Intracellular localization of human Ins(1,3,4,5,6)P5 2-kinase

InsP6 is an intracellular signal with several proposed functions that is synthesized by IP5K [Ins(1,3,4,5,6)P5 2-kinase]. In the present study, we overexpressed EGFP (enhanced green fluorescent protein)–IP5K fusion proteins in NRK (normal rat kidney), COS7 and H1299 cells. The results indicate that there is spatial microheterogeneity in the intracellular localization of IP5K that could also be confirmed for the endogenous enzyme. This may facilitate changes in InsP6 levels at its sites of action. For example, overexpressed IP5K showed a structured organization within the nucleus. The kinase was preferentially localized in euchromatin and nucleoli, and co-localized with mRNA. In the cytoplasm, the overexpressed IP5K showed locally high concentrations in discrete foci. The latter were attributed to stress granules by using mRNA, PABP [poly(A)-binding protein] and TIAR (TIA-1-related protein) as markers. The incidence of stress granules, in which IP5K remained highly concentrated, was further increased by puromycin treatment. Using FRAP (fluorescence recovery after photobleaching) we established that IP5K was actively transported into the nucleus. By site-directed mutagenesis we identified a nuclear import signal and a peptide segment mediating the nuclear export of IP5K.

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A global survey of CRM1-dependent nuclear export sequences in the human deubiquitinase family

Biochemical Journal ◽

10.1042/bj20111300 ◽

2011 ◽

Vol 441 (1) ◽

pp. 209-217 ◽

Cited By ~ 27

Author(s):

Iraia García-Santisteban ◽

Sonia Bañuelos ◽

Jose A. Rodríguez

Keyword(s):

Nuclear Export ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Site Directed Mutagenesis ◽

The Novel ◽

Sequence Motifs ◽

Leptomycin B ◽

Specific Amino Acid ◽

Green Fluorescent ◽

Nuclear Export Receptor

The mechanisms that regulate the nucleocytoplasmic localization of human deubiquitinases remain largely unknown. The nuclear export receptor CRM1 binds to specific amino acid motifs termed NESs (nuclear export sequences). By using in silico prediction and experimental validation of candidate sequences, we identified 32 active NESs and 78 inactive NES-like motifs in human deubiquitinases. These results allowed us to evaluate the performance of three programs widely used for NES prediction, and to add novel information to the recently redefined NES consensus. The novel NESs identified in the present study reveal a subset of 22 deubiquitinases bearing motifs that might mediate their binding to CRM1. We tested the effect of the CRM1 inhibitor LMB (leptomycin B) on the localization of YFP (yellow fluorescent protein)- or GFP (green fluorescent protein)-tagged versions of six NES-bearing deubiquitinases [USP (ubiquitin-specific peptidase) 1, USP3, USP7, USP21, CYLD (cylindromatosis) and OTUD7B (OTU-domain-containing 7B)]. YFP–USP21 and, to a lesser extent, GFP–OTUD7B relocated from the cytoplasm to the nucleus in the presence of LMB, revealing their nucleocytoplasmic shuttling capability. Two sequence motifs in USP21 had been identified during our survey as active NESs in the export assay. Using site-directed mutagenesis, we show that one of these motifs mediates USP21 nuclear export, whereas the second motif is not functional in the context of full-length USP21.

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3′-Untranslated regions of oxidative phosphorylation mRNAs function in vivo as enhancers of translation

Biochemical Journal ◽

10.1042/bj3520109 ◽

2000 ◽

Vol 352 (1) ◽

pp. 109-115 ◽

Cited By ~ 18

Author(s):

Carlo M. Di LIEGRO ◽

Marianna BELLAFIORE ◽

José M. IZQUIERDO ◽

Anja RANTANEN ◽

José M. CUEZVA

Keyword(s):

Oxidative Phosphorylation ◽

Atp Synthase ◽

Fluorescent Protein ◽

Rat Kidney ◽

In Vitro Translation ◽

Mitochondrial Transcription Factor ◽

Normal Rat ◽

Green Fluorescent

Recent findings have indicated that the 3´-untranslated region (3´-UTR) of the mRNA encoding the β-catalytic subunit of the mitochondrial H+-ATP synthase has an in vitro translation-enhancing activity (TEA) [Izquierdo and Cuezva, Mol. Cell. Biol. (1997) 17, 5255–5268; Izquierdo and Cuezva, Biochem. J. (2000) 346, 849–855]. In the present work, we have expressed chimaeric plasmids that encode mRNA variants of green fluorescent protein in normal rat kidney and liver clone 9 cells to determine whether the 3´-UTRs of nuclear-encoded mRNAs involved in the biogenesis of mitochondria have an intrinsic TEA. TEA is found in the 3´-UTR of the mRNAs encoding the α- and β-subunits of the rat H+-ATP synthase complex, as well as in subunit IV of cytochrome c oxidase. No TEA is present in the 3´-UTR of the somatic mRNA encoding rat mitochondrial transcription factor A. Interestingly, the TEA of the 3´-UTR of mRNAs of oxidative phosphorylation is different, depending upon the cell type analysed. These data provide the first in vivo evidence of a novel cell-specific mechanism for the control of the translation of mRNAs required in mitochondrial function.

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The origin of annular junctions: a mechanism of gap junction internalization

Journal of Cell Science ◽

10.1242/jcs.114.4.763 ◽

2001 ◽

Vol 114 (4) ◽

pp. 763-773 ◽

Cited By ~ 15

Author(s):

K. Jordan ◽

R. Chodock ◽

A.R. Hand ◽

D.W. Laird

Keyword(s):

Cell Surface ◽

Gap Junction ◽

Fluorescent Protein ◽

Rat Kidney ◽

Time Lapse ◽

Gap Junctional Intercellular Communication ◽

Gap Junction Channels ◽

Normal Rat ◽

Connexin Proteins ◽

Green Fluorescent

Gap junctional intercellular communication is established when connexin proteins oligomerize into connexon hemichannels, which then pair at the cell surface with connexons from neighboring cells to form functional gap junction channels. Gap junction channels routinely cluster into gap junction plaques, which can exhibit dynamic characteristics while under the frequent processes of formation and removal from the cell surface. We have three lines of evidence to suggest that one mechanism of gap junction removal occurs when one of two contacting cells internalizes the gap junction contribution from both cells. First, in coculture experiments, green fluorescent protein-tagged connexin43 (Cx43-GFP) expressed in normal rat kidney (NRK) cells can be internalized into contacting cells that do not express Cx43-GFP, and the incidences of identifying these internalized structures increase in the presence of lysosomal inhibitors. Secondly, time-lapse imaging of live NRK cells revealed that large areas of gap junction plaques containing Cx43-GFP were internalized as vesicular-like structures into one of two adjacent cells. Finally, when live NRK cells that express endogenous Cx43 were microinjected with anti-Cx43 antibodies, antibody-tagged gap junctions were visualized in cells that contacted the microinjected cell within 3–6.5 hours. Together our results strongly suggest that one mechanism of gap junction removal from the cell surface involves a unique process in which the entire gap junction or a fragment of it is internalized into one of the two contacting cells as an annular junction.

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Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

Biochemical Journal ◽

10.1042/bj3390299 ◽

1999 ◽

Vol 339 (2) ◽

pp. 299-307 ◽

Cited By ~ 33

Author(s):

Arthur L. KRUCKEBERG ◽

Ling YE ◽

Jan A. BERDEN ◽

Karel van DAM

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Green Fluorescent Protein ◽

Glucose Transport ◽

Cellular Localization ◽

Fluorescent Protein ◽

Hexose Transporter ◽

High Concentrations ◽

Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

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Decreased Vancomycin Susceptibility in Staphylococcus aureus Caused by IS256Tempering of WalKR Expression

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00279-13 ◽

2013 ◽

Vol 57 (7) ◽

pp. 3240-3249 ◽

Cited By ~ 34

Author(s):

Christopher R. E. McEvoy ◽

Brian Tsuji ◽

Wei Gao ◽

Torsten Seemann ◽

Jessica L. Porter ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Fluorescent Protein ◽

Site Directed Mutagenesis ◽

Infection Model ◽

Content Type ◽

Reverse Transcription Pcr ◽

Phenotype Analysis ◽

Dosing Regimens ◽

Mrsa Strains ◽

Green Fluorescent

ABSTRACTVancomycin-intermediateStaphylococcus aureus(VISA) strains often arise by mutations in the essential two-component regulatorwalKR; however their impact onwalKRfunction has not been definitively established. Here, we investigated 10 MRSA strains recovered serially after exposure of vancomycin-susceptibleS. aureus(VSSA) JKD6009 to simulated human vancomycin dosing regimens (500 mg to 4,000 mg every 12 h) using a 10-day hollow fiber infection model. After continued exposure to the vancomycin regimens, two isolates displayed reduced susceptibility to both vancomycin and daptomycin, developing independent IS256insertions in thewalKR5′ untranslated region (5′ UTR). Quantitative reverse transcription-PCR (RT-PCR) revealed a 50% reduction inwalKRgene expression in the IS256mutants compared to the VSSA parent. Green fluorescent protein (GFP) reporter analysis, promoter mapping, and site-directed mutagenesis confirmed these findings and showed that the IS256insertions had replaced two SigA-likewalKRpromoters with weaker, hybrid promoters. Removal of IS256reverted the phenotype to VSSA, showing that reduced expression of WalKR did induce the VISA phenotype. Analysis of selected WalKR-regulated autolysins revealed upregulation ofssaAbut no change in expression ofsakandsceDin both IS256mutants. Whole-genome sequencing of the two mutants revealed an additional IS256insertion withinagrCfor one mutant, and we confirmed that this mutation abolishedagrfunction. These data provide the first substantial analysis ofwalKRpromoter function and show that prolonged vancomycin exposure can result in VISA through an IS256-mediated reduction inwalKRexpression; however, the mechanisms by which this occurs remain to be determined.

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Nuclear localization of the Epstein–Barr virus EBNA3B protein

Journal of General Virology ◽

10.1099/vir.0.81640-0 ◽

2006 ◽

Vol 87 (4) ◽

pp. 789-793 ◽

Cited By ~ 7

Author(s):

Anita Burgess ◽

Marion Buck ◽

Kenia Krauer ◽

Tom Sculley

Keyword(s):

Nuclear Localization ◽

Epstein Barr Virus ◽

Fluorescent Protein ◽

Site Directed Mutagenesis ◽

Nuclear Antigen ◽

Nuclear Localization Signals ◽

Barr Virus ◽

Computer Analyses ◽

Epstein Barr ◽

Green Fluorescent

The Epstein–Barr virus nuclear antigen (EBNA) 3B is a hydrophilic, proline-rich, charged protein that is thought to be involved in transcriptional regulation and is targeted exclusively to the cell nucleus, where it localizes to discrete subnuclear granules. Co-localization studies utilizing a fusion protein between enhanced green fluorescent protein (EGFP) and EBNA3B with FLAG-tagged EBNA3A and EBNA3C proteins demonstrated that EBNA3B co-localized with both EBNA3A and EBNA3C in the nuclei of cells when overexpressed. Computer analyses identified four potential nuclear-localization signals (NLSs) in the EBNA3B amino acid sequence. By utilizing fusion proteins with EGFP, deletion constructs of EBNA3B and site-directed mutagenesis, three of the four NLSs (aa 160–166, 430–434 and 867–873) were shown to be functional in truncated forms of EBNA3B, whilst an additional NLS (aa 243–246) was identified within the N-terminal region of EBNA3B. Only two of the NLSs were found to be functional in the context of the full-length EBNA3B protein.

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Developmentally regulated activity of CRM1/XPO1 during early Xenopus embryogenesis

Journal of Cell Science ◽

10.1242/jcs.113.3.451 ◽

2000 ◽

Vol 113 (3) ◽

pp. 451-459 ◽

Cited By ~ 2

Author(s):

M. Callanan ◽

N. Kudo ◽

S. Gout ◽

M. Brocard ◽

M. Yoshida ◽

...

Keyword(s):

Nuclear Export ◽

Fluorescent Protein ◽

Intracellular Localization ◽

Nuclear Export Signal ◽

Specific Inhibitor ◽

Amino Acid Identity ◽

Early Embryogenesis ◽

Leptomycin B ◽

Developmentally Regulated ◽

Neurula Stage

In this work, we have investigated the role of CRM1/XPO1, a protein involved in specific export of proteins and RNA from the nucleus, in early Xenopus embryogenesis. The cloning of the Xenopus laevis CRM1, XCRM1, revealed remarkable conservation of the protein during evolution (96.7% amino acid identity between Xenopus and human). The protein and mRNA are maternally expressed and are present during early embryogenesis. However, our data show that the activity of the protein is developmentally regulated. Embryonic development is insensitive to leptomycin B, a specific inhibitor of CRM1, until the neurula stage. Moreover, the nuclear localization of CRM1 changes concomitantly with the appearance of the leptomycin B sensitivity. These data suggest that CRM1, present initially in an inactive form, becomes functional before the initiation of the neurula stage during gastrula-neurula transition, a period known to correspond to a critical transition in the pattern of gene expression. Finally, we confirmed the gastrula-neurula transition-dependent activation of CRM1 by pull-down experiments as well as by the study of the intracellular localization of a green fluorescent protein tagged with a nuclear export signal motif during early development. This work showed that the regulated activity of CRM1 controls specific transitions during normal development and thus might be a key regulator of early embryogenesis.

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Intracellular localization and in vivo trafficking of p24A and p23

Journal of Cell Science ◽

10.1242/jcs.112.4.537 ◽

1999 ◽

Vol 112 (4) ◽

pp. 537-548 ◽

Cited By ~ 10

Author(s):

R. Blum ◽

F. Pfeiffer ◽

P. Feick ◽

W. Nastainczyk ◽

B. Kohler ◽

...

Keyword(s):

Fluorescent Protein ◽

Intracellular Localization ◽

Maximum Speed ◽

Heterotrimeric G Proteins ◽

P24 Protein ◽

Intermediate Compartment ◽

Copi Vesicle ◽

Green Fluorescent ◽

Golgi Network

Recently, p24A and p23 (also termed Tmp21), two members of the p24 protein family, have been proposed to function as integral receptors for the COPI-vesicle coat. This study describes the intracellular localization and trafficking of p24A in comparison to p23. For immunolocalization of p24A and p23, strong reduction and denaturation conditions were necessary to allow antibody interaction. Both p24A and p23 cycle continuously between intermediate compartment (IC) elements and the cis-Golgi network. In vivo trafficking of p24A and p23 tagged to green fluorescent protein (GFP) revealed that both proteins travel by large (up to 1 micrometer in length) microtubule-dependent pre-Golgi carriers with a maximum speed of up to 1.6 micrometer s-1 from the IC to the Golgi cisternae. Aluminum fluoride, a general activator of heterotrimeric G-proteins, blocked peripheral pre-Golgi movements of GFP-p24A/p23 and inhibited fluorescence recovery after photobleaching in the perinuclear Golgi area. p24A and p23 are predominantly colocalized. Overexpression of GFP-p24A, to an extent which did not destroy the Golgi complex, induced delocalization of part of the proteins into ER elements. This study therefore gives new insights into the localization and trafficking behavior of the two COPI-binding proteins p24A and p23.

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Single-Molecule Imaging and Computational Microscopy Approaches Clarify the Mechanism of the Dimerization and Membrane Interactions of Green Fluorescent Protein

International Journal of Molecular Sciences ◽

10.3390/ijms20061410 ◽

2019 ◽

Vol 20 (6) ◽

pp. 1410 ◽

Cited By ~ 1

Author(s):

Xiaohua Wang ◽

Kai Song ◽

Yang Li ◽

Ling Tang ◽

Xin Deng

Keyword(s):

Molecular Dynamics ◽

Green Fluorescent Protein ◽

Single Molecule ◽

Fluorescent Protein ◽

Hydrophobic Interactions ◽

Site Directed Mutagenesis ◽

Single Amino Acid ◽

Single Mutation ◽

Functional Protein ◽

Green Fluorescent

Green fluorescent protein (GFP) is widely used as a biomarker in living systems; however, GFP and its variants are prone to forming low-affinity dimers under physiological conditions. This undesirable tendency is exacerbated when fluorescent proteins (FP) are confined to membranes, fused to naturally-oligomeric proteins, or expressed at high levels in cells. Oligomerization of FPs introduces artifacts into the measurement of subunit stoichiometry, as well as interactions between proteins fused to FPs. Introduction of a single mutation, A206K, has been shown to disrupt hydrophobic interactions in the region responsible for GFP dimerization, thereby contributing to its monomerization. Nevertheless, a detailed understanding of how this single amino acid-dependent inhibition of dimerization in GFP occurs at the atomic level is still lacking. Single-molecule experiments combined with computational microscopy (atomistic molecular dynamics) revealed that the amino group of A206 contributes to GFP dimer formation via a multivalent electrostatic interaction. We further showed that myristoyl modification is an efficient mechanism to promote membrane attachment of GFP. Molecular dynamics-based site-directed mutagenesis has been used to identify the key functional residues in FPs. The data presented here have been utilized as a monomeric control in downstream single-molecule studies, facilitating more accurate stoichiometry quantification of functional protein complexes in living cells.

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Activity of the renal Na+-K+-2Cl− cotransporter is reduced by mutagenesis of N-glycosylation sites: role for protein surface charge in Cl− transport

AJP Renal Physiology ◽

10.1152/ajprenal.00071.2005 ◽

2006 ◽

Vol 290 (5) ◽

pp. F1094-F1102 ◽

Cited By ~ 49

Author(s):

Anahí Paredes ◽

Consuelo Plata ◽

Manuel Rivera ◽

Erika Moreno ◽

Norma Vázquez ◽

...

Keyword(s):

Confocal Microscopy ◽

Fluorescent Protein ◽

Functional Expression ◽

Site Directed Mutagenesis ◽

Intrinsic Activity ◽

Xenopus Laevis Oocytes ◽

Type I ◽

Wild Type ◽

Glycosylation Sites ◽

Green Fluorescent

The renal-specific Na+-K+-2Cl− cotransporter NKCC2 belongs to the SLC12 gene family; it is the target for loop diuretics and the cause of type I Bartter's syndrome. Because the NKCC2 sequence contains two putative N-linked glycosylation sites, one of which is conserved with the renal Na+-Cl− cotransporter in which glycosylation affects thiazide affinity, we assessed the role of glycosylation on NKCC2 functional properties. One (N442Q or N452Q) or both (N442,452Q) N-glycosylation sites were eliminated by site-directed mutagenesis. Wild-type NKCC2 and mutant clones were expressed in Xenopus laevis oocytes and analyzed by 86Rb+ influx, Western blotting, and confocal microscopy. Inhibition of glycosylation with tunicamycin in wild-type NKCC2-injected oocytes resulted in an 80% reduction of NKCC2 activity. Immunoblot of injected oocytes revealed that glycosylation of NKCC2 was completely prevented in N442,452Q-injected oocytes. Functional activity was reduced by 50% in N442Q- and N452Q-injected oocytes and by 80% in oocytes injected with N442,452Q, whereas confocal microscopy of oocytes injected with wild-type or mutant enhanced green fluorescent protein-tagged NKCC2 clones revealed that surface fluorescence intensity was reduced ∼20% in single mutants and 50% in the double mutant. Ion transport kinetic analyses revealed no changes in cation affinity and a small increase in Cl− affinity by N442Q and N442,452Q. However, a slight decrease in bumetanide affinity was observed. Our data demonstrate that NKCC2 is glycosylated and suggest that prevention of glycosylation reduces its functional expression by affecting insertion into the plasma membrane and the intrinsic activity of the cotransporter.

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