The large intracytoplasmic loop of the glucose transporter GLUT2 is involved in glucose signaling in hepatic cells

The hypothesis that the glucose transporter GLUT2 can function as a protein mediating transcriptional glucose signaling was addressed. To divert the putative interacting proteins from a glucose signaling pathway, two intracytoplasmic domains of GLUT2, the C terminus and the large loop located between transmembrane domains 6 and 7, were transfected into mhAT3F hepatoma cells. Glucose-induced accumulation of two hepatic gene mRNAs (GLUT2 and L-pyruvate kinase) was specifically inhibited in cells transfected with the GLUT2 loop and not with the GLUT2 C terminus. The dual effects of glucose were dissociated in cells expressing the GLUT2 loop; in fact a normal glucose metabolism into glycogen occurred concomitantly with the inhibition of the glucose-induced transcription. This inhibition by the GLUT2 loop could be due to competitive binding of a protein that normally interacts with endogenous GLUT2. In addition, the GLUT2 loop, tagged with green fluorescent protein (GFP), was located within the nucleus, whereas the GFP and GFP-GLUT2 C-terminal proteins remained in the cytoplasm. In living cells, a fraction (50%) of the expressed GFP-GLUT2 loop translocated rapidly from the cytoplasm to the nucleus in response to high glucose concentration and conversely in the absence of glucose. We conclude that, via protein interactions with its large loop, GLUT2 may transduce a glucose signal from the plasma membrane to the nucleus.

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Construction, Verification and Experimental Use of Two Epitope-Tagged Collections of Budding Yeast Strains

Comparative and Functional Genomics ◽

10.1002/cfg.449 ◽

2005 ◽

Vol 6 (1-2) ◽

pp. 2-16 ◽

Cited By ~ 55

Author(s):

Russell Howson ◽

Won-Ki Huh ◽

Sina Ghaemmaghami ◽

James V. Falvo ◽

Kiowa Bower ◽

...

Keyword(s):

Protein Interactions ◽

Fluorescent Protein ◽

Budding Yeast ◽

Affinity Purification ◽

Reading Frame ◽

Yeast Strains ◽

Yeast Community ◽

C Terminus ◽

Protein Properties ◽

Green Fluorescent

A major challenge in the post-genomic era is the development of experimental approaches to monitor the properties of proteins on a proteome-wide level. It would be particularly useful to systematically assay protein subcellular localization, post-translational modifications and protein–protein interactions, both at steady state and in response to environmental stimuli. Development of new reagents and methods will enhance our ability to do so efficiently and systematically. Here we describe the construction of two collections of budding yeast strains that facilitate proteome-wide measurements of protein properties. These collections consist of strains with an epitope tag integrated at the C-terminus of essentially every open reading frame (ORF), one with the tandem affinity purification (TAP) tag, and one with the green fluorescent protein (GFP) tag. We show that in both of these collections we have accurately tagged a high proportion of all ORFs (approximately 75% of the proteome) by confirming expression of the fusion proteins. Furthermore, we demonstrate the use of the TAP collection in performing high-throughput immunoprecipitation experiments. Building on these collections and the methods described in this paper, we hope that the yeast community will expand both the quantity and type of proteome level data available.

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Cellular Targets of Functional and Dysfunctional Mutants of Tobacco Mosaic Virus Movement Protein Fused to Green Fluorescent Protein

Journal of Virology ◽

10.1128/jvi.74.23.11339-11346.2000 ◽

2000 ◽

Vol 74 (23) ◽

pp. 11339-11346 ◽

Cited By ~ 58

Author(s):

Vitaly Boyko ◽

Jessica van der Laak ◽

Jacqueline Ferralli ◽

Elena Suslova ◽

Myoung-Ok Kwon ◽

...

Keyword(s):

Amino Acids ◽

Green Fluorescent Protein ◽

Tobacco Mosaic Virus ◽

Inclusion Bodies ◽

Fluorescent Protein ◽

Movement Protein ◽

Leading Edge ◽

Intercellular Transport ◽

C Terminus ◽

Green Fluorescent

ABSTRACT Intercellular transport of tobacco mosaic virus (TMV) RNA involves the accumulation of virus-encoded movement protein (MP) in plasmodesmata (Pd), in endoplasmic reticulum (ER)-derived inclusion bodies, and on microtubules. The functional significance of these interactions in viral RNA (vRNA) movement was tested in planta and in protoplasts with TMV derivatives expressing N- and C-terminal deletion mutants of MP fused to the green fluorescent protein. Deletion of 55 amino acids from the C terminus of MP did not interfere with the vRNA transport function of MP:GFP but abolished its accumulation in inclusion bodies, indicating that accumulation of MP at these ER-derived sites is not a requirement for function in vRNA intercellular movement. Deletion of 66 amino acids from the C terminus of MP inactivated the protein, and viral infection occurred only upon complementation in plants transgenic for MP. The functional deficiency of the mutant protein correlated with its inability to associate with microtubules and, independently, with its absence from Pd at the leading edge of infection. Inactivation of MP by N-terminal deletions was correlated with the inability of the protein to target Pd throughout the infection site, whereas its associations with microtubules and inclusion bodies were unaffected. The observations support a role of MP-interacting microtubules in TMV RNA movement and indicate that MP targets microtubules and Pd by independent mechanisms. Moreover, accumulation of MP in Pd late in infection is insufficient to support viral movement, confirming that intercellular transport of vRNA relies on the presence of MP in Pd at the leading edge of infection.

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Identification of a Novel Saturable Endoplasmic Reticulum Localization Mechanism Mediated by the C-Terminus of aDictyostelium Protein Disulfide Isomerase

Molecular Biology of the Cell ◽

10.1091/mbc.11.10.3469 ◽

2000 ◽

Vol 11 (10) ◽

pp. 3469-3484 ◽

Cited By ~ 35

Author(s):

Jean Monnat ◽

Eva M. Neuhaus ◽

Marius S. Pop ◽

David M. Ferrari ◽

Barbara Kramer ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Protein Disulfide Isomerase ◽

Fluorescent Protein ◽

Null Mutation ◽

Disulfide Isomerase ◽

Isomerase Activity ◽

C Terminus ◽

Complementary Action ◽

Green Fluorescent ◽

Protein Disulfide

Localization of soluble endoplasmic reticulum (ER) resident proteins is likely achieved by the complementary action of retrieval and retention mechanisms. Whereas the machinery involving the H/KDEL and related retrieval signals in targeting escapees back to the ER is well characterized, other mechanisms including retention are still poorly understood. We have identified a protein disulfide isomerase (Dd-PDI) lacking the HDEL retrieval signal normally found at the C terminus of ER residents in Dictyostelium discoideum. Here we demonstrate that its 57 residue C-terminal domain is necessary for intracellular retention of Dd-PDI and sufficient to localize a green fluorescent protein (GFP) chimera to the ER, especially to the nuclear envelope. Dd-PDI and GFP-PDI57 are recovered in similar cation-dependent complexes. The overexpression of GFP-PDI57 leads to disruption of endogenous PDI complexes and induces the secretion of PDI, whereas overexpression of a GFP-HDEL chimera induces the secretion of endogenous calreticulin, revealing the presence of two independent and saturable mechanisms. Finally, low-level expression of Dd-PDI but not of PDI truncated of its 57 C-terminal residues complements the otherwise lethal yeast TRG1/PDI1 null mutation, demonstrating functional disulfide isomerase activity and ER localization. Altogether, these results indicate that the PDI57 peptide contains ER localization determinants recognized by a conserved machinery present in D. discoideum and Saccharomyces cerevisiae.

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Asc Modulates the Function of NLRC4 in Response to Infection of Macrophages by Legionella pneumophila

mBio ◽

10.1128/mbio.00117-11 ◽

2011 ◽

Vol 2 (4) ◽

Cited By ~ 36

Author(s):

Christopher L. Case ◽

Craig R. Roy

Keyword(s):

Cell Death ◽

Legionella Pneumophila ◽

Fluorescent Protein ◽

Microbial Infection ◽

Content Type ◽

Caspase 1 ◽

Cell Death Pathway ◽

Dependent Cell ◽

Green Fluorescent ◽

In Cells

ABSTRACTNucleotide-binding domain, leucine-rich repeat containing proteins (NLRs) activate caspase-1 in response to a variety of bacterium-derived signals in macrophages. NLR-mediated activation of caspase-1 byLegionella pneumophilaoccurs through both an NLRC4/NAIP5-dependent pathway and a pathway requiring the adapter protein Asc. Both pathways are needed for maximal activation of caspase-1 and for the release of the cytokines interleukin-1β (IL-1β) and IL-18. Asc is not required for caspase-1-dependent pore formation and cell death induced upon infection of macrophages byL. pneumophila. Here, temporal and spatial localization of caspase-1-dependent processes was examined to better define the roles of Asc and NLRC4 during infection. Imaging studies revealed that caspase-1 localized to a single punctate structure in infected cells containing Asc but not in cells lacking this adapter. Both endogenous Asc and ectopically produced NLRC4 tagged with green fluorescent protein (GFP) were found to localize to caspase-1 puncta followingL. pneumophilainfection, suggesting that NLRC4 and Asc coordinate signaling through this complex during caspase-1 activation. Formation of caspase-1-containing puncta correlated with caspase-1 processing, suggesting a role for the Asc/NLRC4/caspase-1 complex in caspase-1 cleavage. In cells deficient for Asc, NLRC4 did not assemble into discrete puncta, and pyroptosis occurred at an accelerated rate. These data indicate that Asc mediates integration of NLR components into caspase-1 processing platforms and that recruitment of NLR components into an Asc complex can dampen pyroptotic responses. Thus, a negative feedback role of complexes containing Asc may be important for regulating caspase-1-mediated responses during microbial infection.IMPORTANCECaspase-1 is a protease activated during infection that is central to the regulation of several innate immune pathways. Studies examining the macromolecular complexes containing this protein, known as inflammasomes, have provided insight into the regulation of this protease. This work demonstrates that the intracellular bacteriumLegionella pneumophilainduces formation of complexes containing caspase-1 by multiple mechanisms and illustrates that an adapter molecule called Asc integrates signals from multiple independent upstream caspase-1 activators in order to assemble a spatially distinct complex in the macrophage. There were caspase-1-associated activities such as cytokine processing and secretion that were controlled by Asc. Importantly, this work uncovered a new role for Asc in dampening a caspase-1-dependent cell death pathway called pyroptosis. These findings suggest that Asc plays a central role in controlling a distinct subset of caspase-1-dependent activities by both assembling complexes that are important for cytokine processing and suppressing processes that mediate pyroptosis.

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Rem2, a new member of the Rem/Rad/Gem/Kir family of Ras-related GTPases

Biochemical Journal ◽

10.1042/bj3470223 ◽

2000 ◽

Vol 347 (1) ◽

pp. 223-231 ◽

Cited By ~ 37

Author(s):

Brian S. FINLIN ◽

Haipeng SHAO ◽

Keiko KADONO-OKUDA ◽

Nan GUO ◽

Douglas A. ANDRES

Keyword(s):

Cellular Localization ◽

Biochemical Characterization ◽

Fluorescent Protein ◽

Dissociation Constants ◽

Intrinsic Rate ◽

Biochemical Properties ◽

Cell Physiology ◽

Gtp Binding ◽

C Terminus ◽

Green Fluorescent

Here we report the molecular cloning and biochemical characterization of Rem2 (for Rem, ad and G-related 2), a novel GTP-binding protein identified on the basis of its homology with the Rem, Rad, Gem and Kir (RGK) family of Ras-related small GTP-binding proteins. Rem2 mRNA was detected in rat brain and kidney, making it the first member of the RGK family to be expressed at relatively high levels in neuronal tissues. Recombinant Rem2 binds GTP saturably and exhibits a low intrinsic rate of GTP hydrolysis. Surprisingly, the guanine nucleotide dissociation constants for both Rem2 and Rem are significantly different than the majority of the Ras-related GTPases, displaying higher dissociation rates for GTP than GDP. Localization studies with green fluorescent protein (GFP)-tagged recombinant protein fusions indicate that Rem2 has a punctate, plasma membrane localization. Deletion of the C-terminal seven amino acid residues that are conserved in all RGK family members did not affect the cellular distribution of the GFP fusion protein, whereas a larger deletion, including much of the polybasic region of the Rem2 C-terminus, resulted in its redistribution to the cytosol. Thus Rem2 is a GTPase of the RGK family with distinctive biochemical properties and possessing a novel cellular localization signal, consistent with its having a unique role in cell physiology.

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Protease-activatable biosensors of SARS-CoV-2 infection for cell-based drug, neutralisation and virological assays

10.1101/2021.03.22.435957 ◽

2021 ◽

Author(s):

Pehuen Pereyra Gerber ◽

Lidia M Duncan ◽

Edward JD Greenwood ◽

Sara Marelli ◽

Adi Naamati ◽

...

Keyword(s):

Fluorescent Protein ◽

Firefly Luciferase ◽

Neutralising Antibodies ◽

Antiviral Therapies ◽

Viral Proteases ◽

Green Fluorescent ◽

High Throughput Screens ◽

Protease Expression ◽

In Cells

The world is in the grip of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, and there is an urgent unmet clinical need for effective antiviral therapies. Many inhibitors of viral enzymes identified in vitro have limited efficacy against viral replication in cells, but conventional plaque assays are impractical for high-throughput screens. In this study, we therefore engineer cell-based biosensors of SARS-CoV-2 infection. Our assays exploit the cleavage of specific oligopeptide linkers by SARS-CoV-2 Main or Papain-like proteases, leading to the activation of green fluorescent protein (GFP) or firefly luciferase-based reporters. First, we characterise these biosensors in cells using recombinant viral proteases. Next, we confirm their ability to detect endogenous viral protease expression during infection with wildtype SARS-CoV-2. Finally, we develop a sensitive luminescent reporter cell line, confirm that it accurately quantitates infectious SARS-CoV-2 virus, and demonstrate its utility for drug screening and titration of neutralising antibodies.

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Probing chemotaxis activity inEscherichia coliusing fluorescent protein fusions

10.1101/367110 ◽

2018 ◽

Author(s):

Clémence Roggo ◽

Jan Roelof van der Meer

Keyword(s):

Escherichia Coli ◽

Protein Interactions ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Bacterial Chemotaxis ◽

Ph Sensitive ◽

Flagellar Motors ◽

Receptor Interactions ◽

Split Egfp ◽

Green Fluorescent

ABSTRACTChemotaxis is based on ligand-receptor interactions that are transmitted via protein-protein interactions to the flagellar motors. Ligand-receptor interactions in chemotaxis can be deployed for the development of rapid biosensor assays, but there is no consensus as to what the best readout of such assays would have to be. Here we explore two potential fluorescent readouts of chemotactically activeEscherichia colicells. In the first, we probed interactions between the chemotaxis signaling proteins CheY and CheZ by fusing them individually with non-fluorescent parts of a ‘split’-Green Fluorescent Protein. Wild-type chemotactic cells but not mutants lacking the CheA kinase produced distinguishable fluorescence foci, two-thirds of which localize at the cell poles with the chemoreceptors and one-third at motor complexes. Cells expressing fusion proteins only were attracted to serine sources, demonstrating measurable functional interactions between CheY~P and CheZ. Fluorescent foci based on stable split-eGFP displayed small fluctuations in cells exposed to attractant or repellent, but those based on an unstable ASV-tagged eGFP showed a higher dynamic behaviour both in the foci intensity changes and the number of foci per cell. For the second readout, we expressed the pH-sensitive fluorophore pHluorin in the cyto- and periplasm of chemotactically activeE. coli. Calibrations of pHluorin fluorescence as a function of pH demonstrated that cells accumulating near a chemo-attractant temporally increase cytoplasmic pH while decreasing periplasmic pH. Both readouts thus show promise as proxies for chemotaxis activity, but will have to be further optimized in order to deliver practical biosensor assays.IMPORTANCEBacterial chemotaxis may be deployed for future biosensing purposes with the advantages of its chemoreceptor ligand-specificity and its minute-scale response time. On the downside, chemotaxis is ephemeral and more difficult to quantitatively read out than, e.g., reporter gene expression. It is thus important to investigate different alternative ways to interrogate chemotactic response of cells. Here we gauge the possibilities to measure dynamic response in theEscherichia colichemotaxis pathway resulting from phosphorylated CheY-CheZ interactions by using (unstable) split-fluorescent proteins. We further test whether pH differences between cyto- and periplasm as a result of chemotactic activity can be measured with help of pH-sensitive fluorescent proteins. Our results show that both approaches conceptually function, but will need further improvement in terms of detection and assay types to be practical for biosensing.

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Na+-dependent phosphate transporters in the murine osteoclast: cellular distribution and protein interactions

AJP Cell Physiology ◽

10.1152/ajpcell.00580.2002 ◽

2003 ◽

Vol 284 (6) ◽

pp. C1633-C1644 ◽

Cited By ~ 37

Author(s):

Mohammed A. Khadeer ◽

Zhihui Tang ◽

Harriet S. Tenenhouse ◽

Maribeth V. Eiden ◽

Heini Murer ◽

...

Keyword(s):

Plasma Membrane ◽

Bone Resorption ◽

Protein Interactions ◽

Cellular Localization ◽

Fluorescent Protein ◽

Basolateral Membrane ◽

Atp Synthesis ◽

Cellular Distribution ◽

Green Fluorescent ◽

Carboxy Terminal

We previously demonstrated that inhibition of Na-dependent phosphate (Pi) transport in osteoclasts led to reduced ATP levels and diminished bone resorption. These findings suggested that Na/Picotransporters in the osteoclast plasma membrane provide Pifor ATP synthesis and that the osteoclast may utilize part of the Pireleased from bone resorption for this purpose. The present study was undertaken to define the cellular localization of Na/Picotransporters in the mouse osteoclast and to identify the proteins with which they interact. Using glutathione S-transferase (GST) fusion constructs, we demonstrate that the type IIa Na/Picotransporter (Npt2a) in osteoclast lysates interacts with the Na/H exchanger regulatory factor, NHERF-1, a PDZ protein that is essential for the regulation of various membrane transporters. In addition, NHERF-1 in osteoclast lysates interacts with Npt2a in spite of deletion of a putative PDZ-binding domain within the carboxy terminus of Npt2a. In contrast, deletion of the carboxy-terminal TRL amino acid motif of Npt2a significantly reduced its interaction with NHERF-1 in kidney lysates. Studies in osteoclasts transfected with green fluorescent protein-Npt2a constructs indicated that Npt2a colocalizes with NHERF-1 and actin at or near the plasma membrane of the osteoclast and associates with ezrin, a linker protein associated with the actin cytoskeleton, likely via NHERF-1. Furthermore, we demonstrate by RT/PCR of osteoclast RNA and in situ hybridization that the type III Na/Picotransporter, PiT-1, is also expressed in mouse osteoclasts. To examine the cellular distribution of PiT-1, we infected mouse osteoclasts with a retroviral vector encoding PiT-1 fused to an epitope tag. PiT-1 colocalizes with actin and is present on the basolateral membrane of the polarized osteoclast, similar to that previously reported for Npt2a. Taken together, our data suggest that association of Npt2a with NHERF-1, ezrin, and actin, and of PiT-1 with actin, may be responsible for membrane sorting and regulation of these Na/Picotransporters in the osteoclast.

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Application of a Chimeric Green Fluorescent Protein to Study Protein-Protein Interactions

BioTechniques ◽

10.2144/97235st02 ◽

1997 ◽

Vol 23 (5) ◽

pp. 864-872 ◽

Cited By ~ 8

Author(s):

N. Garamszegi ◽

Z.P. Garamszegi ◽

M.S. Rogers ◽

S.J. De-Marco ◽

E.E. Strehler

Keyword(s):

Green Fluorescent Protein ◽

Protein Interactions ◽

Fluorescent Protein ◽

Protein Protein Interactions ◽

Green Fluorescent

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Bifunctional fusion proteins containing the sequence of the bradykinin homologue maximakinin: activities at the rat bradykinin B2 receptor

Canadian Journal of Physiology and Pharmacology ◽

10.1139/cjpp-2017-0692 ◽

2018 ◽

Vol 96 (5) ◽

pp. 459-470 ◽

Cited By ~ 1

Author(s):

Xavier Charest-Morin ◽

Robert Lodge ◽

François Marceau

Keyword(s):

Fusion Proteins ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Protein Denaturation ◽

Epifluorescence Microscopy ◽

Terminal Sequence ◽

Protein Ligands ◽

C Terminus ◽

Bona Fide ◽

Green Fluorescent

To support bradykinin (BK) B2 receptor (B2R) detection and therapeutic stimulation, we developed and characterized fusion proteins consisting of the BK homolog maximakinin (MK), or variants, positioned at the C-terminus of functional proteins (enhanced green fluorescent protein (EGFP), the peroxidase APEX2, or human serum albumin (HSA)). EGFP-MK loses its reactivity with anti-BK antibodies and molecular mass as it progresses in the endosomal tract of cells expressing rat B2Rs (immunoblots, epifluorescence microscopy). APEX2-(NG)15-MK is a bona fide agonist of the rat, but not of the human B2R (calcium and c-Fos signaling) and is compatible with the cytochemistry reagent TrueBlue (microscopy), a luminol-based reagent, or 3,3′,5,5′-tetramethylbenzidine (luminescence or colourimetric B2R detection, cell well plate format). APEX2-(NG)15-MK is a non-isotopic ligand suitable for drug discovery via binding competition. Affinity-purified secreted forms of HSA fused with peptides possessing the C-terminal MK or BK sequence failed to stimulate the rat B2R in the concentration range of 50–600 nmol/L. However, the non-secreted construction myc-HSA-MK is a B2R agonist, indicating that protein denaturation made the C-terminal sequence available for receptor binding. Fusion protein ligands of the B2R are stable but subjected to slow intracellular inactivation, strong species specificity, and possible steric hindrance between the receptor and large proteins.

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