Статті в журналах: "Cytosolic export"

1

Banerjee, Tuhina, Lucia Cilenti, Michael Taylor, Adrienne Showman, Suren A. Tatulian, and Ken Teter. "Thermal Unfolding of the Pertussis Toxin S1 Subunit Facilitates Toxin Translocation to the Cytosol by the Mechanism of Endoplasmic Reticulum-Associated Degradation." Infection and Immunity 84, no. 12 (September 19, 2016): 3388–98. http://dx.doi.org/10.1128/iai.00732-16.

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Pertussis toxin (PT) moves from the host cell surface to the endoplasmic reticulum (ER) by retrograde vesicular transport. The catalytic PTS1 subunit dissociates from the rest of the toxin in the ER and then shifts to a disordered conformation which may trigger its export to the cytosol through the quality control mechanism of ER-associated degradation (ERAD). Functional roles for toxin instability and ERAD in PTS1 translocation have not been established. We addressed these issues with the use of a surface plasmon resonance system to quantify the cytosolic pool of PTS1 from intoxicated cells. Only 3% of surface-associated PTS1 reached the host cytosol after 3 h of toxin exposure. This represented, on average, 38,000 molecules of cytosolic PTS1 per cell. Cells treated with a proteasome inhibitor contained larger quantities of cytosolic PTS1. Stabilization of the dissociated PTS1 subunit with chemical chaperones inhibited toxin export to the cytosol and blocked PT intoxication. ERAD-defective cell lines likewise exhibited reduced quantities of cytosolic PTS1 and PT resistance. These observations identify the unfolding of dissociated PTS1 as a trigger for its ERAD-mediated translocation to the cytosol.

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2

Koszinowski, U. "Emptying pandora's box: cytosolic export and MHC degradation." Trends in Microbiology 4, no. 9 (September 1996): 338–39. http://dx.doi.org/10.1016/0966-842x(96)30025-5.

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3

Kehlenbach, Ralph H., Achim Dickmanns, and Larry Gerace. "Nucleocytoplasmic Shuttling Factors Including Ran and CRM1 Mediate Nuclear Export of NFAT In Vitro." Journal of Cell Biology 141, no. 4 (May 18, 1998): 863–74. http://dx.doi.org/10.1083/jcb.141.4.863.

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We have developed a permeabilized cell assay to study the nuclear export of the shuttling transcription factor NFAT, which contains a leucine-rich export signal. The assay uses HeLa cells that are stably transfected with NFAT fused to the green fluorescent protein (GFP). Nuclear export of GFP–NFAT in digitonin-permeabilized cells occurs in a temperature- and ATP-dependent manner and can be quantified by flow cytometry. In vitro NFAT export requires the GTPase Ran, which is released from cells during the digitonin permeabilization. At least one additional rate-limiting export factor is depleted from permeabilized cells by a preincubation at 30°C in the absence of cytosol. This activity can be provided by cytosolic or nucleoplasmic extracts in a subsequent export step. Using this assay, we have purified a second major export activity from cytosol. We found that it corresponds to CRM1, a protein recently reported to be a receptor for certain leucine-rich export sequences. CRM1 appears to be imported into the nucleus by a Ran-dependent mechanism that is distinct from conventional signaling pathways. Considered together, our studies directly demonstrate by fractionation and reconstitution that nuclear export of NFAT is mediated by multiple nucleocytoplasmic shuttling factors, including Ran and CRM1.

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4

Pandey, Alok, Jayashree Pain, Nathaniel Dziuba, Ashutosh K. Pandey, Andrew Dancis, Paul A. Lindahl, and Debkumar Pain. "Mitochondria Export Sulfur Species Required for Cytosolic tRNA Thiolation." Cell Chemical Biology 25, no. 6 (June 2018): 738–48. http://dx.doi.org/10.1016/j.chembiol.2018.04.002.

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5

Ehrnsberger, Hans F., Marion Grasser, and Klaus D. Grasser. "Nucleocytosolic mRNA transport in plants: export factors and their influence on growth and development." Journal of Experimental Botany 70, no. 15 (April 11, 2019): 3757–63. http://dx.doi.org/10.1093/jxb/erz173.

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AbstractIn eukaryotes, the regulated transport of mRNAs from the cell nucleus to the cytosol is a critical step in the expression of protein-coding genes, as it links nuclear mRNA synthesis with cytosolic translation. The pre-mRNAs that are synthesised by RNA polymerase II are processed by 5´-capping, splicing, and 3´-polyadenylation. The multi-subunit THO/TREX complex integrates mRNA biogenesis with their nucleocytosolic transport. Various export factors are recruited to the mRNAs during their maturation, which occurs essentially co-transcriptionally. These RNA-bound export factors ensure efficient transport of the export-competent mRNAs through nuclear pore complexes. In recent years, several factors involved in plant mRNA export have been functionally characterised. Analysis of mutant plants has demonstrated that impaired mRNA export causes defects in growth and development. Moreover, there is accumulating evidence that mRNA export can influence processes such as plant immunity, circadian regulation, and stress responses. Therefore, it is important to learn more details about the mechanism of nucleocytosolic mRNA transport in plants and its physiological significance.

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6

Holaska, James M., Ben E. Black, Dona C. Love, John A. Hanover, John Leszyk, and Bryce M. Paschal. "Calreticulin Is a Receptor for Nuclear Export." Journal of Cell Biology 152, no. 1 (January 8, 2001): 127–40. http://dx.doi.org/10.1083/jcb.152.1.127.

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In previous work, we used a permeabilized cell assay that reconstitutes nuclear export of protein kinase inhibitor (PKI) to show that cytosol contains an export activity that is distinct from Crm1 (Holaska, J.M., and B.M. Paschal. 1995. Proc. Natl. Acad. Sci. USA. 95: 14739–14744). Here, we describe the purification and characterization of the activity as calreticulin (CRT), a protein previously ascribed to functions in the lumen of the ER. We show that cells contain both ER and cytosolic pools of CRT. The mechanism of CRT-dependent export of PKI requires a functional nuclear export signal (NES) in PKI and involves formation of an export complex that contains RanGTP. Previous studies linking CRT to downregulation of steroid hormone receptor function led us to examine its potential role in nuclear export of the glucocorticoid receptor (GR). We found that CRT mediates nuclear export of GR in permeabilized cell, microinjection, and transfection assays. GR export is insensitive to the Crm1 inhibitor leptomycin B in vivo, and it does not rely on a leucine-rich NES. Rather, GR export is facilitated by its DNA-binding domain, which is shown to function as an NES when transplanted to a green fluorescent protein reporter. CRT defines a new export pathway that may regulate the transcriptional activity of steroid hormone receptors.

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7

Tolerico, Leslie H., Ann L. Benko, John P. Aris, David R. Stanford, Nancy C. Martin, and Anita K. Hopper. "Saccharomyces cerevisiae Mod5p-II Contains Sequences Antagonistic for Nuclear and Cytosolic Locations." Genetics 151, no. 1 (January 1, 1999): 57–75. http://dx.doi.org/10.1093/genetics/151.1.57.

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Abstract MOD5 encodes a tRNA modification activity located in three subcellular compartments. Alternative translation initiation generates Mod5p-I, located in the mitochondria and the cytosol, and Mod5p-II, located in the cytosol and nucleus. Here we study the nucleus/cytosol distribution of overexpressed Mod5p-II. Nuclear Mod5p-II appears concentrated in the nucleolus, perhaps indicating that the nuclear pool may have a different biological role than the cytoplasmic and mitochondrial pools. Mod5p contains three motifs resembling bipartite-like nuclear localization sequences (NLSs), but only one is sufficient to locate a passenger protein to the nucleus. Mutations of basic residues of this motif cumulatively contribute to a cytosolic location for the fusion proteins. These alterations also cause decreased nuclear pools of endogenous Mod5p-II. Depletion of nuclear Mod5p-II does not affect tRNATyr function. Despite the NLS, most Mod5p is cytosolic. We assessed whether Mod5p sequences cause a karyophilic reporter to be located in the cytosol. By this assay, Mod5p may contain more than one region that functions as cytoplasmic retention and/or nuclear export sequences. Thus, distribution of Mod5p results from the presence/absence of mitochondrial targeting information and sequences antagonistic for nuclear and cytosolic locations. Mod5p is highly conserved; sequences responsible for subcellular distribution appear to reside in “accessory” motifs missing from prokaryotic counterparts.

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8

Ong, Yan Shan, Bor Luen Tang, Li Shen Loo, and Wanjin Hong. "p125A exists as part of the mammalian Sec13/Sec31 COPII subcomplex to facilitate ER-Golgi transport." Journal of Cell Biology 190, no. 3 (August 2, 2010): 331–45. http://dx.doi.org/10.1083/jcb.201003005.

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Coat protein II (COPII)–mediated export from the endoplasmic reticulum (ER) involves sequential recruitment of COPII complex components, including the Sar1 GTPase, the Sec23/Sec24 subcomplex, and the Sec13/Sec31 subcomplex. p125A was originally identified as a Sec23A-interacting protein. Here we demonstrate that p125A also interacts with the C-terminal region of Sec31A. The Sec31A-interacting domain of p125A is between residues 260–600, and is therefore a distinct domain from that required for interaction with Sec23A. Gel filtration and immunodepletion studies suggest that the majority of cytosolic p125A exists as a ternary complex with the Sec13/Sec31A subcomplex, suggesting that Sec 13, Sec31A, and p125A exist in the cytosol primarily as preassembled Sec13/Sec31A/p125A heterohexamers. Golgi morphology and protein export from the ER were affected in p125A-silenced cells. Our results suggest that p125A is part of the Sec13/Sec31A subcomplex and facilitates ER export in mammalian cells.

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9

Duerden, J. M., and G. F. Gibbons. "Storage, mobilization and secretion of cytosolic triacylglycerol in hepatocyte cultures. The role of insulin." Biochemical Journal 272, no. 3 (December 15, 1990): 583–87. http://dx.doi.org/10.1042/bj2720583.

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Cytosolic triacylglycerol labelled from [3H]oleate accounted for almost 50% (57 +/- 22 nmol/mg of protein) of the total cellular triacylglycerol which was newly synthesized by cultured hepatocytes during a 24 h incubation. Insulin decreased the export of triacylglycerol as very-low-density lipoprotein (VLDL) during this period. This resulted in a sequestration of newly synthesized triacylglycerol in the cytosol, rather than in the particulate fraction of the cell. Longer periods of incubation with [3H]oleate resulted in increased concentrations of newly synthesized triacylglycerol within the cell, most of which (78 +/- 3% after 48 h; 80 +/- 3% after 72 h) was located within the cytosolic fraction. The quantity of newly synthesized triacylglycerol in the cell cytosol was further increased by insulin. During these periods there were decreases in the amounts of triacylglycerol associated with the particulate fraction of the cell, irrespective of the presence or absence of insulin. In no case was a decrease in VLDL triacylglycerol secretion in response to insulin accompanied by an increased triacylglycerol content in the particulate fraction of the cell. In some experiments, the fate of the cytosolic triacylglycerol was studied by pulse labelling with [3H]oleate. In these cases, when insulin was removed from the medium of cells to which they had previously been exposed, more newly synthesized triacylglycerol was secreted compared with cells which had not been exposed to insulin. This extra triacylglycerol was mobilized from the cytosolic rather than from the particulate fraction of the cell. Subsequent addition of insulin to the medium prevented the mobilization of cytosolic triacylglycerol. These results suggest that insulin enhances the storage of hepatocellular triacylglycerol in a cytosolic pool. Deficiency of insulin in the medium stimulates the mobilization of this pool which is channelled into the secretory pathway, entering the extracellular medium as VLDL.

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10

Basu, Somsuvro, Joanne C. Leonard, Nishal Desai, Despoina A. I. Mavridou, Kong Ho Tang, Alan D. Goddard, Michael L. Ginger, Julius Lukeš, and James W. A. Allen. "Divergence of Erv1-Associated Mitochondrial Import and Export Pathways in Trypanosomes and Anaerobic Protists." Eukaryotic Cell 12, no. 2 (December 21, 2012): 343–55. http://dx.doi.org/10.1128/ec.00304-12.

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ABSTRACT In yeast ( Saccharomyces cerevisiae ) and animals, the sulfhydryl oxidase Erv1 functions with Mia40 in the import and oxidative folding of numerous cysteine-rich proteins in the mitochondrial intermembrane space (IMS). Erv1 is also required for Fe-S cluster assembly in the cytosol, which uses at least one mitochondrially derived precursor. Here, we characterize an essential Erv1 orthologue from the protist Trypanosoma brucei (TbERV1), which naturally lacks a Mia40 homolog. We report kinetic parameters for physiologically relevant oxidants cytochrome c and O 2 , unexpectedly find O 2 and cytochrome c are reduced simultaneously, and demonstrate that efficient reduction of O 2 by TbERV1 is not dependent upon a simple O 2 channel defined by conserved histidine and tyrosine residues. Massive mitochondrial swelling following Tb ERV1 RNA interference (RNAi) provides evidence that trypanosome Erv1 functions in IMS protein import despite the natural absence of the key player in the yeast and animal import pathways, Mia40. This suggests significant evolutionary divergence from a recently established paradigm in mitochondrial cell biology. Phylogenomic profiling of genes also points to a conserved role for TbERV1 in cytosolic Fe-S cluster assembly. Conversely, loss of genes implicated in precursor delivery for cytosolic Fe-S assembly in Entamoeba , Trichomonas , and Giardia suggests fundamental differences in intracellular trafficking pathways for activated iron or sulfur species in anaerobic versus aerobic eukaryotes.

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11

Balk, Janneke, Daili J. Aguilar Netz, Katharina Tepper, Antonio J. Pierik, and Roland Lill. "The Essential WD40 Protein Cia1 Is Involved in a Late Step of Cytosolic and Nuclear Iron-Sulfur Protein Assembly." Molecular and Cellular Biology 25, no. 24 (December 15, 2005): 10833–41. http://dx.doi.org/10.1128/mcb.25.24.10833-10841.2005.

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ABSTRACT The assembly of cytosolic and nuclear iron-sulfur (Fe/S) proteins in yeast is dependent on the iron-sulfur cluster assembly and export machineries in mitochondria and three recently identified extramitochondrial proteins, the P-loop NTPases Cfd1 and Nbp35 and the hydrogenase-like Nar1. However, the molecular mechanism of Fe/S protein assembly in the cytosol is far from being understood, and more components are anticipated to take part in this process. Here, we have identified and functionally characterized a novel WD40 repeat protein, designated Cia1, as an essential component required for Fe/S cluster assembly in vivo on cytosolic and nuclear, but not mitochondrial, Fe/S proteins. Surprisingly, Nbp35 and Nar1, themselves Fe/S proteins, could assemble their Fe/S clusters in the absence of Cia1, demonstrating that these components act before Cia1. Consequently, Cia1 is involved in a late step of Fe/S cluster incorporation into target proteins. Coimmunoprecipitation assays demonstrated a specific interaction between Cia1 and Nar1. In contrast to the mostly cytosolic Nar1, Cia1 is preferentially localized to the nucleus, suggesting an additional function of Cia1. Taken together, our results indicate that Cia1 is a new member of the cytosolic Fe/S protein assembly (CIA) machinery participating in a step after Nbp35 and Nar1.

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12

Lu, Zuokun, Han Wang, and TingTing Yu. "The SecB-like chaperone Rv1957 fromMycobacterium tuberculosis: crystallization and X-ray crystallographic analysis." Acta Crystallographica Section F Structural Biology Communications 72, no. 6 (May 23, 2016): 457–61. http://dx.doi.org/10.1107/s2053230x16007287.

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Protein export is important in all bacteria, and bacteria have evolved specialized export machineries to fulfil this task. InMycobacterium tuberculosis, the causative agent of tuberculosis, the general secretion pathway (Sec pathway) is conserved and is essential in performing the export of proteins. The bacterial Sec pathway post-translationally exports unfolded proteins out of the cytoplasm, and the core of the Sec pathway is composed of a heterotrimeric membrane-embedded channel, SecYEG, and two cytosolic components, SecA and SecB. SecB functions by stabilizing unfolded proteins, maintaining them in an export-competent state. Although SecB is mainly found in Proteobacteria, a SecB-like protein, Rv1957, that controls a stress-response toxin–antitoxin system, is found inM. tuberculosis. Rv1957 can also functionally replace theEscherichia coliSecB chaperone bothin vivoandin vitro. In this work, the production, crystallization and X-ray crystallographic analysis of Rv1957 are reported. Notably, diffraction-quality crystals were obtained only at high concentrations of dimethyl sulfoxide,i.e.about 12%(v/v). The crystals of Rv1957 belonged to space groupP212121, with unit-cell parametersa= 64.5,b= 92.0,c= 115.4 Å.

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13

Feng, W., A. L. Benko, J. H. Lee, D. R. Stanford, and A. K. Hopper. "Antagonistic effects of NES and NLS motifs determine S. cerevisiae Rna1p subcellular distribution." Journal of Cell Science 112, no. 3 (February 1, 1999): 339–47. http://dx.doi.org/10.1242/jcs.112.3.339.

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Nucleus/cytosol exchange requires a GTPase, Ran. In yeast Rna1p is the GTPase activating protein for Ran (RanGAP) and Prp20p is the Ran GDP/GTP exchange factor (GEF). RanGAP is primarily cytosolic and GEF is nuclear. Their subcellular distributions led to the prediction that Ran-GTP hydrolysis takes place solely in the cytosol and GDP/GTP exchange solely in the nucleus. Current models propose that the Ran-GTP/Ran-GDP gradient across the nuclear membrane determines the direction of exchange. We provide three lines of evidence that Rna1p enters and leaves the nuclear interior. (1) Rna1p possesses leucine-rich nuclear export sequences (NES) that are able to relocate a passenger karyophilic protein to the cytosol; alterations of consensus residues re-establish nuclear location. (2) Rna1p possesses other sequences that function as a novel nuclear localization sequence able to deliver a passenger cytosolic protein to the nucleus. (3) Endogenous Rna1p location is dependent upon Xpo1p/Crm1p, the yeast exportin for leucine-rich NES-containing proteins. The data support the hypothesis that Rna1p exists on both sides of the nuclear membrane, perhaps regulating the Ran-GTP/Ran-GDP gradient, participating in a complete RanGTPase nuclear cycle or serving a novel function.

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14

Rossig, Claudia, John Gray, Oscar Valdes, Armin Springer, Sachin Rustgi, Diter von Wettstein, Christiane Reinbothe, Joachim Rassow, and Steffen Reinbothe. "PRAT Proteins Operate in Organellar Protein Import and Export in Arabidopsis thaliana." Plants 10, no. 5 (May 11, 2021): 958. http://dx.doi.org/10.3390/plants10050958.

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Chloroplasts need to import preproteins and amino acids from the cytosol during their light-induced differentiation. Similarly, chloroplasts have to export organic matter including proteins and amino acids during leaf senescence. Members of the PRAT (preprotein and amino acid transporter) family are candidate transporters for both processes. Here, we defined the role of two small PRAT gene families, At4g26670 and At5g55510 (HP20 subfamily) versus At3g49560 and At5g24650 (HP30 subfamily) during greening of etiolated plants and during leaf senescence. Using a combination of reverse genetics, protein biochemistry and physiological tools, evidence was obtained for a role of chloroplast HP20, HP30 and HP30-2 in protein, but not amino acid, import into chloroplasts. HP20, HP30 and HP30-2 form larger complexes involved in the uptake of transit sequence-less cytosolic precursors. In addition, we identified a fraction of HP30-2 in mitochondria where it served a similar function as found for chloroplasts and operated in the uptake of transit sequence-less cytosolic precursor proteins. By contrast, HP22 was found to act in the export of proteins from chloroplasts during leaf senescence, and thus its role is entirely different from that of its orthologue, HP20. HP22 is part of a unique protein complex in the envelope of senescing chloroplasts that comprises at least 11 proteins and contains with HP65b (At5g55220) a protein that is related to the bacterial trigger factor chaperone. An ortholog of HP65b exists in the cyanobacterium Synechocystis and has previously been implicated in protein secretion. Whereas plants depleted of either HP22 or HP65b or even both were increasingly delayed in leaf senescence and retained much longer stromal chloroplast constituents than wild-type plants, HP22 overexpressors showed premature leaf senescence that was associated with accelerated losses of stromal chloroplast proteins. Together, our results identify the PRAT protein family as a unique system for importing and exporting proteins from chloroplasts.

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15

Fehr, Marcus, Hitomi Takanaga, David W. Ehrhardt, and Wolf B. Frommer. "Evidence for High-Capacity Bidirectional Glucose Transport across the Endoplasmic Reticulum Membrane by Genetically Encoded Fluorescence Resonance Energy Transfer Nanosensors." Molecular and Cellular Biology 25, no. 24 (December 15, 2005): 11102–12. http://dx.doi.org/10.1128/mcb.25.24.11102-11112.2005.

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ABSTRACT Glucose release from hepatocytes is important for maintenance of blood glucose levels. Glucose-6-phosphate phosphatase, catalyzing the final metabolic step of gluconeogenesis, faces the endoplasmic reticulum (ER) lumen. Thus, glucose produced in the ER has to be either exported from the ER into the cytosol before release into circulation or exported directly by a vesicular pathway. To measure ER transport of glucose, fluorescence resonance energy transfer-based nanosensors were targeted to the cytosol or the ER lumen of HepG2 cells. During perfusion with 5 mM glucose, cytosolic levels were maintained at ∼80% of the external supply, indicating that plasma membrane transport exceeded the rate of glucose phosphorylation. Glucose levels and kinetics inside the ER were indistinguishable from cytosolic levels, suggesting rapid bidirectional glucose transport across the ER membrane. A dynamic model incorporating rapid bidirectional ER transport yields a very good fit with the observed kinetics. Plasma membrane and ER membrane glucose transport differed regarding sensitivity to cytochalasin B and showed different relative kinetics for galactose uptake and release, suggesting catalysis by distinct activities at the two membranes. The presence of a high-capacity glucose transport system on the ER membrane is consistent with the hypothesis that glucose export from hepatocytes occurs via the cytosol by a yet-to-be-identified set of proteins.

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16

Sun, Hong, Yu Huang, Shan Mei, Fengwen Xu, Xiaoman Liu, Fei Zhao, Lijuan Yin, et al. "A Nuclear Export Signal Is Required for cGAS to Sense Cytosolic DNA." Cell Reports 34, no. 1 (January 2021): 108586. http://dx.doi.org/10.1016/j.celrep.2020.108586.

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17

Meuter, S., M. Eberl, and B. Moser. "Prolonged antigen survival and cytosolic export in cross-presenting human T cells." Proceedings of the National Academy of Sciences 107, no. 19 (April 22, 2010): 8730–35. http://dx.doi.org/10.1073/pnas.1002769107.

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18

Cook, Atlanta G., Noemi Fukuhara, Martin Jinek, and Elena Conti. "Structures of the tRNA export factor in the nuclear and cytosolic states." Nature 461, no. 7260 (August 13, 2009): 60–65. http://dx.doi.org/10.1038/nature08394.

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19

Tseng, S. S. I. "Dbp5p, a cytosolic RNA helicase, is required for poly(A)+ RNA export." EMBO Journal 17, no. 9 (May 1, 1998): 2651–62. http://dx.doi.org/10.1093/emboj/17.9.2651.

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20

Kumamoto, Carol A. "SecB protein: A cytosolic export factor that associates with nascent exported proteins." Journal of Bioenergetics and Biomembranes 22, no. 3 (June 1990): 337–51. http://dx.doi.org/10.1007/bf00763171.

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21

van Maris, Antonius J. A., Marijke A. H. Luttik, Aaron A. Winkler, Johannes P. van Dijken, and Jack T. Pronk. "Overproduction of Threonine Aldolase Circumvents the Biosynthetic Role of Pyruvate Decarboxylase in Glucose-Limited Chemostat Cultures of Saccharomyces cerevisiae." Applied and Environmental Microbiology 69, no. 4 (April 2003): 2094–99. http://dx.doi.org/10.1128/aem.69.4.2094-2099.2003.

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ABSTRACT Pyruvate decarboxylase-negative (Pdc−) mutants of Saccharomyces cerevisiae require small amounts of ethanol or acetate to sustain aerobic, glucose-limited growth. This nutritional requirement has been proposed to originate from (i) a need for cytosolic acetyl coenzyme A (acetyl-CoA) for lipid and lysine biosynthesis and (ii) an inability to export mitochondrial acetyl-CoA to the cytosol. To test this hypothesis and to eliminate the C2 requirement of Pdc− S. cerevisiae, we attempted to introduce an alternative pathway for the synthesis of cytosolic acetyl-CoA. The addition of l-carnitine to growth media did not restore growth of a Pdc− strain on glucose, indicating that the C2 requirement was not solely due to the inability of S. cerevisiae to synthesize this compound. The S. cerevisiae GLY1 gene encodes threonine aldolase (EC 4.1.2.5), which catalyzes the cleavage of threonine to glycine and acetaldehyde. Overexpression of GLY1 enabled a Pdc− strain to grow under conditions of carbon limitation in chemostat cultures on glucose as the sole carbon source, indicating that acetaldehyde formed by threonine aldolase served as a precursor for the synthesis of cytosolic acetyl-CoA. Fractionation studies revealed a cytosolic localization of threonine aldolase. The absence of glycine in these cultures indicates that all glycine produced by threonine aldolase was either dissimilated or assimilated. These results confirm the involvement of pyruvate decarboxylase in cytosolic acetyl-CoA synthesis. The Pdc− GLY1 overexpressing strain was still glucose sensitive with respect to growth in batch cultivations. Like any other Pdc− strain, it failed to grow on excess glucose in batch cultures and excreted pyruvate when transferred from glucose limitation to glucose excess.

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22

Tsai, Chi-Lin, Brianne J. Burkinshaw, Natalie C. J. Strynadka, and John A. Tainer. "The Salmonella Type III Secretion System Virulence Effector Forms a New Hexameric Chaperone Assembly for Export of Effector/Chaperone Complexes." Journal of Bacteriology 197, no. 4 (December 8, 2014): 672–75. http://dx.doi.org/10.1128/jb.02524-14.

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Bacteria hijack eukaryotic cells by injecting virulence effectors into host cytosol with a type III secretion system (T3SS). Effectors are targeted with their cognate chaperones to hexameric T3SS ATPase at the bacterial membrane's cytosolic face. In this issue of theJournal of Bacteriology, Roblin et al. (P. Roblin, F. Dewitte, V. Villeret, E. G. Biondi, and C. Bompard, J Bacteriol 197:688–698, 2015,http://dx.doi.org/10.1128/JB.02294-14) show that the T3SS chaperone SigE ofSalmonellacan form hexameric rings rather than dimers when bound to its cognate effector, SopB, implying a novel multimeric association for chaperone/effector complexes with their ATPase.

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23

Catapano, Maria Carmen, Douglas S. Parsons, Radosław Kotuniak, Přemysl Mladěnka, Wojciech Bal, and Wolfgang Maret. "Probing the Structure and Function of the Cytosolic Domain of the Human Zinc Transporter ZnT8 with Nickel(II) Ions." International Journal of Molecular Sciences 22, no. 6 (March 14, 2021): 2940. http://dx.doi.org/10.3390/ijms22062940.

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The human zinc transporter ZnT8 provides the granules of pancreatic β-cells with zinc (II) ions for assembly of insulin hexamers for storage. Until recently, the structure and function of human ZnTs have been modelled on the basis of the 3D structures of bacterial zinc exporters, which form homodimers with each monomer having six transmembrane α-helices harbouring the zinc transport site and a cytosolic domain with an α,β structure and additional zinc-binding sites. However, there are important differences in function as the bacterial proteins export an excess of zinc ions from the bacterial cytoplasm, whereas ZnT8 exports zinc ions into subcellular vesicles when there is no apparent excess of cytosolic zinc ions. Indeed, recent structural investigations of human ZnT8 show differences in metal binding in the cytosolic domain when compared to the bacterial proteins. Two common variants, one with tryptophan (W) and the other with arginine (R) at position 325, have generated considerable interest as the R-variant is associated with a higher risk of developing type 2 diabetes. Since the mutation is at the apex of the cytosolic domain facing towards the cytosol, it is not clear how it can affect zinc transport through the transmembrane domain. We expressed the cytosolic domain of both variants of human ZnT8 and have begun structural and functional studies. We found that (i) the metal binding of the human protein is different from that of the bacterial proteins, (ii) the human protein has a C-terminal extension with three cysteine residues that bind a zinc(II) ion, and (iii) there are small differences in stability between the two variants. In this investigation, we employed nickel(II) ions as a probe for the spectroscopically silent Zn(II) ions and utilised colorimetric and fluorimetric indicators for Ni(II) ions to investigate metal binding. We established Ni(II) coordination to the C-terminal cysteines and found differences in metal affinity and coordination in the two ZnT8 variants. These structural differences are thought to be critical for the functional differences regarding the diabetes risk. Further insight into the assembly of the metal centres in the cytosolic domain was gained from potentiometric investigations of zinc binding to synthetic peptides corresponding to N-terminal and C-terminal sequences of ZnT8 bearing the metal-coordinating ligands. Our work suggests the involvement of the C-terminal cysteines, which are part of the cytosolic domain, in a metal chelation and/or acquisition mechanism and, as now supported by the high-resolution structural work, provides the first example of metal-thiolate coordination chemistry in zinc transporters.

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24

Nair, Devi M., P. Edward Purdue, and Paul B. Lazarow. "Pex7p translocates in and out of peroxisomes in Saccharomyces cerevisiae." Journal of Cell Biology 167, no. 4 (November 15, 2004): 599–604. http://dx.doi.org/10.1083/jcb.200407119.

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Pex7p is the soluble receptor responsible for importing into peroxisomes newly synthesized proteins bearing a type 2 peroxisomal targeting sequence. We observe that appending GFP to Pex7p's COOH terminus shifts Pex7p's intracellular distribution from predominantly cytosolic to predominantly peroxisomal in Saccharomyces cerevisiae. Cleavage of the link between Pex7p and GFP within peroxisomes liberates GFP, which remains inside the organelle, and Pex7p, which exits to the cytosol. The reexported Pex7p is functional, resulting in import of thiolase into peroxisomes and improved growth of the yeast on oleic acid. These results support the “extended shuttle” model of peroxisome import receptor function and open the way to future studies of receptor export.

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25

HAMPTON, B. Mark, Boris ZHIVOTOVSKY, F. G. Andrew SLATER, H. David BURGESS, and Sten ORRENIUS. "Importance of the redox state of cytochrome c during caspase activation in cytosolic extracts." Biochemical Journal 329, no. 1 (January 1, 1998): 95–99. http://dx.doi.org/10.1042/bj3290095.

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The export of cytochrome c from mitochondria to the cytoplasm has been detected during apoptosis. Addition of cytochrome c to cytosolic extracts can activate the caspases, suggesting that this export could be an important intracellular signal for initiating the apoptotic programme. We have investigated the mechanism of caspase activation by cytochrome c. Mitochondrial cytochrome c normally shuttles electrons between complexes III and IV of the electron transport chain. Interaction with these complexes is dependent on electrostatic interactions via a polylysine binding pocket. Cytosolic caspase activation was only observed with intact holocytochrome c, and increasing the ionic composition of the extracts prevented activation, suggesting that stringent allosteric interactions between cytochrome c and other cytoplasmic factors are necessary. Cytochrome c was fully reduced within 5 min of addition to the cytosolic extracts. Potassium ferricyanide could maintain cytochrome c in an oxidized state, but care was taken to use ferricyanide at concentrations where its polyanion effect did not cause interference. The oxidized form of cytochrome c was able to activate the caspases. We conclude that reduced cytochrome c will function in the cytoplasm; however, its reduction is not a critical step, and electron transfer from cytochrome c to its cytoplasmic-binding partner(s) is not necessary in the pathway leading to apoptosis.

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26

Ellis, Mark A., Mark T. Miedel, Christopher J. Guerriero, and Ora A. Weisz. "ADP-ribosylation Factor 1-independent Protein Sorting and Export from thetrans-Golgi Network." Journal of Biological Chemistry 279, no. 50 (September 30, 2004): 52735–43. http://dx.doi.org/10.1074/jbc.m410533200.

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Polarized epithelial cells efficiently sort newly synthesized apical and basolateral proteins into distinct transport carriers that emerge from thetrans-Golgi network (TGN), and this sorting is recapitulated in nonpolarized cells. While the targeting signals of basolaterally destined proteins are generally cytoplasmically disposed, apical sorting signals are not typically accessible to the cytosol, and the transport machinery required for segregation and export of apical cargo remains largely unknown. Here we investigated the molecular requirements for TGN export of the apical marker influenza hemagglutinin (HA) in HeLa cells using anin vitroreconstitution assay. HA was released from the TGN in intact membrane-bound compartments, and export was dependent on addition of an ATP-regenerating system and exogenous cytosol. HA release was inhibited by guanosine 5′-O-(3-thiotriphosphate) (GTPγS) as well as under conditions known to negatively regulate apical transportin vivo, including expression of the acid-activated proton channel influenza M2. Interestingly, release of HA was unaffected by depletion of ADP-ribosylation factor 1, a small GTPase that has been implicated in the recruitment of all known adaptors and coat proteins to the Golgi complex. Furthermore, regulation of HA release by GTPγS or M2 expression was unaffected by cytosolic depletion of ADP-ribosylation factor 1, suggesting that HA sorting remains functionally intact in the absence of the small GTPase. These data suggest that TGN sorting and export of influenza HA does not require classical adaptors involved in the formation of other classes of exocytic carriers and thus appears to proceed via a novel mechanism.

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27

Bajaj Pahuja, Kanika, Jinzhi Wang, Anastasia Blagoveshchenskaya, Lillian Lim, M. S. Madhusudhan, Peter Mayinger, and Randy Schekman. "Phosphoregulatory protein 14-3-3 facilitates SAC1 transport from the endoplasmic reticulum." Proceedings of the National Academy of Sciences 112, no. 25 (June 8, 2015): E3199—E3206. http://dx.doi.org/10.1073/pnas.1509119112.

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Анотація:

Most secretory cargo proteins in eukaryotes are synthesized in the endoplasmic reticulum and actively exported in membrane-bound vesicles that are formed by the cytosolic coat protein complex II (COPII). COPII proteins are assisted by a variety of cargo-specific adaptor proteins required for the concentration and export of secretory proteins from the endoplasmic reticulum (ER). Adaptor proteins are key regulators of cargo export, and defects in their function may result in disease phenotypes in mammals. Here we report the role of 14-3-3 proteins as a cytosolic adaptor in mediating SAC1 transport in COPII-coated vesicles. Sac1 is a phosphatidyl inositol-4 phosphate (PI4P) lipid phosphatase that undergoes serum dependent translocation between the endoplasmic reticulum and Golgi complex and controls cellular PI4P lipid levels. We developed a cell-free COPII vesicle budding reaction to examine SAC1 exit from the ER that requires COPII and at least one additional cytosolic factor, the 14-3-3 protein. Recombinant 14-3-3 protein stimulates the packaging of SAC1 into COPII vesicles and the sorting subunit of COPII, Sec24, interacts with 14-3-3. We identified a minimal sorting motif of SAC1 that is important for 14-3-3 binding and which controls SAC1 export from the ER. This LS motif is part of a 7-aa stretch, RLSNTSP, which is similar to the consensus 14-3-3 binding sequence. Homology models, based on the SAC1 structure from yeast, predict this region to be in the exposed exterior of the protein. Our data suggest a model in which the 14-3-3 protein mediates SAC1 traffic from the ER through direct interaction with a sorting signal and COPII.

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28

Lopez, Sergio, Sofia Rodriguez-Gallardo, Susana Sabido-Bozo, and Manuel Muñiz. "Endoplasmic Reticulum Export of GPI-Anchored Proteins." International Journal of Molecular Sciences 20, no. 14 (July 17, 2019): 3506. http://dx.doi.org/10.3390/ijms20143506.

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Анотація:

Protein export from the endoplasmic reticulum (ER) is an essential process in all eukaryotes driven by the cytosolic coat complex COPII, which forms vesicles at ER exit sites for transport of correctly assembled secretory cargo to the Golgi apparatus. The COPII machinery must adapt to the existing wide variety of different types of cargo proteins and to different cellular needs for cargo secretion. The study of the ER export of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs), a special glycolipid-linked class of cell surface proteins, is contributing to address these key issues. Due to their special biophysical properties, GPI-APs use a specialized COPII machinery to be exported from the ER and their processing and maturation has been recently shown to actively regulate COPII function. In this review, we discuss the regulatory mechanisms by which GPI-APs are assembled and selectively exported from the ER.

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29

Wolff, Georg, Ronald W. A. L. Limpens, Jessika C. Zevenhoven-Dobbe, Ulrike Laugks, Shawn Zheng, Anja W. M. de Jong, Roman I. Koning, et al. "A molecular pore spans the double membrane of the coronavirus replication organelle." Science 369, no. 6509 (August 6, 2020): 1395–98. http://dx.doi.org/10.1126/science.abd3629.

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Анотація:

Coronavirus genome replication is associated with virus-induced cytosolic double-membrane vesicles, which may provide a tailored microenvironment for viral RNA synthesis in the infected cell. However, it is unclear how newly synthesized genomes and messenger RNAs can travel from these sealed replication compartments to the cytosol to ensure their translation and the assembly of progeny virions. In this study, we used cellular cryo–electron microscopy to visualize a molecular pore complex that spans both membranes of the double-membrane vesicle and would allow export of RNA to the cytosol. A hexameric assembly of a large viral transmembrane protein was found to form the core of the crown-shaped complex. This coronavirus-specific structure likely plays a key role in coronavirus replication and thus constitutes a potential drug target.

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30

Richards, S. A., K. M. Lounsbury, K. L. Carey, and I. G. Macara. "A nuclear export signal is essential for the cytosolic localization of the Ran binding protein, RanBP1." Journal of Cell Biology 134, no. 5 (September 1, 1996): 1157–68. http://dx.doi.org/10.1083/jcb.134.5.1157.

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RanBP1 is a Ran/TC4 binding protein that can promote the interaction between Ran and beta-importin /beta-karyopherin, a component of the docking complex for nuclear protein cargo. This interaction occurs through a Ran binding domain (RBD). Here we show that RanBP1 is primarily cytoplasmic, but the isolated RBD accumulates in the nucleus. A region COOH-terminal to the RBD is responsible for this cytoplasmic localization. This domain acts heterologously, localizing a nuclear cyclin B1 mutant to the cytoplasm. The domain contains a nuclear export signal that is necessary but not sufficient for the nuclear export of a functional RBD In transiently transfected cells, epitope-tagged RanBP1 promotes dexamethasone-dependent nuclear accumulation of a glucocorticoid receptor-green fluorescent protein fusion, but the isolated RBD potently inhibits this accumulation. The cytosolic location of RanBP1 may therefore be important for nuclear protein import. RanBP1 may provide a key link between the nuclear import and export pathways.

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31

González-Quiñónez, Nathaly, Ignacio Gutiérrez-Del-Río, Paula García-Cancela, Gemma Fernández-García, Sergio Alonso-Fernández, Paula Yagüe, Álvaro Pérez-Valero, María Montes-Bayón, Felipe Lombó, and Ángel Manteca. "The Modulation of SCO2730/31 Copper Chaperone/Transporter Orthologue Expression Enhances Secondary Metabolism in Streptomycetes." International Journal of Molecular Sciences 22, no. 18 (September 20, 2021): 10143. http://dx.doi.org/10.3390/ijms221810143.

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Streptomycetes are important biotechnological bacteria that produce several clinically bioactive compounds. They have a complex development, including hyphae differentiation and sporulation. Cytosolic copper is a well-known modulator of differentiation and secondary metabolism. The interruption of the Streptomyces coelicolor SCO2730 (copper chaperone, SCO2730::Tn5062 mutant) blocks SCO2730 and reduces SCO2731 (P-type ATPase copper export) expressions, decreasing copper export and increasing cytosolic copper. This mutation triggers the expression of 13 secondary metabolite clusters, including cryptic pathways, during the whole developmental cycle, skipping the vegetative, non-productive stage. As a proof of concept, here, we tested whether the knockdown of the SCO2730/31 orthologue expression can enhance secondary metabolism in streptomycetes. We created a SCO2730/31 consensus antisense mRNA from the sequences of seven key streptomycetes, which helped to increase the cytosolic copper in S. coelicolor, albeit to a lower level than in the SCO2730::Tn5062 mutant. This antisense mRNA affected the production of at least six secondary metabolites (CDA, 2-methylisoborneol, undecylprodigiosin, tetrahydroxynaphtalene, α-actinorhodin, ε-actinorhodin) in the S. coelicolor, and five (phenanthroviridin, alkylresorcinol, chloramphenicol, pikromycin, jadomycin G) in the S. venezuelae; it also helped to alter the S. albus metabolome. The SCO2730/31 consensus antisense mRNA designed here constitutes a tool for the knockdown of SCO2730/31 expression and for the enhancement of Streptomyces’ secondary metabolism.

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32

Auvray, Frédéric, Joanne Thomas, Gillian M. Fraser, and Colin Hughes. "Flagellin polymerisation control by a cytosolic export chaperone1 1Edited by I. B. Holland." Journal of Molecular Biology 308, no. 2 (April 2001): 221–29. http://dx.doi.org/10.1006/jmbi.2001.4597.

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33

Aridor, Meir, Kenneth N. Fish, Sergei Bannykh, Jacques Weissman, Theresa H. Roberts, Jennifer Lippincott-Schwartz, and William E. Balch. "The Sar1 Gtpase Coordinates Biosynthetic Cargo Selection with Endoplasmic Reticulum Export Site Assembly." Journal of Cell Biology 152, no. 1 (January 8, 2001): 213–30. http://dx.doi.org/10.1083/jcb.152.1.213.

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Анотація:

Cargo selection and export from the endoplasmic reticulum is mediated by the COPII coat machinery that includes the small GTPase Sar1 and the Sec23/24 and Sec13/31 complexes. We have analyzed the sequential events regulated by purified Sar1 and COPII coat complexes during synchronized export of cargo from the ER in vitro. We find that activation of Sar1 alone, in the absence of other cytosolic components, leads to the formation of ER-derived tubular domains that resemble ER transitional elements that initiate cargo selection. These Sar1-generated tubular domains were shown to be transient, functional intermediates in ER to Golgi transport in vitro. By following cargo export in live cells, we show that ER export in vivo is also characterized by the formation of dynamic tubular structures. Our results demonstrate an unanticipated and novel role for Sar1 in linking cargo selection with ER morphogenesis through the generation of transitional tubular ER export sites.

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34

Jeckel, D., A. Karrenbauer, KN Burger, G. van Meer, and F. Wieland. "Glucosylceramide is synthesized at the cytosolic surface of various Golgi subfractions." Journal of Cell Biology 117, no. 2 (April 15, 1992): 259–67. http://dx.doi.org/10.1083/jcb.117.2.259.

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In our attempt to assess the topology of glucosylceramide biosynthesis, we have employed a truncated ceramide analogue that permeates cell membranes and is converted into water soluble sphingolipid analogues both in living and in fractionated cells. Truncated sphingomyelin is synthesized in the lumen of the Golgi, whereas glucosylceramide is synthesized at the cytosolic surface of the Golgi as shown by (a) the insensitivity of truncated sphingomyelin synthesis and the sensitivity of truncated glucosylceramide synthesis in intact Golgi membranes from rabbit liver to treatment with protease or the chemical reagent DIDS; and (b) sensitivity of truncated sphingomyelin export and insensitivity of truncated glucosylceramide export to decreased temperature and the presence of GTP-gamma-S in semiintact CHO cells. Moreover, subfractionation of rat liver Golgi demonstrated that the sphingomyelin synthase activity was restricted to fractions containing marker enzymes for the proximal Golgi, whereas the capacity to synthesize truncated glucosylceramide was also found in fractions containing distal Golgi markers. A similar distribution of glucosylceramide synthesizing activity was observed in the Golgi of the human liver derived HepG2 cells. The cytosolic orientation of the reaction in HepG2 cells was confirmed by complete extractability of newly formed NBD-glucosylceramide from isolated Golgi membranes or semiintact cells by serum albumin, whereas NBD-sphingomyelin remained protected against such extraction.

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35

Bankaitis, V. A., D. E. Malehorn, S. D. Emr, and R. Greene. "The Saccharomyces cerevisiae SEC14 gene encodes a cytosolic factor that is required for transport of secretory proteins from the yeast Golgi complex." Journal of Cell Biology 108, no. 4 (April 1, 1989): 1271–81. http://dx.doi.org/10.1083/jcb.108.4.1271.

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We have obtained and characterized a genomic clone of SEC14, a Saccharomyces cerevisiae gene whose product is required for export of yeast secretory proteins from the Golgi complex. Gene disruption experiments indicated that SEC14 is an essential gene for yeast vegetative growth. Nucleotide sequence analysis revealed the presence of an intron within the SEC14 structural gene, and predicted the synthesis of a hydrophilic polypeptide of 35 kD in molecular mass. In confirmation, immunoprecipitation experiments demonstrated SEC14p to be an unglycosylated polypeptide, with an apparent molecular mass of some 37 kD, that behaved predominantly as a cytosolic protein in subcellular fractionation experiments. These data were consistent with the notion that SEC14p is a cytosolic factor that promotes protein export from yeast Golgi. Additional radiolabeling experiments also revealed the presence of SEC14p-related polypeptides in extracts prepared from the yeasts Kluyveromyces lactis and Schizosaccharomyces pombe. Furthermore, the K. lactis SEC14p was able to functionally complement S. cerevisiae sec14ts defects. These data suggested a degree of conservation of SEC14p structure and function in these yeasts species.

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36

Paßvogel, Lars, Barbara G. Klupp, Harald Granzow, Walter Fuchs, and Thomas C. Mettenleiter. "Functional Characterization of Nuclear Trafficking Signals in Pseudorabies Virus pUL31." Journal of Virology 89, no. 4 (December 10, 2014): 2002–12. http://dx.doi.org/10.1128/jvi.03143-14.

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ABSTRACTThe herpesviral nuclear egress complex (NEC), consisting of pUL31 and pUL34 homologs, mediates efficient translocation of newly synthesized capsids from the nucleus to the cytosol. The tail-anchored membrane protein pUL34 is autonomously targeted to the nuclear envelope, while pUL31 is recruited to the inner nuclear membrane (INM) by interaction with pUL34. A nuclear localization signal (NLS) in several pUL31 homologs suggests importin-mediated translocation of the protein. Here we demonstrate that deletion or mutation of the NLS in pseudorabies virus (PrV) pUL31 resulted in exclusively cytosolic localization, indicating active nuclear export. Deletion or mutation of a predicted nuclear export signal (NES) in mutant constructs lacking a functional NLS resulted in diffuse nuclear and cytosolic localization, indicating that both signals are functional. pUL31 molecules lacking the complete NLS or NES were not recruited to the INM by pUL34, while site-specifically mutated proteins formed the NEC and partially complemented the defect of the UL31 deletion mutant. Our data demonstrate that the N terminus of pUL31, encompassing the NLS, is required for efficient nuclear targeting but not for pUL34 interaction, while the C terminus, containing the NES but not necessarily the NES itself, is required for complex formation and efficient budding of viral capsids at the INM. Moreover, pUL31-ΔNLS displayed a dominant negative effect on wild-type PrV replication, probably by diverting pUL34 to cytoplasmic membranes.IMPORTANCEThe molecular details of nuclear egress of herpesvirus capsids are still enigmatic. Although the key players, homologs of herpes simplex virus pUL34 and pUL31, which interact and form the heterodimeric nuclear egress complex, are well known, the molecular basis of this interaction and the successive budding, vesicle formation, and scission from the INM, as well as capsid release into the cytoplasm, remain largely obscure. Here we show that classical cellular targeting signals for nuclear import and export are important for proper localization and function of the NEC, thus regulating herpesvirus nuclear egress.

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37

Stehling, Oliver, Daili J. A. Netz, Brigitte Niggemeyer, Ralf Rösser, Richard S. Eisenstein, Helene Puccio, Antonio J. Pierik, and Roland Lill. "Human Nbp35 Is Essential for both Cytosolic Iron-Sulfur Protein Assembly and Iron Homeostasis." Molecular and Cellular Biology 28, no. 17 (June 23, 2008): 5517–28. http://dx.doi.org/10.1128/mcb.00545-08.

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ABSTRACT The maturation of cytosolic iron-sulfur (Fe/S) proteins in mammalian cells requires components of the mitochondrial iron-sulfur cluster assembly and export machineries. Little is known about the cytosolic components that may facilitate the assembly process. Here, we identified the cytosolic soluble P-loop NTPase termed huNbp35 (also known as Nubp1) as an Fe/S protein, and we defined its role in the maturation of Fe/S proteins in HeLa cells. Depletion of huNbp35 by RNA interference decreased cell growth considerably, indicating its essential function. The deficiency in huNbp35 was associated with an impaired maturation of the cytosolic Fe/S proteins glutamine phosphoribosylpyrophosphate amidotransferase and iron regulatory protein 1 (IRP1), while mitochondrial Fe/S proteins remained intact. Consequently, huNbp35 is specifically involved in the formation of extramitochondrial Fe/S proteins. The impaired maturation of IRP1 upon huNbp35 depletion had profound consequences for cellular iron metabolism, leading to decreased cellular H-ferritin, increased transferrin receptor levels, and higher transferrin uptake. These properties clearly distinguished huNbp35 from its yeast counterpart Nbp35, which is essential for cytosolic-nuclear Fe/S protein assembly but plays no role in iron regulation. huNbp35 formed a complex with its close homologue huCfd1 (also known as Nubp2) in vivo, suggesting the existence of a heteromeric P-loop NTPase complex that is required for both cytosolic Fe/S protein assembly and cellular iron homeostasis.

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38

Novoa, Isabel, Mark G. Rush, and Peter D’Eustachio. "Isolated Mammalian and Schizosaccharomyces pombeRan-binding Domains Rescue S. pombe sbp1 (RanBP1) Genomic Mutants." Molecular Biology of the Cell 10, no. 7 (July 1999): 2175–90. http://dx.doi.org/10.1091/mbc.10.7.2175.

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Анотація:

Mammalian Ran-binding protein-1 (RanBP1) and its fission yeast homologue, sbp1p, are cytosolic proteins that interact with the GTP-charged form of Ran GTPase through a conserved Ran-binding domain (RBD). In vitro, this interaction can accelerate the Ran GTPase-activating protein–mediated hydrolysis of GTP on Ran and the turnover of nuclear import and export complexes. To analyze RanBP1 function in vivo, we expressed exogenous RanBP1, sbp1p, and the RBD of each in mammalian cells, in wild-type fission yeast, and in yeast whose endogenous sbp1 gene was disrupted. Mammalian cells and wild-type yeast expressing moderate levels of each protein were viable and displayed normal nuclear protein import.sbp1 − yeast were inviable but could be rescued by all four exogenous proteins. Two RBDs of the mammalian nucleoporin RanBP2 also rescued sbp1 −yeast. In mammalian cells, wild-type yeast, and rescued mutant yeast, exogenous full-length RanBP1 and sbp1p localized predominantly to the cytosol, whereas exogenous RBDs localized predominantly to the cell nucleus. These results suggest that only the RBD of sbp1p is required for its function in fission yeast, and that this function may not require confinement of the RBD to the cytosol. The results also indicate that the polar amino-terminal portion of sbp1p mediates cytosolic localization of the protein in both yeast and mammalian cells.

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39

Foster, Andrew W., Carl J. Patterson, Rafael Pernil, Corinna R. Hess, and Nigel J. Robinson. "Cytosolic Ni(II) Sensor in Cyanobacterium." Journal of Biological Chemistry 287, no. 15 (February 22, 2012): 12142–51. http://dx.doi.org/10.1074/jbc.m111.338301.

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Efflux of surplus Ni(II) across the outer and inner membranes of Synechocystis PCC 6803 is mediated by the Nrs system under the control of a sensor of periplasmic Ni(II), NrsS. Here, we show that the product of ORF sll0176, which encodes a CsoR/RcnR-like protein now designated InrS (for internal nickel-responsive sensor), represses nrsD (NrsD is deduced to efflux Ni(II) across the inner membrane) from a cryptic promoter between the final two ORFs in the nrs operon. Transcripts initiated from the newly identified nrsD promoter accumulate in response to nickel or cobalt but not copper, and recombinant InrS forms specific, Ni(II)-inhibited complexes with the nrsD promoter region. Metal-dependent difference spectra of Ni(II)- and Cu(I)-InrS are similar to Cu(I)-sensing CsoR and dissimilar to Ni(II)/Co(II)-sensing RcnR, consistent with factors beyond the primary coordination sphere switching metal selectivity. Competition with chelators mag-fura-2, nitrilotriacetic acid, EDTA, and EGTA estimate KD Ni(II) for the tightest site of InrS as 2.05 (±1.5) × 10−14m, and weaker KD Ni(II) for the cells' metal sensors of other types: Zn(II) co-repressor Zur, Co(II) activator CoaR, and Zn(II) derepressor ZiaR. Ni(II) transfer to InrS occurs upon addition to Ni(II) forms of each other sensor. InrS binds Ni(II) sufficiently tightly to derepress Ni(II) export at concentrations below KD Ni(II) of the other sensors.

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40

Parmar, Hirendrasinh B., Christopher Barry, FuiBoon Kai, and Roy Duncan. "Golgi complex–plasma membrane trafficking directed by an autonomous, tribasic Golgi export signal." Molecular Biology of the Cell 25, no. 6 (March 15, 2014): 866–78. http://dx.doi.org/10.1091/mbc.e13-07-0364.

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Анотація:

Although numerous linear motifs that direct protein trafficking within cells have been identified, there are few examples of linear sorting signals mediating directed export of membrane proteins from the Golgi complex to the plasma membrane. The reovirus fusion-associated small transmembrane proteins are simple, single-pass transmembrane proteins that traffic through the endoplasmic reticulum–Golgi pathway to the plasma membrane, where they induce cell–cell membrane fusion. Here we show that a membrane-proximal, polybasic motif (PBM) in the cytosolic tail of p14 is essential for efficient export of p14 from the Golgi complex to the plasma membrane. Extensive mutagenic analysis reveals that the number, but not the identity or position, of basic residues present in the PBM dictates p14 export from the Golgi complex, with a minimum of three basic residues required for efficient Golgi export. Results further indicate that the tribasic motif does not affect plasma membrane retention of p14. Furthermore, introduction of the tribasic motif into a Golgi-localized, chimeric ERGIC-53 protein directs export from the Golgi complex to the plasma membrane. The p14 PBM is the first example of an autonomous, tribasic signal required for Golgi export to the plasma membrane.

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41

Shum, Michael, Chitra A. Shintre, Thorsten Althoff, Vincent Gutierrez, Mayuko Segawa, Alexandra D. Saxberg, Melissa Martinez, et al. "ABCB10 exports mitochondrial biliverdin, driving metabolic maladaptation in obesity." Science Translational Medicine 13, no. 594 (May 19, 2021): eabd1869. http://dx.doi.org/10.1126/scitranslmed.abd1869.

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Анотація:

Although the role of hydrophilic antioxidants in the development of hepatic insulin resistance and nonalcoholic fatty liver disease has been well studied, the role of lipophilic antioxidants remains poorly characterized. A known lipophilic hydrogen peroxide scavenger is bilirubin, which can be oxidized to biliverdin and then reduced back to bilirubin by cytosolic biliverdin reductase. Oxidation of bilirubin to biliverdin inside mitochondria must be followed by the export of biliverdin to the cytosol, where biliverdin is reduced back to bilirubin. Thus, the putative mitochondrial exporter of biliverdin is expected to be a major determinant of bilirubin regeneration and intracellular hydrogen peroxide scavenging. Here, we identified ABCB10 as a mitochondrial biliverdin exporter. ABCB10 reconstituted into liposomes transported biliverdin, and ABCB10 deletion caused accumulation of biliverdin inside mitochondria. Obesity with insulin resistance up-regulated hepatic ABCB10 expression in mice and elevated cytosolic and mitochondrial bilirubin content in an ABCB10-dependent manner. Revealing a maladaptive role of ABCB10-driven bilirubin synthesis, hepatic ABCB10 deletion protected diet-induced obese mice from steatosis and hyperglycemia, improving insulin-mediated suppression of glucose production and decreasing lipogenic SREBP-1c expression. Protection was concurrent with enhanced mitochondrial function and increased inactivation of PTP1B, a phosphatase disrupting insulin signaling and elevating SREBP-1c expression. Restoration of cellular bilirubin content in ABCB10 KO hepatocytes reversed the improvements in mitochondrial function and PTP1B inactivation, demonstrating that bilirubin was the maladaptive effector linked to ABCB10 function. Thus, we identified a fundamental transport process that amplifies intracellular bilirubin redox actions, which can exacerbate insulin resistance and steatosis in obesity.

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42

HANAKA, Hiromi, Takao SHIMIZU, and Takashi IZUMI. "Nuclear-localization-signal-dependent and nuclear-export-signal-dependent mechanisms determine the localization of 5-lipoxygenase." Biochemical Journal 361, no. 3 (January 25, 2002): 505–14. http://dx.doi.org/10.1042/bj3610505.

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Анотація:

5-Lipoxygenase (5-LO) metabolizes arachidonic acid to leukotriene A4, a key intermediate in leukotriene biosynthesis. To explore the molecular mechanisms of its cell-specific localization, a fusion protein between green fluorescent protein (GFP) and human 5-LO (GFP—5LO) was expressed in various cells. GFP—5LO was localized in the cytosol in HL-60 cells and in both the nucleus and the cytosol in RBL (rat basophilic leukaemia) cells, similarly to the native enzyme in these cells. The localization of GFP fusion proteins for mutant 5-LOs in a putative bipartite nuclear localization signal (NLS), amino acids 638–655, in Chinese hamster ovary (CHO)-K1 and Swiss3T3 cells revealed that this motif is important for the nuclear localization of 5-LO. A GFP fusion protein of this short peptide localized consistently in the nucleus. Leptomycin B, a specific inhibitor of nuclear export signal (NES)-dependent transport, diminished the cytosolic localization of 5-LO in HL-60 cells and that of GFP—5LO in CHO-K1 cells, suggesting that an NES-system might also function in determining 5-LO localization. Analysis of the localization of 5-LO during the cell cycle points to a controlled movement of this enzyme. Thus we conclude that a balance of NLS- and NES-dependent mechanisms determines the cell-type-specific localization of 5-LO, suggesting a nuclear function for this enzyme.

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43

Chan, Siu-Kwong, and Gary Struhl. "Evidence that Armadillo Transduces Wingless by Mediating Nuclear Export or Cytosolic Activation of Pangolin." Cell 111, no. 2 (October 2002): 265–80. http://dx.doi.org/10.1016/s0092-8674(02)01037-1.

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Chan, S. K., and G. Struhl. "Evidence that Armadillo Transduces Wingless by Mediating Nuclear Export or Cytosolic Activation of Pangolin." Cell 114, no. 2 (July 2003): 267. http://dx.doi.org/10.1016/s0092-8674(03)00560-9.

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Chen, Yu, Barbara A. Bensing, Ravin Seepersaud, Wei Mi, Maofu Liao, Philip D. Jeffrey, Asif Shajahan, et al. "Unraveling the sequence of cytosolic reactions in the export of GspB adhesin fromStreptococcus gordonii." Journal of Biological Chemistry 293, no. 14 (February 9, 2018): 5360–73. http://dx.doi.org/10.1074/jbc.ra117.000963.

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Watanabe, Makoto, and Günter Blobel. "SecB functions as a cytosolic signal recognition factor for protein export in E. coli." Cell 58, no. 4 (August 1989): 695–705. http://dx.doi.org/10.1016/0092-8674(89)90104-9.

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47

Sachdev, Shrikesh, Sriparna Bagchi, Donna D. Zhang, Angela C. Mings та Mark Hannink. "Nuclear Import of IκBα Is Accomplished by a Ran-Independent Transport Pathway". Molecular and Cellular Biology 20, № 5 (1 березня 2000): 1571–82. http://dx.doi.org/10.1128/mcb.20.5.1571-1582.2000.

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ABSTRACT The inhibitor of kappa B alpha (IκBα) protein is able to shuttle between the cytoplasm and the nucleus. We have utilized a combination of in vivo and in vitro approaches to provide mechanistic insight into nucleocytoplasmic shuttling by IκBα. IκBα contains multiple functional domains that contribute to shuttling of IκBα between the cytoplasm and the nucleus. Nuclear import of IκBα is mediated by the central ankyrin repeat domain. Similar to previously described nuclear import pathways, nuclear import of IκBα is temperature and ATP dependent and is blocked by a dominant-negative mutant of importin β. However, in contrast to classical nuclear import pathways, nuclear import of IκBα is independent of soluble cytosolic factors and is not blocked by the dominant-negative RanQ69L protein. Nuclear export of IκBα is mediated by an N-terminal nuclear export sequence. Nuclear export of IκBα requires the CRM1 nuclear export receptor and is blocked by the dominant-negative RanQ69L protein. Our results are consistent with a model in which nuclear import of IκBα is mediated through direct interactions with components of the nuclear pore complex, while nuclear export of IκBα is mediated via a CRM1-dependent pathway.

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Domínguez, David, Bàrbara Montserrat-Sentís, Ariadna Virgós-Soler, Sandra Guaita, Judit Grueso, Montserrat Porta, Isabel Puig, Josep Baulida, Clara Francí, and Antonio García de Herreros. "Phosphorylation Regulates the Subcellular Location and Activity of the Snail Transcriptional Repressor." Molecular and Cellular Biology 23, no. 14 (July 15, 2003): 5078–89. http://dx.doi.org/10.1128/mcb.23.14.5078-5089.2003.

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ABSTRACT The Snail gene product is a transcriptional repressor of E-cadherin expression and an inducer of the epithelial-to-mesenchymal transition in several epithelial tumor cell lines. This report presents data indicating that Snail function is controlled by its intracellular location. The cytosolic distribution of Snail depended on export from the nucleus by a CRM1-dependent mechanism, and a nuclear export sequence (NES) was located in the regulatory domain of this protein. Export of Snail was controlled by phosphorylation of a Ser-rich sequence adjacent to this NES. Modification of this sequence released the restriction created by the zinc finger domain and allowed nuclear export of the protein. The phosphorylation and subcellular distribution of Snail are controlled by cell attachment to the extracellular matrix. Suspended cells presented higher levels of phosphorylated Snail and an augmented extranuclear localization with respect to cells attached to the plate. These findings show the existence in tumor cells of an effective and fine-tuning nontranscriptional mechanism of regulation of Snail activity dependent on the extracellular environment.

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Navarro, Maria N., Jurgen Goebel, Carmen Feijoo-Carnero, and Doreen A. Cantrell. "The class II Histone deacetylase 7 controls Interleukin 2 receptor expression and proliferation of primary cytotoxic T cells (84.8)." Journal of Immunology 182, no. 1_Supplement (April 1, 2009): 84.8. http://dx.doi.org/10.4049/jimmunol.182.supp.84.8.

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Abstract Regulation of chromatin accessibility via class II histone deacetylases is a critical mechanism controlling gene expression. The paradigm for class II HDAC regulation is that in quiescent lymphocytes these molecules localise to the nucleus and form repressive complexes on chromatin. Antigen receptor activation of Protein Kinase D induces phosphorylation and nuclear export of HDACs thereby relieving their repressive actions on chromatin and gene expression. In this context, the present study has examined the regulation and function the class II histone deacetylase 7 in effector cytotoxic T cells (CTLs). This HDAC has been shown previously to function as a signal dependent repressor of gene transcription during T cell development in the thymus. We now show that HDAC7 is a critical regulator of Interleukin 2 receptor expression and proliferation in primary CTL. Strikingly, there is constitutive and sustained phosphorylation and export of HDAC7 from the nucleus to the cytosol in CTLs. The cytosolic localisation of HDAC7 is determined by the phosphorylation of key serine residues in the molecule S156, S322, S457 and by a balance of src kinase and diacylglycerol signalling pathways that increase its nuclear export and block its nuclear import. An HDAC7 mutant (S156A, S322A, S457A) that cannot be phosphorylated is thus maintained in the nucleus and prevents expression of the Interleukin2 (IL-2) receptor and accordingly inhibits CTL responses to IL-2. HDAC7 thus acts as a molecular switch to mediate IL-2 receptor expression and cellular responsiveness to IL-2 in primary T cells.

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Hewton, Keeley G., Amritpal S. Johal, and Seth J. Parker. "Transporters at the Interface between Cytosolic and Mitochondrial Amino Acid Metabolism." Metabolites 11, no. 2 (February 16, 2021): 112. http://dx.doi.org/10.3390/metabo11020112.

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Mitochondria are central organelles that coordinate a vast array of metabolic and biologic functions important for cellular health. Amino acids are intricately linked to the bioenergetic, biosynthetic, and homeostatic function of the mitochondrion and require specific transporters to facilitate their import, export, and exchange across the inner mitochondrial membrane. Here we review key cellular metabolic outputs of eukaryotic mitochondrial amino acid metabolism and discuss both known and unknown transporters involved. Furthermore, we discuss how utilization of compartmentalized amino acid metabolism functions in disease and physiological contexts. We examine how improved methods to study mitochondrial metabolism, define organelle metabolite composition, and visualize cellular gradients allow for a more comprehensive understanding of how transporters facilitate compartmentalized metabolism.

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