Fusion with Fluorescent Proteins for Subcellular Localization of Enzymes Involved in Plant Alkaloid Biosynthesis

2010 ◽  
pp. 275-290 ◽  
Author(s):  
Patrícia Duarte ◽  
Johan Memelink ◽  
Mariana Sottomayor
Keyword(s):  
Subcellular Localization ◽  
Fluorescent Proteins ◽  
Alkaloid Biosynthesis

scholarly journals Subcellular localization of prion proteins and the 37 kDa/67 kDa laminin receptor fused to fluorescent proteins

2008 ◽  
Vol 1782 (5) ◽  
pp. 335-340 ◽  
Author(s):  
Daphne Nikles ◽  
Karen Vana ◽  
Sabine Gauczynski ◽  
Heike Knetsch ◽  
Heike Ludewigs ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Prion Proteins ◽  
Fluorescent Proteins ◽  
Laminin Receptor

scholarly journals Identifying Genes Involved in Alkaloid Biosynthesis in Vinca minor through Transcriptomics and Gene Co-Expression Analysis

Biomolecules ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 1595
Author(s):  
Emily Amor Stander ◽  
Liuda Johana Sepúlveda ◽  
Thomas Dugé de Bernonville ◽  
Inês Carqueijeiro ◽  
Konstantinos Koudounas ◽  
...  
Keyword(s):  
Candidate Genes ◽  
Biosynthetic Pathway ◽  
Fluorescent Proteins ◽  
Light Exposure ◽  
Indole Alkaloids ◽  
High Light ◽  
Alkaloid Biosynthesis ◽  
Vinca Minor ◽  
Young Leaves ◽  
Derivatives Of

The lesser periwinkle Vinca minor accumulates numerous monoterpene indole alkaloids (MIAs) including the vasodilator vincamine. While the biosynthetic pathway of MIAs has been largely elucidated in other Apocynaceae such as Catharanthus roseus, the counterpart in V. minor remains mostly unknown, especially for reactions leading to MIAs specific to this plant. As a consequence, we generated a comprehensive V. minor transcriptome elaborated from eight distinct samples including roots, old and young leaves exposed to low or high light exposure conditions. This optimized resource exhibits an improved completeness compared to already published ones. Through homology-based searches using C. roseus genes as bait, we predicted candidate genes for all common steps of the MIA pathway as illustrated by the cloning of a tabersonine/vincadifformine 16-O-methyltransferase (Vm16OMT) isoform. The functional validation of this enzyme revealed its capacity of methylating 16-hydroxylated derivatives of tabersonine, vincadifformine and lochnericine with a Km 0.94 ± 0.06 µM for 16-hydroxytabersonine. Furthermore, by combining expression of fusions with yellow fluorescent proteins and interaction assays, we established that Vm16OMT is located in the cytosol and forms homodimers. Finally, a gene co-expression network was performed to identify candidate genes of the missing V. minor biosynthetic steps to guide MIA pathway elucidation.

A simple and effective method for protein subcellular localization using Agrobacterium-mediated transformation of onion epidermal cells

Biologia ◽  
2007 ◽  
Vol 62 (5) ◽  
Author(s):  
Wei Sun ◽  
Ziyi Cao ◽  
Yan Li ◽  
Yanxiu Zhao ◽  
Hui Zhang
Keyword(s):  
Subcellular Localization ◽  
Particle Bombardment ◽  
Fusion Proteins ◽  
Fluorescent Proteins ◽  
Transformed Cells ◽  
Protein Subcellular Localization ◽  
Transformation Protocol ◽  
Agrobacterium Mediated Transformation ◽  
The Mean ◽  
Onion Epidermal Cells

AbstractA modified Agrobacterium-mediated transformation protocol has been successfully used for transient expression of the intrinsically fluorescent proteins and their fusion proteins in onion epidermis. The mean of the transformed cells rate per peel is about 10.5±0.9%, while that of the particle bombardment method is at the range 2.0±0.4%. To compare with the prevailing method of micro-projectile bombardment, the modified Agrobacterium-mediated transformation may provide with higher efficiency and even more simplified manipulability on a lower budget.

scholarly journals Generation of optimized yellow and red fluorescent proteins with distinct subcellular localization

BioTechniques ◽  
2004 ◽  
Vol 36 (3) ◽  
pp. 418-424 ◽  
Author(s):  
Chiga Okita ◽  
Masae Sato ◽  
Timm Schroeder
Keyword(s):  
Subcellular Localization ◽  
Fluorescent Proteins ◽  
Red Fluorescent Proteins

Subcellular localization of acyltransferases for quinolizidine alkaloid biosynthesis in Lupinus

1996 ◽  
Vol 42 (6) ◽  
pp. 1557-1562 ◽  
Author(s):  
Hideyuki Suzuki ◽  
Youichi Koike ◽  
Isamu Murakoshi ◽  
Kazuki Saito
Keyword(s):  
Subcellular Localization ◽  
Alkaloid Biosynthesis ◽  
Quinolizidine Alkaloid

Investigating the functional link between TMEM165 and SPCA1

2019 ◽  
Vol 476 (21) ◽  
pp. 3281-3293 ◽  
Author(s):  
Elodie Lebredonchel ◽  
Marine Houdou ◽  
Hans-Heinrich Hoffmann ◽  
Kateryna Kondratska ◽  
Marie-Ange Krzewinski ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Complete Loss ◽  
Protein Family ◽  
Uncharacterized Protein ◽  
Functional Link ◽  
P Type

TMEM165 was highlighted in 2012 as the first member of the Uncharacterized Protein Family 0016 (UPF0016) related to human glycosylation diseases. Defects in TMEM165 are associated with strong Golgi glycosylation abnormalities. Our previous work has shown that TMEM165 rapidly degrades with supraphysiological manganese supplementation. In this paper, we establish a functional link between TMEM165 and SPCA1, the Golgi Ca2+/Mn2+ P-type ATPase pump. A nearly complete loss of TMEM165 was observed in SPCA1-deficient Hap1 cells. We demonstrate that TMEM165 was constitutively degraded in lysosomes in the absence of SPCA1. Complementation studies showed that TMEM165 abundance was directly dependent on SPCA1's function and more specifically its capacity to pump Mn2+ from the cytosol into the Golgi lumen. Among SPCA1 mutants that differentially impair Mn2+ and Ca2+ transport, only the Q747A mutant that favors Mn2+ pumping rescues the abundance and Golgi subcellular localization of TMEM165. Interestingly, the overexpression of SERCA2b also rescues the expression of TMEM165. Finally, this paper highlights that TMEM165 expression is linked to the function of SPCA1.

Fluorescent proteins for in vivo imaging, where's the biliverdin?

2020 ◽  
Vol 48 (6) ◽  
pp. 2657-2667
Author(s):  
Felipe Montecinos-Franjola ◽  
John Y. Lin ◽  
Erik A. Rodriguez
Keyword(s):  
Hydrogen Peroxide ◽  
In Vivo Imaging ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Imaging ◽  
Infrared Light ◽  
Red Fluorescent Protein ◽  
Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

1019: Visualization of the Neurovascular Bundles and Major Pelvic Ganglion with Fluorescent Proteins after Penile Injection

2006 ◽  
Vol 175 (4S) ◽  
pp. 328-328 ◽  
Author(s):  
Hugo H. Davila ◽  
Maggie Mamcarz ◽  
Irving Nadelhaft ◽  
Raoul Salup ◽  
Jorge Lockhart ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Pelvic Ganglion ◽  
Major Pelvic Ganglion ◽  
Neurovascular Bundles
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