a transfection approach, we obtained the evidence that ILK1 interacts physically with caveolin-1, a 22-kDa integral membrane protein, which is the principal structural and regulatory component of caveolae membranes. By ILK1 deletion mutant analysis, we characterized the caveolin-1-binding domain in the kinase domain of ILK1. In addition, we found that native ILK1 is associated with endogenous caveolin-1 in COS-1 cells. Secondly, transient transfection assays showed that a reduction in caveolin-1 binding leads to a substantial increase in the serine/threonine phosphorylation of ILK1. Thirdly, caveolin-1 and its scaffolding peptide (amino acids 82-101) functionally suppressed the auto-kinase activity of purified recombinant ILK1 protein. Fourthly, the association of ILK1 with caveolin-1 regulated its cytoplasmic retention; if it was not associated with caveolin-1, it was transported to the nucleus. Fifthly, we also noticed the putative nuclear localization sequences (nls) in ILK1 near the caveolin-1-binding domain. Thus, our data indicate that caveolin-1 regulates ILK1 auto-phosphorylation activity and its subcellular localization via a specific protein-protein interaction through blocking the exposure of its putative nls motif.

Amino Acid Motifs↗

PubMed1998

Disruption of a putative SH3 domain and the proline-rich motifs in the 53-kDa substrate of the insulin receptor kinase does not alter its subcellular localization or ability to serve as a substrate.

The recently identified 53-kDa substrate of the insulin receptor family was further characterized in several retroviral-generated stable cell lines overexpressing the wild type and various mutant forms of the protein. To facilitate the study of its subcellular localization in NIH3T3 cells overexpressing insulin receptor, a myc epitope-tag was added to the carboxy terminus of the 53-kDa protein. Like the endogenous protein in Chinese hamster ovary cells, the expressed myc-tagged 53-kDa protein was found partially in the particulate fraction and was tyrosine phosphorylated in insulin-stimulated cells. Immunofluorescence studies showed for the first time that a fraction of the 53-kDa protein was localized to the plasma membrane. Confocal microscopy of cells double-labeled with antibodies to the insulin receptor and the myc epitope showed the two proteins co-localize at the plasma membrane at the level of light microscopy. Further analyses of the protein sequence of the 53-kDa substrate revealed the presence of a putative SH3 domain and two proline-rich regions, putative binding sites for SH3 and WW domains. Disruption of these three motifs by the introduction of previously characterized point mutations did not affect the membrane localization of the 53-kDa protein, its ability to serve as substrate of the insulin receptor, or its colocalization with the insulin receptor, suggesting these domains are not important in the subcellular targeting of the protein and instead may function in the interaction with subsequent signaling proteins.

3T3 Cells↗

PubMed1979

Re-examination of the subcellular localization of thyroxine 5'-deiodination in rat liver.

We describe the existence of at least two thyroxine 5'-deiodinases in rat liver. They co-fractionate with NADPH-cytochrome c reductase, the marker enzyme for membranes of the endoplasmic reticulum. Subcellular-localization studies of the most active microsomal thyroxine 5'-deiodinase were performed under substrate saturation and at optimal pH 6.8. This enzyme was a Km(app.) of about 3 microM-thyroxine and a Vmax. of about 8 ng of tri-iodothyronine/min per mg of protein. Our study confirms in part the earlier reports of microsomal localization of thyroxine 5'-deiodination. However, this process is not mediated by only a single enzyme.

Animals↗

PubMed2000

Functional expression of human DNA topoisomerase I and its subcellular localization in HeLa cells.

DNA topoisomerase (topo) I plays an important role in DNA metabolism by relieving the torsional restraints of DNA topology through ATP-independent single-strand DNA breakage. In the present study, we expressed human topo I in HeLa cells by fusing it to enhanced green fluorescent protein (EGFP). The EGFP-topo I fusion protein is functionally active in that it relaxes supercoiled plasmid DNA; forms complexes with DNA, as revealed by band depletion assays; and increases the sensitivity of cells to topo I inhibitors such as topotecan, as determined by growth inhibition assays. In contrast, a mutant form of the EGFP-topo I fusion protein, in which the active Tyr has been replaced by Phe (Y723F), has no such activities. Furthermore, the fusion protein localizes to the nucleus at interphase and completely associates with chromatids at every stage of mitosis. Of importance, the mutant fusion protein (Y723F) displays a pattern of subcellular localization identical to that of the wild-type fusion protein, although the mutant fusion protein is catalytically inactive. These results suggest that in addition to its role in DNA metabolism, topo I might also play a structural role in chromosomal organization; moreover, the association of topo I with chromosomal DNA is independent of its catalytic activity. Finally, the fusion constructs may provide a useful tool to study drug action in tumor cells, as demonstrated by nucleolar delocalization of the fusion proteins in response to treatment with the topo I inhibitor topotecan.

Amino Acid Substitution↗

PubMed1996

Cellular and subcellular localization of the vasopressin- regulated urea transporter in rat kidney.

The renal urea transporter (RUT) is responsible for urea accumulation in the renal medulla, and consequently plays a central role in the urinary concentrating mechanism. To study its cellular and subcellular localization, we prepared affinity-purified, peptide-derived polyclonal antibodies against rat RUT based on the cloned cDNA sequence. Immunoblots using membrane fractions from rat renal inner medulla revealed a solitary 97-kDa band. Immunocytochemistry demonstrated RUT labeling of the apical and subapical regions of inner medullary collecting duct (IMCD) cells, with no labeling of outer medullary or cortical collecting ducts. Immunoelectron microscopy directly demonstrated labeling of the apical plasma membrane and of subapical intracellular vesicles of IMCD cells, but no labeling of the basolateral plasma membrane. Immunoblots demonstrated RUT labeling in both plasma membrane and intracellular vesicle-enriched membrane fractions from inner medulla, a subcellular distribution similar to that of the vasopressin-regulated water channel, aquaporin-2. In the outer medulla, RUT labeling was seen in terminal portions of short-loop descending thin limbs. Aside from IMCD and descending thin limbs, no other structures were labeled in the kidney. These results suggest that: (i) the RUT provides the apical pathway for rapid, vasopressin-regulated urea transport in the IMCD, (ii) collecting duct urea transport may be increased by vasopressin by stimulation of trafficking of RUT-containing vesicles to the apical plasma membrane, and (iii) the rat urea transporter may provide a pathway for urea entry into the descending limbs of short-loop nephrons.

Animals↗

PubMed1996

Subcellular localization of the type 2 11beta-hydroxysteroid dehydrogenase. A green fluorescent protein study.

11beta-Hydroxysteroid dehydrogenase (11beta-HSD) is thought to confer aldosterone specificity to mineralocorticoid target cells by protecting the inherently non-selective mineralocorticoid receptor (MR) from occupancy by endogenous glucocorticoids. Recently, we characterized a novel isoform of 11beta-HSD in aldosterone target cells, which has high affinity for its substrate, is unidirectional, and prefers NAD as cofactor. In this study we utilized a green fluorescent protein (GFP) technique to determine the subcellular localization of this isoform, 11beta-HSD2. We generated a chimeric gene encoding the full-length rabbit 11beta-HSD2 and, fused to its C terminus, the coding sequence of GFP. This construct was stably transfected into CHO cells. The enzymatic characteristics of the expressed 11beta-HSD2/GFP fusion protein were undistinguishable from those of the native enzyme: high affinity for corticosterone (KM 8-10 nM), NAD dependence, and lack of reductase activity. The intracellular location of the recombinant protein was determined by fluorescence microscopy. 11beta-HSD2-associated fluorescence was observed as a reticular network over the cytoplasm and nuclear envelope, whereas the plasma membrane and the nucleus were negative, suggesting endoplasmic reticulum (ER) localization. Staining of CHO cells expressing 11beta-HSD2/GFP with established subcellular organelle markers revealed a colocalization of 11beta-HSD2/GFP only with ER markers and tubulin. To examine the orientation of 11beta-HSD2 within the ER, we selectively permeabilized CHO cells and stained them with an anti-GFP antibody. Fluorescence microscopy indicated that the C-terminal region of 11beta-HSD2 is on the cytoplasmic surface of the ER membrane, since it was accessible to the GFP antibody. This conclusion was confirmed by trypsin treatment of permeabilized cells followed by Western blotting. The C-terminal region of 11beta-HSD2 was accessible to trypsin, indicating that it is on the cytoplasmic side of the ER membrane. These results indicate that 11beta-HSD2 is localized exclusively to the ER. Since 11beta-HSD2 does not contain any known ER retrieval signal, experiments are currently under way to determine what structural motifs are responsible for its ER localization.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed2003

Subcellular localization and regulation of coenzyme A synthase.

CoA synthase mediates the last two steps in the sequence of enzymatic reactions, leading to CoA biosynthesis. We have recently identified cDNA for CoA synthase and demonstrated that it encodes a bifunctional enzyme possessing 4'-phosphopantetheine adenylyltransferase and dephospho-CoA kinase activities. Molecular cloning of CoA synthase provided us with necessary tools to study subcellular localization and the regulation of this bifunctional enzyme. Transient expression studies and confocal microscopy allowed us to demonstrate that full-length CoA synthase is associated with the mitochondria, whereas the removal of the N-terminal region relocates the enzyme to the cytosol. In addition, we showed that the N-terminal sequence of CoA synthase (amino acids 1-29) exhibits a hydrophobic profile and targets green fluorescent protein exclusively to mitochondria. Further analysis, involving subcellular fractionation and limited proteolysis, indicated that CoA synthase is localized on the mitochondrial outer membrane. Moreover, we demonstrate for the first time that phosphatidylcholine and phosphatidylethanolamine, which are the main components of the mitochondrial outer membrane, are potent activators of both enzymatic activities of CoA synthase in vitro. Taken together, these data provide the evidence that the final stages of CoA biosynthesis take place on mitochondria and the activity of CoA synthase is regulated by phospholipids.

Amino Acid Sequence↗

PubMed2003

Subcellular localization of poly(ADP-ribose) glycohydrolase in mammalian cells.

Posttranslational modification plays important roles in a range of cellular functions. Poly(ADP-ribosyl)ation influences DNA repair, transcription, centrosome duplication, and chromosome stability. Poly(ADP-ribose) attached to acceptor proteins should be properly hydrolyzed by poly(ADP-ribose) glycohydrolase (PARG). However the subcellular localization and the role of PARG have not been well characterized. Here, we transiently expressed GFP- or Myc-tagged human PARG in mammalian cells and revealed that the subcellular distribution of human PARG changes dramatically during the cell cycle. GFP-hPARG is found almost exclusively in the nucleus during interphase. During mitosis, most GFP-hPARG protein localizes to the cytoplasm and hardly any GFP-hPARG protein is found associated with the chromosomes. Furthermore, we found that GFP-hPARG localizes to the centrosomes during mitosis. Our findings suggest that shuttling of PARG between nucleus and cytoplasm and proper control of poly(ADP-ribose) metabolism throughout the cell cycle may play an important role in regulating cell cycle progression and centrosome duplication.

3T3 Cells↗

PubMed1993

Subcellular localization of immunoreactive oxytocin within thymic epithelial cells of the male mouse.

Immunoreactive oxytocin is expressed by thymic epithelial cells, which share properties with neuroendocrine cells. In order to investigate the assumed paracrine secretion of oxytocin, we studied the subcellular localization of immunoreactive oxytocin within thymic tissue and cultured thymic epithelial cells of the male mouse. Three types of immunoreactive cells were distinguished with the electron microscope. Immunoreactive oxytocin was found to be restricted to the cytoplasm by the use of pre- and postembedding methods. Some epithelial cells, especially in the cortex, showed a pronounced labelling of vesicular membranes and membrane tubules of the endoplasmic reticulum. In some cells, keratin filaments were associated with the electron-dense stain. Under culture conditions immunoreactive cells of different shapes were found, all displaying similar patterns of labelling. The contents of different types of vacuoles were only rarely labelled. A special class of immunoreactive exocytotic vesicles could not be identified. Thus, our results do not support neuroendocrine secretion of oxytocin via vesicles of thymic epithelial cells but offer alternative modes of secretion.

Animals↗

PubMed1993

Subcellular localization of alpha-tubulin and opsin mRNA in the goldfish retina using digoxigenin-labeled cRNA probes detected by alkaline phosphatase and HRP histochemistry.

This paper describes a method for non-radioactive in situ hybridization providing subcellular localization of mRNA in 3 microns cryosections. We used two alternative colorimetric reactions to detect digoxigenin-labeled cRNA probes: alkaline phosphatase and HRP (horseradish peroxidase). With some probes the signal with the alkaline phosphatase reaction was intense, and diffusion of the reaction product was noticeable. Using HRP-conjugated antibodies improved the resolution but decreased the sensitivity of the signal. Photoamplification of the HRP reaction product increased the contrast and improved the sensitivity of the technique.

Alkaline Phosphatase↗

PubMedDate not supplied

Characterization and subcellular localization of chlorophyllase from Ginkgo biloba.

Chlorophyllase (Chlase) catalyzes the initial step of chlorophyll (Chl)-degradation, but the physiological significance of this reaction is still ambiguous. Common understanding of its role is that Chlase is involved in de-greening processes such as fruit ripening, leaf senescence, and flowering. But there is a possibility that Chlase is also involved in turnover and homeostasis of Chls. Among the de-greening processes, autumnal coloration is one of the most striking natural phenomena, but the involvement of Chlase during autumnal coloration is not clear. Previously, it was shown that Chlase activity and expression level of the Chlase gene were not increased during autumnal coloration in Ginkgo biloba, indicating that Chlase does not work specially in the de-greening processes in G. biloba. In this study, we characterized the recombinant Chlase and analyzed its subcellular localization to understand the role of the cloned Chlase of G. biloba (GbCLH). GbCLH exhibited its highest activity at pH 7.5, 40 degrees C. Kinetic analysis revealed that GbCLH hydrolyzes pheophytin (Pheo) a and Chl a more rapidly than Pheo b and Chl b. Transient expression analysis of 40 N-terminus amino acids of GbCLH fused with GFP (green fluorescent protein) and subcellular fractionation showed that GbCLH localizes within chloroplasts. Together with our previous results, property of GbCLH and its location within the chloroplasts suggest that GbCLH plays a role in the turnover and homeostasis of Chls in green leaves of G. biloba.

Amino Acid Sequence↗

PubMed2000

Subcellular localization, stability, and trans-cleavage competence of the hepatitis C virus NS3-NS4A complex expressed in tetracycline-regulated cell lines.

A tetracycline-regulated gene expression system and a panel of novel monoclonal antibodies were used to examine the subcellular localization, stability, and trans-cleavage competence of the hepatitis C virus (HCV) NS3-NS4A complex in inducible cell lines. The NS3 serine protease domain and the full-length NS3 protein expressed in the absence of the NS4A cofactor were diffusely distributed in the cytoplasm and nucleus. Coexpression of NS4A, however, directed NS3 to the endoplasmic reticulum (ER) or an ER-like modified compartment, as demonstrated by colocalization with 3,3'-dihexyloxacarbocyanine iodide, protein disulfide isomerase, and calnexin, as well as subcellular fractionation analyses. In addition, coexpression with NS4A dramatically increased the intracellular stability of NS3 (mean protein half-life of 26 versus 3 h) and allowed for NS4A-dependent trans-cleavage at the NS4B-NS5A junction. Deletion analyses revealed that the hydrophobic amino-terminal domain of NS4A was required for ER targeting of NS3. These results demonstrate the importance of studying HCV proteins in their biological context and define a well-characterized cell culture system for further analyses of the NS3-NS4A complex and the evaluation of novel antiviral strategies against hepatitis C.

Animals↗

PubMed2002

Subcellular localization of the yeast proteome.

Protein localization data are a valuable information resource helpful in elucidating eukaryotic protein function. Here, we report the first proteome-scale analysis of protein localization within any eukaryote. Using directed topoisomerase I-mediated cloning strategies and genome-wide transposon mutagenesis, we have epitope-tagged 60% of the Saccharomyces cerevisiae proteome. By high-throughput immunolocalization of tagged gene products, we have determined the subcellular localization of 2744 yeast proteins. Extrapolating these data through a computational algorithm employing Bayesian formalism, we define the yeast localizome (the subcellular distribution of all 6100 yeast proteins). We estimate the yeast proteome to encompass approximately 5100 soluble proteins and >1000 transmembrane proteins. Our results indicate that 47% of yeast proteins are cytoplasmic, 13% mitochondrial, 13% exocytic (including proteins of the endoplasmic reticulum and secretory vesicles), and 27% nuclear/nucleolar. A subset of nuclear proteins was further analyzed by immunolocalization using surface-spread preparations of meiotic chromosomes. Of these proteins, 38% were found associated with chromosomal DNA. As determined from phenotypic analyses of nuclear proteins, 34% are essential for spore viability--a percentage nearly twice as great as that observed for the proteome as a whole. In total, this study presents experimentally derived localization data for 955 proteins of previously unknown function: nearly half of all functionally uncharacterized proteins in yeast. To facilitate access to these data, we provide a searchable database featuring 2900 fluorescent micrographs at http://ygac.med.yale.edu.

Algorithms↗

PubMed1992

Identification and subcellular localization of the Q gene product of visna virus.

The genome of the sheep visna lentivirus contains an open reading frame, Q, which has a coding potential of 230 amino acid residues. This paper reports the identification and the subcellular localization of the Q ORF-encoded protein detected in lysates of visna virus-infected sheep choroid plexus cells. Sera from sheep either experimentally or naturally infected with visna virus reacted with the bacterially synthesized Q protein indicating that the in vivo expressed Q product is immunogenic. Antibodies raised against a synthetic N-terminal peptide, reacted with either the bacterial Q or the in vitro translated Q protein as well as with the Q protein expressed during cellular infection. This 29 kDa protein is detectable late in the lytic viral cycle, i.e., 72 hr postinfection, and this expression correlates with the late transcription of its 4.8-kb mRNA. These results provide evidence for the first time that the Q ORF is a late gene of visna virus and that the Q protein is located in the cytosol compartment, without evidence of accumulation at the cell membrane, or in cell-free virion particles.

Animals↗

PubMed2000

Cellular and subcellular localization of catalase in the heart of transgenic mice.

Previous studies have described a cardiac-specific, catalase-overexpressing transgenic mouse model that was used to study myocardial oxidative injury. This study was undertaken to demonstrate cellular and subcellular localization of catalase in the hearts of transgenic mice. By the light microscopic immunoperoxidase method, we found that the overexpressed catalase was exclusively localized in cardiomyocytes. The ratios of immunoreactive cardiomyocytes in the heart were quite different among three transgenic lines examined but agreed with the elevated levels of catalase activity. In the cardiac blood vessels, positive cells were found in the walls of pulmonary veins and the vena cava, which consist of cardiomyocytes, but not in the pulmonary arteries, aorta, or cardiac valves. The electron microscopic immunogold method revealed that the elevated catalase was in sarcoplasm, nucleus, and peroxisomes, but not in mitochondria. In contrast to these distributions, catalase in the non-transgenic cardiomyocytes was in peroxisomes only. In addition, the number and size of peroxisomes in the transgenic cardiomyocytes were markedly increased, but no other ultrastructural changes were observed in comparison with those of non-transgenic mice. These results demonstrated that the elevated catalase in transgenic mouse heart is localized in cardiomyocytes and is distributed to peroxisomal and extraperoxisomal, but not mitochondrial, compartments.

Animals↗

PubMed1984

Accumulation, subcellular localization and release of propranolol from synaptosomes of rat cerebral cortex.

Propranolol is accumulated at several adrenergic neuroeffector junctions after chronic oral administration in the dog, and is released subsequently during sympathetic nerve stimulation. In the present study, the accumulation, subcellular localization and release of propranolol was examined in rat cortical synaptosomes. Synaptosomal propranolol accumulation was rapid and attained equilibrium within 1 min. Propranolol uptake increased in a nonlinear manner with increasing drug concentration in the medium, but could not be fully saturated over the concentration range studied (10(-7) - 10(-3) M). Uptake was unaffected by cocaine or ouabain and showed no stereoselectivity. Subsynaptosomal fractionation of propranolol-loaded synaptosomes revealed that the drug was concentrated principally in fractions enriched in synaptic plasma membranes and synaptic storage vesicles. Exposure of propranolol-loaded synaptosomes to elevated potassium evoked a concentration-dependent increase in propranolol overflow, which was not seen in mitochondrial fractions, myelin fractions or in freeze-thawed synaptosomal preparations. Veratridine was also effective in promoting propranolol overflow in a concentration-dependent manner. The increase in propranolol overflow induced by elevated potassium was significantly reduced, but not completely inhibited, in a calcium-free, ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid-supplemented medium. These results indicate that propranolol may be accumulated by neuronal tissue and stored at sites from which release may occur in response to depolarizing stimuli. The data further suggest that propranolol release in the synaptosome preparation may occur by both calcium-dependent and calcium-independent processes.

Animals↗