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Showing papers on "Cellular compartment published in 2010"


Journal ArticleDOI
02 Aug 2010-Small
TL;DR: Quantitative evaluation of gold nanoparticles exposed to human alveolar epithelial cells at the air-liquid interface reveals that NP surface coatings modulate endocytotic uptake pathways and cellular NP trafficking.
Abstract: This study addresses the cellular uptake and intracellular trafficking of 15-nm gold nanoparticles (NPs), either plain (i.e., stabilized with citrate) or coated with polyethylene glycol (PEG), exposed to human alveolar epithelial cells (A549) at the air-liquid interface for 1, 4, and 24 h. Quantitative analysis by stereology on transmission electron microscopy images reveals a significant, nonrandom intracellular distribution for both NP types. No particles are observed in the nucleus, mitochondria, endoplasmic reticulum, or golgi. The cytosol is not a preferred cellular compartment for both NP types, although significantly more PEG-coated than citrate-stabilized NPs are present there. The preferred particle localizations are vesicles of different sizes ( 1000 nm). This is observed for both NP types and indicates a predominant uptake by endocytosis. Subsequent inhibition of caveolin- and clathrin-mediated endocytosis by methyl-beta-cyclodextrin (MbetaCD) results in a significant reduction of intracellular NPs. The inhibition, however, is more pronounced for PEG-coated than citrate-stabilized NPs. The latter are mostly found in larger vesicles; therefore, they are potentially taken up by macropinocytosis, which is not inhibited by MbetaCD. With prolonged exposure times, both NPs are preferentially localized in larger-sized intracellular vesicles such as lysosomes, thus indicating intracellular particle trafficking. This quantitative evaluation reveals that NP surface coatings modulate endocytotic uptake pathways and cellular NP trafficking. Other nonendocytotic entry mechanisms are found to be involved as well, as indicated by localization of a minority of PEG-coated NPs in the cytosol.

325 citations


Journal ArticleDOI
TL;DR: In this article, the authors identify an essential function of the conserved cytosolic monothiol glutaredoxins Grx3 and Grx4 in intracellular iron trafficking and sensing.

259 citations


Journal ArticleDOI
TL;DR: This work highlights both fundamental similarities and important differences in the processes that lead to the modified membrane compartments in cells infected by distinct groups of positive-sense RNA viruses.
Abstract: Like other positive-strand RNA viruses, alphaviruses replicate their genomes in association with modified intracellular membranes. Alphavirus replication sites consist of numerous bulb-shaped membrane invaginations (spherules), which contain the double-stranded replication intermediates. Time course studies with Semliki Forest virus (SFV)-infected cells were combined with live-cell imaging and electron microscopy to reveal that the replication complex spherules of SFV undergo an unprecedented large-scale movement between cellular compartments. The spherules first accumulated at the plasma membrane and were then internalized using an endocytic process that required a functional actin-myosin network, as shown by blebbistatin treatment. Wortmannin and other inhibitors indicated that the internalization of spherules also required the activity of phosphatidylinositol 3-kinase. The spherules therefore represent an unusual type of endocytic cargo. After endocytosis, spherule-containing vesicles were highly dynamic and had a neutral pH. These primary carriers fused with acidic endosomes and moved long distances on microtubules, in a manner prevented by nocodazole. The result of the large-scale migration was the formation of a very stable compartment, where the spherules were accumulated on the outer surfaces of unusually large and static acidic vacuoles localized in the pericentriolar region. Our work highlights both fundamental similarities and important differences in the processes that lead to the modified membrane compartments in cells infected by distinct groups of positive-sense RNA viruses.

184 citations


Journal ArticleDOI
TL;DR: Proteomic studies reveal that, although many of the proteins found to associate with CLDs are connected to lipid metabolism, storage, and homeostasis, there are also proteins with no obvious connection to the classical function and typically associated with other cellular compartments.

144 citations


Journal ArticleDOI
TL;DR: Shifts in the distribution of GPD1p to different cellular compartments in response to changing cellular status suggests a role for Gpd1p in the spatial regulation of redox potential, a process critical to cell survival, especially under the complex stress conditions expected to occur in the wild.

82 citations


Journal ArticleDOI
TL;DR: This review focuses on the membrane recycling pathway and proteins that support the recruitment and redistribution of H,K-ATPase-rich membranes, including those involved in signal transduction, membrane targeting, docking, and fusing, in addition to the integral role of the actin cytoskeleton and its associated proteins in the process of membrane recycling.
Abstract: The gastric parietal cell was the first system where a regulated membrane recycling hypothesis was proposed as the principal means for moving molecular transporters between cellular compartments. Upon stimulation, massive membrane flow from an endosomal compartment of tubulovesicle membranes to the apical secretory surface places the ATP-driven pumps in position to secrete a solution of strong acid in collaboration with several other membrane transporters. This review focuses on the membrane recycling pathway and proteins that support the recruitment and redistribution of H,K-ATPase-rich membranes, including those involved in signal transduction, membrane targeting, docking, and fusing, in addition to the integral role of the actin cytoskeleton and its associated proteins in the process of membrane recycling. Although much of the evidence discussed here comes from parietal cell studies, other physiological transport systems, as well as less complex cellular and in vitro models, are examined and cited for generality of principle.

63 citations


Journal Article
TL;DR: The mechanisms of intracellular CYP transport, and the implications of the presence of CYP proteins in extra-ER compartments for drug metabolism and toxicity are discussed.
Abstract: Cytochrome P450 (CYP) is a large family of well-conserved integral membrane proteins localized primarily in the membrane of the endoplasmic reticulum (ER), where these enzymes metabolize a variety of both endogenous and exogenous compounds. It has become apparent that these microsomal CYP proteins are also present in other cellular compartments, such as the cell surface and in mitochondria, where the enzymes display catalytic activity toward CYP-specific substrates, in some cases with altered substrate specificity. CYP-drug adducts exposed at the cell surface are important mediators of idiosyncratic drug toxicities. Therefore, understanding the molecular mechanisms responsible for directing these microsomal CYPs to other, non-ER cellular compartments is important. These alternatively localized CYPs should be considered as possible drug targets and as important factors during drug discovery and development, as the detoxification capacity is lower in the compartments where such CYP proteins are located. This review discusses the mechanisms of intracellular CYP transport, and the implications of the presence of CYP proteins in extra-ER compartments for drug metabolism and toxicity.

62 citations


Journal ArticleDOI
04 Feb 2010-Langmuir
TL;DR: This single vesicle-based assay of a large number of liposomes enabled us to examine the volume dependence of enclosed reactions in detail, revealing that the presence of specific lipid affected the specific kinetic parameters of encapsulated reactions.
Abstract: One of the important characteristics of the cellular system is that interactions between the plasma membrane and water-soluble molecules in the cytoplasm are enhanced by the confinement of the molecules to the small volume of the intracellular space. Studying this effect in a model cell system, we measured the time evolution of an enzymatic hydrolysis reaction and a cell-free protein synthesis reaction taking place in giant liposomes having various size and phospholipid compositions by a flow cytometry. This single vesicle-based assay of a large number of liposomes enabled us to examine the volume dependence of enclosed reactions in detail, revealing that the presence of specific lipid affected the specific kinetic parameters of encapsulated reactions.

59 citations


Journal ArticleDOI
TL;DR: The first in vivo use of these novel roGFP1 variants for the measurement of redox conditions within the ER and cytosol in the yeast Pichia pastoris is reported, and it is shown that the ER, which is required for oxidative protein folding, is indeed more oxidizing than the cytOSol.
Abstract: Methods for in vivo monitoring of redox changes in different cellular compartments have been developed in recent years, and are mostly based on redox-sensitive variants of the green fluorescent protein (GFP). However, due to the thermodynamic stability of the introduced reactive disulfide bond, these sensors are limited to reducing compartments such as the cytosol and the mitochondria, and are not suited for more oxidizing environments such as the endoplasmic reticulum (ER). To overcome this problem, a family of redox-sensitive GFP variants that differed in their midpoint potential has been developed by the group of Remington (University of Oregon) and tested in vitro. Here, we report the first in vivo use of these novel roGFP1 variants for the measurement of redox conditions within the ER and cytosol in the yeast Pichia pastoris. With the fluorescence data obtained, it was possible to determine the reduction potential of the two compartments. Thereby, we could show that the ER, which is required for oxidative protein folding, is indeed more oxidizing than the cytosol. Contrary to previous results with roGFP, the optimized roGFP1_iE and roGFP1_iL constructs were not completely oxidized, and are therefore useful sensors for monitoring the ER under conditions when it is even more oxidized.

52 citations


Journal ArticleDOI
29 Nov 2010-PLOS ONE
TL;DR: This work has identified a novel binding partner, translin-associated factor X (TRAX), a cytosolic protein that can transit into the nucleus that competes with plasma-membrane bound Gαq for PLC β1 binding thus stabilizing PLCβ1 in other cellular compartments.
Abstract: Mammalian phospholipase Cβ1 (PLCβ1) is activated by the ubiquitous Gαq family of G proteins on the surface of the inner leaflet of plasma membrane where it catalyzes the hydrolysis of phosphatidylinositol 4,5 bisphosphate. In general, PLCβ1 is mainly localized on the cytosolic plasma membrane surface, although a substantial fraction is also found in the cytosol and, under some conditions, in the nucleus. The factors that localize PLCβ1in these other compartments are unknown. Here, we identified a novel binding partner, translin-associated factor X (TRAX). TRAX is a cytosolic protein that can transit into the nucleus. In purified form, PLCβ1 binds strongly to TRAX with an affinity that is only ten-fold weaker than its affinity for its functional partner, Gαq. In solution, TRAX has little effect on the membrane association or the catalytic activity of PLCβ1. However, TRAX directly competes with Gαq for PLCβ1 binding, and excess TRAX reverses Gαq activation of PLCβ1. In C6 glia cells, endogenous PLCβ1 and TRAX colocalize in the cytosol and the nucleus, but not on the plasma membrane where TRAX is absent. In Neuro2A cells expressing enhanced yellow and cyano fluorescent proteins (i.e., eYFP- PLCβ1 and eCFP-TRAX), Forster resonance energy transfer (FRET) is observed mostly in the cytosol and a small amount is seen in the nucleus. FRET does not occur at the plasma membrane where TRAX is not found. Our studies show that TRAX, localized in the cytosol and nucleus, competes with plasma-membrane bound Gαq for PLCβ1 binding thus stabilizing PLCβ1 in other cellular compartments.

45 citations


Journal ArticleDOI
02 Sep 2010-PLOS ONE
TL;DR: It is suggested that lipid and apolipoprotein moieties of oxLDL-IC traffic to separate cellular compartments, and that HSP70/70B' might sequester the lipid moiety of oxPLD-IC in the endosomal compartment, which could ultimately influence macrophage function and survival.
Abstract: Background Oxidized low-density lipoproteins (oxLDL) and oxLDL-containing immune complexes (oxLDL-IC) contribute to formation of lipid-laden macrophages (foam cells). It has been shown that oxLDL-IC are considerably more efficient than oxLDL in induction of foam cell formation, inflammatory cytokines secretion, and cell survival promotion. Whereas oxLDL is taken up by several scavenger receptors, oxLDL-IC are predominantly internalized through the FCγ receptor I (FCγ RI). This study examined differences in intracellular trafficking of lipid and apolipoprotein moieties of oxLDL and oxLDL-IC and the impact on oxidative stress. Methodology/Findings Fluorescently labeled lipid and protein moieties of oxLDL co-localized within endosomal and lysosomal compartments in U937 human monocytic cells. In contrast, the lipid moiety of oxLDL-IC was detected in the endosomal compartment, whereas its apolipoprotein moiety advanced to the lysosomal compartment. Cells treated with oxLDL-IC prior to oxLDL demonstrated co-localization of internalized lipid moieties from both oxLDL and oxLDL-IC in the endosomal compartment. This sequential treatment likely inhibited oxLDL lipid moieties from trafficking to the lysosomal compartment. In RAW 264.7 macrophages, oxLDL-IC but not oxLDL induced GFP-tagged heat shock protein 70 (HSP70) and HSP70B', which co-localized with the lipid moiety of oxLDL-IC in the endosomal compartment. This suggests that HSP70 family members might prevent the degradation of the internalized lipid moiety of oxLDL-IC by delaying its advancement to the lysosome. The data also showed that mitochondrial membrane potential was decreased and generation of reactive oxygen and nitrogen species was increased in U937 cell treated with oxLDL compared to oxLDL-IC. Conclusions/Significance Findings suggest that lipid and apolipoprotein moieties of oxLDL-IC traffic to separate cellular compartments, and that HSP70/70B' might sequester the lipid moiety of oxLDL-IC in the endosomal compartment. This mechanism could ultimately influence macrophage function and survival. Furthermore, oxLDL-IC might regulate the intracellular trafficking of free oxLDL possibly through the induction of HSP70/70B'.

Journal ArticleDOI
TL;DR: Results show that TeA causing cell necrosis of host-plants is a result of direct oxidative damage from chloroplast-mediated ROS eruption.

Journal ArticleDOI
TL;DR: The functional role of FAT/CD36 in lipid transport seems primarily to be allocated to the plasma membrane in skeletal muscle, whereas the mitochondrial marker F1ATPase-β was clearly detected using immunoblotting.

Journal ArticleDOI
TL;DR: It appears that proteins already well known for an ROS-unrelated specific function in one compartment participate in the ROS response within another compartment, which may represent an important mechanism of intracellular communication between different cellular compartments.
Abstract: With the appearance of oxygen and the development of aerobic life on earth, reactive oxygen species (ROS) became important factors influencing a number of processes within a cell. Although initially considered unwanted and harmful by-products of a number of cellular reactions, the last decade has shown that ROS can also act as signalling molecules mediating changes in O2 tension, as well as the response to hormones, growth factors, and mechanical or chemical stress. Different ROS-generating and ROS-degrading systems in different intracellular compartments seem to play an important role. In line with this, it appears that proteins already well known for an ROS-unrelated specific function in one compartment participate in the ROS response within another compartment. Thus, it is easy to envision that redox changes in different compartments and resulting changes in ROS levels may represent an important mechanism of intracellular communication between different cellular compartments. Antioxid. Redox S...

Journal ArticleDOI
TL;DR: The important functional role of pan-sodium channel and isoform-specific partners in regulating sodium current density and gating properties is reviewed.

Journal ArticleDOI
TL;DR: The picture emerging is that pathogenic challenge triggers a complex network of lipid hydrolysis events across the cellular compartments resulting in changes in membrane structures and release of signal precursors involved in the building-up of an adequate immune response.
Abstract: Lipid acyl hydrolases (LAH) have received recently increased attention in the context of plant defense. Multiple structurally unrelated gene families have been annotated in Arabidopsis as encoding potential lipid deacylating enzymes with numerous members being transcriptionally activated upon biotic stress. Confirming in silico predictions, experimental data have illustrated the wide subcellular distribution of LAHs indicating they likely interact with distinct membrane systems to initiate specific cellular responses. While recombinant LAHs are active in vitro on a small set of polar lipids, precise knowledge of in vivo substrates and hydrolysis products is generally lacking. Functional analysis of a few LAHs has revealed their roles in initiating oxylipin biosynthesis, cell death execution, signalling or direct antimicrobial activity. The picture emerging is that pathogenic challenge triggers a complex network of lipid hydrolysis events across the cellular compartments resulting in changes in membrane st...

Journal ArticleDOI
TL;DR: Unlike chalcone 1, the combretastatin A4 analogous platinum complex 2 is highly cell line specific, is taken up via cell-controlled transporters and induces apoptosis by triggering multiple targets.

Journal ArticleDOI
TL;DR: A specific isoform of kinesin‐1 is required for microtubule‐dependent recruitment of Na,K‐ATPase to the plasma membrane, which is of physiological significance.
Abstract: Recruitment of the Na,K-ATPase to the plasma membrane of alveolar epithelial cells results in increased active Na+ transport and fluid clearance in a process that requires an intact microtubule network. However, the microtubule motors involved in this process have not been identified. In the present report, we studied the role of kinesin-1, a plus-end microtubule molecular motor that has been implicated in the movement of organelles in the Na,K-ATPase traffic. We determined by confocal microscopy and biochemical assays that kinesin-1 and the Na,K-ATPase are present in the same membranous cellular compartment. Knockdown of kinesin-1 heavy chain (KHC) or the light chain-2 (KLC2), but not of the light chain-1 (KLC1), decreased the movement of Na,K-ATPase-containing vesicles when compared to sham siRNA-transfected cells (control group). Thus, a specific isoform of kinesin-1 is required for microtubule-dependent recruitment of Na,K-ATPase to the plasma membrane, which is of physiological significance—Trejo, H....

Journal ArticleDOI
TL;DR: The N-terminal sequence of the soluble mitochondrial matrix Grx2 is determined by mass spectrometry and the exact cleavage site by Mitochondrial Processing Peptidase is determined, showing that evolutionary diversification of GrX2 structural and functional properties are strictly dependent on the sequences of the targeting signal peptide.

Journal ArticleDOI
TL;DR: It is shown that DRAQ5 intercalation rapidly modifies both the localization and the mobility properties of several DNA-binding proteins such as histones, DNA repair, replication and transcription factors, by stimulating a release of these proteins from their substrate.

01 Oct 2010
TL;DR: The study uncovers an important role of monothiol glutaredoxins in cellular iron metabolism, with a surprising connection to cellular redox and sulfur metabolisms.
Abstract: Iron is an essential nutrient for cells. It is unknown how iron, after its import into the cytosol, is specifically delivered to iron-dependent processes in various cellular compartments. Here, we identify an essential function of the conserved cytosolic monothiol glutaredoxins Grx3 and Grx4 in intracellular iron trafficking and sensing. Depletion of Grx3/4 specifically impaired all iron-requiring reactions in the cytosol, mitochondria, and nucleus, including the synthesis of Fe/S clusters, heme, and di-iron centers. These defects were caused by impairment of iron insertion into proteins and iron transfer to mitochondria, indicating that intracellular iron is not bioavailable, despite highly elevated cytosolic levels. The crucial task of Grx3/4 is mediated by a bridging, glutathione-containing Fe/S center that functions both as an iron sensor and in intracellular iron delivery. Collectively, our study uncovers an important role of monothiol glutaredoxins in cellular iron metabolism, with a surprising connection to cellular redox and sulfur metabolisms.

Journal ArticleDOI
TL;DR: In this paper, the authors used adenovirus-mediated gene transfer of the two transgenes in neonatal rat cardiac myocytes, assessed cellular distribution and performed selected biochemical assays.

Journal ArticleDOI
TL;DR: Experimental evidence obtained supports the model of the GS/GOGAT cycle in developing pine seedlings that accounts for the stoichiometric balance of metabolites.
Abstract: In higher plants, ammonium is assimilated into amino acids through the glutamine synthetase (GS)⁄glutamate synthase (GOGAT) cycle. This metabolic cycle is distributed in different cellular compartments in conifer seedlings: glutamine synthesis occurs in the cytosol and glutamate synthesis within the chloroplast. A method for preparing intact chloroplasts of pine cotyledons is presented with the aim of identifying a glutamine‐glutamate translocator. Glutamine‐glutamate exchange has been studied using the double silicone layer system, suggesting the existence of a translocator that imports glutamine into the chloroplast and exports glutamate to the cytoplasm. The translocator identified is specific for glutamine and glutamate, and the kinetic constants for both substrates indicate that it is unsaturated at intracellular concentrations. Thus, the experimental evidence obtained supports the model of the GS⁄GOGAT cycle in developing pine seedlings that accounts for the stoichiometric balance of metabolites. As a result, the efficient assimilation of free ammonia produced by photorespiration, nitrate reduction, storage protein mobilisation, phenylpropanoid pathway or S-adenosylmethionine synthesis is guaranteed.

Book ChapterDOI
01 Jan 2010
TL;DR: The aim of this chapter is to provide a review of the current literature as it pertains to Entamoeba histolytica organelles, protein transport, and nutrient uptake, specifically by examining the Golgi, ER, mitosome, phagocyTosis, and endocytosis.
Abstract: Publication of the Entamoeba histolytica genome has provided strong support for cell biology studies on this lower eukaryote, in which clear cellular compartments involving vesicular trafficking have not yet been well established. Protein trafficking through the endoplasmic reticulum and the Golgi apparatus is an important issue linked to pathogenesis due to the important number of factors secreted by this parasite to accomplish cell killing, tissue invasion, and the onset of inflammation in humans. The advancements in the cellular and molecular tools available have allowed previously unidentifiable organelles to be identified based on gene homologies and video microscopy. The aim of this chapter is to provide a review of the current literature as it pertains to Entamoeba histolytica organelles, protein transport, and nutrient uptake, specifically by examining the Golgi, ER, mitosome, phagocytosis, and endocytosis.

Book ChapterDOI
01 Jan 2010
TL;DR: This chapter emphasizes the key stress signals in various cellular compartments and focuses on current knowledge of the sensing mechanisms of the mitochondria, a crucial center for stress detection and processing of stress signals arising in other cellular compartment.
Abstract: Publisher Summary This chapter emphasizes the key stress signals in various cellular compartments and focuses on current knowledge of the sensing mechanisms. The mitochondria are a crucial center for stress detection and processing of stress signals arising in other cellular compartments. Mitochondria activate apoptosis in response to external signals or stresses detected within the mitochondrion. The p53 in the cytosol can localize to the mitochondria and instigate apoptosis. Metabolic and other stresses, such as ionizing and some types of ultraviolet (UV) radiation may increase the mitochondrial reactive nitrogen species (ROS) production rate beyond the detoxification capacity. Elevated metabolic activity increases electron leakage from complex I and subsequent ROS production. The ER is where secretory proteins are processed, a variety of posttranslational modifications are made, and it is the site of intracellular Ca 2+ storage. Stresses affecting Ca 2+ levels or the redox balance are the most notable ER stress signals that impact protein folding. Ca 2+ is essential for a number of enzymes, including some involved in protein processing. Signal transduction can occur through precise protein to protein relays, or through less specific mechanisms where the concentration of a molecule is changed and sensors respond to the change in concentration gradient. The archetypical protein based method for conveying a highly specific signal is through phosphorelays where proteins pass a phosphate through a transduction pathway. Protein modification is a major mechanism for stress signal propagation within the cell and other methods that proteins use for passing messages includes acetylation, ISGylation, nitrosylation, ROS generation, and sumolyation.

Book ChapterDOI
01 Jan 2010
TL;DR: The data strongly argue for a major involvement of autophagy in the pathogenesis of NCL, although it remains largely unknown what signaling is essential for autophagosome formation.
Abstract: Autophagy is a mechanism by which parts of a cell that are old and unneeded are segregated inside structures called “autophagosomes”. The materials ingested by this autophagy are brought to cellular compartments called “lysosomes,” which are specific intracellular compartments for degradation, and the degraded products are re-used for cell metabolism. We have shown that, in mice, deficiency in lysosomal proteinases such as cathepsin D or cathepsins B and L induces the accumulation of lysosomes containing ceroid-lipofuscin; the phenotypes of these mice resemble those of neuronal ceroid lipofuscinosis (NCL). In these mutant mice, the accumulation of abnormal lysosomal structures appears in accordance with an increase in the amount of membrane-bound microtubule associated protein 1 light chain 3 (LC3), a marker of “autophagosomes” in neurons. Such autophagosomes often contain granular osmiophilic deposits, a hallmark of NCL, together with part of the cytoplasm, which contains undigested materials. These data strongly argue for a major involvement of autophagy in the pathogenesis of NCL, although it remains largely unknown what signaling is essential for autophagosome formation.

Journal ArticleDOI
TL;DR: Experimental evidence is provided to indicate that micronuclei arising from the genomic instability inherent to cancer cells may copurify with plasma membrane fractions on sucrose gradients and mass spectrometric analysis confirmed the presence of nuclear proteins as well as membrane and associated signaling proteins in sucrose gradient-enriched preparations.

Dissertation
16 Jun 2010
TL;DR: The presented work aimed to identify the mechanisms underlying the intracellular localization of the SUMO E1 complex and suggest that thioester charged Ubc9 may shuttle to and allow efficient SUMOylation both in the nuclear and cytoplasmic compartment.
Abstract: Dynamic posttranslational modification with ubiquitin related proteins of the SUMO (small ubiquitin-related modifier) family is an important cellular mechanism to alter the activity, abundance or localization of proteins. Reversible covalent attachment of SUMO to target proteins requires the catalytic activities of an E1-activating enzyme, an E2-conjugating enzyme, one of several E3 ligases as well as specific isopeptidases. Consistant with the existence of nuclear and cytoplasmic SUMO substrates, the components of the enzymatic machinery are also found in both compartments. This raises the interesting question how these pools are generated. The presented work aimed to identify the mechanisms underlying the intracellular localization of the SUMO E1 complex. Since both subunits, Aos1 and Uba2, are predominantly localized in the nucleus, I initially determined the generation of the nuclear pool. I demonstrated that nuclear import can occur in two ways: Aos1 and Uba2 can be imported independently by distinct nuclear localization signals (NLSs), and the assembled complex can be imported by the NLS of Uba2. In both cases the import is mediated by the receptor importin beta and the adaptor protein importin alpha. Functional studies concerning the generation of the minor cytoplasmic pool of Aos1/Uba2 indicated that the proteins are not actively exported from the nucleus into the cytoplasm. Since the nuclear and cytoplasmic pools of E1 are not subjected to frequent exchange and the cytoplasmic fraction of E1 is very small I reasoned that the SUMO activating acitvity in the cytoplasm would be constantly low. This raised the question whether the E1 s intracellular localization is at all of general importance for SUMOylation in different cellular compartments. With yeast UBA2 shuffle strains, in which the endogenous UBA2 gene is deleted and cells are kept alive by a removable exogenous copy, I studied the ability of cytoplasmic E1 to substitute for endogenous E1. Surprisingly, deletion of endogenous E1 can be rescued both by exogenous cytoplasmic or nuclear E1. Yeast strains with either cytoplasmic or nuclear E1 exhibit similar modification patterns, indicating that the localization of E1 might not determine compartment specific SUMOylation. These findings suggest that thioester charged Ubc9 may shuttle to and allow efficient SUMOylation both in the nuclear and cytoplasmic compartment.

Book ChapterDOI
01 Jan 2010
TL;DR: It must be recognized that metabolism is not a subject to be ignored as history, but metabolism and bioenergetics are indispensable aspects of the expression of genetic information, development and differentiation, and ultimately the phenomenon of senescence and death.
Abstract: Publisher Summary This chapter focuses on signals originating from mitochondria that are primarily small molecules with targets in the cytosol and the nucleus. It begins with a discussion of some general themes in the current literature. Following this, it presents few examples of specific signaling pathways, where the understanding has advanced significantly in recent years. The small molecules (ATP/ADP/AMP, NAD+/NADH, GSH/GSSG) that are continuously turned over in the cytosol and in the mitochondrial matrix are now recognized to be not only cofactors driving endergonic reactions, or donors/acceptors of electrons in redox reactions. In combination with Ca+2, NO, cAMP, and cGMP they serve as allosteric effectors and in feedback mechanisms to control metabolic flux through numerous pathways, and they can also act as powerful elements in the modulation of gene expression. Major players involved in signaling pathways leading to transcriptional regulation have been identified: AMPK, PGC-1, PPARγ, and the sirtuins. Finally, the study states that it must be recognized that metabolism is not a subject to be ignored as history, but metabolism and bioenergetics are indispensable aspects of the expression of genetic information, development and differentiation, and ultimately the phenomenon of senescence and death. A challenge of the future is to measure accurate concentrations of each of these components in the various cellular compartments, to observe absolute and relative changes, and to incorporate all of these reactions into a computer model.

Book ChapterDOI
01 Jan 2010
TL;DR: In cases when the antigens of interest are located in different cellular compartments or in different cells, one should use immunofluorescent labeling with fluorophore-conjugated antibodies (see Chap. 8), which is inapplicable for co-localization of a pair of antigen located in the same cellular compartment.
Abstract: Immunoenzyme staining can be utilized for simultaneous localization of two or more tissue antigens in cases when the antigens of interest are located in different cellular compartments or in different cells. This approach is, however, inapplicable for co-localization of a pair of antigens located in the same cellular compartment, because of massive deposits of chromogen molecules covering the antigen targeted by the enzyme marker. This renders the second antigen of interest inaccessible for the second primary antibody, and makes the targeting of a pair of antigens in the same cellular compartment (nucleus, membrane, cytoplasm) impossible. In such situations, one should use immunofluorescent labeling with fluorophore-conjugated antibodies (see Chap. 8).