TY - JOUR T1 - Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers JF - bioRxiv DO - 10.1101/023119 SP - 023119 AU - Anton Khmelinskii AU - Matthias Meurer AU - Chi-Ting Ho AU - Birgit Besenbeck AU - Bernd Bukau AU - Axel Mogk AU - Michael Knop Y1 - 2015/01/01 UR - http://biorxiv.org/content/early/2015/07/23/023119.abstract N2 - 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 were constructed by combining different slower-maturing red fluorescent proteins (redFPs) with the same faster-maturing superfolder green fluorescent protein (sfGFP). Towards a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing 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 towards 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 design of new tFTs. ER -