scholarly journals TOR1 and TOR2 Have Distinct Locations in Live Cells

2008 ◽  
Vol 7 (10) ◽  
pp. 1819-1830 ◽  
Author(s):  
Thomas W. Sturgill ◽  
Adiel Cohen ◽  
Melanie Diefenbacher ◽  
Mark Trautwein ◽  
Dietmar E. Martin ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Essential Gene ◽  
Control Cell ◽  
Variable Region ◽  
Live Cells ◽  
Cellular Processes ◽  
C Terminus ◽  
Cold Sensitive ◽  
Green Fluorescent

ABSTRACT TOR is a structurally and functionally conserved Ser/Thr kinase found in two multiprotein complexes that regulate many cellular processes to control cell growth. Although extensively studied, the localization of TOR is still ambiguous, possibly because endogenous TOR in live cells has not been examined. Here, we examined the localization of green fluorescent protein (GFP) tagged, endogenous TOR1 and TOR2 in live S. cerevisiae cells. A DNA cassette encoding three copies of green fluorescent protein (3XGFP) was inserted in the TOR1 gene (at codon D330) or the TOR2 gene (at codon N321). The TORs were tagged internally because TOR1 or TOR2 tagged at the N or C terminus was not functional. The TOR1 D330-3XGFP strain was not hypersensitive to rapamycin, was not cold sensitive, and was not resistant to manganese toxicity caused by the loss of Pmr1, all indications that TOR1-3XGFP was expressed and functional. TOR2-3XGFP was functional, as TOR2 is an essential gene and TOR2 N321-3XGFP haploid cells were viable. Thus, TOR1 and TOR2 retain function after the insertion of 748 amino acids in a variable region of their noncatalytic domain. The localization patterns of TOR1-3XGFP and TOR2-3XGFP were documented by imaging of live cells. TOR1-3XGFP was diffusely cytoplasmic and concentrated near the vacuolar membrane. The TOR2-3XGFP signal was cytoplasmic but predominately in dots at the plasma membrane. Thus, TOR1 and TOR2 have distinct localization patterns, consistent with the regulation of cellular processes as part of two different complexes.

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

2000 ◽  
Vol 74 (23) ◽  
pp. 11339-11346 ◽  
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.

scholarly journals C-terminal tripeptide Ser-Asn-Leu (SNL) of human D-aspartate oxidase is a functional peroxisome-targeting signal

1998 ◽  
Vol 336 (2) ◽  
pp. 367-371 ◽  
Author(s):  
Leen AMERY ◽  
Chantal BREES ◽  
Myriam BAES ◽  
Chiaki SETOYAMA ◽  
Retsu MIURA ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Fluorescent Protein ◽  
Consensus Sequence ◽  
C Terminus ◽  
Targeting Signal ◽  
Fluorescent Pattern ◽  
Green Fluorescent ◽  
Positively Charged

The functionality of the C-terminus (Ser-Asn-Leu; SNL) of human d-aspartate oxidase, an enzyme proposed to have a role in the inactivation of synaptically released d-aspartate, as a peroxisome-targeting signal (PTS1) was investigated in vivoand in vitro. Bacterially expressed human d-aspartate oxidase was shown to interact with the human PTS1-binding protein, peroxin protein 5 (PEX5p). Binding was gradually abolished by carboxypeptidase treatment of the oxidase and competitively inhibited by a Ser-Lys-Leu (SKL)-containing peptide. After transfection of mouse fibroblasts with a plasmid encoding green fluorescent protein (GFP) extended by PKSNL (the C-terminal pentapeptide of the oxidase), a punctate fluorescent pattern was evident. The modified GFP co-localized with peroxisomal thiolase as shown by indirect immunofluorescence. On transfection in fibroblasts lacking PEX5p receptor, GFP–PKSNL staining was cytosolic. Peroxisomal import of GFP extended by PGSNL (replacement of the positively charged fourth-last amino acid by glycine) seemed to be slower than that of GFP–PKSNL, whereas extension by PKSNG abolished the import of the modified GFP. Taken together, these results indicate that SNL, a tripeptide not fitting the PTS1 consensus currently defined in mammalian systems, acts as a functional PTS1 in mammalian systems, and that the consensus sequence, based on this work and that of other groups, has to be broadened to (S/A/C/K/N)-(K/R/H/Q/N/S)-L.

scholarly journals Sexual Pheromone Induces Diffusion of the Pheromone-Homologous Polypeptide in the Extracellular Matrix of Volvox carteri

2007 ◽  
Vol 6 (11) ◽  
pp. 2157-2162 ◽  
Author(s):  
Koichi Ishida
Keyword(s):  
Extracellular Matrix ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Sexual Pheromone ◽  
Volvox Carteri ◽  
Terminal Domain ◽  
Induction Signal ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT The C-terminal domain of pherophorin II is homologous to the sexual pheromone of Volvox carteri and is released from other domains during sexual induction. Green fluorescent protein fused to the C terminus of pherophorin II was located at the extracellular matrix directly surrounding the gonidium, the final target of the sexual-induction signal.

scholarly journals Utilization of green fluorescent protein as a marker for studying the expression and turnover of the monocarboxylate permease Jen1p of Saccharomyces cerevisiae

2002 ◽  
Vol 363 (3) ◽  
pp. 737-744 ◽  
Author(s):  
Sandra PAIVA ◽  
Arthur L. KRUCKEBERG ◽  
Margarida CASAL
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Reporter Protein ◽  
C Terminus ◽  
Lactate Transporter ◽  
Green Fluorescent ◽  
Endocytic Pathways

Green fluorescent protein (GFP) from Aequorea victoria was used as an in vivo reporter protein when fused to the C-terminus of the Jen1 lactate permease of Saccharomyces cerevisiae. The Jen1 protein tagged with GFP is a functional lactate transporter with a cellular abundance of 1670 molecules/cell, and a catalytic-centre activity of 123s−1. It is expressed and tagged to the plasma membrane under induction conditions. The factors involved in proper localization and turnover of Jen1p were revealed by expression of the Jen1p—GFP fusion protein in a set of strains bearing mutations in specific steps of the secretory and endocytic pathways. The chimaeric protein Jen1p—GFP is targeted to the plasma membrane via a Sec6-dependent process; upon treatment with glucose, it is endocytosed via END3 and targeted for degradation in the vacuole. Experiments performed in a Δdoa4 mutant strain showed that ubiquitination is associated with the turnover of the permease.

scholarly journals Green Fluorescent Protein-Dal80p Illuminates up to 16 Distinct Foci That Colocalize with and Exhibit the Same Behavior as Chromosomal DNA Proceeding through the Cell Cycle of Saccharomyces cerevisiae

2001 ◽  
Vol 183 (15) ◽  
pp. 4636-4642 ◽  
Author(s):  
MacKenzie Distler ◽  
Ajit Kulkarni ◽  
Rajendra Rai ◽  
Terrance G. Cooper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Green Fluorescent Protein ◽  
Leucine Zipper ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Live Cells ◽  
Nitrogen Catabolite Repression ◽  
Chromosomal Dna ◽  
Green Fluorescent

ABSTRACT Four GATA family DNA binding proteins mediate nitrogen catabolite repression-sensitive transcription in Saccharomyces cerevisiae. Gln3p and Gat1p are transcriptional activators, while Dal80p and Deh1p repress Gln3p- and Gat1p-mediated transcription by competing with these activators for binding to DNA. Strong Dal80p binding to DNA is thought to result from C-terminal leucine zipper-mediated dimerization. Many Dal80p binding site-homologous sequences are relatively evenly distributed across the S. cerevisiae genome, raising the possibility that Dal80p might be able to “stain” DNA. We demonstrate that cells containing enhanced green fluorescent protein-Dal80p (EGFP-Dal80p) exhibit up to 16 fluorescent foci that colocalize with DAPI (4′,6′-diamidino-2-phenylindole)-positive material and follow DNA movement through the cell cycle, suggesting that EGFP-Dal80p may indeed be useful for monitoring yeast chromosomes in live cells and in real time.

scholarly journals Functional Regions of the Pseudomonas aeruginosa Cytotoxin ExoU

2005 ◽  
Vol 73 (1) ◽  
pp. 573-582 ◽  
Author(s):  
Shira D. P. Rabin ◽  
Alan R. Hauser
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Hela Cells ◽  
Fluorescent Protein ◽  
Host Cells ◽  
Phospholipase Activity ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT ExoU, a potent patatin-like phospholipase, causes rapid cell death following its injection into host cells by the Pseudomonas aeruginosa type III secretion system. To better define regions of ExoU required for cytotoxicity, transposon-based linker insertion mutagenesis followed by site-directed mutagenesis of individual residues was employed by using a Saccharomyces cerevisiae model system. Random insertion of five amino acids identified multiple regions within ExoU that are required for cell killing. Five regions were chosen for further characterization: three corresponded to the oxyanion hole, hydrolase motif, and catalytic aspartate motif of the patatin-like domain within the N-terminal half of ExoU; one corresponded to an uncharacterized part of the patatin-like domain; and one corresponded to a region near the C terminus. Specific individual amino acid substitutions in each of the four N-terminal regions prevented killing of yeast and significantly reduced phospholipase activity. Whereas five amino acid insertions in the fifth region near the C terminus markedly reduced cytotoxicity and phospholipase activity, substitution of individual amino acids did not abolish either activity. To determine whether each of the five identified regions of ExoU was also essential for cytotoxicity in human cells, representative mutant forms of ExoU fused to green fluorescent protein were expressed in HeLa cells. These variants of ExoU were readily visualized and caused minimal cytotoxicity to HeLa cells, while wild-type ExoU fused to green fluorescent protein induced significant cell lysis and no detectable fluorescence. Thus, a minimum of five regions, including one which is well removed from the patatin-like domain, are required for the cytotoxicity and phospholipase activity of ExoU.

scholarly journals A single-wavelength flow cytometric approach using redox-sensitive green fluorescent protein probes for measuring redox stress in live cells

BioTechniques ◽  
2021 ◽  
Author(s):  
Elizabeth R Denn ◽  
Joseph M Schober
Keyword(s):  
Oxidative Stress ◽  
Green Fluorescent Protein ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Recovery Phase ◽  
Live Cells ◽  
Redox Stress ◽  
Green Fluorescent

Cellular redox changes are common in apoptosis, immune function, signaling pathways and cancer. The authors aimed to develop a single-wavelength method using the superior fluorescence sensitivity of a flow cytometer for measuring redox-sensitive green fluorescent protein signal during oxidative stress in cell lines. The single-wavelength method was able to discern small differences in oxidative stress between cell lines and between the cytoplasmic and mitochondrial compartments within the same cell line. In Chinese hamster ovary cells, the mitochondrial matrix compartment was more sensitive to oxidative stress compared with MDA-MB-231 cells, and the rapid changes in redox state were followed by a slow recovery phase. The authors conclude that this simplified method is useful and preferred for studies where alterations in overall redox-sensitive green fluorescent protein expression are controlled.

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