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

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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HIV-1 Vpr Oligomerization but Not That of Gag Directs the Interaction between Vpr and Gag

Journal of Virology ◽

10.1128/jvi.01691-09 ◽

2009 ◽

Vol 84 (3) ◽

pp. 1585-1596 ◽

Cited By ~ 20

Author(s):

Joëlle V. Fritz ◽

Denis Dujardin ◽

Julien Godet ◽

Pascal Didier ◽

Jan De Mey ◽

...

Keyword(s):

Plasma Membrane ◽

Fluorescent Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Cell Physiology ◽

Fluorescence Lifetime Imaging ◽

M Cell ◽

C Terminus ◽

Green Fluorescent ◽

Hiv 1

ABSTRACT During HIV-1 assembly, the viral protein R (Vpr) is incorporated into newly made viral particles via an interaction with the C-terminal domain of the Gag polyprotein precursor Pr55Gag. Vpr has been implicated in the nuclear import of newly made viral DNA and subsequently in its transcription. In addition, Vpr can affect the cell physiology by causing G2/M cell cycle arrest and apoptosis. Vpr can form oligomers, but their roles have not yet been investigated. We have developed fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer-based assays to monitor the interaction between Pr55Gag and Vpr in HeLa cells. To that end, we used enhanced green fluorescent protein-Vpr that can be incorporated into the virus and tetracysteine (TC)-tagged Pr55Gag-TC. This TC motif is tethered to the C terminus of Pr55Gag and does not interfere with Pr55Gag trafficking and the assembly of virus-like particles (VLPs). Results show that the Pr55Gag-Vpr complexes accumulated mainly at the plasma membrane. In addition, results with Pr55Gag-TC mutants confirm that the 41LXXLF domain of Gag-p6 is essential for Pr55Gag-Vpr interaction. We also report that Vpr oligomerization is crucial for Pr55Gag recognition and its accumulation at the plasma membrane. On the other hand, Pr55Gag-Vpr complexes are still formed when Pr55Gag carries mutations impairing its multimerization. These findings suggest that Pr55Gag-Vpr recognition and complex formation occur early during Pr55Gag assembly.

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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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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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Complex regulation and nuclear localization of JRK protein

Biochemical Society Transactions ◽

10.1042/bst0320920 ◽

2004 ◽

Vol 32 (6) ◽

pp. 920-923 ◽

Cited By ~ 3

Author(s):

R. Waldron ◽

T. Moore

Keyword(s):

Cellular Localization ◽

Fluorescent Protein ◽

Binding Protein ◽

Distinct Pattern ◽

Nuclear Bodies ◽

Null Mouse ◽

Fluorescence Confocal Microscopy ◽

Pml Nuclear Bodies ◽

Generalized Epilepsy ◽

Green Fluorescent

The mouse jerky gene and its human orthologue, JRK/JH8, encode a putative DNA-binding protein with homology to the CENP-B (centromere-binding protein B). Disruption of the mouse jerky gene by transgene insertion causes generalized recurrent seizures reminiscent of human idiopathic generalized epilepsy. In addition (and similar to a cenp-b null mouse) jerky null mice exhibit postnatal weight loss and reduced fertility. Using fluorescence confocal microscopy, the cellular localization of a JRK–GFP fusion (where GFP stands for green fluorescent protein) was investigated in HeLa cells. JRK–GFP has a dynamic expression pattern in the interphase nucleus, localizing in a small number of punctate nuclear foci and in the nucleolus. The JRK–GFP foci number changes during the cell cycle, but a distinct pattern of three JRK–GFP foci is observed at G2. The endogenous protein behaves in a similar manner to the GFP-fusion protein. JRK–GFP was found to co-localize with CREST antigens (which recognize the centromere-binding proteins, CENP-A, -B and -C) through S and G2 phases of interphase and co-localized completely with a subset of PML nuclear bodies at G2. We speculate that JRK protein associates with a specific chromosomal centromeric locus in G2, where it associates fully with PML bodies. Research is underway to identify this locus.

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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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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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The C-Terminal Domain of MinC Inhibits Assembly of the Z Ring in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00666-06 ◽

2006 ◽

Vol 189 (1) ◽

pp. 236-243 ◽

Cited By ~ 43

Author(s):

Daisuke Shiomi ◽

William Margolin

Keyword(s):

Escherichia Coli ◽

Cell Division ◽

Fluorescent Protein ◽

Additional Function ◽

Ring Assembly ◽

C Terminus ◽

Min Proteins ◽

Z Ring ◽

Ftsz Assembly ◽

Green Fluorescent

ABSTRACT In Escherichia coli, the Min system, consisting of three proteins, MinC, MinD, and MinE, negatively regulates FtsZ assembly at the cell poles, helping to ensure that the Z ring will assemble only at midcell. Of the three Min proteins, MinC is sufficient to inhibit Z-ring assembly. By binding to MinD, which is mostly localized at the membrane near the cell poles, MinC is sequestered away from the cell midpoint, increasing the probability of Z-ring assembly there. Previously, it has been shown that the two halves of MinC have two distinct functions. The N-terminal half is sufficient for inhibition of FtsZ assembly, whereas the C-terminal half of the protein is required for binding to MinD as well as to a component of the division septum. In this study, we discovered that overproduction of the C-terminal half of MinC (MinC122-231) could also inhibit cell division and that this inhibition was at the level of Z-ring disassembly and dependent on MinD. We also found that fusing green fluorescent protein to either the N-terminal end of MinC122-231, the C terminus of full-length MinC, or the C terminus of MinC122-231 perturbed MinC function, which may explain why cell division inhibition by MinC122-231 was not detected previously. These results suggest that the C-terminal half of MinC has an additional function in the regulation of Z-ring assembly.

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The sustainability of interactions between the orexin-1 receptor and β-arrestin-2 is defined by a single C-terminal cluster of hydroxy amino acids and modulates the kinetics of ERK MAPK regulation

Biochemical Journal ◽

10.1042/bj20041745 ◽

2005 ◽

Vol 387 (3) ◽

pp. 573-584 ◽

Cited By ~ 58

Author(s):

Sandra MILASTA ◽

Nicholas A. EVANS ◽

Laura ORMISTON ◽

Shelagh WILSON ◽

Robert J. LEFKOWITZ ◽

...

Keyword(s):

Amino Acids ◽

Fluorescent Protein ◽

Weak Interactions ◽

Dependent Manner ◽

Wild Type ◽

Erk Mapk ◽

Hydroxy Amino ◽

C Terminus ◽

Green Fluorescent ◽

Kinetics Of

The orexin-1 receptor interacts with β-arrestin-2 in an agonist-dependent manner. In HEK-293T cells, these two proteins became co-internalized into acidic endosomes. Truncations from the C-terminal tail did not prevent agonist-induced internalization of the orexin-1 receptor or alter the pathway of internalization, although such mutants failed to interact with β-arrestin-2 in a sustained manner or produce its co-internalization. Mutation of a cluster of three threonine and one serine residue at the extreme C-terminus of the receptor greatly reduced interaction and abolished co-internalization of β-arrestin-2–GFP (green fluorescent protein). Despite the weak interactions of this C-terminally mutated form of the receptor with β-arrestin-2, studies in wild-type and β-arrestin-deficient mouse embryo fibroblasts confirmed that agonist-induced internalization of this mutant required expression of a β-arrestin. Although without effect on agonist-mediated elevation of intracellular Ca2+ levels, the C-terminally mutated form of the orexin-1 receptor was unable to sustain phosphorylation of the MAPKs (mitogen-activated protein kinases) ERK1 and ERK2 (extracellular-signal-regulated kinases 1 and 2) to the same extent as the wild-type receptor. These studies indicate that a single cluster of hydroxy amino acids within the C-terminal seven amino acids of the orexin-1 receptor determine the sustainability of interaction with β-arrestin-2, and indicate an important role of β-arrestin scaffolding in defining the kinetics of orexin-1 receptor-mediated ERK MAPK activation.

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Plant Hormones Differentially Control the Sub-Cellular Localization of Plasma Membrane Microdomains during the Early Stage of Soybean Nodulation

Genes ◽

10.3390/genes10121012 ◽

2019 ◽

Vol 10 (12) ◽

pp. 1012 ◽

Cited By ~ 1

Author(s):

Zhenzhen Qiao ◽

Prince Zogli ◽

Marc Libault

Keyword(s):

Plasma Membrane ◽

Cellular Localization ◽

Fluorescent Protein ◽

Early Stage ◽

Cellular Level ◽

Infection Thread ◽

Membrane Microdomains ◽

Symbiotic Interaction ◽

Green Fluorescent ◽

Plasma Membrane Microdomains

Phytohormones regulate the mutualistic symbiotic interaction between legumes and rhizobia, nitrogen-fixing soil bacteria, notably by controlling the formation of the infection thread in the root hair (RH). At the cellular level, the formation of the infection thread is promoted by the translocation of plasma membrane microdomains at the tip of the RH. We hypothesize that phytohormones regulate the translocation of plasma membrane microdomains to regulate infection thread formation. Accordingly, we treated with hormone and hormone inhibitors transgenic soybean roots expressing fusions between the Green Fluorescent Protein (GFP) and GmFWL1 or GmFLOT2/4, two microdomain-associated proteins translocated at the tip of the soybean RH in response to rhizobia. Auxin and cytokinin treatments are sufficient to trigger or inhibit the translocation of GmFWL1 and GmFLOT2/4 to the RH tip independently of the presence of rhizobia, respectively. Unexpectedly, the application of salicylic acid, a phytohormone regulating the plant defense system, also promotes the translocation of GmFWL1 and GmFLOT2/4 to the RH tip regardless of the presence of rhizobia. These results suggest that phytohormones are playing a central role in controlling the early stages of rhizobia infection by regulating the translocation of plasma membrane microdomains. They also support the concept of crosstalk of phytohormones to control nodulation.

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