RT Journal Article SR Electronic T1 Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers JF bioRxiv FD Cold Spring Harbor Laboratory SP 023119 DO 10.1101/023119 A1 Anton Khmelinskii A1 Matthias Meurer A1 Chi-Ting Ho A1 Birgit Besenbeck A1 Julia Füller A1 Marius K. Lemberg A1 Bernd Bukau A1 Axel Mogk A1 Michael Knop YR 2015 UL http://biorxiv.org/content/early/2015/11/12/023119.abstract AB 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.Abbreviations tFT – tandem fluorescent protein timerFP – fluorescent proteingreenFP – green fluorescent proteinredFP – red fluorescent protein