scholarly journals Functional Characterization of a Drought-Responsive Invertase Inhibitor from Maize (Zea mays L.)

2019 ◽  
Vol 20 (17) ◽  
pp. 4081 ◽  
Author(s):  
Lin Chen ◽  
Xiaohong Liu ◽  
Xiaojia Huang ◽  
Wei Luo ◽  
Yuming Long ◽  
...  
Keyword(s):  
Cell Wall ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Protein Fusion ◽  
Drought Treatment ◽  
Vacuolar Invertase ◽  
Maize Leaves ◽  
Green Fluorescent ◽  
Proteinaceous Inhibitor

Invertases (INVs) play essential roles in plant growth in response to environmental cues. Previous work showed that plant invertases can be post-translationally regulated by small protein inhibitors (INVINHs). Here, this study characterizes a proteinaceous inhibitor of INVs in maize (Zm-INVINH4). A functional analysis of the recombinant Zm-INVINH4 protein revealed that it inhibited both cell wall and vacuolar invertase activities from maize leaves. A Zm-INVINH4::green fluorescent protein fusion experiment indicated that this protein localized in the apoplast. Transcript analysis showed that Zm-INVINH4 is specifically expressed in maize sink tissues, such as the base part of the leaves and young kernels. Moreover, drought stress perturbation significantly induced Zm-INVINH4 expression, which was accompanied with a decrease of cell wall invertase (CWI) activities and an increase of sucrose accumulation in both base parts of the leaves 2 to 7 days after pollinated kernels. In summary, the results support the hypothesis that INV-related sink growth in response to drought treatment is (partially) caused by a silencing of INV activity via drought-induced induction of Zm-INVINH4 protein.

scholarly journals Characterization of sarR, a Modulator ofsar Expression in Staphylococcus aureus

2001 ◽  
Vol 69 (2) ◽  
pp. 885-896 ◽  
Author(s):  
Adhar Manna ◽  
Ambrose L. Cheung
Keyword(s):  
Staphylococcus Aureus ◽  
Fluorescent Protein ◽  
Regulatory Protein ◽  
Virulence Determinants ◽  
Protein Fusion ◽  
Putative Gene ◽  
P2 Promoter ◽  
Green Fluorescent ◽  
Overlapping Transcripts

ABSTRACT The expression of virulence determinants in Staphylococcus aureus is controlled by global regulatory loci (e.g.,sar and agr). The sar locus is composed of three overlapping transcripts (sar P1, P3, and P2 transcripts from P1, P3, and P2 promoters, respectively), all encoding the 372-bp sarA gene. The level of SarA, the major regulatory protein, is partially controlled by the differential activation of sar promoters. We previously partially purified a ∼12 kDa protein with a DNA-specific column containing asar P2 promoter fragment. In this study, the putative gene, designated sarR, was identified and found to encode a 13.6-kDa protein with homology to SarA. Transcriptional and immunoblot studies revealed the sarR gene to be expressed in other staphylococcal strains. Recombinant SarR protein bound sarP1, P2, and P3 promoter fragments in gel shift and footprinting assays. A sarR mutant expressed a higher level of P1 transcript than the parent, as confirmed by promoter green fluorescent protein fusion assays. As the P1 transcript is the predominant sartranscript, we confirmed that the sarR mutant expressed more SarA than the parental strain. We thus proposed that SarR is a regulatory protein that binds to the sar promoters to down-regulate P1 transcription and the ensuing SarA protein expression.

scholarly journals A Mutation in a Novel Yeast Proteasomal Gene,RPN11/MPR1, Produces a Cell Cycle Arrest, Overreplication of Nuclear and Mitochondrial DNA, and an Altered Mitochondrial Morphology

1998 ◽  
Vol 9 (10) ◽  
pp. 2917-2931 ◽  
Author(s):  
Teresa Rinaldi ◽  
Carlo Ricci ◽  
Danilo Porro ◽  
Monique Bolotin-Fukuhara ◽  
Laura Frontali
Keyword(s):  
Mitochondrial Dna ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Mitochondrial Morphology ◽  
Nonpermissive Temperature ◽  
Protein Fusion ◽  
In The Wild ◽  
A Cell ◽  
Green Fluorescent ◽  
Cytoplasmic Structures

We report here the functional characterization of an essentialSaccharomyces cerevisiae gene, MPR1, coding for a regulatory proteasomal subunit for which the name Rpn11p has been proposed. For this study we made use of thempr1-1 mutation that causes the following pleiotropic defects. At 24°C growth is delayed on glucose and impaired on glycerol, whereas no growth is seen at 36°C on either carbon source. Microscopic observation of cells growing on glucose at 24°C shows that most of them bear a large bud, whereas mitochondrial morphology is profoundly altered. A shift to the nonpermissive temperature produces aberrant elongated cell morphologies, whereas the nucleus fails to divide. Flow cytometry profiles after the shift to the nonpermissive temperature indicate overreplication of both nuclear and mitochondrial DNA. Consistently with the identification of Mpr1p with a proteasomal subunit, the mutation is complemented by the human POH1proteasomal gene. Moreover, the mpr1-1 mutant grown to stationary phase accumulates ubiquitinated proteins. Localization of the Rpn11p/Mpr1p protein has been studied by green fluorescent protein fusion, and the fusion protein has been found to be mainly associated to cytoplasmic structures. For the first time, a proteasomal mutation has also revealed an associated mitochondrial phenotype. We actually showed, by the use of [rho°] cells derived from the mutant, that the increase in DNA content per cell is due in part to an increase in the amount of mitochondrial DNA. Moreover, microscopy of mpr1-1 cells grown on glucose showed that multiple punctate mitochondrial structures were present in place of the tubular network found in the wild-type strain. These data strongly suggest that mpr1-1 is a valuable tool with which to study the possible roles of proteasomal function in mitochondrial biogenesis.

Four novel splice variants of sulfonylurea receptor 1

2002 ◽  
Vol 283 (2) ◽  
pp. C587-C598 ◽  
Author(s):  
Annette Hambrock ◽  
Regina Preisig-Müller ◽  
Ulrich Russ ◽  
Anke Piehl ◽  
Peter J. Hanley ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Splice Variants ◽  
Functional Characterization ◽  
Pcr Analysis ◽  
Sulfonylurea Receptor ◽  
Transmembrane Segments ◽  
Sulfonylurea Receptor 1 ◽  
Green Fluorescent ◽  
Splice Forms

ATP-sensitive K+ (KATP) channels are composed of pore-forming Kir6.x subunits and regulatory sulfonylurea receptor (SUR) subunits. SURs are ATP-binding cassette proteins with two nucleotide-binding folds (NBFs) and binding sites for sulfonylureas, like glibenclamide, and for channel openers. Here we report the identification and functional characterization of four novel splice forms of guinea pig SUR1. Three splice forms originate from alternative splicing of the region coding for NBF1 and lack exons 17 (SUR1Δ17), 19 (SUR1Δ19), or both (SUR1Δ17Δ19). The fourth (SUR1C) is a COOH-terminal SUR1-fragment formed by exons 31–39 containing the last two transmembrane segments and the COOH terminus of SUR1. RT-PCR analysis showed that these splice forms are expressed in several tissues with strong expression of SUR1C in cardiomyocytes. Confocal microscopy using enhanced green fluorescent protein-tagged SUR or Kir6.x did not provide any evidence for involvement of these splice forms in the mitochondrial KATP channel. Only SUR1 and SUR1Δ17 showed high-affinity binding of glibenclamide ( K d≈ 2 nM in the presence of 1 mM ATP) and formed functional KATPchannels upon coexpression with Kir6.2.

scholarly journals Green fluorescent protein-based assays for high-throughput functional characterization and ligand-binding studies of biotin protein ligase

2016 ◽  
Vol 8 (2) ◽  
pp. 418-424 ◽  
Author(s):  
Samuel P. Askin ◽  
Thomas E. H. Bond ◽  
Patrick M. Schaeffer
Keyword(s):  
Green Fluorescent Protein ◽  
Ligand Binding ◽  
High Throughput ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Binding Studies ◽  
Biotin Protein Ligase ◽  
Green Fluorescent

Rapid functional characterization of GFP-tagged biotin protein ligase (BirA-GFP) with a high-throughput DSF-GTP assay.

Functional characterization of a vacuolar invertase from Solanum lycopersicum: Post-translational regulation by N-glycosylation and a proteinaceous inhibitor

Biochimie ◽  
2014 ◽  
Vol 101 ◽  
pp. 39-49 ◽  
Author(s):  
Alexandra S. Tauzin ◽  
Gerlind Sulzenbacher ◽  
Mickael Lafond ◽  
Véronique Desseaux ◽  
Ida Barbara Reca ◽  
...  
Keyword(s):  
Solanum Lycopersicum ◽  
Translational Regulation ◽  
Functional Characterization ◽  
Vacuolar Invertase ◽  
Proteinaceous Inhibitor

scholarly journals Characterization of a Serine Proteinase Mediating Encystation of Acanthamoeba

2008 ◽  
Vol 7 (9) ◽  
pp. 1513-1517 ◽  
Author(s):  
Eun-Kyung Moon ◽  
Dong-Il Chung ◽  
Yeon-Chul Hong ◽  
Hyun-Hee Kong
Keyword(s):  
Fluorescent Protein ◽  
Serine Proteinase ◽  
Tissue Destruction ◽  
Protein Fusion ◽  
Granulomatous Amoebic Encephalitis ◽  
Causative Agents ◽  
Amoebic Keratitis ◽  
Green Fluorescent ◽  
Interfering Rna

ABSTRACT Members of the genus Acanthamoeba, amphizoic protozoan parasites, are causative agents of granulomatous amoebic encephalitis and amoebic keratitis. Proteinases play a role in various biologic actions in Acanthamoeba, including host tissue destruction, pathogenesis, and digestion of phagocytosed food. Interestingly, we found that encystation of Acanthamoeba was inhibited by the serine proteinase inhibitor phenylmethanesulfonyl fluoride. In this study, we characterize a serine proteinase that is involved in mediating the encystation of Acanthamoeba. This encystation-mediating serine proteinase (EMSP) is shown to be highly expressed during encystation by real-time PCR and Western blot analysis. Chemically synthesized small interfering RNA against EMSP inhibited the expression of EMSP mRNA and significantly reduced the encystation efficiency of Acanthamoeba. An EMSP-enhanced green fluorescent protein fusion protein localized to vesicle-like structures within the amoeba. Using LysoTracker analysis, these vesicular structures were confirmed to be lysosomes. After incubation of the transfected amoeba in encystment media, small fluorescent vesicle-like structures gathered and formed ball-like structures, which were identified as colocalizing with the autophagosome. Taken together, these results indicate that EMSP plays an important role in the differentiation of Acanthamoeba by promoting autolysis.

scholarly journals Biochemical and functional characterization of a mitochondrial citrate carrier in Arabidopsis thaliana

2020 ◽  
Vol 477 (9) ◽  
pp. 1759-1777 ◽  
Author(s):  
Danielle S. Brito ◽  
Gennaro Agrimi ◽  
Lennart Charton ◽  
Dominik Brilhaus ◽  
Maria Gabriella Bitetto ◽  
...  
Keyword(s):  
Gene Expression Analysis ◽  
Nitrogen Assimilation ◽  
Fluorescent Protein ◽  
Developmental Stages ◽  
Functional Characterization ◽  
Radicle Growth ◽  
Citrate Carrier ◽  
Green Fluorescent ◽  
Lower Expression

A homolog of the mitochondrial succinate/fumarate carrier from yeast (Sfc1p) has been found in the Arabidopsis genome, named AtSFC1. The AtSFC1 gene was expressed in Escherichia coli, and the gene product was purified and reconstituted in liposomes. Its transport properties and kinetic parameters demonstrated that AtSFC1 transports citrate, isocitrate and aconitate and, to a lesser extent, succinate and fumarate. This carrier catalyzes a fast counter-exchange transport as well as a low uniport of substrates, exhibits a higher transport affinity for tricarboxylates than dicarboxylates, and is inhibited by pyridoxal 5′-phosphate and other inhibitors of mitochondrial carriers to various degrees. Gene expression analysis indicated that the AtSFC1 transcript is mainly present in heterotrophic tissues, and fusion with a green-fluorescent protein localized AtSFC1 to the mitochondria. Furthermore, 35S-AtSFC1 antisense lines were generated and characterized at metabolic and physiological levels in different organs and at various developmental stages. Lower expression of AtSFC1 reduced seed germination and impaired radicle growth, a phenotype that was related to reduced respiration rate. These findings demonstrate that AtSFC1 might be involved in storage oil mobilization at the early stages of seedling growth and in nitrogen assimilation in root tissue by catalyzing citrate/isocitrate or citrate/succinate exchanges.

scholarly journals Functional characterization of green fluorescent protein-profilin fusion proteins

2000 ◽  
Vol 267 (16) ◽  
pp. 5247-5256 ◽  
Author(s):  
Nina Wittenmayer ◽  
Martin Rothkegel ◽  
Brigitte M. Jockusch ◽  
Kathrin Schlüter
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Green Fluorescent

scholarly journals Functional characterization of the Beet necrotic yellow vein virus RNA-5-encoded p26 protein: evidence for structural pathogenicity determinants

2005 ◽  
Vol 86 (7) ◽  
pp. 2115-2125 ◽  
Author(s):  
Didier Link ◽  
Laure Schmidlin ◽  
Audrey Schirmer ◽  
Elodie Klein ◽  
Mathieu Erhardt ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Chenopodium Quinoa ◽  
Yellow Vein ◽  
Virus Rna ◽  
Infected Cells ◽  
Green Fluorescent ◽  
Necrotic Symptoms

A Beet necrotic yellow vein virus isolate containing a fifth RNA is present in the Pithiviers area of France. A full-length cDNA clone of RNA-5 was obtained and placed under the control of a T7-RNA-pol promoter that allowed the production of infectious transcripts. ‘Pithiviers' isolate-specific necrotic symptoms were obtained on Chenopodium quinoa when RNA-5-encoded p26 was expressed either from RNA-5 or from an RNA-3-derived replicon. By using haemagglutinin- and green fluorescent protein-tagged constructs, virally expressed p26-fusion proteins induced the same necrotic local lesions on host plants and were localized mainly in the nucleus of infected cells. Deletion mutagenesis permitted identification of two domains, responsible respectively for nuclear export and cytoplasmic retention of the p26 mutated proteins. By using a yeast two-hybrid system, Gal4DB–p26 protein self-activated transcription of the His3 reporter gene. The p26 transcription-activation domain was located within its first 55 aa and has been studied by alanine scanning. Resulting p26 mutants were tested for their capability to induce necrotic symptoms and to localize in the nuclear compartment.

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