Analysis of LhcSR3, a protein essential for feedback de-excitation in the green alga Chlamydomonas reinhardtii.
In photosynthetic organisms, feedback dissipation of excess absorbed light energy balances harvesting of light with metabolic energy consumption. This mechanism prevents photodamage caused by reactive oxygen species produced by the reaction of chlorophyll (Chl) triplet states with O₂. Plants have been found to perform the heat dissipation in specific proteins, binding Chls and carotenoids (Cars), that belong to the Lhc family, while triggering of the process is performed by the PsbS subunit, needed for lumenal pH detection. PsbS is not found in algae, suggesting important differences in energy-dependent quenching (qE) machinery. Consistent with this suggestion, a different Lhc-like gene product, called LhcSR3 (formerly known as LI818) has been found to be essential for qE in Chlamydomonas reinhardtii. In this work, we report the production of two recombinant LhcSR isoforms from C. reinhardtii and their biochemical and spectroscopic characterization. We found the following: (i) LhcSR isoforms are Chl a/b- and xanthophyll-binding proteins, contrary to higher plant PsbS; (ii) the LhcSR3 isoform, accumulating in high light, is a strong quencher of Chl excited states, exhibiting a very fast fluorescence decay, with lifetimes below 100 ps, capable of dissipating excitation energy from neighbor antenna proteins; (iii) the LhcSR3 isoform is highly active in the transient formation of Car radical cation, a species proposed to act as a quencher in the heat dissipation process. Remarkably, the radical cation signal is detected at wavelengths corresponding to the Car lutein, rather than to zeaxanthin, implying that the latter, predominant in plants, is not essential; (iv) LhcSR3 is responsive to low pH, the trigger of non-photochemical quenching, since it binds the non-photochemical quenching inhibitor dicyclohexylcarbodiimide, and increases its energy dissipation properties upon acidification. This is the first report of an isolated Lhc protein constitutively active in energy dissipation in its purified form, opening the way to detailed molecular analysis. Owing to its protonatable residues and constitutive excitation energy dissipation, this protein appears to merge both pH-sensing and energy-quenching functions, accomplished respectively by PsbS and monomeric Lhcb proteins in plants.
Dipartimento di Biotecnologie, Università di Verona, Verona, Italy.
Giulia
G
Bonente
Matteo
M
Ballottari
Thuy B
TB
Truong
Tomas
T
Morosinotto
Tae K
TK
Ahn
Graham R
GR
Fleming
Krishna K
KK
Niyogi
Roberto
R
Bassi
D000042:Absorption; D020418:Algal Proteins / Q000378:metabolism*; D000595:Amino Acid Sequence; D016825:Chlamydomonas reinhardtii / Q000378:metabolism*; D002734:Chlorophyll / Q000378:metabolism*; D025461:Feedback, Physiological; D005453:Fluorescence; D008027:Light; D045342:Light-Harvesting Protein Complexes / Q000378:metabolism; D014975:Lutein / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D058849:Protein Refolding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism*; D016415:Sequence Alignment; D013816:Thermodynamics; D020524:Thylakoids / Q000378:metabolism; D024341:Xanthophylls / Q000378:metabolism*
D020418:Algal Proteins; D045342:Light-Harvesting Protein Complexes; D020033:Protein Isoforms; D011994:Recombinant Proteins; D024341:Xanthophylls; D002734:Chlorophyll; C005613:violaxanthin; C011947:neoxanthin; D014975:Lutein
Science. 2003 Mar 7;299(5612):1572-5
12624266
Science. 2008 May 9;320(5877):794-7
18467588
Biochemistry. 2003 Apr 15;42(14):4226-34
12680777
J Biol Chem. 1999 Apr 9;274(15):10458-65
10187836
Plant Mol Biol. 2000 Jan;42(2):303-16
10794530
J Biol Chem. 2001 Sep 21;276(38):35924-33
11454869
Nature. 2007 Nov 22;450(7169):575-8
18033302
Nature. 2004 Mar 18;428(6980):287-92
15029188
Proc Natl Acad Sci U S A. 1998 Oct 13;95(21):12719-23
9770552
Proc Natl Acad Sci U S A. 1997 Dec 9;94(25):14162-7
9391170
Eur J Biochem. 2004 Dec;271(23-24):4659-65
15606753
Photosynth Res. 2007 Oct;94(1):67-78
17638112
Plant Physiol. 2007 Apr;143(4):1802-16
17307901
J Biol Chem. 2002 Sep 27;277(39):36253-61
12095992
J Biol Chem. 1999 Oct 15;274(42):29613-23
10514429
Biophys J. 2000 Oct;79(4):1706-17
11023879
FEBS Lett. 1994 Oct 3;352(3):265-70
7925984
Plant Cell. 2006 Nov;18(11):3106-20
17114352
J Biol Chem. 2004 Jun 25;279(26):26823-9
15070896
Nature. 2009 Nov 26;462(7272):518-21
19940928
J Biol Chem. 2007 Mar 23;282(12):8947-58
17229724
Biochemistry. 2004 Jul 27;43(29):9467-76
15260489
Nature. 1970 Aug 15;227(5259):680-5
5432063
Biochim Biophys Acta. 2002 Oct 3;1556(1):29-40
12351216
J Mol Biol. 2001 Dec 14;314(5):1157-66
11743731
Biochemistry. 2007 Apr 24;46(16):4745-54
17402710
J Biol Chem. 2005 Sep 2;280(35):31050-8
15983042
Nature. 1994 Feb 17;367(6464):614-21
8107845
J Biol Chem. 2004 May 28;279(22):22866-74
15033974
FEBS Lett. 1997 Feb 3;402(2-3):151-6
9037185
J Biol Chem. 2009 May 29;284(22):15255-66
19307183
Eur J Biochem. 2001 Jan;268(2):260-7
11168359
BMC Plant Biol. 2006 Dec 27;6:32
17192177
Plant Physiol. 2006 Mar;140(3):793-804
16524980
J Biol Chem. 1999 Oct 22;274(43):30987-94
10521495
FEBS Lett. 2007 Oct 2;581(24):4704-10
17850797
Proc Natl Acad Sci U S A. 1999 Aug 31;96(18):10056-61
10468561
Biophys J. 2007 Oct 1;93(7):2418-28
17545247
Biochemistry. 2006 Feb 7;45(5):1490-8
16445291
Science. 2007 Oct 12;318(5848):245-50
17932292
J Biol Chem. 2002 Oct 4;277(40):36913-20
12114527
Photochem Photobiol. 1991 Sep;54(3):433-43
1784642
Proc Natl Acad Sci U S A. 2010 Jun 15;107(24):11128-33
20505121
Biochemistry. 2004 Jul 6;43(26):8281-9
15222740
J Biol Chem. 1991 May 5;266(13):8136-42
2022634
Photosynth Res. 2000;64(2-3):221-31
16228460
J Biol Chem. 2008 Mar 7;283(10):6184-92
18079125
Proteomics. 2007 Nov;7(21):3964-79
17922516
Biochemistry. 2001 Oct 23;40(42):12552-61
11601979
Chemphyschem. 2010 Apr 26;11(6):1289-96
20127930
Plant Cell. 2009 Jun;21(6):1798-812
19549928
Biochemistry. 1998 Jan 27;37(4):1143-9
9454607
Eur J Biochem. 1996 May 15;238(1):112-20
8665927
Plant Mol Biol. 1996 Nov;32(3):461-73
8980495
Biochemistry. 2002 Jun 11;41(23):7334-43
12044165
Plant Cell. 2008 Apr;20(4):1012-28
18381925
J Biol Chem. 2009 Mar 20;284(12):8103-13
19129188
Biochim Biophys Acta. 2010 Aug;1797(8):1449-57
20388491
Genome. 2010 Jan;53(1):68-78
20130750
Biophys J. 1998 Dec;75(6):3064-77
9826626
Plant Cell. 1997 Aug;9(8):1369-1380
12237386
EMBO J. 2001 Jul 16;20(14):3623-30
11447103
J Biol Chem. 2003 May 23;278(21):19190-8
12601013
Planta. 1991 Feb;183(3):423-33
24193753
Plant Cell. 1998 Jul;10(7):1121-34
9668132
Nature. 2000 Jan 27;403(6768):391-5
10667783
J Biol Chem. 2002 Jun 21;277(25):22750-8
11934892
Biochim Biophys Acta. 2010 Feb;1797(2):212-21
19853576
Proc Natl Acad Sci U S A. 1998 Oct 27;95(22):13324-9
9789087
Plant Cell. 2005 Apr;17(4):1217-32
15749754
Biophys J. 1999 Apr;76(4):2238-48
10096919
J Biol Chem. 2001 Jun 22;276(25):22154-9
11301335
J Phys Chem B. 2008 Oct 23;112(42):13418-23
18826191
Funct Plant Biol. 2002 Oct;29(10):1131-1139
32689566
J Biol Chem. 2010 May 28;285(22):17020-8
20360011
Appl Environ Microbiol. 2000 Jan;66(1):64-72
10618204
Plant Cell. 2007 Mar;19(3):1048-64
17351115
J Biol Chem. 2008 Feb 8;283(6):3550-3558
17991753
FEBS Lett. 1999 Jul 30;456(1):1-6
10452518
Proc Natl Acad Sci U S A. 1975 Jun;72(6):2175-9
1056023
Plant Physiol. 2009 Feb;149(2):1061-75
19011000
J Biol Chem. 2008 Mar 28;283(13):8434-45
18070876
Science. 1989 Dec 15;246(4936):1503-4
17756009
Nature. 2005 Feb 24;433(7028):892-5
15729347
Nature. 2003 Dec 11;426(6967):630-5
14668855
Proc Natl Acad Sci U S A. 1997 Jul 8;94(14):7667-72
11038558
J Biol Chem. 1995 Apr 14;270(15):8474-81
7721743
Plant J. 2007 Jun;50(5):795-809
17553115
Anal Biochem. 1987 Nov 1;166(2):368-79
2449095
Photochem Photobiol. 2008 Nov-Dec;84(6):1359-70
19067957
J Biol Chem. 1999 Nov 19;274(47):33510-21
10559236
J Biol Chem. 2005 May 27;280(21):20612-9
15788395
Science. 2005 Jan 21;307(5708):433-6
15662017
Photosynth Res. 2001;67(1-2):139-45
16228323
Plant Cell. 2002 Aug;14(8):1801-16
12172023