Abstract
pH is one of the most critical physiological parameters determining vital cellular activities, such as photosynthetic performance. Fluorescent sensor proteins capable of measuring in situ pH in animal cells have been reported. However, these proteins require an excitation laser for pH measurement that may affect photosynthetic performance and induce autofluorescence from chlorophyll. As a result, it is not possible to measure the intracellular or intraorganelle pH changes in plants. To overcome this problem, we developed a luminescent pH sensor by fusing the luminescent protein Nanoluc to a uniquely designed pH-sensitive GFP variant protein. In this system, an excitation laser is unnecessary because the fused GFP variant reports on the luminescent signal by bioluminescence resonance energy transfer from Nanoluc. The ratio of two luminescent peaks from the sensor protein was approximately linear with respect to pH in the range of 7.0 to 8.5. We designated this sensor protein as “luminescent pH indicator protein” (Luphin). We applied Luphin to the in situ pH measurement of a photosynthetic organism under fluctuating light conditions, allowing us to successfully observe the cytosolic pH changes associated with photosynthetic electron transfer in the cyanobacterium Synechocystis sp. PCC 6803. Detailed analyses of the mechanisms of the observed estimated pH changes in the cytosol in this alga suggested that the photosynthetic electron transfer is suppressed by the reduced plastoquinone pool under light conditions. These results indicate that Luphin may serve as a helpful tool to further illuminate pH-dependent processes throughout the photosynthetic organisms.
Highlights
Gene-encoded pH-sensitive fluorescent proteins are one of the possible solutions for this challenge
Because the light intensity of the luminescent proteins is weak enough and does not influence the proton gradient formation across thylakoid membranes coupled with photosynthetic electron transfer reactions, the luminescent protein was thought to be a good tool for the GFP-based pH sensor proteins
Because protonated chromophores are dominant at lower pH conditions, the 509 nm emission caused by bioluminescent resonance energy transfer (BRET) will be weak enough at lower pH, when the GFP variant is fused with Nluc
Summary
A luminescent Nanoluc-GFP fusion protein enables readout of cellular pH in photosynthetic organisms. It is not possible to measure the intracellular or intraorganelle pH changes in plants To overcome this problem, we developed a luminescent pH sensor by fusing the luminescent protein Nanoluc to a uniquely designed pH-sensitive GFP variant protein. Detailed analyses of the mechanisms of the observed estimated pH changes in the cytosol in this alga suggested that the photosynthetic electron transfer is suppressed by the reduced plastoquinone pool under light conditions These results indicate that Luphin may serve as a helpful tool to further illuminate pH-dependent processes throughout the photosynthetic organisms. We fused pH-sensitive GFP variant protein and NanoLuc (Nluc) and obtained the desired pH sensor protein Luphin (luminescent pH indicator protein) that enabled the measurement of the linear signal changes from pH 7 to 8.5. It is still difficult to measure pH changes in the thylakoid lumen, we have successfully used this sensor protein to observe unexpected pH changes in the cytoplasm of cyanobacteria during the light–dark transition
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