title: A super-ecliptic, pHluorin-mKate2, tandem fluorescent protein-tagged human LC3 for the monitoring of mammalian autophagy.
abstract: Tandem fluorescent protein-tagged LC3s that were comprised of a protein tag that emits green fluorescence (e.g., EGFP or mWasabi) fused with another tag that emits red fluorescence (e.g. mCherry or TagRFP) were used for monitoring the maturation step of mammalian autophagosomes. A critical point for this tandem fluorescent-tagged LC3 was the sensitivity of green fluorescence at an acidic pH. EGFP and mWasabi continue to emit a weak, but significant, fluorescence at a pH of approximately 6. To overcome this issue, we focused on super-ecliptic pHluorin, which is a more pH-sensitive GFP variation. The green fluorescence of EGFP and mWasabi in the cells was still observed at weakly acidic levels (pH 6.0-6.5). In contrast, the fluorescence of pHluorin was more significantly quenched at pH 6.5, and was almost completely abolished at pH 5.5-6.0, indicating that pHluorin is more suitable for use in a tandem fluorescent protein-tag for monitoring autophagy. A pHluorin-mKate2 tandem fluorescence protein showed pH-sensitive green fluorescence and pH-resistant far-red fluorescence. We therefore generated expression plasmids for pHluorin-mKate2-tagged human LC3 (PK-hLC3), which could be used as a modifier for LC3-lipidation. The green and far-red fluorescent puncta of PK-hLC3 were increased under starvation conditions. Puncta that were green-negative, but far-red positive, were increased when autolysosomes accumulated, but few puncta of the mutant PK-hLC3ΔG that lacked the carboxyl terminal Gly essential for autophagy were observed in the cells under the same conditions. These results indicated that the PK-hLC3 were more appropriate for the pH-sensitive monitoring of the maturation step of autophagosomes.
authors.affiliation: Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan; Laboratory of Proteomics and Biomolecular Science, Research Support Center, Juntendo University Graduate School of Medicine, Bunkyo, Tokyo, Japan.
authors.forename: Isei
authors.initials: I
authors.lastname: Tanida
authors.affiliation: Laboratory of Proteomics and Biomolecular Science, Research Support Center, Juntendo University Graduate School of Medicine, Bunkyo, Tokyo, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Ueno
authors.affiliation: Department of Cellular and Molecular Neuropathology, Juntendo University School of Medicine, Bunkyo, Tokyo, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Uchiyama
mesh_terms: D000143:Acids / Q000378:metabolism; D000818:Animals; D001343:Autophagy; D045744:Cell Line, Tumor; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008164:Luminescent Proteins / Q000378:metabolism*; D008322:Mammals / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D000143:Acids; D008164:Luminescent Proteins; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D050505:Mutant Proteins; C485187:PHluorin; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins