Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers
Tandem fluorescent protein timers (tFTs)-fusions of red and green fluorescent proteins-facilitate studies of intracellular protein dynamics. Various green fluorescent proteins are resistant to proteasomal degradation, and incomplete degradation affects tFT behavior. Simple guidelines are given for t...
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| Main Authors: | , , , , , , , , |
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| Format: | Article (Journal) |
| Language: | English |
| Published: |
2016 Jan 15
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| In: |
Molecular biology of the cell
Year: 2016, Volume: 27, Issue: 2, Pages: 360-370 |
| ISSN: | 1939-4586 |
| DOI: | 10.1091/mbc.E15-07-0525 |
| Online Access: | Verlag, kostenfrei, Volltext: http://dx.doi.org/10.1091/mbc.E15-07-0525 Verlag, kostenfrei, Volltext: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4713137/ 
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| Author Notes: | Anton Khmelinskii, Matthias Meurer, Chi-Ting Ho, Birgit Besenbeck, Julia Füller, Marius K. Lemberg, Bernd Bukau, Axel Mogk, and Michael Knop |
| Summary: | Tandem fluorescent protein timers (tFTs)-fusions of red and green fluorescent proteins-facilitate studies of intracellular protein dynamics. Various green fluorescent proteins are resistant to proteasomal degradation, and incomplete degradation affects tFT behavior. Simple guidelines are given for the design of new tFTs., Tandem fluorescent protein timers (tFTs) report on protein age through time-dependent change in color, which can be exploited to study protein turnover and trafficking. Each tFT, composed of two fluorescent proteins (FPs) that differ in maturation kinetics, is suited to follow protein dynamics within a specific time range determined by the maturation rates of both FPs. So far, tFTs have been constructed by combining slower-maturing red fluorescent proteins (redFPs) with the faster-maturing superfolder green fluorescent protein (sfGFP). Toward a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing green fluorescent proteins (greenFPs) while keeping the slower-maturing redFP constant (mCherry). Our results indicate that the greenFP maturation kinetics influences the time range of a tFT. Moreover, we observe that commonly used greenFPs can partially withstand proteasomal degradation due to the stability of the FP fold, which results in accumulation of tFT fragments in the cell. Depending on the order of FPs in the timer, incomplete proteasomal degradation either shifts the time range of the tFT toward slower time scales or precludes its use for measurements of protein turnover. We identify greenFPs that are efficiently degraded by the proteasome and provide simple guidelines for the design of new tFTs. |
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| Item Description: | Gesehen am 07.08.2017 Epub 2015 Nov 25 |
| Physical Description: | Online Resource |
| ISSN: | 1939-4586 |
| DOI: | 10.1091/mbc.E15-07-0525 |