Method for Real-Time Monitoring of Protein Degradation at the Single Cell Level

Zhang, Lijuan; Gurskaya, Nadya G.; Merzlyak, Ekaterina M.; Staroverov, Dmitry B; Mudrik, N. N.; Samarkina, Olga N; Vinokurov, Leonid M.; Lukyanov, Sergey; Lukyanov, Konstantin A.

doi:10.2144/000112453

Cited by 78 publications

(68 citation statements)

References 14 publications

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“…Monitoring of the degradation of transiently expressed proteins in BY-2 cells was based in part on methods described for other fluorescent protein-based degradation assays developed for mammalian cultured cells (37)(38)(39). Briefly, BY-2 cells were bombarded biolistically as described above except that: (i) cells were incubated in MS medium rather than transformation buffer (to reduce any potential cellular damage from plasmolysis); and (ii) 30 min after bombardment, cells were transferred from Whatman paper, resuspended in MS medium in a culture flask, and then incubated under standard conditions for maintaining BY-2 cell cultures (i.e.…”

Section: Methodsmentioning

confidence: 99%

Temperature-sensitive Post-translational Regulation of Plant Omega-3 Fatty-acid Desaturases Is Mediated by the Endoplasmic Reticulum-associated Degradation Pathway

O'Quin

Bourassa

Zhang

et al. 2010

Journal of Biological Chemistry

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Changes in ambient temperature represent a major physiological challenge to membranes of poikilothermic organisms. In plants, the endoplasmic reticulum (ER)-localized omega-3 fatty-acid desaturases (Fad3) increase the production of polyunsaturated fatty acids at cooler temperatures, but the FAD3 genes themselves are typically not up-regulated during this adaptive response. Here, we expressed two closely related plant FAD3 genes in yeast cells and found that their enzymes produced significantly different amounts of omega-3 fatty acids and that these differences correlated to differences in rates of protein turnover. Domain-swapping and mutagenesis experiments revealed that each protein contained a degradation signal in its N terminus and that the charge density of a PEST-like sequence within this region was largely responsible for the differences in rates of protein turnover. The half-life of each Fad3 protein was increased at cooler temperatures, and protein degradation required specific components of the ER-associated degradation pathway including the Cdc48 adaptor proteins Doa1, Shp1, and Ufd2. Expression of the Fad3 proteins in tobacco cells incubated with the proteasomal inhibitor MG132 further confirmed that they were degraded via the proteasomal pathway in plants. Collectively, these findings indicate that Fad3 protein abundance is regulated by a combination of cis-acting degradation signals and the ubiquitin-proteasome pathway and that modulation of Fad3 protein amounts in response to temperature may represent one mechanism of homeoviscous adaptation in plants.

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Section: Methodsmentioning

confidence: 99%

Temperature-sensitive Post-translational Regulation of Plant Omega-3 Fatty-acid Desaturases Is Mediated by the Endoplasmic Reticulum-associated Degradation Pathway

O'Quin

Bourassa

Zhang

et al. 2010

Journal of Biological Chemistry

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“…Dendra2 is a mutated form of green fluorescent protein (GFP) that is expressed as a green fluorophore that can be photoconverted to a red fluorophore. Similar to GFP, protein folding occurs rapidly for Dendra2, but the subsequent spontaneous formation of its fluorophore through cyclization and oxidation of amino acid side chains is slow, requiring approximately 90 min (Zhang et al 2007). In order to visualize the differential impact of GA on newly made and preexisting ERBB3, individual MCF7 cells expressing ERBB3-Dendra2 were imaged prior to photoconversion and the reemergence of green fluorescent ERBB3-Dendra2 after photoconversion was evaluated in the presence or absence of GA.…”

Section: Nascent Erbb3 Is Ga Sensitivementioning

confidence: 99%

“…Additional insight into the time window of GA sensitivity is provided by the time delay between the synthesis and rapid folding of Dendra2 and the slow maturation of its fluorophore (Zhang et al 2007). Due to this delay, most of the green fluorescent protein that reemerges after photoconversion was already synthesized prior to the addition of either GA or CHX.…”

Section: Nascent Erbb3 Is Ga Sensitivementioning

confidence: 99%

Geldanamycin selectively targets the nascent form of ERBB3 for degradation

Gerbin

Landgraf

2010

Cell Stress and Chaperones

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Heat shock protein 90 (HSP90) targets a broad spectrum of client proteins with divergent modes of interaction and consequences. The homologous epidermal growth factor receptor (EGFR) and ERBB2 receptors as well as kinase-deficient mutants thereof differ in their requirement for HSP90 in the nascent versus mature state of the receptor. Specific features of the kinase domain have been implicated for the selective association of HSP90 with mature ERBB2. We evaluated the role of HSP90 for the homologous ERBB3 receptor. ERBB3 is naturally kinase deficient, a central mediator in cell survival and stress response and the primary dimerization partner for ERBB2 in signaling. Cellular studies indicate that, similar to EGFR, the geldanamycin (GA) sensitivity of ERBB3 and HSP90 binding resides in the nascent state and is dependent on the presence of the kinase domain of ERBB3. Furthermore, despite its intrinsic lack of kinase activity and in contrast to the reported GA sensitivity of mature and kinase-deficient EGFR, the GA sensitivity of the nascent state of ERBB3 appears to be exclusive. Geldanamycin disrupts the interaction of ERBB3 and HSP90 and inhibits ERBB3 maturation at an early stage of synthesis, prior to export from the ER. Studies with a photo-convertible fusion protein of ERBB3 suggest geldanamycin sensitivity at a later stage in maturation, possibly through the putative role of HSP90 in structural proofreading.

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“…This reaction is non-oxidative and requires no molecular oxygen. A number of Kaede-like proteins were characterized and shown to be a useful tool to study movements of cells, organelles and proteins [10,[14][15][16][17][18][19], to perform ultra-high resolution fluorescence imaging [20], and to visualize protein degradation in living cells [21].…”

Section: Introductionmentioning

confidence: 99%

Synthesis and properties of the red chromophore of the green-to-red photoconvertible fluorescent protein Kaede and its analogs

Yampolsky

Kislukhin

Amatov

et al. 2008

Bioorganic Chemistry

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SummaryGreen Fluorescent Protein (GFP) and homologous proteins possess a unique pathway of chromophore formation based on autocatalytic modification of their own amino acid residues. Greento-red photoconvertible fluorescent protein Kaede carries His-Tyr-Gly chromophore-forming triad.Here we describe synthesis of Kaede red chromophore (2-[(1E)-2-(5-imidazolyl)ethenyl]-4-(phydroxybenzylidene)-5-imidazolone) and its analogs that can be potentially formed by natural amino acid residues. Chromophores corresponding to the following tripeptides were obtained: His-Tyr-Gly, Trp-Tyr-Gly, Phe-Trp-Gly, Tyr-Trp-Gly, Asn-Tyr-Gly, Phe-Tyr-Gly, and Tyr-Tyr-Gly. In basic conditions they fluoresced red with relatively high quantum yield (up to 0.017 for Trp-derived compounds). The most red-shifted absorption peak at 595 nm was found for the chromophore TrpTyr-Gly in basic DMSO. Surprisingly, in basic DMF non-aromatic Asn-derived chromophore AsnTyr-Gly demonstrated the most red-shifted emission maximum at 642 nm. Thus, Asn residue may be a promising substituent, which can potentially diversify posttranslational chemistry in GFP-like proteins.

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Method for Real-Time Monitoring of Protein Degradation at the Single Cell Level

Cited by 78 publications

References 14 publications

Temperature-sensitive Post-translational Regulation of Plant Omega-3 Fatty-acid Desaturases Is Mediated by the Endoplasmic Reticulum-associated Degradation Pathway

Temperature-sensitive Post-translational Regulation of Plant Omega-3 Fatty-acid Desaturases Is Mediated by the Endoplasmic Reticulum-associated Degradation Pathway

Geldanamycin selectively targets the nascent form of ERBB3 for degradation

Synthesis and properties of the red chromophore of the green-to-red photoconvertible fluorescent protein Kaede and its analogs

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