RT Journal Article
SR Electronic
T1 Folding latency of fluorescent proteins affects the mitochondrial localization of fusion proteins
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 768432
DO 10.1101/768432
A1 Sayaka Kashiwagi
A1 Yoichiro Fujioka
A1 Aya O. Satoh
A1 Aiko Yoshida
A1 Mari Fujioka
A1 Prabha Nepal
A1 Atsushi Tsuzuki
A1 Ozora Aoki
A1 Sarad Paudel
A1 Hitoshi Sasajima
A1 Yusuke Ohba
YR 2019
UL http://biorxiv.org/content/early/2019/09/13/768432.abstract
AB The discovery of fluorescent proteins (FPs) has revolutionized cell biology. The fusion of targeting sequences to FPs enables the investigation of cellular organelles and their dynamics; however, occasionally, such fluorescent fusion proteins (FFPs) exhibit behavior different from that of the native proteins. Here, we constructed a color pallet comprising different organelle markers and found that FFPs targeted to the mitochondria were mislocalized when fused to certain types of FPs. Such FPs included several variants of Aequorea victoria green FP (aqGFP) and a monomeric variant of the red FP. Because the FFPs that are mislocalized include FPs with faster maturing or folding mutations, the increase in the maturation rate is likely to prevent their expected localization. Indeed, when we reintroduced amino acid substitutions so that the FP sequences were equivalent to that of wild-type aqGFP, FFP localization to the mitochondria was significantly enhanced. Moreover, similar amino acid substitutions improved the localization of mitochondria-targeted pHluorin, which is a pH-sensitive variant of GFP, and its capability to monitor pH changes in the mitochondrial matrix. Our findings demonstrate the importance of selecting FPs that maximize FFP function.aqAequorea victoriaDsDiscosoma sea anemonesEEA1early endosomal antigen 1ERendoplasmic reticulumFCCPp-trifluoromethoxyphenylhydrazoneFFPfluorescent fusion proteinsFPfluorescent proteinsFRETFörster resonance energy transferGalTβ1,4-galactose transferaseGFPgreen fluorescent proteinH2Bhistone 2BKRasCTC-terminal hypervariable region of K-Ras 4BRFPizedred fluorescence-proteinizedsfGFPsuperfolder GFPTOMtranslocase of outer membrane