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PubMed2005

Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania) amazonensis promastigotes.

Here we report the subcellular localization of an intracellular serine protease of 68 kDa in axenic promastigotes of Leishmania (Leishmania) amazonensis, using subcellular fractionation, enzymatic assays, immunoblotting, and immunocytochemistry. All fractions were evaluated by transmission electron microscopy and the serine protease activity was measured during the cell fractionation procedure using alpha-N-r-tosyl-L-arginine methyl ester (L-TAME) as substrate, phenylmethylsulphone fluoride (PMSF) and L-1-tosylamino-2-phenylethylchloromethylketone (TPCK) as specific inhibitors. The enzymatic activity was detected mainly in a membranous vesicular fraction (6.5-fold enrichment relative to the whole homogenate), but also in a crude plasma membrane fraction (2.0-fold). Analysis by SDS-PAGE gelatin under reducing conditions demonstrated that the major proteolytic activity was found in a 68 kDa protein in all fractions studied. A protein with identical molecular weight was also recognized in immunoblots by a polyclonal antibody against serine protease (anti-SP), with higher immunoreactivity in the vesicular fraction. Electron microscopic immunolocalization using the same polyclonal antibody showed the enzyme present at the cell surface, as well as in cytoplasmic membranous compartments of the parasite. Our findings indicate that the internal location of this serine protease in L. amazonensis is mainly restricted to the membranes of intracellular compartments resembling endocytic/exocytic elements.

Animals↗

PubMed1999

Subcellular localization patterns and their relationship to photodynamic activity of pyropheophorbide-a derivatives.

To determine if subcellular localization is important to photodynamic therapy (PDT) efficacy, an in vitro fluorescence microscopy study was conducted with a congeneric series of pyropheophorbide-a derivatives in human pharyngeal squamous cell carcinoma (FaDu) cells and murine radiation-induced fibrosarcoma (RIF) mutant cells. In the FaDu cells the octyl, decyl and dodecyl ether derivatives localized to the lysosomes at extracellular concentrations less than needed to produce a 50% cell kill (LD50). At extracellular concentrations equal or greater than the LD50 the compounds localized mainly to mitochondria. The propyl, pentyl, hexyl and heptyl ether derivatives localized mainly to the mitochondria at all concentrations studied. This suggested that mitochondria are a sensitive PDT target for these derivatives. Similar experiments were performed with two Photofrin-PDT resistant RIF cell lines, one of which was found to be resistant to hexyl ether derivative (C6) mediated-PDT and the other sensitive to C6-PDT relative to the parent line. At extracellular concentrations of C6 below the LD50 of each cell line, the mutants exhibited lysosomal localization. At concentrations above these values the patterns shifted to a mainly mitochondrial pattern. In these cell lines mitochondrial localization also correlated with PDT sensitivity. Localization to mitochondria or lysosomes appeared to be affected by the aggregation state of the congeners, all of which are highly aggregated in aqueous medium. Monomers apparently were the active fraction of these compounds because equalizing the extracellular monomer concentrations produced equivalent intracellular concentrations, photoxicity and localization patterns. Compounds that were mainly aggregates localized to the lysosomes where they were rendered less active. Mitochondria appear to be a sensitive target for pyropheophorbide-a-mediated photodamage, and the degree of aggregation seems to be a determinant of the localization site.

Animals↗

PubMed1996

Subcellular localization of spinach cysteine synthase isoforms and regulation of their gene expression by nitrogen and sulfur.

Subcellular localization and regulation of the spinach (Spinacia oleracea) cysteine synthase (O-acetyl-L-serine[thiol]-lyase, EC 4.2.99.8) isoforms (CysA, CysB, and CysC) were determined in transgenic tobacco (Nicotiana tabacum) and in spinach cell cultures. The 5' regions of CysB and CysC encoding the chloroplastic (CysB-TP) and the putative mitochondrial (CysC-TP) transit peptide (TP) sequences were fused to a bacterial beta-glucuronidase gene (gus) and expressed in tobacco under the control of the cauliflower mosaic virus 35S promoter. Subcellular fractionation of transgenic tobacco showed transportation of beta-glucuronidase proteins to chloroplasts by CysB-TP and to mitochondria by CysC-TP, respectively, indicating that both presequences were sufficient to act specifically as chloroplastic and mitochondrial TPs in vivo. The mRNA expression patterns of CysA (cytoplasmic form), CysB, and CysC genes under nitrogen- and sulfur-starved conditions were characterized in spinach cell cultures. In sulfur-starved cells, only slight differences (approximately 1.2- to 1.5-fold) in the mRNA levels of CysA and CysB were observed during the short-term (0-24 h) cultivation periods compared with cells grown in Murashige-Skoog medium. However, under nitrogen and nitrogen/sulfur double-deficient stress conditions, mRNA levels of CysC increased up to 500% of the original level within 72 h.

Base Sequence↗

PubMedDate not supplied

Studies on the subcellular localization of the porphycene CPO.

This study was designed to provide more detailed information on the subcellular sites of binding of the porphycene, termed 9-capronyloxytetrakis (methoxyethyl) porphycene (CPO), with a fluorescence resonance energy transfer (FRET) technique. The proximity of CPO to two fluorescent probes was determined: nonyl acridine orange (NAO), a dye with specific affinity for the mitochondrial lipid cardiolipin, and dihexa-oxacarbocyanine iodide (DiOC6), an agent that labels the endoplasmic reticulum (ER). FRET spectra indicated energy transfer between DiOC6 and CPO but no significant transfer between NAO and CPO. These results confirm data obtained by fluorescence microscopy, suggesting a similar pattern of subcellular localization by CPO and DiOC6 but not by CPO and NAO. However, when cells containing CPO were irradiated and then loaded with NAO, FRET between the two fluorophores was observed. Hence, a relocalization of CPO can occur during irradiation. These data provide an explanation for recent studies on CPO-catalyzed photodamage to both ER and mitochondrial Bcl-2.

Acridine Orange↗

PubMed2005

Prediction of subcellular localization using sequence-biased recurrent networks.

MOTIVATION: Targeting peptides direct nascent proteins to their specific subcellular compartment. Knowledge of targeting signals enables informed drug design and reliable annotation of gene products. However, due to the low similarity of such sequences and the dynamical nature of the sorting process, the computational prediction of subcellular localization of proteins is challenging. RESULTS: We contrast the use of feed forward models as employed by the popular TargetP/SignalP predictors with a sequence-biased recurrent network model. The models are evaluated in terms of performance at the residue level and at the sequence level, and demonstrate that recurrent networks improve the overall prediction performance. Compared to the original results reported for TargetP, an ensemble of the tested models increases the accuracy by 6 and 5% on non-plant and plant data, respectively. AVAILABILITY: The Protein Prowler incorporating the recurrent network predictor described in this paper is available online at http://pprowler.imb.uq.edu.au/

Algorithms↗

PubMed1995

Subcellular localization of and photosensitization by protoporphyrin IXhuman keratinocytes and fibroblasts cultivated with 5-aminolevulinic acid.

The subcellular localization of protoporphyrin (PP) has been studied by microspectrofluorometric techniques in NCTC 2544 keratinocytes incubated with 5-aminolevulinic acid (ALA) for times up to 42 h. Whereas the plasma membrane shows strong staining, fluorescent spots are observed within the cytoplasm especially in the perinuclear region. Although the topographic pattern of the PP distribution does not change much with the incubation time with ALA, the fluorescence spectra suggest that the PP microenvironments are quite different at short and long incubation times. Addition of 18 microM desferrioxamine almost doubles the ALA-induced PP concentration. Colocalization experiments with rhodamine 123, a mitochondrial probe, and lucifer yellow (LY) or neutral red (NR), two lysosome probes, demonstrate that at least some of these spots are of lysosomal origin. Study of the time evolution of the NR fluorescence under irradiation with visible light in the presence and absence of ALA demonstrates that lysosomes are damaged cells that have synthesized PP. No PP fluorescence can be detected in mitochondria after incubation with ALA. However, photosensitization of mitochondria occurs under irradiation with visible light. Very little formation of lipofuscins by photosensitization with exogenous PP or ALA-induced PP is observed with the NCTC 2544 keratinocytes, as compared to normal human fibroblasts.

Aminolevulinic Acid↗

PubMed1994

Subcellular localization and translocation of the receptor for N-formylmethionyl-leucyl-phenylalanine in human neutrophils.

The subcellular localization of N-formylmethionyl-leucyl-phenylalanine (fMLP) receptors in human neutrophils was investigated. The fMLP receptor was detected with a high-affinity, photoactivatable, radioiodinated derivative of N-formyl-methionyl-leucyl-phenylalanyl-lysine (fMLFK). Neutrophils were disrupted by nitrogen cavitation and fractionated on Percoll density gradients. fMLP receptors were located in the beta-band containing gelatinase and specific granules, and in the gamma-band containing plasma membrane and secretory vesicles. Plasma membranes and secretory vesicles were separated by high-voltage free-flow electrophoresis, and secretory vesicles were demonstrated to be highly enriched in fMLP receptors. The receptors found in secretory vesicles translocated fully to the plasma membrane upon stimulation with inflammatory mediators. The receptor translocation from the beta-band indicated that the receptor present there was mainly located in gelatinase granules. A 25 kDa fMLP-binding protein was found in the beta-band. Immunoprecipitation revealed that this protein was identical with NGAL (neutrophil gelatinase-associated lipocalin), a novel protein found in specific granules. In summary, we demonstrate that the compartment in human neutrophils that is mobilized most easily and fastest, the secretory vesicle, is a major reservoir of fMLP receptors. This explains the prompt and extensive upregulation of fMLP receptors on the neutrophil surface in response to inflammatory stimuli.

Cell Fractionation↗

PubMed1990

Hepatic subcellular localization of cresylbenzodioxaphosphorin oxide (CBDP)-sensitive soman binding sites.

The toxicity of the organophosphorus poison soman (pinacolylmethylphosphonofluoridate) is attributable to its irreversible inhibition of the enzyme acetylcholinesterase. In addition, soman binds irreversibly to a number of noncholinesterase tissue binding sites which appear to be its major means of in vivo detoxification. This study was conducted to determine the hepatic subcellular localization of these sites. Subcellular fractions of liver from male Sprague-Dawley rats (200-250 g) were prepared by differential and isopycnic density gradient centrifugation. The binding of [14C]soman to these subcellular fractions was determined in the presence and absence of cresylbenzodioxaphosphorin oxide (CBDP), a compound that binds irreversibly to the noncholinesterase soman binding sites. Crude fractionation of liver homogenates into nuclear, mitochondrial, microsomal, and soluble fractions revealed that 78% of the total CBDP-sensitive binding activity was localized in the nuclear and microsomal fractions. Further purification of these fractions indicated that all of the homogenate binding activity could be accounted for in the purified microsomal fraction. When purified liver microsomes were solubilized and fractionated on linear sucrose gradients, 90% of the CBDP-sensitive soman binding activity cosedimented with carboxylesterase activity which suggests that these binding sites are carboxylesterase.

Animals↗

PubMed2004

Subcellular localization and mobilization of carcinoembryonic antigen-related cell adhesion molecule 8 in human neutrophils.

The subcellular localization and mobilization of carcinoembryonic antigen-related cell adhesion molecule 8 (CEACAM8) was investigated quantitatively in human neutrophils. In resting neutrophils the majority of CEACAM8 was present in the secondary granules, and a small amount of CEACAM8 was present in a light membrane fraction. Stimulation of the neutrophils with phorbol 12-myristate 13-acetate caused a dramatic increase in the content of CEACAM8 in the light membrane fraction, suggesting a translocation of CEACAM8 to the plasma membrane from intracellular pools. The cellular content of CEACAM8 in the neutrophils was estimated to be 82.4 +/- 8.9 ng/10(6) cells (mean +/- SE, n = 10). Administration of granulocyte colony-stimulating factor (G-CSF) to healthy individuals resulted in an increased content of CEACAM8 in neutrophils on day 1, which decreased on day 4. However, the content of CEACAM8 in the light membrane fraction was increased on day 4, possibly due to the stimulation by induced secondary cytokines, such as tumour necrosis factor-alpha (TNF-alpha). This study establishes the secondary granules as the major intracellular pools of CEACAM8 in human neutrophils, from which it may translocate to the plasma membranes upon stimulation of the cells. The translocation of CEACAM8 seen in vivo after G-CSF administration is probably indirect and caused by cytokines such as TNF-alpha.

Adult↗

PubMed1998

Changes in subcellular localization of metabotropic glutamate receptor subtypes during postnatal development of mouse thalamus.

High resolution immunoelectron microscopy was used to study subcellular localization patterns of three metabotropic glutamate receptor subtypes (mGluR1alpha, mGluR5, and mGluR2/3) during postnatal development of mouse ventral posterior (VP) thalamic nucleus. Immunoreactivity for all three mGluRs was detected from birth (postnatal day 0, P0), but mGluR1alpha showed dramatic changes in localization with age. In the first postnatal week, mGluR1alpha immunoreactivity was mainly found in proximal dendrites and somata and not usually associated with synaptic contacts. From the second postnatal week, it became concentrated in distal dendrites and preferentially associated with corticothalamic (RS) synapses. mGluR5 immunoreactivity was weaker than mGluR1alpha immunoreactivity at all postnatal ages and showed a similar change in subcellular distribution to that of mGluR1alpha. It was also localized in astrocytic processes. mGluR2/3 immunoreactivity was mainly localized in astrocytic processes surrounding neuronal somata and synapses and this pattern was consistently maintained through all postnatal ages. A small number of presynaptic axon terminals were labeled for mGluR2/3 immunoreactivity and formed asymmetrical synapses. This study demonstrates that Group I mGluR proteins (mGluR1alpha and mGluR5) become redistributed in association with the development of corticothalamic function as demonstrated physiologically, whereas Group II mGluR proteins (mGluR2/3) are mainly associated with neuroglia.

Aging↗

PubMed1983

Subcellular localization and tissue distribution of sialic acid precursor-forming enzymes.

The enzymes UDP-N-acetylglucosamine pyrophosphorylase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine kinase, N-acetylglucosamine kinase and N-acetylglucosamine 2-epimerase, which are involved in the metabolism of N-acetylneuraminic acid, were studied in rat with regard to their subcellular localization and tissue distribution. The subcellular distribution studies in liver indicated that the enzymes are localized in the soluble cell fraction. In other tissues the comparison of enzyme activities in homogenates with that in high-speed supernatants led to a similar conclusion. UDP-N-acetylglucosamine pyrophosphorylase, N-acetylglucosamine kinase and N-acetylglucosamine 2-epimerase were detected in almost all tissues studied. UDP-N-acetylglucosamine 2-epimerase and N-acetylmannosamine kinase, two enzymes considered to be key enzymes in the N-acetylneuraminic acid biosynthesis, were detected only in sialoglycoprotein-secreting tissues, i.e. liver, salivary gland and intestinal mucosa. The low activity of the key enzymes in other tissues suggests that the biosynthetic pathway of N-acetylneuraminic acid is not the same in various tissues.

Animals↗

PubMed2005

Synaptic and subcellular localization of A-kinase anchoring protein 150 in rat hippocampal CA1 pyramidal cells: Co-localization with excitatory synaptic markers.

Excitatory and inhibitory ionotropic receptors are regulated by protein kinases and phosphatases, which are localized to specific subcellular locations by one of several anchoring proteins. One of these is the A-kinase anchoring protein (AKAP150), which confers spatial specificity to protein kinase A and protein phosphatase 2B in the rat brain. The distribution of AKAP150 was examined at rat hippocampal CA1 pyramidal cell asymmetric and symmetric post-synaptic densities and with respect to the distribution of markers of excitatory (vesicular glutamate transporter 1, glutamate receptor subunit 1) and inhibitory receptors (vesicular GABA transporter, GABA receptor type A beta2/3 subunits, gephyrin) and the Golgi marker, trans-Golgi network glycoprotein 38. AKAP150 was close to asymmetric synapses, consistent with numerous molecular and biochemical studies suggesting its interaction with components of the excitatory postsynaptic density. In contrast, we did not find AKAP150-immunoreactivity associated with inhibitory synapses in rat CA1 neurons, despite reports demonstrating an in vitro interaction between AKAP150 and GABA receptor type A receptor beta subunits, and the reported co-localization of these proteins in rat hippocampal cultures. There was some overlap between AKAP150 and GABA receptor type A receptor beta2/3-immunoreactivity intracellularly in perinuclear clusters. These findings support previous work indicating the integration of kinase and phosphatase activity at excitatory synapses by AKAP150, but do not support a role for selective targeting of AKAP150 and its accompanying proteins to inhibitory synapses.

A Kinase Anchor Proteins↗

PubMed1977

Subcellular localization of the alterations in phosphatidylinositol metabolism following glucose-induced insulin release from rat pancreatic islets.

The subcellular localization of the incorporation of 2-(3H)-myoinositol into lipids has been studied in isolated pancreatic islets of the rat. The recovery of lipid-bound myoinositol increased with time in the nuclear, mitochondrial, microsomal, and secretory granule fractions. The utilization of a filtration technique for the more complete separation of mitochondrial and secretory granule elements permitted us to show that the recovery of lipid-bound 2-(3H)-myoinositol increased most rapidly in the secretory granule fraction. A 30-minute exposure of prelabeled islets to a stimulatory concentration of D-glucose (3.0 mg./ml.) resulted in a statistically significant decrease in the amount of lipid-bound 2-(3H)-myoinositol that was recovered from the secretory granule fraction (p less than 0.001). In contrast, exposure of islets to the elevated glucose concentration had no statistically significant effect on the recovery of lipid-bound radioactivity from other subcellular fractions. Since the majority of lipid-bound radioactivity associated with the secretory granule fraction could be recovered with the presumptive secretory granule membranes, these data suggest that the hydrolysis of phosphatidylinositol that accompanies glucose-induced insulin secretion from the rat pancreatic islet may be localized to the beta granule and, in particular, to its limiting membrane.

Animals↗

PubMed1976

Esterolytic activities of rat intestinal mucosa. 1. Characterization, cellular distribution and subcellular localization of a glycerol-ester hydrolase.

The preferential cellular distribution in the villus tip and the subcellular localization in the endoplasmic reticulum of an intestinal glycerol-ester hydrolase from rat mucosa are described. The enzyme is shown not to be from either pancreatic or bacterial origin; it catalyzes the hydrolysis of short- and medium chain triglycerides and of p-nitrophenylacetate. Contrarily to the specificity found for the pig intestinal lipase (Serrero, Négrel and Ailhaud, 1975), no activity is detectable against acylCoA; a thiolester hydrolase different from the glycerol-ester hydrolase was demonstrated after differential solubilization and chromatographic separation. A high proportion of glycerol-ester hydrolase is present in the intestinal lumen; its possible complementary role in lipid degradation is discussed.

Aging↗

PubMed1998

Subcellular localization of Bacillus subtilis SMC, a protein involved in chromosome condensation and segregation.

We have investigated the subcellular localization of the SMC protein in the gram-positive bacterium Bacillus subtilis. Recent work has shown that SMC is required for chromosome condensation and faithful chromosome segregation during the B. subtilis cell cycle. Using antibodies against SMC and fluorescence microscopy, we have shown that SMC is associated with the chromosome but is also present in discrete foci near the poles of the cell. DNase treatment of permeabilized cells disrupted the association of SMC with the chromosome but not with the polar foci. The use of a truncated smc gene demonstrated that the C-terminal domain of the protein is required for chromosomal binding but not for the formation of polar foci. Regular arrays of SMC-containing foci were still present between nucleoids along the length of aseptate filaments generated by depleting cells of the cell division protein FtsZ, indicating that the formation of polar foci does not require the formation of septal structures. In slowly growing cells, which have only one or two chromosomes, SMC foci were principally observed early in the cell cycle, prior to or coincident with chromosome segregation. Cell cycle-dependent release of stored SMC from polar foci may mediate segregation by condensation of chromosomes.

Animals↗

PubMed1988

Different subcellular localization of neurotensin-receptor and neurotensin-acceptor sites in the rat brain dopaminergic system.

The subcellular localization of neurotensin-receptor sites (NT2 sites) and neurotensin-acceptor sites (NT1 sites) was studied in rat caudate-putamen by isopycnic centrifugation in sucrose density gradients. [3H]Neurotensin binding to NT2 sites occurred as a major peak at higher sucrose densities, colocalized with [3H]dopamine uptake, and as a small peak at a lower density; whereas binding to NT1 sites occurred as a single large peak at an intermediate density. 6-Hydroxydopamine lesions of the median forebrain bundle resulted in a total loss of NT2 sites in the caudate-putamen but did not affect NT2 sites in the nucleus accumbens and the olfactory tubercle. NT1 sites were not affected. Kainic acid injections into the rat caudate-putamen led to a partial decrease of NT1 sites in this region 5 days later. After a few weeks they returned to normal. Therefore NT2 sites are probably associated with presynaptic nigrostriatal dopaminergic terminals in the caudate-putamen but not in the nucleus accumbens and the olfactory tubercle. A possible association of NT1 sites with glial cells is suggested.

Animals↗

PubMed1981

Subcellular localization of the env-related glycoproteins in Friend erythroleukemia cells.

A scheme was developed for the subcellular fractionation of murine erythroleukemia cells transformed by Friend leukemia virus. The subcellular localization of the env-related glycoproteins was determined by immune precipitation with antiserum against gp70, the envelope glycoprotein of the helper virus, followed by gel electrophoresis. In cells labeled for 2 h with [35S]methionine, the glycoprotein encoded by the defective spleen focus-forming virus, gp55SFFV, was found primarily in the nuclear fraction and in fractions containing dense cytoplasmic membranes such as endoplasmic reticulum. A similar distribution was noted for gp85env, the precursor to gp70. The concentration of viral glycoproteins in the nuclear fraction could not be accounted for by contamination with endoplasmic reticulum. In pulse-chase experiments, neither glycoprotein underwent major redistribution. However, labeled gp85env disappeared from intracellular membranes with a half-time of 30 min to 1 h, whereas labeled gp55SFFV was stable during a 2-h chase. In plasma membrane preparations with very low levels of contamination with endoplasmic reticulum, gp70 was the major viral env-related glycoprotein detected; a minor amount of gp55SFFV and no gp85env could be detected. The unexpected result of these experiments is the amount of viral glycoproteins found in the nuclear fraction. Presence of viral proteins in the nucleus could be relevant to the mechanism of viral leukemogenesis.

Animals↗

PubMed1978

Subcellular localization of two long-chain acyl-coenzyme-A synthetases in Candida lipolytica.

Studies have been made on the subcellular localization of two long-chain acyl-coenzyme-A synthetases as well as glycerolphosphate acyltransferase and the acyl-CoA-oxidizing system in Candida lipolytica grown on oleic acid. Acyl-CoA synthetase I is distributed among different subcellular fractions, including microsomes and mitochondria where glycerolphosphate acyltransferase is located. On the other hand, acyl-CoA synthetase II is localized in microbodies where the acyl-CoA-oxidizing system is located. These results support our previous conclusion that acyl-CoA synthetase I is responsible for the production of acyl-CoA to be utilized for the synthesis of cellular lipids, while acyl-CoA synthetase II provides acyl-CoA that is exclusively degraded via beta-oxidation.

Candida↗