scholarly journals Tracking the subcellular protein localization process by combining the time-gated method and photoconvertible fluorescence protein

2020 ◽  
Vol 32 (1) ◽  
pp. 27-30
Author(s):  
Momoko Sakata ◽  
Yutaka Kodama
Keyword(s):  
Protein Localization ◽  
Subcellular Protein Localization ◽  
Fluorescence Protein

scholarly journals Improved fusion protein expression of EGFP via the mutation of both Kozak and the initial ATG codon

2007 ◽  
Vol 12 (3) ◽  
Author(s):  
Chao Dai ◽  
Zhijian Cao ◽  
Yingliang Wu ◽  
Hong Yi ◽  
Dahe Jiang ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Expression Vector ◽  
Fusion Gene ◽  
Protein Localization ◽  
Green Fluorescence Protein ◽  
Green Fluorescence ◽  
Fusion Protein Expression ◽  
Subcellular Protein Localization ◽  
Egfp Fusion Protein ◽  
Fluorescence Protein

AbstractSince its discovery, green fluorescence protein (GFP) has been used as a reporter in a broad range of applications, including the determination of gene expresion in diverse organisms, and subcellular protein localization. pEGFP-N1 is a eukayotic expression vector encoding EGFP, the MCS of which locates at the N terminus of EGFP. In this study, the cDNA sequence of scorpion toxin BmKK2 was inserted into the XhoI-HindIII cut of pEGFP-N1 to construct a toxin-EGFP fusion gene (named pEGFP-BmKK2). Fluorescence imaging revealed that HEK 293T cells that were transfected by pEGFP-BmKK2 emitted green fluorescence. Transcription of pEGFP-BmKK2 was confirmed by RT-PCR. However, western blotting analysis showed that the transfected HEK 293T cells expressed mostly EGFP, but little toxin-EGFP fusion protein, implying that pEGFP-N1 cannot be used as a fusion expression vector for subcellular protein localization for the BmKK2 gene. Consequently, two modified recombinant vectors (pEGFP-BmKK2-M1 and pEGFP-BmKK2-M2) were constructed based on pEGFP-BmKK2. This greatly improved the expression of toxin-EGFP fusion protein from pEGFP-BmKK2-M2.

TheArabidopsis Localizome: Subcellular Protein Localization and Interactions inARABIDOPSIS

2008 ◽  
pp. 61-81
Author(s):  
Georgios Kitsios ◽  
Nicolas Tsesmetzis ◽  
Max Bush ◽  
John H. Doonan
Keyword(s):  
Protein Localization ◽  
Subcellular Protein Localization

scholarly journals MultiLoc2: integrating phylogeny and Gene Ontology terms improves subcellular protein localization prediction

2009 ◽  
Vol 10 (1) ◽  
Author(s):  
Torsten Blum ◽  
Sebastian Briesemeister ◽  
Oliver Kohlbacher
Keyword(s):  
Gene Ontology ◽  
Protein Localization ◽  
Subcellular Protein Localization ◽  
Localization Prediction

Barcodes for subcellular protein localization

2019 ◽  
Vol 3 (9) ◽  
pp. 673-675 ◽  
Author(s):  
Yizhe Zhang ◽  
Alden Moss ◽  
Kristine Tan ◽  
Amy E. Herr
Keyword(s):  
Protein Localization ◽  
Subcellular Protein Localization

scholarly journals High-throughput subcellular protein localization using cell arrays

2005 ◽  
Vol 33 (6) ◽  
pp. 1407 ◽  
Author(s):  
D. Vanhecke ◽  
Y.-H. Hu ◽  
H. Lehrach ◽  
M. Janitz
Keyword(s):  
High Throughput ◽  
Protein Localization ◽  
Subcellular Protein Localization ◽  
Cell Arrays

scholarly journals A Genomic Study of the Bipolar Bud Site Selection Pattern inSaccharomyces cerevisiae

2001 ◽  
Vol 12 (7) ◽  
pp. 2147-2170 ◽  
Author(s):  
Li Ni ◽  
Michael Snyder
Keyword(s):  
Site Selection ◽  
Protein Modification ◽  
Protein Localization ◽  
Green Fluorescence Protein ◽  
Green Fluorescence ◽  
Genome Wide ◽  
A Genome ◽  
Genomic Study ◽  
Fluorescence Protein ◽  
Bud Site

A genome-wide screen of 4168 homozygous diploid yeast deletion strains has been performed to identify nonessential genes that participate in the bipolar budding pattern. By examining bud scar patterns representing the sites of previous cell divisions, 127 mutants representing three different phenotypes were found: unipolar, axial-like, and random. From this screen, 11 functional classes of known genes were identified, including those involved in actin-cytoskeleton organization, general bud site selection, cell polarity, vesicular transport, cell wall synthesis, protein modification, transcription, nuclear function, translation, and other functions. Four characterized genes that were not known previously to participate in bud site selection were also found to be important for the haploid axial budding pattern. In addition to known genes, we found 22 novel genes (20 are designated BUD13-BUD32) important for bud site selection. Deletion of one resulted in unipolar budding exclusively from the proximal pole, suggesting that this gene plays an important role in diploid distal budding. Mutations in 20 other novelBUD genes produced a random budding phenotype and one produced an axial-like budding defect. Several of the novel Bud proteins were fused to green fluorescence protein; two proteins were found to localize to sites of polarized cell growth (i.e., the bud tip in small budded cells and the neck in cells undergoing cytokinesis), similar to that postulated for the bipolar signals and proteins that target cell division site tags to their proper location in the cell. Four others localized to the nucleus, suggesting that they play a role in gene expression. The bipolar distal marker Bud8 was localized in a number of mutants; many showed an altered Bud8-green fluorescence protein localization pattern. Through the genome-wide identification and analysis of different mutants involved in bipolar bud site selection, an integrated pathway for this process is presented in which proximal and distal bud site selection tags are synthesized and localized at their appropriate poles, thereby directing growth at those sites. Genome-wide screens of defined collections of mutants hold significant promise for dissecting many biological processes in yeast.

scholarly journals Glycolytic flux-signaling controls mouse embryo mesoderm development

2021 ◽  
Author(s):  
Hidenobu Miyazawa ◽  
Marteinn T. Snaebjornsson ◽  
Nicole Prior ◽  
Eleni Kafkia ◽  
Henrik M Hammarén ◽  
...  
Keyword(s):  
Soluble Fraction ◽  
Protein Localization ◽  
Proteome Analysis ◽  
Glycolytic Flux ◽  
Mesoderm Development ◽  
Subcellular Protein Localization ◽  
Fructose 1,6 Bisphosphate ◽  
Post Implantation ◽  
Medium Supplementation

How cellular metabolic state impacts cellular programs is a fundamental, unresolved question. Here we investigated how glycolytic flux impacts embryonic development, using presomitic mesoderm (PSM) patterning as the experimental model. First, we identified fructose 1,6-bisphosphate (FBP) as an in vivo sentinel metabolite that mirrors glycolytic flux within PSM cells of post-implantation mouse embryos. We found that medium-supplementation with FBP, but not with other glycolytic metabolites, such as fructose 6-phosphate and 3-phosphoglycerate, impaired mesoderm segmentation. To genetically manipulate glycolytic flux and FBP levels, we generated a mouse model enabling the conditional overexpression of dominant active, cytoplasmic Pfkfb3 (cytoPfkfb3). Overexpression of cytoPfkfb3 indeed led to increased glycolytic flux/FBP levels and caused an impairment of mesoderm segmentation, paralleled by the downregulation of Wnt-signaling, reminiscent of the effects seen upon FBP-supplementation. To probe for mechanisms underlying glycolytic flux-signaling, we performed subcellular proteome analysis and revealed that cytoPfkfb3 overexpression altered subcellular localization of certain proteins, including glycolytic enzymes, in PSM cells. Specifically, we revealed that FBP supplementation caused depletion of Pfkl and Aldoa from the nuclear-soluble fraction. Combined, we propose that FBP functions as a flux-signaling metabolite connecting glycolysis and PSM patterning, potentially through modulating subcellular protein localization.

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