RRC ID 69994
Author Kashiwagi S, Fujioka Y, Satoh AO, Yoshida A, Fujioka M, Nepal P, Tsuzuki A, Aoki O, Paudel S, Sasajima H, Ohba Y.
Title Folding Latency of Fluorescent Proteins Affects the Mitochondrial Localization of Fusion Proteins.
Journal Cell Struct Funct
Abstract 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 (avGFP) 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 avGFP, 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.Key words: fluorescent protein, organelle, fusion protein, mitochondria.
Volume 44(2)
Pages 183-194
Published 2019-12-26
DOI 10.1247/csf.19028
PMID 31735740
MeSH Animals Green Fluorescent Proteins / chemistry* Green Fluorescent Proteins / metabolism* HEK293 Cells HeLa Cells Humans Hydrozoa Mitochondria / metabolism* Protein Folding* Recombinant Fusion Proteins / chemistry* Recombinant Fusion Proteins / metabolism*
IF 2.429
DNA material pFX-Tom20-SECFP (RDB19410) pFX-SECFP-Sec61β (RDB19411) pFX-GalT-SECFP (RDB19412) pFX-β-tubulin-SECFP (RDB19413) pFX-H2B-SECFP (RDB19414) pFX-mito-EGFP (RDB19415) pFX-EGFP-EEA1 (RDB19416) pFX-EGFP-Rab5 (RDB19417) pFX-EGFP-Rab7 (RDB19418) pFX-EGFP-Rab11 (RDB19419)