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Results for “Subcellular localization”

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PubMed1977

Subcellular localization of monoamine oxidase in bovine thalamus tissue using immunoferritin conjugates.

A combination of discontinuous sucrose gradient analysis and polyacrylamide electrophoresis was used to isolate one of the multiple forms of monoamine oxidase (MAO) from bovine thalamus. This substance (the principle form and the most anodic of the five MAO forms observed) was used as a basis for an immunoferritin-electron microscope approach to determine the subcellular localization of MAO in thalamus. This form of MAO, as well as antigenically related forms, was found to reside mainly on the outer mitochondrial membrane. In addition, the action of SDS on solubilization and interconversion of MAO forms was studied and found to be dependent on the concentration and time of reaction of SDS with thalamus tissue.

Animals↗

PubMed2000

Regulator of G protein signaling 8 (RGS8) requires its NH2 terminus for subcellular localization and acute desensitization of G protein-gated K+ channels.

Functional roles of the NH(2)-terminal region of RGS (regulators of G protein signaling) 8 in G protein signaling were studied. The deletion of the NH(2)-terminal region of RGS8 (DeltaNRGS8) resulted in a partial loss of the inhibitory function in pheromone response of yeasts, although Galpha binding was not affected. To examine roles in subcellular distribution, we coexpressed two fusion proteins of RGS8-RFP and DeltaNRGS8-GFP in DDT1MF2 cells. RGS8-RFP was highly concentrated in nuclei of unstimulated cells. Coexpression of constitutively active Galpha(o) resulted in translocation of RGS8 protein to the plasma membrane. In contrast, DeltaNRGS8-GFP was distributed diffusely through the cytoplasm in the presence or absence of active Galpha(o). When coexpressed with G protein-gated inwardly rectifying K(+) channels, DeltaNRGS8 accelerated both turning on and off similar to RGS8. Acute desensitization of G protein-gated inwardly rectifying K(+) current observed in the presence of RGS8, however, was not induced by DeltaNRGS8. Thus, we, for the first time, showed that the NH(2) terminus of RGS8 contributes to the subcellular localization and to the desensitization of the G protein-coupled response.

Active Transport, Cell Nucleus↗

PubMed1989

Human platelet-derived mitogens. II. Subcellular localization of insulinlike growth factor I to the alpha-granule and release in response to thrombin.

Platelets contain mitogenic activities for MCF-7 human breast cancer cells when assayed under serum-free chemically defined conditions. Purification from outdated human platelets identified insulinlike growth factor I (IGF-I) as the most potent breast cancer cell mitogen in lysates (Karey KP, Sirbasku DA: see accompanying article, this issue). In this study the release and subcellular localization of IGF-I was investigated. Degranulation of platelets by thrombin treatment caused release of lysosomal enzymes (beta-glucuronidase and N-acetyl-D-glucosaminidase), alpha-granule proteins (beta-thromboglobulin and fibrinogen) as well as mitogenic activity for MCF-7 cells and IGF-I as measured by radioimmunoassay (RIA) and radioreceptor assay. Release of mitogenic activity and immunologically identified IGF-I was induced tenfold over controls by thrombin and was nearly complete as compared to platelets disrupted by repeated freezing and thawing. Disruption of platelets by nitrogen cavitation followed by separation of the organelles by sucrose density gradient sedimentation showed that IGF-I and mitogenic activity localized predominantly to fractions containing alpha-granules rather than soluble cellular components, lysosomes, or dense granules. The morphology of MCF-7 cells in serum-free medium supplemented with supernatants from thrombin-treated platelets also indicated the release of important cell-adhesion factors for human breast cancer cells.

Blood Platelets↗

PubMed1998

Rigor-type mutation in the kinesin-related protein HsEg5 changes its subcellular localization and induces microtubule bundling.

HsEg5 is a human kinesin-related motor protein essential for the formation of a bipolar mitotic spindle. It interacts with the mitotic centrosomes in a phosphorylation-dependent manner. To investigate further the mechanisms involved in targetting HsEg5 to the spindle apparatus, we expressed various mutants of HsEg5 in HeLa cells. All these mutants share a mutation of Thr-112 in the N-terminal motor domain, resulting in the inactivation of the ATP binding domain. In vitro, the HsEg5-T112N mutant motor domain showed a nucleotide-independent microtubule association, typical of a kinesin protein binding to microtubules in a rigor state. In vivo, overexpression of the HsEg5 rigor mutant in HeLa cells induced, in interphase, microtubule bundling, and, in mitosis, the formation of monopolar mitotic spindles similar to those observed after microinjection of anti-HsEg5 antibodies. Localization of the HsEg5 rigor mutant on cytoplasmic microtubules did not require the C-terminal tail domain but was lost when the stalk domain was also deleted. Sucrose gradient centrifugation experiments showed that microtubule bundling was most likely caused by the binding of HsEg5 mutants in a dimeric state. These results demonstrate that the precise subcellular localization of HsEg5 in vivo is regulated not only by the phosphorylation of the tail domain but also by the oligomeric state of the protein.

Adenosine Triphosphate↗

PubMed1996

Mutations within the Ran/TC4 GTPase. Effects on regulatory factor interactions and subcellular localization.

Ran, a member of the Ras superfamily of GTPases, is predominantly localized in the nucleus and is a necessary component in the active transport of proteins through nuclear pores. Disruption of Ran function affects the regulation of mitosis, DNA synthesis, and RNA processing and export. To explore the mechanisms of Ran function, mutants of the Ran GTPase were characterized, several of which are capable of dominantly interfering with nuclear protein import. Unlike wild-type Ran, the putative gain-of-function mutant (G19V Ran) was not sensitive to the exchange factor, RCC1. In addition the G19V Ran and effector domain mutants (L43E and E46G Ran) were not sensitive to the GTPase-activating protein, Fug1. Epitope-tagged G19V Ran and L43E Ran isolated from transfected BHK21 cells were each about 50% GTP-bound, whereas the wild-type and a C-terminal deletion mutant (Delta-DE Ran) were primarily bound to GDP. While G19V Ran interacted with known Ran-binding proteins and with an isolated Ran-binding domain, the T24N Ran did not, and binding by L43E Ran was substantially reduced. Wild-type HA1-tagged Ran expressed in BHK21 cells was nuclear, whereas the G19V, T24N, L43E, and E46G forms of Ran were predominantly localized at the nuclear envelope, and Delta-DE Ran was primarily cytosolic. Similar results were observed when permeabilized BHK21 cells were incubated with extracts of COS cells expressing the mutants. Thus mutations that affect the interaction of Ran with regulatory proteins and effectors can disrupt the normal subcellular localization of Ran, lending support for the current model of Ran-mediated nuclear import.

Animals↗

PubMed1995

Subcellular localization of pyruvate dehydrogenase dihydrolipoamide acetyltransferase in human intrahepatic biliary epithelial cells.

In previous histological studies, biliary epithelial cells (BEC) in the liver of patients with primary biliary cirrhosis (PBC), but not controls, reacted strongly with antibodies specific for the major autoantigen associated with PBC, the E2 component of pyruvate dehydrogenase complex (PDC-E2). In this study we have used transmission electron microscopy (TEM) to document the precise subcellular localization of PDC-E2 in BEC. Two antibodies which recognize PDC-E2 were used: affinity-purified anti-PDC-E2 raised in rabbits; and human antibody from the serum of patients with PBC, affinity-purified against human heart PDC. The intracellular localization of antibody binding was determined by laser scanning confocal microscopy and TEM. Both antibodies bound to the inner membrane of mitochondria in BEC isolated from both patients with PBC and controls, but binding to the external aspect of the plasma membrane was observed only in BEC from patients with PBC. Surface antigen expression in PBC may make BEC immunological targets.

Adult↗

PubMed1998

Regulation of bHLH-PAS protein subcellular localization during Drosophila embryogenesis.

The Drosophila Single-minded and Tango basic-helix-loop-helix-PAS protein heterodimer controls transcription and embryonic development of the CNS midline cells, while the Trachealess and Tango heterodimer controls tracheal cell and salivary duct transcription and development. Expression of both single-minded and trachealess is highly restricted to their respective cell lineages, however tango is broadly expressed. The developmental control of subcellular localization of these proteins is investigated because of their similarity to the mammalian basic-helix-loop-helix-PAS Aromatic hydrocarbon receptor whose nuclear localization is dependent on ligand binding. Confocal imaging of Single-minded and Trachealess protein localization indicate that they accumulate in cell nuclei when initially synthesized in their respective cell lineages and remain nuclear throughout embryogenesis. Ectopic expression experiments show that Single-minded and Trachealess are localized to nuclei in cells throughout the ectoderm and mesoderm, indicating that nuclear accumulation is not regulated in a cell-specific fashion and unlikely to be ligand dependent. In contrast, nuclear localization of Tango is developmentally regulated; it is localized to the cytoplasm in most cells except the CNS midline, salivary duct, and tracheal cells where it accumulates in nuclei. Genetic and ectopic expression experiments indicate that Tango nuclear localization is dependent on the presence of a basic-helix-loop-helix-PAS protein such as Single-minded or Trachealess. Conversely, Drosophila cell culture experiments show that Single-minded and Trachealess nuclear localization is dependent on Tango since they are cytoplasmic in the absence of Tango. These results suggest a model in which Single-minded and Trachealess dimerize with Tango in the cytoplasm of the CNS midline cells and trachea, respectively, and the dimeric complex accumulates in nuclei in a ligand-independent mode and regulates lineage-specific transcription. The lineage-specific action of Single-minded and Trachealess derives from transcriptional activation of their genes in their respective lineages, not from extracellular signaling.

Animals↗

PubMed2003

Subcellular localization of rickettsial invasion protein, InvA.

To understand further the molecular basis of rickettsial host cell invasion, Rickettsia prowazekii invasion gene homolog (invA) has been characterized. Our previous experiments have shown that InvA is an Ap5A pyrophosphatase, a member of the Nudix hydrolase family, which is up-regulated during the internalization, early growth phase, and exit steps during rickettsial mammalian cell infection. In addition to the molecular characterization, subcellular localization of InvA was investigated. InvA-specific antibodies were raised in mice and used for immunoelectron microscopy. The generated antibodies were shown to recognize InvA and by immunogold labeling showed InvA in the cytoplasm of rickettsiae. A cytoplasmic location for InvA would allow for a rapid response to any internal substance and efficient functioning in hydrolysis of toxic metabolic by-products that are accumulated in the rickettsial cytoplasm during host cell invasion. Protecting bacteria from a hazardous environment could enhance their viability and allow them to remain metabolically active, which is a necessary step for the rickettsial obligate intracellular lifestyle.

Animals↗

PubMed2001

WFS1 (Wolfram syndrome 1) gene product: predominant subcellular localization to endoplasmic reticulum in cultured cells and neuronal expression in rat brain.

Wolfram (DIDMOAD) syndrome is an autosomal recessive neurodegenerative disorder accompanied by insulin-dependent diabetes mellitus and progressive optic atrophy. Recent positional cloning led to identification of the WFS1 (Wolfram syndrome 1) gene, a member of a novel gene family of unknown function. In this study, we generated a specific antibody against the C-terminus of the WFS1 protein and investigated its subcellular localization in cultured cells. We also studied its distribution in the rat brain. Biochemical studies indicated the WFS1 protein to be an integral, endoglycosidase H-sensitive membrane glycoprotein that localizes primarily in the endoplasmic reticulum (ER). Consistent with this, immunofluorescence cell staining of overexpressed WFS1 showed a characteristic reticular pattern over the cytoplasm and overlapped with the ER marker staining. No co-localization of WFS1 with mitochondria argues against an earlier clinical hypothesis that Wolfram syndrome is a mitochondria-mediated disorder. In the rat brain, at both the protein and mRNA level, WFS1 was found to be present predominantly in selected neurons in the hippocampus CA1, amygdaloid areas, olfactory tubercle and superficial layer of the allocortex. These expression sites, i.e. components of the limbic system or structures closely associated with this system, may be involved in the psychiatric, behavioral and emotional abnormalities characteristic of this syndrome. ER localization of WFS1 suggests that this protein plays an as yet undefined role in membrane trafficking, protein processing and/or regulation of ER calcium homeostasis. These studies represent a first step toward the characterization of WFS1 protein, which presumably functions to maintain certain populations of neuronal and endocrine cells.

Animals↗

PubMed1984

A simple method to determine whole cell uptake of radiolabelled oestrogen and progesterone and their subcellular localization in breast cancer cell lines in monolayer culture.

Specific uptake of tritiated 17 beta-oestradiol and R5020, a synthetic progestin, in breast cancer cell lines ( MCF7 and T47D) growing in monolayer culture in multiwell plates has been shown. Binding characteristics, calculated by Scatchard analysis, indicate the presence of steroid receptors of similar affinities and capacities to those already obtained with broken cell preparations. Lysis of the cells by treatment with a hypotonic buffer reveals the subcellular localization of the receptors so the method can be used to study receptor dynamics such as nuclear translocation and processing. Cell growth can be measured by DNA determination directly in the multiwell plates. Thus, the method provides a convenient way of studying the effects of steroid hormones (or any antihormone or chemotherapeutic agent) on growth and receptor content of breast cancer cells in monolayer culture.

Breast Neoplasms↗

PubMed2004

Subcellular localization of WD40 repeat 1 protein in PC12 rat pheochromocytoma cells.

The dynamics of actin filament protein is crucial for various physiological processes of the cells. Among the proteins correlating with actin dynamics, a novel 67-kDa WD40 repeat protein 1 (WDR1) was the vertebrate homologue of actin-interacting protein 1 (Aip1). Even though previous studies have provided the clues on the function of WDR1 in specific organs under pathological conditions, the exact subcellular localization of WDR1 is not known. Therefore, in the present study, we undertook to determine the distribution of WDR1 within PC12 pheochromocytoma cells (PC12 cells) using light and electron microscopic techniques. Double immunocytochemistry clearly showed that WDR1 immunoreactivities (IRs) were co-localized with anti-actin antibody, suggesting the involvement of WDR1 in actin dynamics. WDR1 immunoreactivities (IRs) in PC12 cells showed different distribution patterns as nerve growth factor (NGF) concentrations varied. During active proliferation, the distribution of WDR1 IRs seemed to be similar to those found in cortical actin patches, whereas WDR1 IR was observed in cytoplasmic actin cables after PC12 cells were induced to differentiate by treating with NGF. Though further studies are necessary to determine the function of WDR1, the current data represents a first step towards the in vitro study of WDR1 protein.

Actins↗

PubMed2001

Pin1 regulates turnover and subcellular localization of beta-catenin by inhibiting its interaction with APC.

Phosphorylation on a serine or threonine residue preceding proline (Ser/Thr-Pro) is a key regulatory mechanism, and the conformation of certain phosphorylated Ser/Thr-Pro bonds is regulated specifically by the prolyl isomerase Pin1. Whereas the inhibition of Pin1 induces apoptosis, Pin1 is strikingly overexpressed in a subset of human tumours. Here we show that Pin1 regulates beta-catenin turnover and subcellular localization by interfering with its interaction with adenomatous polyposis coli protein (APC). A differential-display screen reveals that Pin1 increases the transcription of several beta-catenin target genes, including those encoding cyclin D1 and c-Myc. Manipulation of Pin1 levels affects the stability of beta-catenin in vitro. Furthermore, beta-catenin levels are decreased in Pin1-deficient mice but are increased and correlated with Pin1 overexpression in human breast cancer. Pin1 directly binds a phosphorylated Ser-Pro motif next to the APC-binding site in beta-catenin, inhibits its interaction with APC and increases its translocation into the nucleus. Thus, Pin1 is a novel regulator of beta-catenin signalling and its overexpression might contribute to the upregulation of beta-catenin in tumours such as breast cancer, in which APC or beta-catenin mutations are not common.

Adenomatous Polyposis Coli↗

PubMed2001

Subcellular localization of the Snf1 kinase is regulated by specific beta subunits and a novel glucose signaling mechanism.

The Snf1/AMP-activated protein kinase family has broad roles in transcriptional, metabolic, and developmental regulation in response to stress. In Saccharomyces cerevisiae, Snf1 is required for the response to glucose limitation. Snf1 kinase complexes contain the alpha (catalytic) subunit Snf1, one of the three related beta subunits Gal83, Sip1, or Sip2, and the gamma subunit Snf4. We present evidence that the beta subunits regulate the subcellular localization of the Snf1 kinase. Green fluorescent protein fusions to Gal83, Sip1, and Sip2 show different patterns of localization to the nucleus, vacuole, and/or cytoplasm. We show that Gal83 directs Snf1 to the nucleus in a glucose-regulated manner. We further identify a novel signaling pathway that controls this nuclear localization in response to glucose phosphorylation. This pathway is distinct from the glucose signaling pathway that inhibits Snf1 kinase activity and responds not only to glucose but also to galactose and sucrose. Such independent regulation of the localization and the activity of the Snf1 kinase, combined with the distinct localization of kinases containing different beta subunits, affords versatility in regulating physiological responses.

AMP-Activated Protein Kinases↗

PubMed2001

Subcellular localization and characterization of chorismate synthase in the apicomplexan Plasmodium falciparum.

The resurgence of drug-resistant apicomplexa, in particular Plasmodium falciparum, the most fatal human malarial parasite, has focused attention on the recent discovery of the shikimate pathway in these organisms, as it may provide the urgently required, novel drug targets resulting from the absence of this pathway in mammals. The direction of a parasiticidal drug design programme obviously requires knowledge of the subcellular localization and indeed full characterization of the possible enzyme targets. Here, we report the cloning and characterization of chorismate synthase from P. falciparum and present the first biochemical and immunological studies of an enzyme of the shikimate pathway from an apicomplexan parasite. We show that this chorismate synthase does not possess an intrinsic flavin reductase activity and is therefore monofunctional like the plant and bacterial chorismate synthases. Highest immunological cross-reactivity was found with a plant chorismate synthase. However, in contrast to the plant enzyme, which is located to the plastid, P. falciparum chorismate synthase is found in the parasite cytosol, akin to the fungal enzymes that possess an intrinsic flavin reductase activity (i.e. are bifunctional). Thus, P. falciparum chorismate synthase has a combination of properties that distinguishes it from other described chorismate synthases.

Amino Acid Sequence↗

PubMed1994

Overexpressed protein kinase C-delta and -epsilon subtypes in NIH 3T3 cells exhibit differential subcellular localization and differential regulation of sodium-dependent phosphate uptake.

To examine the biological properties of protein kinase C (PKC)-delta and -epsilon NIH 3T3 cells were stably transfected with metallothionein-based expression vectors that overexpressed these isoforms. In addition to their inducibility by Zn2+, the protein levels of these two PKC subtypes, but not that of endogenous PKC-alpha, increased with increasing cell density. An unexpected role for Mg2+ in the subcellular localization of PKC-delta was found. This isoenzyme was predominantly membrane-associated when cell fractionation was carried out in the absence of Mg2+ but cytosolic when the fractionation was performed in the presence of 10 mM Mg2+. In contrast, the predominant localization of cytosolic PKC-alpha and of membrane-associated PKC-epsilon was not influenced by Mg2+. In vivo and in vitro studies of [3H]phorbol 12,13-dibutyrate binding in the overexpressing cell lines confirmed the cytosolic localization of PKC-alpha, the membrane-associated state of PKC-epsilon, and the presence of PKC-delta at both locations. Readdition of serum for 5 min to serum-starved, quiescent cell lines initiated the redistribution of PKC-alpha to the particulate fraction, while the location of PKC-delta and PKC-epsilon was not affected. Zn(2+)-induced overexpression of PKC-delta- and PKC-epsilon-stimulated sodium-dependent phosphate uptake. Overexpression of PKC-delta caused an increase in the Vmax of Na+/P(i) uptake, while overexpression of PKC-epsilon resulted in a decrease in Km for orthophosphate. A further stimulation of Na+/P(i) uptake in the overexpressing cells could be achieved by phorbol ester activation of endogenous PKC-alpha. These results suggest that each of the three PKC isotypes contribute to the regulation of sodium-dependent phosphate uptake, but through distinct mechanisms.

3T3 Cells↗

PubMed2002

Intracellular membrane trafficking pathways in bone-resorbing osteoclasts revealed by cloning and subcellular localization studies of small GTP-binding rab proteins.

A variety of intracellular membrane trafficking pathways are involved in establishing the polarization of resorbing osteoclasts and regulating bone resorption activities. Small GTP-binding proteins of rab family have been implicated as key regulators of membrane trafficking in mammalian cells. Here we used a RT-PCR-based cloning method and confocal laser scanning microscopy to explore the expression array and subcellular localization of rab proteins in osteoclasts. Rab1B, rab4B, rab5C, rab7, rab9, rab11B, and rab35 were identified from rat osteoclasts in this study. Rab5C may be associated with early endosomes, while rab11B is localized at perinuclear recycling compartments and may function in the ruffled border membrane turnover and osteoclast motility. Interestingly, late endosomal rabs, rab7, and rab9, were found to localize at the ruffled border membrane indicating a late endosomal nature of this specialized plasma membrane domain in resorbing osteoclasts. This also suggests that late endocytotic pathways may play an important role in the secretion of lysosomal enzymes, such as cathepsin K, during bone resorption.

Amino Acid Sequence↗

PubMed1982

Subcellular localization of the tRNA processing enzyme, tRNA nucleotidyltransferase, in Xenopus laevis oocytes and in somatic cells.

Earlier studies of the tRNA processing enzyme, tRNA nucleotidyltransferase, in rat liver cells suggested that the enzyme was absent from nuclei, whereas more recent studies with tRNA transcription-processing systems from Xenopus laevis oocytes indicated that the enzyme was present in germinal vesicles of these cells. In order to resolve this apparent discrepancy, the subcellular localization of tRNA nucleotidyltransferase in oocytes was directly determined, and its distribution in somatic cells was re-examined. Oocyte germinal vesicles, isolated manually, contained about 25% of the total tRNA nucleotidyltransferase activity of the cell, but the enzyme rapidly leaked out of these nuclei during storage. In contrast, purified nuclei isolated from rat or Xenopus liver using a variety of aqueous procedures were devoid of tRNA nucleotidyltransferase activity. Since rapidly-isolated, crude nuclear preparations from liver contained substantially more of the enzyme than could be accounted for by cytoplasmic contamination, it appears that the enzyme leaks out of the somatic cell nuclei during the lengthy times required for their purification. The implications of these results for the development of tRNA processing systems from somatic cell nuclei are discussed.

Animals↗

PubMed1993

Hydrophobic coiled-coil domains regulate the subcellular localization of human heat shock factor 2.

HSF2, one of the heat shock transcription factors in mammalian cells, is localized to the cytoplasm during normal growth and moves to the nucleus upon activation. Heat shock transcription factors in metazoans contain four hydrophobic heptad repeat sequences, three in the amino terminus and one in the carboxy terminus, which are predicted to form alpha-helical coiled-coil structures analogous to the leucine zipper. Here, we show that point mutations in either of two amino-terminal zippers or in the carboxy-terminal zipper disrupt normal localization of HSF2 and cause it to be constitutively nuclear. We demonstrate further that two sequences immediately surrounding the amino-terminal zipper domain are required for nuclear localization. These sequences fit the consensus for a bipartite nuclear localization signal (NLS). We suggest that interactions between the amino- and carboxy-terminal zippers normally mask the NLS sequences of HSF2 and that these interactions are disrupted upon activation to expose the NLS sequences and allow transport of HSF2 to the nucleus. We conclude that zipper domains can regulate subcellular localization.

Amino Acid Sequence↗