A new fluorescence-based method to monitor the pH in the thylakoid lumen using GFP variants

Yang, Hong; Pu, Xiaojun; Wang, Lu; Liu, Li; Theg, Steven M.

doi:10.1016/j.bbrc.2016.12.032

Title: A new fluorescence-based method to monitor the pH in the thylakoid lumen using GFP variants

Journal Article · Thu Dec 08 00:00:00 EST 2016 · Biochemical and Biophysical Research Communications

DOI:https://doi.org/10.1016/j.bbrc.2016.12.032· OSTI ID:1490415

Yang, Hong ^[1];

^[1]; Wang, Lu ^[1]; Liu, Li ^[2];

^[3]

Chinese Academy of Sciences, Kunming (China)
Chinese Academy of Sciences, Kunming (China); University of California, Davis, CA (United States)
University of California, Davis, CA (United States)

The ΔpH-dependent/Tat pathway is unique for using only the proton motive force for driving proteins transport across the thylakoid membrane in chloroplasts. 9-aminoacridine fluorescence quenching is widely used to monitor the ΔpH developed across the thylakoid membrane in the light. However, this method suffers from limited sensitivity to low ΔpH values and to spurious fluorescence signals due to membrane binding. In order to develop a more sensitive method for monitoring the real pH of the thylakoid lumen without these problems we transformed Arabidopsis thaliana with a ratiometric pH-sensitive GFP variant (termed pHluorin) targeted to the lumen by the prOE17 transit peptide. Positive transgenic plants displayed localization of pHluorin in the chloroplast by confocal microscopy, and fractionation experiments revealed that it is in the lumen. The pHluorin signal was the strongest in very young plants and diminished as the plants matured. The pHluorin released from the lumen displayed the expected fluorescence intensity changes in response to pH titration. Here, the fluorescence signal in isolated chloroplasts responded to illumination in a manner consistent with light-dependent lumen acidification. Future experiments will exploit the use of this new pH-indicating probe of the thylakoid lumen to examine the influence of the thylakoid ΔpH on ATP synthesis and protein transport.

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Research Organization:: Univ. of California, Davis, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences & Biosciences Division; USDOE

Grant/Contract Number:: FG02-03ER15405

OSTI ID:: 1490415

Alternate ID(s):: OSTI ID: 1413373

Journal Information:: Biochemical and Biophysical Research Communications, Vol. 486, Issue 1; ISSN 0006-291X

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 5 works

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59 BASIC BIOLOGICAL SCIENCES
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