The Green Fluorescent Protein

Tsien, Roger Y.

doi:10.1146/annurev.biochem.67.1.509

Cited by 5,635 publications

(5,357 citation statements)

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“…The extraordinary sensitivity of fluorescent techniques was responsible for the widespread use of FRET for detection of protein interactions, a trend that was highly accentuated with the use of fluorescent proteins and genetic engineering (Tsien 1998). However, the application of FRET methodologies to the study of association between membrane components has been generally limited to high-affinity interactions, such as protein–protein interactions or those typically observed for membrane proteins and specifically bound lipids.…”

Section: Discussionmentioning

confidence: 99%

Quantification of protein–lipid selectivity using FRET

Loura

Prieto²,

Fernandes

2009

Eur Biophys J

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Membrane proteins exhibit different affinities for different lipid species, and protein–lipid selectivity regulates the membrane composition in close proximity to the protein, playing an important role in the formation of nanoscale membrane heterogeneities. The sensitivity of Förster resonance energy transfer (FRET) for distances of 10 Å up to 100 Å is particularly useful to retrieve information on the relative distribution of proteins and lipids in the range over which protein–lipid selectivity is expected to influence membrane composition. Several FRET-based methods applied to the quantification of protein–lipid selectivity are described herein, and different formalisms applied to the analysis of FRET data for particular geometries of donor–acceptor distribution are critically assessed.

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Section: Discussionmentioning

confidence: 99%

Quantification of protein–lipid selectivity using FRET

Loura

Prieto²,

Fernandes

2009

Eur Biophys J

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“…The advent of live cell imaging and GFP-labelling technologies in the 1990s ( Tsien, 1998) have greatly facilitated the study of proteasome dynamics in yeast and mammalian cells. Through these non-invasive techniques, the localization of the proteasome in growing yeast and highly proliferating cancer cells has been elucidated to be primarily nuclear ( Enenkel et al , 1998; Laporte et al , 2008; McDonald & Byers, 1997; Russell et al , 1999).…”

Section: Introductionmentioning

confidence: 99%

Intracellular Dynamics of the Ubiquitin-Proteasome-System

Chowdhury

Enenkel

2015

F1000Res

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The ubiquitin-proteasome system is the major degradation pathway for short-lived proteins in eukaryotic cells. Targets of the ubiquitin-proteasome-system are proteins regulating a broad range of cellular processes including cell cycle progression, gene expression, the quality control of proteostasis and the response to geno- and proteotoxic stress. Prior to degradation, the proteasomal substrate is marked with a poly-ubiquitin chain. The key protease of the ubiquitin system is the proteasome. In dividing cells, proteasomes exist as holo-enzymes composed of regulatory and core particles. The regulatory complex confers ubiquitin-recognition and ATP dependence on proteasomal protein degradation. The catalytic sites are located in the proteasome core particle. Proteasome holo-enzymes are predominantly nuclear suggesting a major requirement for proteasomal proteolysis in the nucleus. In cell cycle arrested mammalian or quiescent yeast cells, proteasomes deplete from the nucleus and accumulate in granules at the nuclear envelope (NE) / endoplasmic reticulum (ER) membranes. In prolonged quiescence, proteasome granules drop off the NE / ER membranes and migrate as stable organelles throughout the cytoplasm, as thoroughly investigated in yeast. When quiescence yeast cells are allowed to resume growth, proteasome granules clear and proteasomes are rapidly imported into the nucleus.Here, we summarize our knowledge about the enigmatic structure of proteasome storage granules and the trafficking of proteasomes and their substrates between the cyto- and nucleoplasm.Most of our current knowledge is based on studies in yeast. Their translation to mammalian cells promises to provide keen insight into protein degradation in non-dividing cells which comprise the majority of our body’s cells.

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“…For example, green fluorescent protein (GFP) and its variants have made a significant impact on cell biology as in vivo reporters of protein localization and gene expression [1,2]. Continued development of novel and improved fluorescent protein probes will increase the repertoire of available fluorescent probes, and contribute to new discoveries about cell structure and function [3].…”

Section: Introductionmentioning

confidence: 99%

Formation of fluorescent proteins by the attachment of phycoerythrobilin to R-phycoerythrin alpha and beta apo-subunits

Isailović

Sultana

Phillips

et al. 2006

Analytical Biochemistry

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Formation of fluorescent proteins was explored after incubation of recombinant apo-subunits of phycobiliprotein R-phycoerythrin with phycoerythrobilin chromophore. Alpha and beta apo-subunit genes of R-phycoerythrin from red algae Polisiphonia boldii were cloned in plasmids pET-21d (+). Hexa-histidine tagged apo-alpha and apo-beta subunits were expressed in Escherichia coli. Although expressed apo-subunits formed inclusion bodies, fluorescent holo-subunits were constituted after incubation of Escherichia coli cells with phycoerythrobilin. Subunits contained both phycoerythrobilin and urobilin chromophores. Fluorescence and differential interference contrast microscopy showed polar location of holo-subunit inclusion bodies in bacterial cells. Cells containing fluorescent holo-subunits were several times brighter than control cells as found by fluorescence microscopy and flow cytometry. Addition of phycoerythrobilin to cells did not show cytotoxic effects in contrast to expression of proteins in inclusion bodies. In an attempt to improve solubility, Rphycoerythrin apo-subunits were fused to maltose binding protein and incubated with phycoerythrobilin both in vitro and in vivo. Highly-fluorescent soluble fusion proteins were formed containing phycoerythrobilin as the sole chromophore. Fusion proteins were localized by fluorescence microscopy either throughout Escherichia coli cells or at cell poles. Flow cytometry showed that cells containing fluorescent fusion proteins were up to ten times brighter than control cells. Results indicate that fluorescent proteins formed by attachment of phycoerythrobilin to expressed apo-subunits of phycobiliproteins can be used as fluorescent probes for analysis of cells by microscopy and flow cytometry. A unique property of these fluorescent reporters is their utility in both properly folded (soluble) subunits and subunits aggregated in inclusion bodies.

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The Green Fluorescent Protein

Cited by 5,635 publications

References 116 publications

Quantification of protein–lipid selectivity using FRET

Quantification of protein–lipid selectivity using FRET

Intracellular Dynamics of the Ubiquitin-Proteasome-System

Formation of fluorescent proteins by the attachment of phycoerythrobilin to R-phycoerythrin alpha and beta apo-subunits

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