Folding latency of fluorescent proteins affects the mitochondrial localization of fusion proteins

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. * aq : Aequorea victoria Ds : Discosoma sea anemones EEA1 : early endosomal antigen 1 ER : endoplasmic reticulum FCCP : p-trifluoromethoxyphenylhydrazone FFP : fluorescent fusion proteins FP : fluorescent proteins FRET : Förster resonance energy transfer GalT : β1,4-galactose transferase GFP : green fluorescent protein H2B : histone 2B KRasCT : C-terminal hypervariable region of K-Ras 4B RFPized : red fluorescence-proteinized sfGFP : superfolder GFP TOM : translocase of outer membrane