Photosynthesis Research 82: 241–263, 2004. 2004 Kluwer Academic Publishers. Printed in the Netherlands.
241
Review
Structure, function and assembly of Photosystem II and its light-harvesting proteins Jun Minagawa1,* & Yuichiro Takahashi2 1
Institute of Low Temperature Science, Hokkaido University, N19 W8, Sapporo 060-0819, Japan; Department of Biology, Faculty of Science, Okayama University, 3-1-1 Tsushima-naka, Okayama 700-8530, Japan; *Author for correspondence (e-mail:
[email protected]; fax: +81-11-7065493) 2
Received 4 December 2003; accepted in revised form 19 July 2004
Key words: antenna, assembly, Chlamydomonas reinhardtii, LHCII, light-harvesting complex, Photosystem II
Abstract Photosystem II (PSII) is a multisubunit chlorophyll–protein complex that drives electron transfer from water to plastoquinone using energy derived from light. In green plants, the native form of PSII is surrounded by the light-harvesting complex (LHCII complex) and thus it is called the PSII–LHCII supercomplex. Over the past several years, understanding of the structure, function, and assembly of PSII and LHCII complexes has increased considerably. The unicellular green alga Chlamydomonas reinhardtii has been an excellent model organism to study PSII and LHCII complexes, because this organism grows heterotrophically and photoautotrophically and it is amenable to biochemical, genetic, molecular biological and recombinant DNA methodology. Here, the genes encoding and regulating components of the C. reinhardtii PSII–LHCII supercomplex have been thoroughly catalogued: they include 15 chloroplast and 20 nuclear structural genes as well as 13 nuclear genes coding for regulatory factors. This review discusses these molecular genetic data and presents an overview of the structure, function and assembly of PSII and LHCII complexes. Abbreviations: Chl – chlorophyll; Cyt – cytochrome; LHC – the light-harvesting complex; LHCII – proteins constitute major antenna complex for Photosystem II; NPQ – non-photochemical quenching; P680 – primary electron donor in PSII; Pheo – pheophytin a; PheoD1 – pheophytin on D1 as an intermediate electron acceptor of PSII; PSII – Photosystem II; PQ – plastoquinone; QA – the primary electron acceptor of PSII; QB – the secondary electron acceptor of PSII; RC – reaction center for Photosystem II; SRP – signal recognition particle; TMH – transmembrane helix
Introduction Photosystem II (PSII) is a large multisubunit chlorophyll–protein complex embedded in the thylakoid membranes of oxygen-evolving photosynthetic organisms. Its native form is the PSII– LHCII supercomplex that consists of more than 30 proteins (Figure 1). The supercomplex collects light energy, converts it into electro-chemical
energy and drives electron transfer from water to plastoquinone. At the center of this complex is the reaction center (RC), which is composed of a D1/ D2 heterodimer and a few intrinsic low molecular weight polypeptides. Surrounding RC are other subunits to form the ‘core’ complex, which includes the core antenna composed of chlorophyll (Chl)-a binding proteins, CP43 and CP47, that mediate excitation energy to RC, four lumenal
242
Figure 1. Subunit structure of PSII–LHCII supercomplex. Green polypeptides bind Chl a and b-carotene; yellow green polypeptides associate Chl a and b and xanthophylls. The capital letters in black and green represent nuclear-encoded and chloroplast-encoded psb genes, respectively. The RC consists of two homologous D1 and D2 proteins encoded by the chloroplast psbA and psbD genes, respectively, and several small polypeptides such as Cyt b559 and PsbI, PsbT and PsbW. The PSII core complex is composed of the RC, two core antenna proteins, CP43 and CP47, and several additional small polypeptides. The PSII–LHCII supercomplex contains PSII core complex and minor and major antennae. PsbZ is at the interface between PSII core and minor antenna and PsbS is in a peripheral region of the supercomplex.
extrinsic proteins that protect and facilitate oxygen-evolving reactions, and several other low molecular weight subunits. Chlorophytes have additional outer antenna or LHCII complex composed of proteins that bind Chl a and b. The LHCII complex is primarily composed of major antenna proteins, as well as less abundant minor antenna proteins. Sequencing of C. reinhardtii chloroplast genome has been completed (BK000554, Maul et al. 2002). The draft C. reinhardtii genomic sequence ver. 2.0 (http://genome.jgi-psf.org/chlre2) is now available and can be searched using nucleotide sequences from original and EST clones or other previously unidentified nucleotide sequences. In addition, high resolution X-ray crystal structures were recently reported for PSII–LHCII complexes; the structure of PSII core complex from Thermosynechococcus elongatus at 3.8 A˚ was reported by Zouni et al. (2001) and at 3.5 A˚ by Ferreira et al. (2004), PSII core complex from Thermosynechococcus vulcanus at 3.7 A˚ was reported by Kamiya and Shen (2003), and the structure of LHCII from spinach at 2.72 A˚ was reported by Liu et al. (2004). The first half of this review describes the structure, function, and assembly of PSII core complex and the second half describes LHCII complex; recent genetic characterization of the
components that constitute or regulate C. reinhardtii PSII and LHCII complexes is emphasized. Other recent reviews provide complimentary information on detailed structure/function and assembly of PSII (Erickson 1998; Ruffle and Sayre 1998), engineering of the chloroplast encoded proteins (Xiong and Sayre 2004), assembly of LHC and greening (Hoober et al. 1998), state transition (Kruse 2001), functional proteomics (Hippler et al. 2002), and genomics of LHC superfamily (Elrad and Grossman 2004).
Structure and function of PSII Functional organization of Photosystem II reaction center PSII is a multimeric protein complex with more than 20 subunits and approximately 50 cofactors which catalyzes the oxidation of water and the reduction of plastoquinone using energy derived from light. The primary photochemical and subsequent electron transfer reactions occur on RC composed of D1/D2 heterodimer and Cyt b559 (Nanba and Satoh 1987). When the primary donor, P680, is excited by light energy captured by antenna pigments, the primary charge separation
243 takes place between P680 and the intermediate acceptor, Pheo (a pheophytin a); this reaction generates the radical pair (P680+/Pheo)). Reduced Pheo transfers an electron to the primary acceptor, QA (a tightly bound plastoquinone), to generate QA), and subsequently reduces the secondary acceptor, QB (a loosely bound plastoquinone). The second photochemical reaction coupled with two protonations generates doubly reduced QB, QBH2 (a plastoquinol), which is released from the binding pocket and is replaced with a plastoquinone in the lipid bilayer of the thylakoid membranes. QA is bound to the binding pocket on D2, and QB binds to D1. The non-heme iron (Fe2+) lies between QA and QB and is ligated by His-215 and His-272 on D1 and His-213 and His-268 on D2. The Fe2+ is essential for the electron transfer from QA to QB, but is not directly involved in the redox reaction (Diner and Petrouleas 1987). On the donor side, P680+ is reduced by the immediate electron donor, YZ (Tyr-161 on D1), and the resulting neutral radical YZ• is reduced by an electron from a cluster of four manganese atoms (Mn-cluster) involved in oxygen evolution. A mutation of Tyr161 on D1 abolishes oxygen-evolving activity in Synechocystis sp. PCC6803 (Debus et al. 1988b) and Chlamydomonas (Minagawa et al. 1996) while substitution of Tyr-160 on D2 inactivates YD but not PSII activity (Debus et al. 1988a; Vermaas et al. 1988). The Mn-cluster accumulates four oxidizing equivalents to split two water molecules into one oxygen molecule and four protons. This linear electron transfer reaction in PSII catalyzes the light-induced water–plastoquinone oxidation– reduction with a high quantum efficiency. In addition to the main linear electron transfer, it has been proposed that there is a low quantum yield cyclic electron transfer around PSII, which mediates electron flow from QB to P680 (Diner and Rappaport 2002). The cyclic electron transfer may protect PSII against photoinhibition by preventing over-reduction of QA and QB on the acceptor side and accumulation of long-lived P680+ on the donor side. The 3D X-ray crystal structure of the cyanobacterial PSII core complex clearly revealed that the D1/D2 heterodimer contains six Chl a, two Pheo a, two b-carotenes, and one or two plastoquinone molecules (Zouni et al. 2001; Kamiya and Shen 2003; Ferreira et al. 2004). Kamiya and Shen placed cis- and trans-b-carotenes near the special
pair while Ferreira et al. assigned them as trans-bcarotene. D1 and D2 are homologous hydrophobic proteins with five transmembrane helices with diffuse mobility during SDS-polyacrylamide gel electrophoresis (Chua and Gillham 1977). The Chl a dimer, so-called special pair, is ligated by His198 on D1 and His197 on D2 and lies in the interface between D1 and D2 on the lumenal side. These two Chl molecules are designated PD1 and PD2. Two other Chl a molecules, which are the accessory Chls, ChlD1 and ChlD2, are symmetrically located on D1 and D2 in close proximity to PD1 and PD2, respectively. It is proposed that the excitation energy in the RC is broadly distributed over these four Chl molecules (Durrant et al. 1995) while the cation radical of P680 (P680+) is delocalized on PD1 (Diner et al. 2001). ChlD1 is proposed to be in an excited triplet state when P680+/Pheo) cannot relax to form P680+/QA) (Noguchi 2002). Two extra Chl a molecules, ChlZD1 and ChlZD2, which are coordinated by symmetrically related His-118 on D1 and His-117 on D2 are the peripheral accessory Chls (Zouni et al. 2001; Kamiya and Shen 2003; Ferreira et al. 2004). They are structurally in close association with the core antennae, CP43 and CP47, and are involved in energy transfer from CP43 and CP47 to RC. ChlZD2 may also be involved in a cyclic electron transfer around PSII proceeding from QB to P680 through Cyt b559, ChlZD2, and a carotenoid (Car) (Diner and Rappaport 2002). Indeed, the 3D structure of the PSII core complex reveals that ChlZD2 is located near Cyt b559 (Zouni et al. 2001; Kamiya and Shen 2003; Ferreira et al. 2004). However, Kamiya and Shen (2003) claim that Cyt b559–Car–P680 and ChlZD2–Car–P680 are equally plausible secondary electron transfer pathway(s), while Cyt b559–ChlZD2–Car–P680 is less likely since Cyt b559 and ChlZD2 are closer to Car. In C. reinhardtii, substitution of Gln or Asn for His117 (D2-H117Q or D2-H117N) impaired the structure and oxidation of ChlZD2 and reduced the energy transfer from antenna complexes to RC (Ruffle et al. 2001; Wang et al. 2002). One of the two Pheo a molecules functions as the intermediate acceptor, PheoD1, and is proposed to be hydrogen-bonded by Glu-130 on the hydrophobic transmembrane B-helix of D1. In contrast, the other Pheo a molecule, which is symmetrically located in RC, is not photochemically active and is proposed to be hydrogen-bonded by Gln-129 on
244 the transmembrane B-helix of D2. QA is bound in a pocket in the interhelical region connecting helix D and E of D2 and QB is located symmetrically, with respect to QA, in a pocket in the corresponding interhelical region of D1. Among the redox components of PSII, YZ and YD are unique in that they are Tyr residues on D1 (Tyr-161) and D2 (Tyr-160), respectively. YZ is located in close proximity to PD1 and functions as an immediate electron donor to P680+, while YD is in the vicinity of PD2 and is not involved in the main electron transfer on the donor side of PSII. The Mn-cluster consists of four Mn atoms coordinated to Asp, Glu, and His residues on D1/ D2 heterodimer. Three Mn ions form the corner of an isosceles triangle and the fourth Mn ion is near the center of the triangle (Zouni et al. 2001). Ca2+, which is essential for the oxygen-evolution, was also observed in the 3.5 A˚ structure reported by Ferreira et al. (2004), and a cubane-like Mn3CaO4 cluster linked to a fourth Mn has been proposed. However, mutants of these amino acid residues in D1 and D2 from Chlamydomonas and Synechocystis sp. PCC6803 suggested that several alternative amino acid residues in the hydrophilic domains of D1 and D2 may function as ligands to the Mn-cluster (Debus 2001). The recent detailed 3D structure around the Mn-cluster of PSII complex suggested additional amino acid residues as potential ligands for the Mn-cluster, including Asp-170, Glu-189 or His-190, His-332 or Glu-333, His-337, and Ala-344 on D1 (Kamiya and Shin 2003) and Asp-170, Glu-189, His-332, Glu-333, Asp-342 on D1 and E354 on CP43 (Ferreira et al. 2004). Ala-344 is the carboxyl-terminus of the mature D1 polypeptide and its carboxyl group may be a ligand for the Mn-cluster. This result is consistent with the fact that the carboxyl-terminal extension must be processed prior to assembly of the Mn-cluster (Nixon et al. 1992; Hatano-Iwasaki et al. 2000). Cyt b559, which consists of a- and b-subunits (encoded by psbE and psaF, respectively), is intimately associated with RC. The heme is coordinated by two His residues on the a- and b-subunits and is located in a transmembrane region near the stromal side. As already described above, this is consistent with the involvement of Cyt b559 in cyclic electron transfer around PSII. In Chlamydomonas and Synechocystis, deletion of one Cyt b559 subunit inhibits formation of PSII, indicating
that Cyt b559 is essential for the assembly and/or stability of PSII (Morais et al. 1998; Pakrasi et al. 1988). A rapid method for purifying high quality PSII complex was recently developed based on recombinant DNA methods and nickel affinity chromatography. Minagawa and coworkers attached a hexahistidine-tag to the C-terminus of D2 (Sugiura et al. 1998) and CP47 (Suzuki et al. 2003) of C. reinhardtii and demonstrated that nickel column chromatography is an effective purification method for the PSII complex containing these recombinant proteins. This technique has since been used to purify many other PSII-related proteins from various photosynthetic organisms. Functional organization of core antennae RC is surrounded by the two core antennae, CP43 and CP47. As described above, ChlZD1 and ChlZD2 are in close proximity to the core antennae and play essential role in energy transfer from CP43 and CP47 to RC. The 3D structure of the PSII core complex reveals that CP43 and CP47 contain six transmembrane helices and bind 13–14 and 14–17 Chl a molecules, respectively (Vasil’ev et al. 2001, Kamiya and Shen 2003, Ferreira et al. 2004). The Chl a molecules are likely to be bound mostly through His ligands in the transmembrane helices of CP43 and CP47. In addition, three and two all trans-b-carotenes are assigned on CP43 and CP47, respectively, and there might be more carotenes on the core antenna complexes (Ferreira et al. 2004). Most b-carotenes are in close contact with Chl a head group, which is required for high efficiency in transferring energy from b-carotene to Chl a and quenching Chl triplets. Small or extrinsic polypeptides Table 1 lists C. reinhardtii genes that encode components of the PSII and LHCII complexes. There are 15 chloroplast and 8 nuclear genes that encode PSII core components. All polypeptides encoded in the chloroplast genome have at least one transmembrane helix (TMH) and are integral membrane polypeptides. The extrinsic polypeptides (PsbO, PsbP, PsbQ, and PsbR) are encoded by nuclear genes and are located on the lumenal side of PSII.
C_1460035 C_1460005 C_10030 C_530002 –
Nuclear
Nuclear
Nuclear
Nuclear
–
Nuclear
LhcbM8
LhcbM9
Lhcb4
Lhcb5
Lhcb6
C_20371
38,236
–
30,714
29,938
27,077
29,761
26,686
26,961
28,687
24,615
27,380
26,650
27,565
6565
33,926
9883
12,141
–
–
–
3636
26,133
15,139
21,824
25,899
30,523
5210
3761
4428
4757
4290
4242
9439
4981
9304
39,447
50,639
3
–
3
3
3
3
3
3
3
3
3
3
3
Unknown
CP24
CP26
CP29
LHCII type I (LhcII-1.5)
LHCII type I (LhcII-1.4)
LHCII type III (LhcII-3.2)
LHCII type I (LhcII-1.1)
LHCII type II (LhcII-2)
LHCII type I (LhcII-1.2)
LHCII type I (LhcII-1.3)
LHCII type III (LhcII-3.1)
LHCII type IV (LhcII-4)
Stabilization of supercomplex
Unknown
1e 2
Stabilization of ChlZD2
Dimerization
O2 evolving enhancer, Cyt c550
O2 evolving enhancer
Unknown
Repair of damaged PS II
Photoprotection
1
1
–
–
–
1
4
Unknown
b
This study, blast hit for M24072?
This study, no blast hit for At1g15820
AB050007
AB051207
This study, blast hit for M24072
AF479778
AF330793
M24072
AF104631
AF104630
AB051204
AB051205
AB051206
BK000554
This study, blast hit for At1g67740
This study, blast hit for BAA10311
AF170026
This study, no blast hit for D45178
This study, no blast hit for D84098
This study, no blast hit for At1g51400
AJ296291
This study, blast hit for At1g44575
This study, blast hit for U86018
X13832
O2 evolving enhancer
0d 1
M15187
Binding of Ca2+ and Cl)
0d
L13303 X13826
Unknown Mn-stabilization
U81552
X66250
X53413
AF025877
BK000554
Z15133
X80195
X80196
X04147
X13879
M84022
X014242
Reference
1
Stabilization of dimer
Assembly or stabilization of dimer
Assembly or stabilization
Assembly of supercomplex
Assembly, stabilization of ChlZD1
Assembly or stabilization
b-subunit of Cyt b559
a-subunit of Cyt b559
Primary reaction (D2)
Light-harvesting (CP43)
Light-harvesting (CP47)
Primary reaction (D1)
Function
0d
1
1
1
1
1
1
1
1
5
6
6
5
TMH
Sequence coordinate of chloroplast-encoded genes (BK000554) or genemodel names of nuclear-encoded genes (http://genome.jgi-psf.org/chlre2) are shown. Predicted MW of precursor. c psbA is duplicated. d These proteins lack a TMH and bind to the lumenal side of the core complex. e This gene is predicted to encode four single-TMH polypeptides (PsbY-A1, -A2, -A3, and -A4), expressed as a polyprotein.
a
C_70041
Nuclear
LhcbM7
C_20371
C_290023
Nuclear
PsbX
Nuclear
C_50023
Nuclear
PsbW
LhcbM6
C_197000
–
PsbV
C_110177
–
–
PsbU
C_1460037
–
–
PsbTn
Nuclear
–
Chloroplast
PsbT
Nuclear
79825-79730
Nuclear
PsbS
LhcbM5
C_280068
Nuclear
PsbR
LhcbM4
C_3560004
Nuclear
PsbQ
C_2050001
C_1340006
Nuclear
PsbP
C_70161
C_880018
Nuclear
PsbO
Nuclear
77583-77717 C_940002
Chloroplast
PsbN
Nuclear
64301-64197
Chloroplast
PsbM
LhcbM3
102894-103028
Chloroplast
PsbL
LhcbM2
11842-11982
Chloroplast
PsbK
C_1190021
172315-172467
Chloroplast
PsbJ
Nuclear
126668-126781
Chloroplast
PsbI
LhcbM1
77013-76747
Chloroplast
PsbH
64951-64763
102148-102282
Chloroplast
PsbF
C_320026
94840-94592
Chloroplast
PsbE
Nuclear
175632-174574
Chloroplast
PsbD
Chloroplast
187025-188410
Chloroplast
PsbC
PsbY
81803-80277
Chloroplast
PsbB
PsbZ
39,042
55413-48775, 138790-145428c
Chloroplast
PsbA 56,106
MWb
Positiona
Genome
Gene
Table 1. Genes encoding components of PS II–LHC II supercomplex
245
246 Nuclear-encoded lumenal polypeptides (PsbO, PsbP, PsbQ, PsbR) On the lumenal side of PSII, at least three extrinsic polypeptides, PsbO, PsbP, and PsbQ, function as an oxygen evolving enhancer in chlorophytes. PsbO is crucial for oxygen evolution in C. reinhardtii (Mayfield et al. 1987a); however, it is not required in Synechocystis sp. PCC6803, in which a deficiency in PsbO has a minor effect on PSII function (Burnap and Sherman 1991). It should be noted that a recent crystal structure of the PSII core from Thermosynechococcus vulcanus showed structural similarity between PsbO and bacterial porins (Kamiya and Shin 2003); this suggests that PsbO may form a water channel for the Mn-complex. Ferreira et al. (2004) pointed out that despite the structural similarity between PsbO and bacterial porins, the putative water channel in PsbO is not an open tube. Two other extrinsic polypeptides, PsbP and PsbQ, are only present in chlorophytes and are likely to be functional substitutes for the extrinsic PsbU (12 kDa polypeptide) and PsbV (Cyt c550) polypeptides found in cyanobacteria, glaucophytes, rhodophytes and those in the Chl a/c lineage. A C. reinhardtii mutant BF25 lacking PsbP had very low rates of oxygen evolution, but could grow photoautotrophically (Mayfield et al. 1987b). A similar mutant strain was also deficient in photoactivation, which suggests that PsbP sequesters chloride ions during PSII assembly (Rova et al. 1996). Suzuki et al. (2003) observed that PsbO, PsbP, and PsbQ can bind PSII independently in C. reinhardtii. In contrast, PsbP cannot directly associate with PSII in higher plants, where it interacts with PSII through PsbO, and PsbQ functionally associates with PSII only in the presence of both PsbO and PsbP (Miyao and Murata 1989). Recently, mass spectrometric analyses indicate that cyanobacterial PSII includes not only PsbU and PsbV but a homolog of PsbQ (Kashino et al. 2002). Higher plants have two additional nuclear-encoded polypeptides on the lumenal side of the PSII core complex, 3 kDa PsbTn and 10 kDa PsbR. The former protein is extrinsically localized to the lumenal side, but its function is not known (Kapazoglou et al. 1995). PsbR is also extrinsically localized to the lumenal side but is anchored to the membrane with a TMH on its C-terminal side (Lautner et al. 1988). It has been suggested that
PsbR provides a binding site for PsbP. PsbR antisense inhibited acceptor-side electron transfer in Arabidopsis, although the mechanism for this effect is not understood (Stockhaus et al. 1990). The EST library and draft genomic sequence of C. reinhardtii do not have homologs (E-value less than 0.1) for Synechocystis psbU, psbV and Arabidopsis psbTn, but a homolog of psbR (E-value 5.4e)15 for U86018) was found in the nuclear genome (C_3560004) by searching with a rice cDNA sequence (Table 1). The C. reinhardtii PsbR sequence is shown aligned with homologues from vascular plants in Figure 2a. The presence of this gene in a green alga suggests that it evolved early in the chlorophytic lineage. Chloroplast-encoded intrinsic polypeptides (PsbH, PsbI, PsbJ, PsbK, PsbL, PsbM, PsbN, PsbT, PsbZ) The psbH gene is located in a highly conserved chloroplast-encoded gene cluster including psbBpsbT-psbH-petB-petD. Chlamydomonas PsbH is phosphorylated on a Thr2, the second amino acid from the N-terminus (Bennett 1977; Dedner et al. 1988). However, this Thr is absent from cyanobacteria, because PsbH is truncated at the amino terminus (Mayes and Barber 1991). The functional significance of the phosphorylation is unclear, because PSII is synthesized normally and is functional in a Chlamydomonas Thr–Ala substitution mutant (O’Connor et al. 1998). PsbH is found in the PSII core complex. A DpsbH mutant of C. reinhardtii lacks PSII complex (Summer et al. 1997; O’Connor et al. 1998). But, a cyanobacterial DpsbH mutant grows photosynthetically at slower rate than wild-type (Mayes et al. 1993). In the cyanobacterial mutant, electron transfer from QA to QB is impaired. Single particle analysis of PSII core complex containing PsbH labeled with gold cluster localized this polypeptide near Cyt b559 (Bu¨chel et al. 2001). However, the 3D structure of PSII core complex at 3.5 A˚ resolution positioned PsbH in a gap between D2 and CP47 and PsbX between PsbH and Cyt b559 (Ferreira et al. 2004). PsbI is a component of RC in higher plants and cyanobacteria, indicating that it is intimately associated with the D1/D2 heterodimer (Ikeuchi and Inoue 1988). The C. reinhardtii DpsbI mutant grows slowly photosynthetically and accumulates a reduced amount of functional PSII complex,
247 indicating that PsbI is not essential for PSII function but is required for efficient assembly and/ or stability of the complex (Ku¨nstner et al. 1995). A cyanobacterial DpsbI mutant has a less severe phenotype and accumulates PSII complex at 70%– 75% of wild-type level (Ikeuchi et al. 1995). This polypeptide is positioned on the periphery of D1 and appears to stabilize ChlZD1 (Ferreira et al. 2004). It is interesting to note that PsbX localizes to the opposite side of RC with respect to PsbI and thus is positioned close to ChlZD2. PsbJ is weakly associated with the PSII core complex, suggesting that it is in a peripheral domain of the core complex. In fact, this polypeptide is assigned to the TMH between Cyt b559 and PsbK. A cyanobacterial DpsbJ mutant accumulates less PSII complex than wild-type (Lind et al. 1993). In mature leaves of tobacco DpsbJ mutant, LHCII complex remains detached from PSII core complex, suggesting that PsbJ is required for the assembly of PSII–LHCII supercomplex (Suorsa et al. 2004). This gene has not been disrupted in Chlamydomonas. PsbK is synthesized as a precursor with an N-terminal extension required for its integration into PSII (Murata et al. 1988; Ikeuchi et al. 1991). PsbK is detected in the highly purified PSII core complex from Chlamydomonas (de Vitry et al. 1991). Recent studies suggest that PsbK is tightly associated with CP43 and involved in stabilizing the association of CP43 to PSII core complex (Sugimoto and Takahashi 2003). PsbK is assigned to the TMH with a characteristic bend near CP43 due to two Pro residues (Kamiya and Shen 2003). The Chlamydomonas DpsbK mutant does not grow photosynthetically and expresses PSII proteins at 10%–20% of the wild-type level (Takahashi et al. 1994). PsbL is a component of the PSII core complex in higher plants and is detected in small amounts in cyanobacterial RC (Ikeuchi et al. 1989). Cyanobacterial DpsbL mutants do not grow photosynthetically and contain reduced levels of PSII proteins (Anbudurai and Pakrasi 1993). Reconstitution of PSII with recombinant PsbL suggested that this polypeptide is involved in electron transfer from Yz to P680+ (Hoshida et al. 1997). A tobacco DpsbL mutant accumulates neither PSII core dimer nor PSII–LHCII supercomplex (Suorsa et al. 2004). Chlamydomonas DpsbL mutants have not been generated.
PsbM is a component of the PSII core complex in C. reinhardtii (de Vitry et al. 1991), whose function is unknown. PsbN is detected only in the cyanobacterial PSII core complex (Ikeuchi et al. 1989) and is not essential for the function or assembly of PSII (Mayes et al. 1993). Neither DpsbM nor DpsbN strains of Chlamydomonas have been characterized and little is known about PsbN, except that it may be positioned near PsbK in cyanobacterial PSII (Ferreira et al. 2004). The psbT gene was originally designated as ycf8 and the gene product was detected by immunochemical methods in Chlamydomonas (Monod et al. 1994). PsbT is expressed in wild-type cells and is enriched in the PSII core complex (Monod et al. 1994), but it is detected at very low levels in PSIIdeficient mutants. Recent biochemical analyses revealed that the majority of the PsbT protein copurifies with the RC and the remainder is solubilized during purification of PSII (Ohnishi and Takahashi 2001). A Chlamydomonas DpsbT mutant grows photosynthetically in low light and accumulates functional PSII complex at a nearly wild-type level; however, the DpsbT mutant is photosensitive and does not grow in strong light (Monod et al. 1994). Three TMHs at the dimer interface of the PSII core complex are assigned to PsbL, PsbM and PsbT (Ferreira et al. 2004). These polypeptides are thought to play a role in stability of the dimer. PsbT and PsbL are in the vicinity of the QA-binding pocked on D2. QA-deficient PSII core complex accumulates during PSII purification from a Chlamydomonas DpsbT mutant, suggesting that PsbT is required for stability of QA (Ohnishi, Kashino, Satoh and Takahashi, unpublished result). The PsbZ gene (first identified as ycf9) encodes a polypeptide with two putative TMHs. PsbZ copurifies with PSII core complex in Chlamydomonas and it is expressed in LHCII-deficient mutants but not in PSII-deficient mutants (Swiatek et al. 2001). However, Ruf et al. (2000) reported that PsbZ is detected in purified LHCII in tobacco. DpsbZ mutants of Chlamydomonas and tobacco show decreased expression of CP26 (Ruf et al. 2000; Swiatek et al. 2001) and slightly decreased expression of CP29. In tobacco, PsbZdeficiency causes instability of the PSII–LHCII supercomplex (Swiatek et al. 2001). Thus, PsbZ appears to lie between PSII and LHCII complexes and may stabilize the PSII–LHCII supercomplex.
248
Figure 2. Sequence alignment of PsbR (a), PsbX (b), and PsbY(c). Sequences were aligned using Clustal W (Thompson et al.1994). Note that PsbY sequences from O. sinensis (Os) and Synechocystis sp. PCC6803 (Sy) were aligned against more C-terminal regions within the C. reinhardtii (Cr) and A. thaliana (At) PsbY sequences (c). Gaps (—) were introduced to maximize homology. Hydrophobic domains predicted by hydrophobicity analysis software SOSUI (Hirokawa et al. 1998) or Topred II (Claros and von Heijne 1994) are indicated in shadow font. N-terminal sequences of mature PsbR from spinach (Lautner et al. 1988), PsbX from wheat (Ikeuchi et al. 1989), and PsbY from A. thaliana (Thompson et al. 1999) were used to predict the mature proteins. Amino acid sequences were from the following databases: Genbank – At.PsbR (P27202), At.PsbX (At2g06520), At.PsbY (At1g67740), Os.PsbX (P49509), Os.PsbY (P49543), So.PsbR (P10690), Sy.PsbX (BAA10311), Sy.PsbY (P73676); JGI – Cr.PsbR(C_3560004), Cr.PsbX (C_50023), Cr.PsbY (C_320026).
249 This polypeptide is also detected in the peripheral region of CP43 in cyanobacterial PSII (Ferreira et al. 2004). Nuclear-encoded intrinsic polypeptides (PsbW, PsbX, PsbY) PsbW is a 6.1 kDa protein originally thought to be in close proximity to RC (Irrgang et al. 1995) and to play a central role in stabilizing the PSII dimer (Thidholm et al. 2002). The location of PsbW became controversial when Hiyama et al. (2000) reported that it was copurified with PSI from various plant species. However, a recent study used immunochemical methods to demonstrate that C. reinhardtii PsbW is exclusively found in PSII (Bishop et al. 2003). PsbX is a 4.1 kDa protein with a single TMH located in the vicinity of ChlZD2 in a symmetrical relationship with PsbI (Ferreira et al. 2004). PsbY is found in higher plants and is a homolog of Ycf32 in Cyanobacteria and Chromophyta. The latter is expressed as a precursor polyprotein which is processed into two Ycf32-related proteins (PsbY-A1 and PsbY-A2), each of which contains a single TMH (Thompson et al. 1999). PsbX and PsbY have been detected in oxygen-evolving PSII core complexes (Kim et al. 1996; Gau et al 1998). The psbX and psbY (or ycf32) genes are nuclear in cyanobacteria and higher plants, cyanelle in glaucophytes, and in the plastid genomes of chromophytes. C. reinhardtii homologues of the Synechocystis PsbX (BAA10311) and Arabidopsis psbY (At1g67740) genes were identified in the draft C. reinhardtii genomic sequence ver. 2.0 as C_50023 and C_320026, respectively (Table 1, Figures 2b and c), indicating that psbX and psbY were transferred to the nuclear genome before green algae and higher plants diverged. In the case of psbY (ycf32), the duplication(s) event that created a polyprotein gene occurred at the time of or after transfer. However, the primary structure of the newly identified C. reinhardtii PsbY is unusual in that it encodes four Ycf32-related proteins: PsbY-A1, -A2, -A3, and -A4 (Figure 2c). Although deletion mutants of psbX and psbY did not affect PSII assembly or photoautotrophic growth of Synechocystis (Funk 2000; Meetam et al. 1999), it has been proposed that PsbX plays a role in binding or turnover of plastoquinones at the QB site and PsbY plays a role in Mn-binding (Gau et al 1998; Katoh and Ikeuchi 2001).
It is noteworthy that PsbR, PsbW, PsbX, and PsbY contain bipartite transit peptides in higher plants and translocated to the thylakoid membrane by a spontaneous or other unknown mechanism that is not mediated by Sec, cpSRP/Alb3, or Tat. In contrast, LHC proteins are translocated by a cpSRP/Alb3-dependent pathway (Woolhead et al. 2001). The C. reinhardtii genes encoding PsbR, PsbW, PsbX, and PsbY also contain bipartite-type transit presequences (Figures 2a–c). Assembly of PSII complex The structure and composition of PSII have been studied extensively, but much less is known about the assembly of its components into a functional multimeric protein complex. Because of the structural complexity of PSII, its assembly is likely to be a multi-step process assisted by molecular chaperones or other factors (Baena-Gonza´lez and Aro 2002; Erickson 1998; Hippler et al. 2002). One approach to dissecting the steps of this assembly process is to identify and characterize partial assembly products or assembly intermediates and to analyze greening process of dark-grown Chlamydomonas y-1 mutant or etioplasts of higher plants (Figure 3). In barley etioplasts in which no Chls and functional PSII complexes are formed, D2 and Cyt b559 accumulate and appear to form a precomplex (Figure 3–1) (Mu¨ller and Eichacker, 1999). Upon illumination, Chls and D1 are synthesized and integrated into the precomplex (Figure 3-2). It appears that the precomplex serves as a primary acceptor for D1 during translation (Zhang et al. 1999), which is consistent with the observations that translation of D1 is reduced in the absence of D2 (Erickson et al. 1986) and Cyt b559 deficient mutant accumulates no PSII complex (Morais et al. 1994). Several factors involved in the early steps of D1 assembly have been reported so far. Hydrophobic D1 is synthesized on membraneassociated polysomes (Herrin et al. 1981; Zhang et al. 2000) and the targeting and insertion of nascent chain of D1 on 70S ribosome to thylakoid membranes are mediated by chloroplast-localized signal recognizing particle, cpSRP54 (Nilsson et al. 1999). Strong interaction of a chloroplast homologue of bacterial essential translocon component SecY, cpSecY, with the membrane-associated polysomes suggests that cpSecY is involved in
250 directing the D1 nascent chains to the location in PSII complex (Zhang et al. 2001). The thylakoid membrane-localized Alb3, which is required for the normal accumulation of LHC (Bellafiore et al. 2002), is also involved in a early step of PSII assembly by interacting with D1 (Ossenbu¨hl et al. 2004). Cofactors such as Chl a are also co-translationally integrated into D1 (Mullet et al. 1990) and the association of Chl a stabilizes D1 (Mu¨ller and Eichacker, 1999). The other Chl-binding subunits, CP43 and CP47, are most probably synthesized in a similar manner. Zerges and Rochaix (1998) observed that there is a low-density membrane fraction associated with thylakoid membranes that is similar to an inner envelope membrane; this membrane fraction is enriched in factors that promote and regulate translation of proteins targeted to the thylakoid membrane. Thus, the PSII complex might be assembled at a specific site on the inner envelope associated with thylakoid membranes. D1 is synthesized as a precursor (pD1) with a carboxyl-terminal extension that is removed during assembly by protease CtpA (Anbudurai et al. 1994). A nuclear C. reinhardtii homolog of ctpA (C_270080) was identified by searching with the Arabidopsis genomic sequence (E-value 4.4e)25 for At5g46390) (Table 2). The cleavage between Ala-344 and Ser-345 of pD1 is not essential for
assembly of the PSII core complex, but it is essential for assembly of the Mn cluster (Nixon et al. 1992). Pulse-labeling of thylakoid proteins and subsequent fractionation of the thylakoid extracts revealed that the processing of pD1 occurs during assembly of CP43 into the core complex in wild-type cells (Zhang et al. 2000). In C. reinhardtii, substitution of Ser-345 with Pro decreases the rate of pD1 processing and assembly of the functional Mn-cluster 100-fold, indicating that pD1 can be matured after insertion into the PSII core complex (Hatano-Iwasaki et al. 2000). Several small polypeptides (PsbI, PsbT, and PsbW) are assembled into the D1/D2 heterodimer to form RC (Figure 3-2). The DpsbI and DpsbT mutants form functional PSII at 20%–30% and at 90% of the wild-type level, respectively (Monod et al. 1994; Ku¨nstner et al. 1995). Thus, PsbI and PsbT are not essential for the assembly of functional PSII, but they are required for efficient assembly. PsbW is thought to be incorporated at a later step in assembly (see below). In subsequent assembly steps, CP47 and CP43 are integrated into the RC (Figure 3-3). CP43 accumulates at 28%–36% of wild-type level in the absence of D1, D2 or CP47; when CP43 is absent, D1, D2, and CP47 accumulate in stoichiometric amounts at 12%–16% of the wild-type level (de Vitry et al. 1989; Sugimoto and Takahashi 2003).
Figure 3. Model for assembly of the PSII–LHCII supercomplex. Proposed assembly steps are as follows: (1) formation of a precomplex consisting of Cyt b559 and D2, (2) integration of D1 into the precomplex to form RC, (3) integration of CP47 into the RC to form RC–CP47 sub-complex, (4) assembly of CP43–PsbK sub-complex, (5) integration of CP43–PsbK into RC–CP47 to form PSII core complex and binding extrinsic polypeptides on the lumenal side of PSII core complex, (6) dimerization of PSII core complex, (7) synthesis of major and minor antenna complexes independently of the assembly of PSII core complex, (8) association of PSII core complex dimer and outer antenna to form PSII–LHCII supercomplex.
251 These observations suggest that D1, D2, and CP47 form a transient assembly intermediate, RC-CP47 (Figure 3-2) and that CP43 is synthesized independently. PsbK is tightly associated with CP43 (Sugimoto and Takahashi 2003), and the PsbK–CP43 sub-complex could integrate into RC–CP47 (Figure 3-3). The association of CP43 with the PSII core complex is inefficient in the Chlamydomonas DpsbK mutant (Sugimoto and Takahashi 2003). Several small polypeptides as well as the extrinsic polypeptides, PsbO, PsbP, PsbQ (and probably PsbR) and the Mn cluster are also assembled into the PSII core complex. In Chlamydomonas y-1 mutants, Chls or the photosynthetic apparatus are not synthesized in the dark, and greening is initiated by exposure to light. When y-1 cells are grown in the dark, synthesis of major PSII polypeptides (i.e., D1, D2, CP43 and CP47 and LHCII polypeptides) is transiently terminated in a light-regulated manner. Extrinsic polypeptides PsbO, PsbP, and PsbQ do accumulate in the dark, probably in the lumen or on the lumenal side of premature membranes before the PSII core complex is synthesized (Malno¨e et al. 1988). Mutants deficient in PsbO, Fud44, synthesize PSII polypeptides D1, D2, CP43 and CP47 at the wild-type rate but they accumulate at a significantly reduced level, indicating that PsbO is essential for the assembly and/or stability of PSII polypeptides (Mayfield et al. 1987a). In contrast, deficiency in PsbP (BF25 mutant) does not affect the assembly of normal amounts
of PSII, but oxygen evolving capacity is 5% of wild-type (Mayfield et al. 1987b). The highly purified PSII core complex is a monomer (de Vitry et al. 1991) but the PSIILHCII supercomplex is a dimer (Nield et al. 2000b) (Figure 3-4), indicating that dimerization occurs after the monomer is assembled. In higher plants, the dimeric PSII core complex is stabilized by PsbW (Shi et al. 2000), suggesting that PsbW is integrated at this step. The dimeric structure may also be stabilized by PsbL, PsbM, and/or PsbT at the dimer interface. In tobacco DpsbL mutant, no PSII core dimers are detected (Suorsa et al. 2004). Analyses of PSII-deficient and Chl b-deficient mutants revealed that PSII core complex and LHCII are synthesized independently and that formation of the dimer supercomplex is the last step of assembly (Figure 3-5). This step is facilitated by the chloroplast-encoded PsbJ and PsbZ (Swiatek et al. 2001; Suorsa et al. 2004). In addition to the factors involved in the early steps of PSII assembly described above, it is presumed that more molecular chaperones facilitate this multi-step assembly process, but little is known about such factors. The high fluorescence Arabidopsis mutant hcf136 is defective in production of PSII but not in synthesis of chloroplastencoded proteins (Meurer et al. 1998). HCF136 is localized to the lumen and may be a central factor in biogenesis of the PSII complex. In vivo pulselabeling of chloroplast-encoded proteins in the hcf136 mutant revealed that the assembly of the
Table 2. Genes involved in the regulation of PSII-LHCII supercomplex
a b
Gene
Genome
Positiona
MWb
Function
Reference for the initial identifier
LI818-1 LI818-2 LI818-3 LI818-4 CtpA DegP2-1 DegP2-2 DegP2-3 FtsH2(VAR2) FtsH5(VAR1) Hcf136 HSP70B Stt7
Nuclear Nuclear Nuclear Nuclear Nuclear Nuclear Nuclear Nuclear Nuclear Nuclear Nuclear Nuclear Nuclear
C_13190001 C_400109 C_400023 C_400069 C_270080 C_680053 C_280028 C_240116 C_10225 C_1620016 C_270022 C_750041 C_660011
27,557 27,573 28,224 28,224 46,398 66,136 32,117 51,734 74,385 77,547 43,155 71,969 80,745
Signal transduction Signal transduction Signal transduction Signal transduction C-terminal processing of D1 Cleavage of damaged D1 Cleavage of damaged D1 Cleavage of damaged D1 Chaperon, repair of D1 Chaperon, repair of D1 Assembly Chaperon, protection or repair of D1 Phosphorylation in state transition
X95307 This study, This study, This study, This study, This study, This study, This study, This study, This study, This study, X96502 AY220122
Genemodel names of nuclear-encoded genes (http://genome.jgi-psf.org/cholre2) are shown. Predicted MW of precursor.
blast blast blast blast blast blast blast blast blast blast
hit hit hit hit hit hit hit hit hit hit
for for for for for for for for for for
X95307 X95307 X95307 At5g46390 At2g47940 At2g47940 At2g47940 At2g30950 At5g42270 At5g23120
252 D1/D2 heterodimer is impaired and unassembled PSII proteins accumulate (Plu¨cken et al. 2002). In addition, HCF136 interacts with PSII assembly intermediates. Thus, HCF136 may be a chaperonlike assembly factor. Arabidopsis hcf136 was used to identify a C. reinhardtii homolog C_270022 (E-value 0 for At5g23120) in the C. reinhardtii draft nuclear genome sequence (Table 2). Turnover of PSII in strong light Although light is essential for photosynthesis, excessive light causes photoinhibition and decreases the efficiency of energy production. PSII is the main target of light-induced damage leading to photoinhibition. PSII activity is relatively constant under conditions of low and moderate light, because light-induced damage can be repaired quickly enough to prevent deleterious effects. However, under strong light conditions, the amount of photodamage to PSII exceeds the capacity for repair. When C. reinhardtii cells are irradiated with 4000 lmol photons m)2 s)1, PSII activity is reduced by 50% (Ohnishi and Takahashi 2001). To recover from photoinhibition, damaged D1 protein must be degraded and replaced by undamaged D1 with its cofactors; other less essential PSII components also need to be replaced. Light-induced turnover of D1 transiently produces a D1-depleted PSII complex, and releases lumenal extrinsic polypeptides into the lumen or retains them attached loosely to the thylakoids. However, degradation and synthesis of PSII components proceed simultaneously and intermediates of the repair process are not observed (van Wijk et al. 1994). The damage-repair cycle is distinct from assembly of PSII complex; in particular, D1-depleted PSII core complex is not an assembly intermediate during de novo synthesis of PSII (Figure 3). PsbT, which binds to PSII–RC, is required for growth of C. reinhardtii in strong light, although it is not essential for function and assembly of PSII (Monod et al. 1994). DpsbT mutant cells fail to repair damaged PSII efficiently, suggesting that it may facilitate replacement of photodamaged D1 and/or activate cofactors in the PSII complex (Ohnishi and Takahashi 2001). It seems likely that PsbT is involved in efficient reactivation of QA photoreduction (Ohnishi, Kashino, Satoh and Takahashi, unpublished results).
Some cellular components protect PSII against photoinhibition. Repair of PSII after photoinhibition requires that photodamaged D1 be proteolyzed and then replaced with a functional D1 molecule. Two proteases, DegP2 and FtsH, have been identified that degrade photodamaged D1 (Lindahl et al. 2000; Haußu¨hl et al. 2001; Kanervo et al. 2003; Silva et al. 2003). Degp2 is a GTPdependent protease that is associated with the stromal side of the non-appressed region of thylakoid membranes in higher plants (Haußu¨hl et al. 2001). Homologs of DegP2 were identified in the C. reinhardtii nuclear genome (C_680053, C_280028, and C_240116) by searching with the Arabidopsis nuclear DegP2 sequence (E-value < 1.1e)18 for At2g47940) (Table 2). FtsH is an ATP-dependent zinc metalloprotease that may also play a role in degrading photodamaged D1 (Lindahl et al. 2000). In Arabidopsis thaliana, two homologs of FtsH, FtsH2 and FtsH5, form an oligomer (Sakamoto et al. 2003). Antibodies to Arabidopsis FtsH cross-react with proteins in the thylakoid membranes of Chlamydomonas (Takahashi and Sakamoto, unpublished results), and homologs of Arabidopsis FtsH2 (At2g30950) and FtsH5 (At5g42270) were identified in the nuclear genome of C. reinhardtii (C_10225 with E-value 0 and C_1620016 with E-value 0, respectively) (Table 2). In C. reinhardtii, overexpression of heat shock protein 70 (HSP70B) in the chloroplast protects against photoinhibition, possibly by preventing light-induced damage of PSII or facilitating repair of such damage (Schroda et al. 1999). A C. reinhardtii gene encoding HSP70B has been deposited in Genbank (X96502, Table 2).
Structure and function of LHCII complex Organization of the light-harvesting complex for PSII Eukaryotic Chl-binding light-harvesting complexes have been characterized in various organisms including Chl a/b-containing chlorophytes, Chl a-containing rhodophytes, as well as those in Chl a/c lineage (Durnford et al. 1999). Structural studies of these proteins show they are monophyletic and share a common architecture (Green and Durnford 1996). The light-harvesting complex for PSII in chlorophytes are generally divided into
253 three parts: (1) the core antenna (Chl a-containing CP47 and CP43) immediately associated with RC, (2) the minor antenna (CP29, CP26, and CP24 in vascular plants) lying at the interface between (1) and (3) (Harrer et al. 1998: Yakushevska et al. 2003), and (3) the major antenna (LHCII) reside at the periphery of PSII–LHCII supercomplex (see also Figure 1). ‘LHCII complex’ or ‘outer antenna’ commonly denotes the antenna for PSII which are made up of Chl a/b-binding LHC proteins, namely (2) and (3) above. Nield et al. (2000b) described the 3D dimer structure of PSII–LHCII supercomplex from C. reinhardtii, indicating that it is structurally similar to PSII–LHCII in vascular plants with a few exceptions. A recent high resolution X-ray crystal structure suggests that the LHCII proteins in higher plants forms trimers (Liu et al. 2004). Each LHCII polypeptide has 3 TMHs and binds 8 Chl a, 6 Chl b, 2 luteins and one 90 -cis-neoxanthin. The monomer–monomer interface accommodates phosphatidylglycerol and violaxanthin. The phosphatidylglycerol may stabilize the trimer, and it has been proposed that the violaxanthin molecule, a xanthophyll-cycle carotenoid, traps energy during quenching. Genes encoding the major antenna proteins LhcbM6 (LhcII-1.1), originally designated cabII-1, was the first Lhc gene isolated from C. reinhardtii (Imbault et al. 1988) (Note: Recently, a nomenclature for the Lhc-related genes in C. reinhardtii was proposed by Grossman, Hippler and Minagawa (in preparation). This new nomenclature is adopted throughout this article. The conventional gene names based on Teramoto et al. (2001) are also indicated in parentheses for consistency.) Since then, additional Lhc genes have been identified in C. reinhardtii using EST databases and other molecular genetic methods (Minagawa et al. 2001, Teramoto et al. 2001, Elrad et al. 2002). BLAST homology search versus the C. reinhardtii genomic sequence ver. 2.0 (Table 1) predicts 12 Lhcb genes. Figure 4 shows a neighbor-joining phylogenetic tree for nine major antenna proteins (LHCII), three minor antenna proteins (CP29, CP26, and a tentatively assigned minor antenna protein encoded by C_20371), and four copies of a distantly related LHC protein, LI818, in C. reinhardtii. The tree has four distinct branches for
major antenna proteins, corresponding to Type I (LhcbM6, LhcbM4, LhcbM3, LhcbM8, LhcbM9), Type II (LhcbM5), Type III (LhcbM2, LhcbM7), and Type IV (LhcbM1). The four subgroups (types) of the major antenna proteins do not exactly correlate with each of the three types of major antenna proteins in higher plants (Teramoto et al. 2001). Thus, the genes encoding major antenna proteins may have diversified into four and three types in Chlorophyceae (green algae) and Streptophyta (terrestrial plants and their closest green algal relatives, charophytes), respectively, after the split between Chlorophyceae and Streptophyta. Figure 5 shows an alignment of the 12 predicted antenna proteins for the LHCII complex in C. reinhardtii. The average sequence identity between a representative Type I protein LhcbM6 (LhcII-1) and LhcM5 (LhcII-2), LhcbM2 (LhcII3.1), or LhcbM1 (LhcII-4) is 80%, 77%, or 74%, respectively. Multiple genes within each type may reflect recent gene duplication event(s). Type I includes five duplicate genes with an average sequence identity of 93%. Genes encoding LhcbM4 (LhcII-1.2), LhcbM8 (LhcII-1.4), and LhcbM9 (LhcII-1.5) are located on the same scaffold in the draft C. reinhardtii genomic sequence ver. 2.0 (scaffold 146). The former two genes lie only 4.2 kb apart and share 92% identity. LhcbM4 (LhcII-1.2) and LhcbM9 (LhcII-1.5) (newly identified in this study) are 99% identical. The Type III major antenna proteins, LhcbM2 (LhcII-3.1) and LhcbM7 (LhcII-3.2) are also located on the same scaffold (scaffold 70) and share 99% identity. Type II and Type IV major antenna proteins are encoded by a single gene, LhcbM5 (LhcII-2) and LhcbM1 (LhcII-4), respectively. Genes encoding the minor antenna proteins Genes encoding two minor antenna proteins, CP26 (p10) and CP29 (p9), were isolated previously (Minagawa et al. 2001; Teramoto et al. 2001); the deduced primary sequences are 50% and 47–89% identical with their counterparts in A. thaliana, respectively. While the two minor antenna proteins are conserved in Chlorophyceae and Streptophyta, a homolog (E-value less than 0.1) for another minor antenna protein CP24 has not been detected in C. reinhardtii EST or genomic sequences. However, there is one C. reinhardtii
254
Figure 4. Unrooted phylogenetic tree of LHC proteins associated with C. reinhardtii PSII . Neighbor joining-distance tree was constructed with bootstrap values (% of 1000 replicates) displayed at the appropriate nodes. Amino acid sequences were from the following databases: Genbank – LI818-1 (CAA64632), LhcbM1 (BAB64418), LhcbM2 (BAB64417), LhcbM3 (BAB64416), LhcbM4 (AAD03731), LhcbM5 (AAD03732), LhcbM6 (AAA33082), LhcbM7 (AAK01125), LhcbM8 (AAL88457), Lhcb4 (BAB64419), Lhcb5 (BAB20613); JGI – LI818-2 (C_400109), LI818-3 (C_400023), LI818-4 (C_400069), LhcbM9 (C_1460005), C_20371. The entire coding regions including transit peptides were used to align amino acid sequences using Clustal W.
hypothetical protein encoded by a gene model C_20371 with 20%–30% identity to other minor antenna proteins in C. reinhardtii and A. thaliana (Figures 4 and 5); a possible counterpart for this gene in the nuclear genome of A. thaliana is At1g76570. If this gene product is a bona fide component of the LHCII complex, it will be called Lhcb7. As mentioned above, genes encoding major antenna proteins diverged into several ‘types’ after the phylogenetic separation of Chlorophyceae and Streptophyta (Teramoto et al. 2001). Lhcb4, Lhcb5, and possibly Lhcb6, encoding CP29, CP26, and CP24, may have emerged before the split, since they are conserved between Chlorophyceae and Streptophyta. The conservation of CP29 and CP26 argues in favor of specific role(s) for each one. Lhcb5 encoding CP26 was duplicated not and has not diverged in C. reinhardtii and A. thaliana. Lhcb4 encoding CP29 was triplicated only in A. thaliana (Jansson 1999). The absence of CP24 in C. reinhardtii may indicate that this gene was
Figure 5. Sequence alignment of C. reinhardtii LHCII-related proteins. Sequences were aligned using Clustal W. Gaps (—) were introduced to maximize homology. Boxed regions correspond to putative membrane-spanning (A–C, thick line) and amphipathic (D–E, thin line) helices (Liu et al. 2004). Residues contribute their side chains or backbone carbonyls to coordinate the central Mg of Chl in LHCII (Liu et al. 2004) are shaded in black or grey, respectively. The trimer motif (WYGPDR, Hobe et al. 1995) and L18 domain (VDPLYPGGSFDPLGLADD, DeLille et al. 2000) are indicated; conserved residues are shaded.
255 originally present but then lost, or that it never diverged in green algae. A line of evidence supporting the former hypothesis has been presented in a phylogenic tree by Tokutsu et al. (2003). An alternative scenario would be that C. reinhardtii CP24 diverged rapidly after the split, retaining very little homology with the prototypical CP24 sequence at present. Polypeptides associated with LHCII complex C. reinhardtii LHCII complex and PSII–LHCII supercomplexes have been characterized biochemically using electrophoretic and centrifugal techniques. Bassi and Wollman (1991) identified five LHC proteins including p10, p11, p13, p16, and p17 in purified LHCII fraction. (Note: nomenclature used for these proteins is after Chua and Bennoun 1975.) One additional LHC protein, p9, fractionates with PSII membranes, bringing the total to six LHC proteins associated with PSII. p11, p13, p16 and p17 oligomerize into the high molecular weight form of LHC, suggesting that these four polypeptides constitute the major antenna. p9 and p10 only exist in a low molecular weight form, suggesting they may be homologs of CP29 and CP26. p9 and p10 also have low Chl b content and exhibit red-shifted absorption maxima at 676–677 nm (Bassi and Wollman 1991). Later, Minagawa et al. (2001) identified C. reinhardtii p10 as CP26 based on partial amino acid sequences. Recently, Hippler and colleagues reported a detailed two-dimensional map of C. reinhardtii LHC proteins associated with PSI and PSII based on tandem mass spectrometry data (Stauber et al. 2003). They compared the experimentally determined amino acid sequences of 11 LHC proteins in the LHCII complex with the amino acid sequences deduced from EST data. This procedure resulted in nearly unequivocal identification of the six LHCII proteins including LhcbM6 (LhcII-1.1), LhcbM4 (LhcII-1.2), LhcbM3 (LhcII-1.3), LhcbM2 (LhcII3.1) or LhcbM7 (LhcII-3.2), LhcbM1 (LhcII-4), and two minor antenna proteins CP26 and CP29. LhcbM5 (LhcII-2), LhcbM8 (LhcII-1.4), and LhcbM9 (LhcII-1.5) were not present on this protein map. Another analysis based on denaturing SDS-PAGE data (similar to the method used by Bassi and Wollman, 1991) identified protein sequences for major antenna proteins Type I,
Type IV, and Type III, corresponding to p11/p13, p16, and p17, respectively (Seguchi, Hayakawa and Minagawa, in preparation). LhcbM5 (LhcII2) was not identified in this study. These results confirm that the predominant components of the C. reinhardtii LHCII complex are the major antenna proteins Type I, Type III, and Type IV, which brings into question the role, level of expression and/or stability of the major antenna protein Type II, LhcbM5 (LhcII-2). Hippler and his colleagues (Hippler et al. 2001; Stauber et al. 2003) determined the N-terminal amino acid sequences of several LHCII proteins: [KALQVTCKATGKK] and [APKSS] for LhcbM6 (LhcII-1.1), [SSGVEFYGPNR] for LhcbM5 (LhcII-2), [QAPASSGIEFYGPNR], [PASSGIEFYGPNR], and [SSGIEYGPNR] for LhcbM3 (LhcII-1.3), and [IEWYGDPR] for LhcbM2 (LhcII-3.1)/LhcbM7 (LhcII-3.2). They suggested that the mature form of LHCII proteins could have multiple N-termini due to differential N-terminal processing. The significance of the alternate processing sites for these proteins is currently unknown, but may relate to assembly or regulation of the LHCII complex. CP26 exists predominantly as a monomer, but CP26 trimerization was observed in an Arabidopsis strain that lacks one polypeptide of major antenna complexes (Ruban et al. 2003). This interesting result might have been expected, because CP26 has the ‘trimer motif’ WYGPDR which is found in other LHCII proteins (Hobe et al. 1995). C. reinhardtii CP26 also carries this motif and may also trimerize under certain conditions (Minagawa et al. 2001) (Figure 5). Photoacclimation of LHCII complex For photosynthetic organisms, the ability to survive fluctuations in quality and quantity of light is crucial; to do this, organisms must ensure that lightharvesting capacity and energy utilization are modified in response to environmental conditions. Capture of excessive light energy under conditions of strong light causes photoinhibition, as discussed above. Photosynthetic organisms survive under variable light conditions by the process known as photoacclimation. Three well-studied photoacclimation mechanisms are induced under conditions of strong light: (1) down-regulation of light-harvesting complex (antenna size adjustment), (2) a
256 rapid decrease in absorption cross-section of PSII-LHCII supercomplex by redistributing lightharvesting antennae (state-transition), (3) nonphotochemical quenching (NPQ) to decrease photosynthetic efficiency. These mechanisms are described below in the context of the C. reinhardtii LHCII complex. Antenna size adjustment Transcriptional regulation of C. reinhardtii Lhc genes plays a central role in antenna size adjustment. Many factors affect transcription of Lhcb genes including circadian rhythm (Kindle 1987), redox state of the plastoquinone pool (Escoubas et al. 1995), Chl synthesis (Johanningmeier and Howell 1984), and intensity of incident light (Elrad et al. 2002; Teramoto et al. 2002; Durnford et al. 2003). These factors also influence gene expression in higher plants, in which light-dependent regulation of Lhcb is mediated by the red light receptor phytochrome. However, transcription of C. reinhardtii Lhcb genes is not regulated by phytochrome (Kindle 1987). Regulatory cis-acting DNA sequence elements (i.e., GT-1-binding sites, I-Boxes, and G-Boxes) are thought to exist in higher plants (Terzaghi and Cashmore 1995), but are not present in the 50 -UTR of light-regulated genes in C. reinhardtii (Hahn and Ku¨ck 1999). These observations indicate that light-responsive gene expression is mediated by different mechanisms in C. reinhardtii and higher plants. Teramoto et al. (2002) investigated expression of six Lhcb genes including LhcbM1 (LhcII-4), LhcbM2 (LhcII-3.1), LhcbM3 (LhcII-1.3), LhcbM6 (LhcII-1.1), Lhcb4, and Lhcb5 under conditions of variable light intensity. All Lhcb genes are downregulated coordinately by strong light. The light intensity that triggers down-regulation decreases when cells are grown at lower temperature or lower ambient CO2 concentration, suggesting that excitation energy, CO2 assimilation and transcription of Lhcb genes are maintained in balance by a negative feedback mechanism. Escoubas et al. (1995) suggested that reduction in the plastoquinone pool might act as an initial signal for this negative-feedback loop in the green alga D. tertiolecta. They observed that expression of Lhcb is enhanced under high light when electron transfer from QA to QB is inhibited, but this effect is repressed when oxidation of plastoquinol is inhibited at the Cyt bf complex. Teramoto et al. (2002)
reported that negative-feedback was only marginally affected by inhibitors of electron-transport or mutations in PSI and/or PSII. These results led them to conclude that expression of the major antenna proteins is loosely regulated by a redoxdependent mechanism and primarily regulated by a redox-independent mechanism. Redox-independent regulation has also been described for other light-responsive genes in C. reinhardtii (Im and Grossman 2001) and D. salina (Masuda et al. 2003). Two possible candidates for the redoxindependent signal have been proposed; reactive oxygen species or precursors of Chl synthesis. Numerous Chl molecules bound to the LHCII complex could be a potent source of reactive oxygen species, and precursors of Chl synthesis may also be affected by reactive oxygen species. The kinetics of LhcbM gene expression were studied following a shift to strong light by Durnford et al. (2003) and Elrad and Grossman (2004). The results indicate a rapid decrease in expression during the first 2 h, followed by recovery from 6 to 24 h. Durnford et al. (2003) also observed that the stability of LhcbM transcripts did not change significantly during exposure to strong light, suggesting that expression of LhcbM genes is controlled predominantly at the level of transcription. The Lhcb4 and Lhcb5 transcripts are coordinately regulated with transcripts of LhcbM genes, although they are less light-responsive during light-shift (Teramoto et al. 2002) and their steadystate level is not dramatically altered after photoacclimation (Minagawa et al. 2001). Schrager et al. (2001) reported that the Lhcb4 transcript was present in a C. reinhardtii cDNA library from nutrient-starved cells (Stress I), which could indicate less strict regulation of genes encoding minor antenna proteins. State transition and phosphorylation A mechanism for rapid acclimation to variable light under redox control is referred to as a state transition. This process is correlated with redistribution of peripheral antenna proteins and thus excitation energy from PSII to PSI, and vice versa, and involves phosphorylation/dephosphorylation and migration of a fraction of the LHCII proteins (Wollman and Delepelaire 1984). The phosphorylation reaction is regulated by the redox state of the plastoquinone pool; in particular, excessive stimulation of PSII activates thylakoid protein kinase
257 through the plastoquinol molecule on the Cyt bf complex. Phosphorylated LHCII proteins dissociate from PSII and reassociate with PSI during transition from state I to II. (Vener et al. 1997). Protein kinases that phosphorylate LHCII proteins have been identified. Kohorn and his colleagues identified three thylakoid associated kinases, TAK1, TAK2, and TAK3 (Snyders and Kohorn 1999) and showed that TAK1-deficiency abolishes state transitions in A. thaliana (Snyders and Kohorn 2001). In C. reinhardtii, a chloroplast kinase was recently described that plays a crucial role in state transition-related phosphorylation (Depe`ge et al. 2003). In the mutant stt7 (Table 1), phosphorylation of LHCII proteins was not observed and cells were unable to undergo transition from state I to state II. This suggests strongly that the Stt7 kinase participates the state transition or the signal transduction cascade for the state transition in C. reinhardtii. LHC proteins that are reversibly phosphorylated in state II include p9, p10, and p11 (de Vitry and Wollman 1988; Fleischmann et al. 1999), corresponding to CP29, CP26 and the major antenna proteins Type I. Stauber et al. (2003) also identified LhcbM3 (LhcII-1.3) as a target of phosphorylation. The significance of phosphorylation of minor antenna proteins CP29 and CP26 is not known. qE quenching and PsbS The minor antenna proteins have been studied extensively because they contain a high content of xanthophyll-cycle pigments, which are thought to
play a role in thermal dissipation (Bassi et al. 1993). In fact, Crimi et al. (2001) demonstrated that binding of zeaxanthin decreases the lifetime of Chl fluorescence in CP29, suggesting xanthophylldependent quenching of excess excitation energy (qE quenching). However, decreased expression of A. thaliana Lhcb4 and Lhcb5 does not significantly alter capacity for NPQ (Andersson et al. 2001). This suggests that these minor antenna proteins are not essential for NPQ. Niyogi and his colleagues searched extensively for NPQ-defective mutants of C. reinhardtii and A. thaliana. Several mutants were identified, but they do not map to CP29, CP26, or CP24 (Baroli and Niyogi 2000), indicating that NPQ is a complex process involving protein factors that have not yet been fully characterized and the sites for NPQ are much more complicated than originally proposed. Light harvesting and photoprotection are not yet well understood processes. In addition, the functional role of each LHCII protein has not been elucidated. In this regard, C. reinhardtii npq5 could provide a clue to explore the specific role of each major antenna protein as well as a clue to reveal the mechanism to induce qE quenching. The npq5 mutant was generated by non-targeted gene-tagging in C. reinhardtii, followed by a screen for inability to establish rapid, reversible NPQ of Chl fluorescence (Niyogi et al. 1997; Elrad et al. 2002). The tagged npq5 locus maps to LhcbM1 (LhcII-4), and the wild-type genomic fragment for this locus complements the npq5 phenotype. Thus, a major antenna protein Type
Figure 6. Sequence alignment of PsbS homologs. The C. reinhardtii PsbS sequence is aligned to Arabidopsis thaliana (At) and Spinacia oleracea (spinach, So) PsbS sequences. Hydrophobic domains predicted by the hydrophobicity analysis software SOSUI (Hirokawa et al. 1998) or Topred II (Claros and von Heijne 1994) are shown with shadow font. The N-terminal sequence of mature PsbS from A. thaliana (Li et al. 2002) was used to predict the N-termini of other proteins. Amino acid sequences were from the following databases: Genbank – At.PsbS (At1g44575), So.PsbS (CAA48557); JGI – Cr.PsbS (C_280068).
258 IV (LhcbM1, Lhc II-4) is implicated as a critical player in thermal dissipation. It is now established that the PSII light-harvesting antenna can exist in two different states in vivo: a light-harvesting state and a quenched state that dissipates excess excitation energy. It is clear that the quenched state is not induced in the absence of LhcbM1 (LhcII-4). Detailed structural and biochemical studies on the LHCII complex in npq5 could provide new insights into mechanisms of photoprotection. It should be noted that a structure-based non-photochemical quenching model was recently proposed based on the high resolution structure of spinach LHCII (Liu et al. 2004). This model suggests that a violaxanthin molecule binds to the monomer-monomer interface, so that only trimer forms of LHCII proteins can accommodate this xanthophyll-cycle pigment. PsbS is another PSII-associated polypeptide that is critical for thermal dissipation. Li et al. (2000) demonstrated that A. thaliana npq4 mutants are deficient in psbS. Although it is now obvious that this protein is involved in high energy quenching of Chl fluorescence, the nature of this protein is as yet unknown. It was identified as a distant relative to the LHC protein superfamily, classified as a Chl/carotenoid-binding protein and therefore called CP22. However, recent studies have called into question its pigment-binding ability (Dominici et al. 2002). It interacts weakly with the LHCII complex, is not present in the PSII–LHCII supercomplex, and is located in the LHCII-rich regions that interconnect the supercomplex in the membrane (Nield et al. 2000a). The deduced amino acid sequence of PsbS indicates four TMH, instead of three TMH as in other LHC proteins, and most of the Chl ligands are not conserved. A BLAST search using Arabidopsis PsbS (E-value 1.2e)32 for At1g44575) revealed a probable homolog in C. reinhardtii genome sequence v2.0 (Table 1). It shares 45% identity over 180 amino acids with Arabidopsis PsbS (Figure 6), which includes the proposed pH sensing glutamate residues (Li et al. 2004). However, its sequence has not been identified in >100,000 C. reinhardtii EST entries (Teramoto et al. 2001; Elrad et al. 2002) and the protein has not been detected on thylakoid membrane in C. reinhardtii. Thus, expression of the putative Chlamydomonas PsbS homolog needs to be verified as well as its putative role in high energy dissipation.
Genes involved in regulation of LHCII complex LI818 is distantly related to the green algal LHC superfamily (Figure 4). LI818 was originally described in Chlamydomonas eugametos by Gagne´ and Guertin (1992) as an LHC-related gene whose expression is regulated by a non-photosynthetic photoreceptor and that is induced transiently at the onset of light exposure. This regulatory mechanism differs from regulatory mechanisms of other C. reinhardtii Lhc genes (Savard et al. 1996). Four copies of LI818 gene are present in the nuclear C. reinhardtii genomic sequence v.2.0 (C_13190001, C_400109, C_400023, and C_400069, see Table 1; note: Since C_400023 and C_400069 are located on the same scaffold side by side and almost identical, the distinctions between the two genes could be the consequence of sequencing errors or mistaken assemblies.) LI818 is very strongly induced by exposure to strong light by a redox-independent mechanism (Elrad and Grossman 2004). Although it seems likely that LI818 plays a role in redoxindependent signaling in response to strong light, the precise function of LI818 is still unknown. A recent study identified a gene that may regulate integration of LHCII proteins into the thylakoid membrane (Bellafiore 2002). This gene maps to Alb3, which encodes a chloroplast homolog of Oxa1p in plant mitochondria and YidC in E. coli, proteins that are required for the insertion of a SRP-dependent protein such as Lhcb4.1 and Lhcb5 in higher plants (Woolhead et al. 2001). The C. reinhardtii Alb3 mutant expresses a 10-fold reduced level of LHCII proteins, suggesting that Alb3 and probably cpSRP facilitate translocation of the major antenna proteins in C. reinhardtii. In fact, the ‘L18 domain’ is conserved in LHC proteins associated with PSII in C. reinhardtii (Figure 5), and it is required in order to form a transit complex for cpSRP and LHC proteins during reconstitution of pea LHC proteins (DeLille et al. 2000).
Acknowledgements Sequence data for the nuclear genome were produced by the US Department of Energy Joint Genome Institute, http://www.jgi.doe.gov/ and are provided for use in this publication/correspondence only. We are grateful to Dr Arthur Grossman for sending us a copy of the manuscript
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