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Title: Identification of pH-sensing Sites in the Light Harvesting Complex Stress-related 3 Protein Essential for Triggering Non-photochemical Quenching in Chlamydomonas reinhardtii

Light harvesting complex stress-related 3 (LHCSR3) is the protein essential for photoprotective excess energy dissipation (non-photochemical quenching, NPQ) in the model green alga Chlamydomonas reinhardtii. Activation of NPQ requires low pH in the thylakoid lumen, which is induced in excess light conditions and sensed by lumen-exposed acidic residues. In this work we have used site-specific mutagenesis in vivo and in vitro for identification of the residues in LHCSR3 that are responsible for sensing lumen pH. Lumen-exposed protonatable residues, aspartate and glutamate, were mutated to asparagine and glutamine, respectively. By expression in a mutant lacking all LHCSR isoforms, residues Asp 117, Glu 221, and Glu 224 were shown to be essential for LHCSR3-dependent NPQ induction in C. reinhardtii. Analysis of recombinant proteins carrying the same mutations refolded in vitro with pigments showed that the capacity of responding to low pH by decreasing the fluorescence lifetime, present in the wild-type protein, was lost. Consistent with a role in pH sensing, the mutations led to a substantial reduction in binding the NPQ inhibitor dicyclohexylcarbodiimide.
Authors:
 [1] ;  [2] ;  [3] ;  [4] ;  [5] ;  [6] ;  [1] ;  [4]
  1. Univ. of Verona (Italy). Dept. of Biotechnology
  2. Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst. Dept. of Plant and Microbial Biology
  3. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division; Univ. of California, Berkeley, CA (United States). Graduate Group in Applied Science and Technology
  4. Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst. Dept. of Plant and Microbial Biology; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division
  5. Univ. of Verona (Italy). Dept. of Biotechnology; Sorbonne Univ., Paris (France). Lab. of Computational and Quantitative Biology
  6. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division; Univ. of California, Berkeley, CA (United States). Graduate Group in Applied Science and Technology. Dept. of Chemistry
Publication Date:
Grant/Contract Number:
AC02-05CH11231; 2012XSAWYM
Type:
Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 291; Journal Issue: 14; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Research Org:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States); Univ. of Verona (Italy)
Sponsoring Org:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22); Ministry of Education, Universities and Research (Italy)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; fluorescence; photosynthesis; photosynthetic pigment; photosystem II; plant biochemistry; non-photochemical quenching; photoprotection
OSTI Identifier:
1414754