Cyanobacterial high-light-inducible proteins — Protectors of chlorophyll–protein synthesis and assembly

    Cyanobacterial high-light-inducible proteins - protectors of chlorophyllprotein synthesis and assembly Josef Komenda, Roman Sobotka PII: DOI: Reference:

S0005-2728(15)00175-9 doi: 10.1016/j.bbabio.2015.08.011 BBABIO 47521

To appear in:

BBA - Bioenergetics

Received date: Revised date: Accepted date:

17 June 2015 28 August 2015 30 August 2015

Please cite this article as: Josef Komenda, Roman Sobotka, Cyanobacterial high-lightinducible proteins - protectors of chlorophyll-protein synthesis and assembly, BBA - Bioenergetics (2015), doi: 10.1016/j.bbabio.2015.08.011

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ACCEPTED MANUSCRIPT Cyanobacterial high-light-inducible proteins - protectors of chlorophyll-protein synthesis and assembly

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Josef Komenda1,2* and Roman Sobotka1,2

Institute of Microbiology, Laboratory of Photosynthesis, Centre Algatech, 37901 Třeboň, Faculty of Science, University of South Bohemia, 370 05 České Budějovice, Czech Republic

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Czech Republic

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* To whom correspondence should be addressed: [email protected]

Abstract

Cyanobacteria contain a family of genes encoding one-helix high-light-inducible proteins (Hlips)

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that are homologous to light harvesting chlorophyll a/b-binding proteins of plants and algae. Based on various experimental approaches, a spectrum of functions that includes regulation of chlorophyll biosynthesis, transient chlorophyll binding, quenching of singlet oxygen and nonphotochemical quenching of absorbed energy is ascribed to these proteins. However, these

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functions had not been supported by conclusive experimental evidence until recently when it became clear that Hlips are able to quench absorbed light energy and assist during terminal

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step(s) of the chlorophyll biosynthesis and early stages of Photosystem II assembly. In this review we summarize and discuss the present knowledge about Hlips and provide a model of how individual members of the Hlip family operate during the biogenesis of chlorophyll-proteins, namely Photosystem II.

Abbreviations: -car, β-carotene; Cab, chlorophyll a/b binding; Chl, chlorophyll; Chlide, chlorophyllide; CP43m, CP47m, D1m and D2m, CP43, CP47, D1 and D2 assembly modules, respectively; Elip, early light-induced protein; FeCh, ferrochelatase; Hlip, high-light-inducible protein; Lhc, lightharvesting complex; Lil, Lhc-like proteins; Ohp, one-helix protein; Pchlide, protochlorophyllide; Proto, protoporphyrin IX; PSI, photosystem I; PSII, photosystem II; RC, reaction center; Scp, small Cab-like protein; Sep, stress-enhanced protein.

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Keywords: chlorophyll; cyanobacteria; high-light-inducible protein; photosynthesis; small Cab-like protein;

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Synechocystis sp. PCC 6803;

INTRODUCTION

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Algae and plants possess membrane embedded light harvesting complexes (Lhcs) that collect energy from photons and transfer it predominantly to Photosystem II (PSII) for driving oxygen

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evolution, but there is also a distinct group of Lhcs associated with Photosystem I (PSI) [1-3]. PSII and PSI are chlorophyll (Chl)-binding protein complexes that perform light-induced charge separation and drive photosynthetic electron transfer in oxygenic phototrophs. All known Lhcs

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contain three transmembrane -helices and bind Chl a, other Chl molecules (b or c), carotenoids and lipids [4, 5]. The pigment composition is species dependent and variability exists even

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among the Lhc complexes from a single organism [6]. However, the binding motif for Chl a is highly conserved and frequently occurring even outside of the Lhc family. This finally led to the recognition of a much wider superfamily of Chl a/b binding (Cab) proteins, which is very likely derived from a common ancestor and apart from the ‘true’ Lhcs includes single, double, triple

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and quadruple helix Lhc-like proteins (for reviews see [7, 8]). The role of these proteins is generally poorly understood but they are predicted to play a role in photoprotection (see below).

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Cyanobacteria, the only prokaryotic oxygenic phototrophs, harvest most of light energy via membrane attached phycobilisomes, a large assemblage of proteins with covalently bound phycobilins [9]. These pigment-protein complexes function as the main antenna system for PSII and also partly for PSI [10, 11]. Cyanobacteria lack true Lhcs but their genomes contain a family of genes coding for single transmembrane helix proteins that show similarity to the first or third helix of Lhc. The first cyanobacterial gene for such a Cab-like protein was identified in Synechococcus PCC 7942 two decades ago and according to its strong induction under increased irradiance it was named high-light-inducible protein (Hlip; [12]). The complete repertoire of Hlip-encoding hli genes was subsequently identified in the fully sequenced genome of the cyanobacterium Synechocystis PCC 6803 (hereafter Synechocystis) [13, 14]. These genes code for four small Hlips named HliAD and an Hlip domain is also found in the C-terminus of the ferrochelatase (FeCh) enzyme. According to their similarity to helixes of members of the Cab

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ACCEPTED MANUSCRIPT protein family Hlips are also called small Cab–like proteins (Scps). Since then, hli/scp genes have been identified in all cyanobacterial species with known genome sequences. The number of hli/scp genes in cyanobacterial genomes is variable but even the primitive thylakoid-less

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cyanobacterium Gloeobacter violaceus possesses at least five genes encoding Hlip homologues.

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Numerous functions have been ascribed to Hlips ranging from a role in regulation of Chl biosynthesis [15-17] to transient binding of Chl and carotenoids [18], Chl recycling [19], non-

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photochemical energy quenching [20] and scavenging of singlet oxygen [21]. Until recently no direct evidence existed about pigment binding to Hlips but in the last two years convincing

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evidence about binding of Chl and carotenoids to Hlips and their stoichiometry has been reported [22, 23]. It has also become clear that Hlips are involved in the process of biogenesis of Chl-

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binding proteins [24].

HLIP GENES AND CLUSTERS AND REGULATION OF THEIR EXPRESSION

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As already noted above the hli genes are generally considered to be ubiquitous in cyanobacteria (see Cyanobase). An extensive analysis of the hli family has been performed for seven cyanobacterial species including four marine cyanobacteria adapted to high, moderate or low light, and three freshwater strains. Notably, the high-light grown marine cyanobacterium

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Prochlorococcus MED4 with the smallest genome contains twice as many hli genes (>20) as any of the other six studied species and some of them possibly have arisen from recent duplication

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events [25]. Moreover, the cluster analysis has demonstrated specificity of some hli genes for marine or freshwater species and, in accordance with this, hli genes of freshwater unicellular species are more closely related to the hli genes from freshwater filamentous species than to the hli genes from unicellular marine strains [25]. From the first description of a hli gene the expression profiles connected this gene family with stress conditions. In pioneering work Dolganov et al. [12] monitored expression of the GUS reporter fused to the hliA gene in Synechococcus PCC 7942 and showed an increase directly proportional to the intensity of white light and indirectly proportional to the wavelength of irradiation, with the strongest induction detected under UV-A light [14]. Later, He et al. [26] showed an increased level of Hlips under various stress conditions including high irradiance, nitrogen or sulfur depletion and low temperature. Low-intensity blue or UV-A light [27, 28] and the deletion of PSI [14] also induce the hli gene expression in Synechocystis. In marine

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ACCEPTED MANUSCRIPT Prochlorococcus species the expression of hli genes also responds to nitrogen deficiency [29] or viral infection [30]. A further analysis of the hli expression in Synechocystis revealed that it is under the

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control of the sensory histidine kinase Hik33 [31, 32] and orthologues of this kinase most

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probably regulate hli expression in other cyanobacterial species too. Interestingly, Hik33 is also essential for high-light-induced synthesis of the additional copies of the D1 subunit of PSII and

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FtsH2 (Slr0228) and FtsH3 (Slr1604) proteases. All these proteins are essential for maintaining the activity of PSII under high irradiance. These results provided the first clue that the function of

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Hlips is connected to PSII. This essential membrane complex is sensitive to a light-induced loss of photochemical activity that is caused by damage to D1, the central subunit carrying many

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cofactors and pigments required for PSII photochemistry (for review see [33]). The loss of PSII activity can be restored via the so called PSII repair cycle, which consists of several steps (for review see [34]). These include monomerization and partial disassembly of PSII, degradation of

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the damaged D1 protein catalyzed by the heterohexamer of FtsH2 and FtsH3 proteases [35] and its replacement with the newly synthesized copy, and reassembly and reactivation of PSII. Many cyanobacteria contain additional copies of the psbA gene encoding the D1 protein and like expression of the hli genes, the expression of these psbA copies is also induced by high light

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under regulation by Hik33 [36]. A mutant lacking Hik33 is aberrant for the expression of many genes including those encoding Hlips [37]; it contains higher transcript levels of the hli genes in

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low light but in high light this level increases only moderately [37].

ESTABLISHING A LINK BETWEEN HLIPS AND PSII All studies dealing with the location of Hlips have been exclusively performed using strains of Synechocystis PCC 6803. The results of Funk and Vermaas [14] showed significant accumulation of Hlips in PSI-less strains, indicating that the proteins are not associated with PSI. The first attempts to isolate and localize Hlips have been performed using variants expressing these small proteins fused to a His-tag at the C-terminus [26]. This approach revealed that the two larger Synechocystis Hlips (HliA and HliB), which differ just in seven amino acid residues in the total 70 amino-acid chains, exhibit similar fast kinetics of appearance and disappearance. It led to the suggestion that HliA and HliB might be redundant copies with the same function. The smaller HliC also appeared very quickly after exposure of cells to high irradiance while the appearance of

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ACCEPTED MANUSCRIPT HliD was slower. Following the transfer of cells from high to low light both HliC and HliD persisted in the cells much longer than the two remaining Hlips [26]. The different kinetics already indicated that the individual Hlips may have different locations and this was supported by

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a fractionation experiment. Solubilization of cellular membranes and subsequent fractionation of

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Hlip-associated complexes by gel filtration showed the HliA and HliB polypeptides in the 100kDa fraction, whereas the HliC and HliD polypeptides were in the 50-kDa fraction [26]. These

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data suggested that Hlips function as complexes and that the similar Hlip pairs may be associated with each other.

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The first comprehensive localization study of the HliA and HliB proteins has been performed by Promnares et al. [38] who unequivocally proved association of both Hlips with

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PSII. By combining a variety of PSII mutants and several biochemical methods the authors demonstrated that the HliA/B binding site is on the CP47 antenna and is located in the vicinity of a small PSII transmembrane polypeptide PsbH (see Fig. 1 for a hypothetical model of the Hlip-

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CP47 structure). A subsequent study by Yao et al. [39] extended the knowledge about Hlip localization showing a close relationship between HliB and HliC and their binding to PSII. The results show that HliC is not required for binding of HliB to CP47 and there is also a link between HliA and HliC as the former disappears in the HliC-less mutant.

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In contrast to the other Hlips, HliD was not found by Yao et al. [39] in any of the PSII complexes containing CP47 including the assembly intermediate of PSII lacking CP43 (RC47).

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Nevertheless, the presence of HliC in PSII and the overexpression of HliD in the strain overexpressing HliC [40] indeed suggested a link between HliD and PSII. In order to understand this link it is important to have some knowledge about the biogenesis of PSII. According to the current model, the PSII complex is assembled from four relatively independent modules. Each module (m) consists of one large Chl-binding PSII subunit (D1, D2, CP43 and CP47) containing pigments and other cofactors which associates with adjoining small subunits. Modules are assembled in a stepwise manner in the order D1m + D2m + CP47m + CP43m [34]. Mutants lacking the CP47 antenna accumulate two RCII sub-complexes consisting of D1m, D2m and the assembly factor Ycf48 (Slr2034, [41]). The recent work of Knoppová et al [22] described and characterized the larger of these two complexes, designated RCII*, which contains an additional assembly factor Ycf39 (Slr0399) and tightly bound HliD and HliC. Using a D2-less strain helped to further clarify that Ycf39 and Hlips also bind to the unassembled D1m. In summary, all

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ACCEPTED MANUSCRIPT Synechocystis small Hlips (HliA-D) have been clearly demonstrated to associate with PSII complexes, either with the complete core complexes, with the assembly intermediate RC47 already containing the CP47m (HliA/B) or with the earlier assembly intermediates before CP47m

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is attached (HliC/D).

THE PHOTOPROTECTIVE ROLE OF HLIPS DURING PSII BIOGENESIS

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Hlips do not play an essential role in cyanobacteria as the Synechocystis mutants lacking all small Hlips [26] or all five Hlips including the FeCh C-terminal Hlip/Cab domain [18], are

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viable. However, they are highly sensitive to increased irradiance or oxidative stress [21, 26], which clearly suggests that these proteins play an important role during acclimation to stress

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conditions.

Binding of HliC and HliD to the putative short-chain dehydrogenase Ycf39 enabled isolation of the Ycf39-Hlip complex using the Flag-tagged version of Ycf39 [22, 23]. Although it

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has been speculated that Hlips bind Chls and carotenoids [18] and the reconstitution of pigment binding to Synechocystis Hlips produced by in vitro translation also provided some support for it [42], characterization of the isolated Flag-Ycf39-Hlip complex provided the first unequivocal evidence for pigment binding to Hlips. The Ycf39-Hlip complex contains Chl and β-carotene (-

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car) in the ratio 3:1 [23] which corresponds well to the pigment binding observed in the two homologous transmembrane helixes I and III of the plant Lhc proteins [5]. Since Ycf39 does not

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contain pigments [22, 23] all Chl and -car molecules are clearly bound to Hlips. In Lhc, two of the four Chl a molecules bound to the conserved ExxH/NxR motif are coordinated by glutamatearginine ion pairs [5] and to achieve such a configuration, Hlips have to form an oligomer. We have already proposed a hypothetical model for the Hlip dimer containing six Chl and two ß-car molecules [23]. In Figure 1, the Hlip dimer represents the HliA/B pair but we expect that such a cross-shape of two Hlip helixes is typical for the whole Hlip family. The Ycf39-Hlip complex has been shown to efficiently quench energy absorbed by Chl and characterization of this quenching by femtosecond absorption spectroscopy provided the convincing proof-of-principle for the direct energy transfer from the excited state of Chl to the S1 energy state of the bound β-car [23]. The described mechanism provides explanation for two proposed functions of Hlips. The complex binds Chl and therefore can act as the proposed scavenger of released Chl, which would otherwise be lethal for cells due to its photodynamic

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ACCEPTED MANUSCRIPT action and generation of singlet oxygen [21]. If Chls bound to Hlips are excited, the energy is efficiently quenched preventing the generation of singlet oxygen. Attachment of Ycf39-Hlip also abolishes Chl fluorescence of the D1-D2 assembly intermediate complex RCII* [22]. This means

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that the β-car of Hlips also quenches the energy absorbed by the Chl bound to this PSII assembly

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complex. Previously, a function of Hlips in non-photochemical energy quenching in Synechocystis cells was proposed by Havaux et al. [20] based on characterization of the hli-

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quadruple deletion mutant. Experimental evidence demonstrating that the D1 protein is the binding partner of Ycf39-Hlip [22] suggests that β-car bound to Hlips either interacts directly

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with Chls bound to D1 or the energy is first transferred to Chls bound to Hlips and then quenched. Within the D1-D2 RCII complex the Chls of the primary donor have absorption

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maxima close to 680 nm making the direct energy transfer from P680 to Chls of Hlips (absorbing at 674 nm [22]) improbable. In addition, the location of P680 inside the RCII* complex would not allow for energy transfer to distant Hlip pigments. In contrast, the peripheral Chl bound to

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His118 of D1 has an absorption maximum around 671 nm [43] and represents a good candidate for the Chl interacting with Hlip pigments. The co-isolation of the Ycf39-Hlip complex with the unassembled D1m in the absence of D2m also indicate that D1 binds Chl already before its attachment to D2m and binding of Hlips provides similar protection to D1m as to the D1-D2

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assembly complex RCII* [43] (Fig.2).

In the time course of PSII assembly, the next step after the formation of the RCII

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complex from D1m and D2m is the attachment of CP47m. In the resulting RC47 complex the Ycf39-Hlip complex is no longer present but CP47m can now bind HliA, HliB and HliC and accordingly these Hlips have been detected in the isolated RC47 complex [44]. Therefore, it is probable that the protective function of HliC and HliD bound to D1m in the RCII complex is taken over by HliA, HliB and HliC now bound to CP47m. With knowledge of the detailed structure of the cyanobacterial PSII complex (including the precise location of Chl and β-car molecules [45-47]) and using our model of pigment location in the Hlip pair [23] we drew a hypothetical model of the CP47m-HliA/B complex (Fig. 1). The model assumes interaction of the Chls and/or -car of the Hlip pair with exposed Chls of CP47, for instance Chl 620. Here, it is important to note that Chls in the CP47 antenna form two layers, one close to the stromal and the second close to the lumenal side of the membrane. Chl 620 and other exposed Chls are mostly located on the lumenal side of the membrane allowing efficient quenching of energy from the

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ACCEPTED MANUSCRIPT lumenal pigment layer of PSII, while the stromal side could be quenched by the red fluorescence emitting Chl bound to His114 of CP47 and stabilized by hydrogen bond from Thr5 of PsbH [48]. Hlips remain associated with the PSII core complex even after attachment of CP43m.

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This indicates that Hlips bound to CP47 may even quench energy coming from Chls bound to the

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other PSII Chl-binding proteins. This may easily happen due to the fast energy transfer and energy equilibrium within PSII as its charge separation system acts as a shallow trap [43]. In this

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context it is interesting that the unassembled CP43m, which is rather abundant in Synechocystis cells, does not seem to bind Hlips [49]. On the other hand, it can transiently associate with PSI,

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which acts as an effective quencher of the CP43m fluorescence [49]. It is therefore possible that PSI photoprotects CP43m and CP43-specific Hlips are not required. It is still not clear whether

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association of Hlips with the PSII core complexes happens only during the de novo assembly process or PSII repair under stress conditions, or whether Hlips also directly bind to completely assembled PSII complexes after the transfer of cells to increased irradiance.

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The evidence for Chl and β-car binding to HliC and HliD in Synechocystis and the related ability to perform non-photochemical quenching of energy absorbed by Chl justifies the function of Hlips in the transient binding of Chl released from other Chl-binding proteins. However, for this function, direct and convincing evidence is still missing. Mutant cells lacking PSI and Hlips

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exposed to high irradiance show changes in the Chl fluorescence spectra which can be attributed to a release of Chl during PSII damage and these changes are accompanied by a high rate of

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singlet oxygen generation [21]. In contrast, the PSI-less cells containing Hlips evolve much less singlet oxygen under high irradiance and Chl fluorescence does not show the presence of free Chls fluorescing at shorter wavelengths [21]. These data support the ability of Hlips to bind Chl released from PSII but do not directly show that this Chl is then reused for newly synthesized PSII Chl-binding proteins. Neither is it clear whether all such Chls bound to Hlips can only be reused after removal of phytol and its re-binding [50], or whether they can also be reused directly. Association of Hlips with the PSII complexes and the related Hlip-mediated photoprotection of PSII is most probably related to the presence of hli genes in genomes of some marine cyanophages [51]. These organisms also contain psbA and psbD genes coding for the D1 and D2 subunits of PSII [52] and expression of these genes and synthesis of the viral D1 and D2 proteins in the host cyanobacterial cells have been demonstrated [53]. It is probable that synthesis

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ACCEPTED MANUSCRIPT of the phage D1 and D2 proteins supports survival of host cells until the cyanophage particles are matured. We believe that presence of the phage hli genes and their putative expression in the host cell has the same purpose, i.e. support of the host photosynthesis via protection of the newly

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formed PSII complexes of viral origin which give energy for the survival of infected cells until

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the phage particles are matured and released following lysis of the host cells.

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THE FUNCTION OF HLIPS IN CHLOROPHYLL METABOLISM Apart from their presence in the RCII* complex HliC, and especially HliD, also strongly

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associate with Chl synthase (ChlG), the enzyme attaching phytol or geranyl-geraniol to chlorophyllide (Chlide) [24]. The ChlG-Hlip complex was co-purified with the YidC insertase,

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participating in the synthesis of membrane proteins, and with ribosomes, which implies involvement of Hlips at the very early steps in the biogenesis of Chl-binding proteins. During this process Hlips might protect components of the machinery for biosynthesis of Chl-binding

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proteins against photodamage mediated by accidentally released Chls or Chl synthesis intermediates. The possible role of Hlips in Chl biosynthesis is in agreement with earlier results [15-18]. The machinery for synthesis of membrane proteins seems to be intimately interconnected with enzymes of the tetrapyrrole biosynthesis pathway and together these have

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been proposed to form a large complex implicated in the synthesis of Chl-binding proteins [54]. The most convincing data about regulation of Chl biosynthesis by Hlips have been

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obtained using Synechocystis mutants lacking PSI. These strains are very sensitive to light [55] and therefore, it is rather easy to envisage that the accumulation of certain Chl biosynthesis intermediates (especially in the absence of HliD) may result from photodamage or destabilization of a particular enzyme of the pathway. Indeed, PSI-less strains lacking HliD and HliC exhibit inhibition of early steps of tetrapyrrole biosynthesis [15] and the absence of HliD also leads to the accumulation of Chlide in the wild-type [24] as well as in the PSI-less strain [18]. Given the binding of HliD to ChlG, these results support the key role of ChlG in re-binding of phytol to recycled Chlide. As well as biosynthesis, the life-time of Chl in Synechocystis cells is also dependent on the presence of Hlips [18, 19]. Characterization of various multiple hli deletion mutants points to the specific importance of HliC in Chl recycling since deletion of this gene leads to a more significant shortening of the Chl life-time than inactivation of other hli genes [18, 19]. The role of

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ACCEPTED MANUSCRIPT HliC is unclear. It can be detected together with HliD bound to ChlG, D1m or RCII* but also binds together with HliB to RC47 [39]. It is the shortest Hlip in Synechocystis and its sequence is rather similar to that of the Hlip domain of FeCh, possibly suggesting some mutual relationship.

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Its short N-terminus in comparison with the other Hlips may allow for better accessibility of the

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pigment binding sites for proteins/pigments from the stromal side. So, in a HliC homodimer or in its hetorodimer with the other Hlips, pigment binding could be destabilized allowing pigment

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delivery to proteins with higher affinity wherever it is needed.

Although for the clarity of this review it is useful to separate Hlip functions to

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photoprotection during PSII biogenesis and a regulatory/protective function related to Chl metabolism, there is very likely a strong overlap. We assume that Hlips generally operate at the

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interface between the last steps of Chl biosynthesis/re-cycling and Chl-binding protein synthesis and assembly. It has been already proposed that all these processes are located in a distinct membrane domain called biogenesis centre [34, 56, 57]. Figure 2 summarizes the current

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knowledge about the participation of individual Synechocystis Hlips in the synthesis of PSII Chlbinding proteins and their assembly.

FUSION OF HLIPS WITH FERROCHELATASE AND WITH OTHER PROTEINS

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Most Hlip genes encode small proteins with a relative molecular weight not exceeding 7 kDa and one transmembrane helix with residues involved in pigment binding. However, there are also

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genes coding for larger proteins and a closer look at the sequences indicate that these proteins are fusions of Hlips with other proteins. The typical example is a Hlip attached to the C-terminus of the FeCh [14], an essential enzyme catalyzing the insertion of iron into Protoporphyrin IX (Proto, for review see [58]), which is the last step of heme biosynthesis. Proto is the last common precursor of both Chl and heme and, apparently, FeCh serves an important regulatory role at this branch point. A reduced FeCh activity has drastic consequences on the regulation of the whole tetrapyrrole pathway in Synechocystis but causes no obvious decrease in the heme or phycobilisome levels [59, 60]. On the other hand, inhibition of the Chl biosynthesis branch in various PSII mutants [61] as well as in the PSI-less strains leads to a strong upregulation of the FeCh activity in the cell (Sobotka R, unpublished data). This is caused by an increased level of FeCh meaning that the FeCh Hlip/Cab domain is also upregulated. The high FeCh activity might lead to a higher production of heme, which can operate as a feedback regulatory molecule and

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ACCEPTED MANUSCRIPT inhibits early steps of the tetrapyrrole biosynthesis in order to avoid the accumulation of toxic biosynthesis intermediates [62]. This strategic position of FeCh at the branching point between the Chl and heme biosynthesis led to a speculation that Chl binding to the Hlip/Cab domain of the

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protein may play a regulatory role which contributes to the optimal partition of Proto between the

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two main branches of tetrapyrrole biosynthesis [15, 61]. A proposed scenario has been based on the idea that putatively empty Chl binding sites in the FeCh Hlip domain partly inhibits the FeCh

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activity. In contrast, their occupation, when enough Chl is available, may activate the enzyme to redirect Proto molecules to heme synthesis and thereby block the whole pathway.

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The later analysis of the Synechocystis mutant expressing truncated FeCh lacking the Hlip/Cab domain however did not provide any support for this model [59]. The mutant exhibited

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a similar FeCh content and activity though the truncated enzyme was monomeric and not dimeric like the wild-type full-length FeCh. Phenotypic analysis revealed that the mutant lacking the FeCh Hlip domain had unaltered pigment composition under low-light intensities but was

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sensitive to high light and accumulated Chlide [59]. The study also showed the importance of the linker between the enzyme and the Hlip domain (residues 324-347 of FeCh). A mutant lacking both linker and Hlip domain of FeCh had residual iron chelating activity although the truncated FeCH was still associated with the membrane [60]. As already noted the FeCh enzyme is a global

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regulator of the tetrapyrrole pathway and it is not difficult to imagine that an accidental fusion between FeCh and an Hlip (e.g. HliC) ensures that both proteins are always co-expressed and co-

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located. The fusion might better synchronize the overall flow through the tetrapyrrole pathway with the requirement of the Chl branch, which is given by the momentary synthesis of Chlbinding proteins and by the rate of Chl re-cycling. An accidental fusion between the FeCh and Hlips with no affect on FeCh activity is in line with the finding that in some cyanobacteria (Gloeobacter violaceus, Pseudanabaena sp. PCC 6802, Pseudanabaena biceps, Synechococcus sp. PCC 7502) FeCh does not contain the Hlip domain, instead the protein with high similarity to the Hlip domain of Synechocystis FeCh is encoded by a separate gene. The Hlip domain is also missing in the FeCh of Synechococcus sp. JA-2-3B'a(2-13), however, in this cyanobacterium a gene for the Hlip domain is fused to a gene encoding a homologue of the Synechocystis Ssl2148 protein (cyb1999, [63]). The function of the Ssl2148 protein in Synechocystis is unknown and consequently the importance of this gene fusion remains enigmatic. There are two possibilities explaining why some proteins become mutually

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ACCEPTED MANUSCRIPT fused. They can be involved in the same physiological process or biochemical pathway when their function in this process is closely connected (for instance two enzymes catalyzing consecutive steps in the biosynthesis pathway). Alternatively, they can be components of distinct

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biochemical processes that occur in the same cellular compartment or location and the protein

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fusion is beneficial for the proper function of both neighbouring processes.

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ARE HLIPS ASSOCIATED WITH PHOTOSYSTEM I?

Despite the early reports and later convincing evidence about the association of Hlips with

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PSII, several reports have also provided contradictory experimental data apparently supporting their binding to PSI rather than PSII. He at al. [64] supposedly identified Hlips in trimeric PSI

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complexes separated by sucrose density gradient centrifugation. However, this method has only a limited resolution and the authors did not provide any evidence that their PSI fraction does not contain any oligomeric PSII complexes. In addition, they used previously constructed strains with

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His6-tagged versions of Hlips [26], which might be mislocated due to the presence of the tag on the lumenal side of the protein. In an attempt to reconcile this result we analyzed the strain containing C-terminally His-tagged HliB (used in [26] and [64]) together with a strain lacking HliC and HliD, following exposure to increased irradiance. Using two dimensional blue

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native/SDS electrophoresis (Fig. 3), the HliA protein was expectedly detected in monomeric and dimeric PSII core complexes, in RC47 and also in a large supercomplex at the beginning of the

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native gel that contained both PSII and PSI. This supercomplex has been shown to contain PSII assembly factors, such as Psb27, and a role in PSII biogenesis has been recently proposed [49]. The trimer of PSI was located in the vicinity of the supercomplex but was clearly separated from the supercomplex by native electrophoresis and was free of HliA. In contrast to HliA, taggedHliB was found only in the PSII-PSI supercomplex and nowhere else. This result shows that Cterminally His-tagged HliB is absent in the PSI trimer and, unlike the native HliB protein, is also absent in PSII complexes with the exception of the PSII-PSI supercomplex. Comparison of the analysis of the HliC/HliD-less mutant and HliB-His mutant also indicates that unlike HliB, HliA does not bind to the CP47m, and therefore HliA and HliB might not be fully interchangeable. Thus, we believe that unlike the N-terminal His6 tag [38], the C-terminal His6 tag may cause a mislocalisation of Hlips, and results obtained using strains expressing these proteins should be taken with extreme caution. Another unusual result obtained in the study of Wang et al. [64] was

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ACCEPTED MANUSCRIPT a high-light-induced association of HliA and HliB with the stomatin protein (Slr1128), PSI and IsiA. The last protein is the CP43-like antenna induced under iron depletion or oxidative stress [65], which is usually associated with PSI trimers, and the PSI-IsiA supercomplex has

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approximately the same size as the PSII-PSI supercomplex [66]. Moreover, large ring-like

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complexes of the Slr1128 stomatin are also similar in size and Slr1128 have been shown to nonspecifically interact with the Ni-affinity column used for isolation of the His-tagged proteins [67].

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Thus, PSII-PSI supercomplexes containing C-terminally tagged HliB, PSI-IsiA supercomplexes and Slr1128 complexes overlap on native gels or sucrose gradients and their co-localization in the

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study of Wang et al. could be coincidental and artefactual. A similar problem might concern the following study [68], in which HliA and HliB have been found together with Slr1128, the PSI

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subunit PsaD and IsiA. We never experienced any accumulation of detectable amounts of IsiA under high light conditions [69] and it indicates that cultures used in [64] and [68], in which IsiA is easily detected, are most probably subjected to additional stresses than just high light.

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Nevertheless, we do not entirely exclude that Hlips interact with PSI. The isolated flagtagged Ycf39-Hlip complex is always co-isolated with a small amount of PSI trimer [22]. Given the proposed role of Ycf39 in the recycling of Chl, this co-isolation may reflect a transient association of both complexes during the transfer of Chl from the PSI trimer to the RCII

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complex. Indeed, in Synechocystis cells the PSI trimer is supposed to accept most of the newly synthesized Chl,which must then be redistributed to other Chl-binding proteins [69]. Thus, the

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main cyanobacterial membrane complex that binds Hlips is PSII, in accordance with the proven photoprotective function of Hlips. PSII is the most light-sensitive photosynthetic complex which requires high level protection against photodamage [33]. In contrast, PSI is much more stable, can be photoinhibited only under specific conditions (for instance at low temperature [70]) and the need for its photoprotection is rather limited. Nevertheless, an indirect or transient association of Hlips with PSI, for instance during the transfer of Chls from PSI to PSII, cannot be excluded although conclusive evidence is missing.

WHAT PROTEINS FULFILL THE HLIP FUNCTION IN PLANTS? The synthesis and assembly of PSII in algae and plants is considered to rely on fairly similar molecular machinery to that found in cyanobacteria [34] and one would therefore expect that the machinery is also protected by a set of Hlip-like proteins. Indeed, photosynthetic eukaryotes also

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ACCEPTED MANUSCRIPT contain Lhc-like proteins, which are unlikely to be involved in light-harvesting and could serve similar functions to Hlips in cyanobacteria. However, the situation in eukaryotes is made quite complex by the presence of a broader spectrum of Lhc-like proteins, which includes single-helix

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(Ohp) [71], double-helix (Sep or Lil), triple-helix (Elip) and quadruple-helix (PsbS-like) proteins

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(for review see [72]).

The function of Ohps in plants and algae is unclear. In the genomes of Arabidopsis and

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other photosynthetic eukaryotes two groups of ohp genes, ohp1 and ohp2, have been identified. The Ohp1 proteins are similar to Hlips; they are also induced by high irradiance or by other

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stresses [71] and their association with PSII is highly probable. On the other hand, Ohp2 proteins are distributed ubiquitously across photosynthetic eukaryotes but are missing in cyanobacteria

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[73]. They are induced by high irradiance but not by other stresses and unlike Hlip/Ohp1, they possess a short C-terminal hydrophobic element, which might be embedded in the thylakoid membrane [73]. The protein has been found in association with PSI-enriched fractions obtained

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using sucrose density gradient centrifugation [74]. However, the localization of Ohp2 with PSI needs further confirmation. PSII dimers and their supercomplexes in plants have a similar size to PSI supercomplexes [75] and insufficient detection of PSII subunits in the gradient performed in the study has not excluded the possibility that the protein associates with large PSII-containing

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complexes.

The best characterized Lhc-like proteins in plants are Elips and the Lil3 proteins. Elips are

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three-helix proteins and their expression is strikingly similar to Hlips. They accumulate during exposure of the plant to a variety of stress conditions that result in excessive excitation pressure [76-78]. Elips have been reported to bind Chl a and lutein and considered to be involved in photoprotection [79]. Although the elimination of both the elip genes present in the Arabidopsis genome did not modify the sensitivity to photoinhibition or photooxidation, or the ability to recover from light stress [80], overexpression of the Elip2 reduced the levels of Chl precursors and Chl-binding protein complexes [81]. This indicates that Elips are involved in the regulation of the Chl metabolism rather than in the protection of PSII biogenesis. The two transmembrane Lil3 proteins have been identified in Arabidopsis in association with a late enzyme of the Chl biosynthesis pathway, geranyl-geranyl reductase (ChlP) [82], which reduces geranyl-geraniol to phytol. This reaction can occur either before the phytol is attached to Chlide or after formation of geranyl-geranyl Chl [82]. Lil3 proteins are not induced

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ACCEPTED MANUSCRIPT under increased irradiance and thus they are likely not directly involved in photoprotection. A mutant lacking Lil3 is however chlorotic and grows showly due to destabilization of the ChlP enzyme and a consequent decrease in the availability of phytol and tocopherol [82]. The stability

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of ChlP in lil3 mutants is compromised even under low-light intensities, which resembles the

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constantly reduced ChlG content in the Synechocystis HliD-less strain [24]. The following study of the Tanaka group demonstrated that ChlP can be stabilized by anchoring this enzyme to the

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membrane, even in the absence of Lil3 [83]. As the authors did not detect pigments on the purified Lil3 they suggested that the function of this protein is to target and anchor ChlP to a

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membrane domain [83]. However, the Lil3 proteins contain a conserved Chl-binding motif and have previously been proposed to bind newly synthesized Chl during photomorphogenesis [84].

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The ChlP cooperates with ChlG for Chl phytylation [85] and thus the question whether Lil3 can bind pigments and quench absorbed energy like HliD/C is still open. Very recently, MorkJansson et al. [86] identified Lil3 in pigment-containing complexes with the latter enzymes of the

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Chl biosynthesis pathway, the D2 protein and the Psb29 antenna, suggesting a protective role of Lil3 during Chl-protein biosynthesis akin to that of cyanobacterial Hlips.

CONCLUSION

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Hlips are cyanobacterial proteins binding Chl and -car. They are mostly bound to various complexes of PSII including assembly intermediates and modules, in which they fulfill a

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photoprotective function based on energy transfer from Chl excited states to the S1 state of -car. They also play an important function in stabilizing Chl synthase, the last enzyme of the Chl biosynthesis pathway. Although Hlips are important for the biogenesis and function of the cyanobacterial photosynthetic apparatus, this process can occur in their absence, albeit with lower efficiency, especially under stress conditions. Despite recent progress in the knowledge of the structure and function of Hlips, many questions related to these proteins still remain unanswered. We still do not know whether the PSII modules CP43m and D2m bind Hlips or not, and if Hlips are needed for the synthesis of PSI and the stress-induced CP43-like protein IsiA. It also remains to be clarified whether Hlips can directly bind Chl released from degraded Chl-binding proteins and which protein(s) catalyze(s) phytol removal during Chl recycling.

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[78] M. Heddad, I. Adamska, Light Stress-Regulated Two-Helix Proteins in Arabidopsis thaliana Related to the Chlorophyll a/b-Binding Gene Family, Proc. Natl. Acad. Sci. U.S.A., 97 (2000) 3741-3746. [79] I. Adamska, M. Roobol-Boza, M. Lindahl, B. Andersson, Isolation of pigment-binding early lightinducible proteins from pea, Eur. J. Biochem., 260 (1999) 453-460. [80] S. Rossini, A.P. Casazza, E.C. Engelmann, M. Havaux, R.C. Jennings, C. Soave, Suppression of both ELIP1 and ELIP2 in Arabidopsis does not affect tolerance to photoinhibition and photooxidative stress, Plant Physiol., 141 (2006) 1264-1273. [81] T. Tzvetkova-Chevolleau, F. Franck, A.E. Alawady, L. Dall'Osto, F. Carriere, R. Bassi, B. Grimm, L. Nussaume, M. Havaux, The light stress-induced protein ELIP2 is a regulator of chlorophyll synthesis in Arabidopsis thaliana, Plant J., 50 (2007) 795-809. [82] R. Tanaka, M. Rothbart, S. Oka, A. Takabayashi, K. Takahashi, M. Shibata, F. Myouga, R. Motohashi, K. Shinozaki, B. Grimm, A. Tanaka, LIL3, a light-harvesting-like protein, plays an essential role in chlorophyll and tocopherol biosynthesis, Proc. Natl. Acad. Sci. USA, 107 (2010) 16721-16725. [83] K. Takahashi, A. Takabayashi, A. Tanaka, R. Tanaka, Functional analysis of light-harvesting-like protein 3 (LIL3) and its light-harvesting chlorophyll-binding motif in Arabidopsis, J. Biol. Chem., 289 (2014) 987-999. [84] V. Reisinger, M. Ploscher, L.A. Eichacker, Lil3 assembles as chlorophyll-binding protein complex during deetiolation, FEBS Lett., 582 (2008) 1547-1551. [85] W. Rudiger, S. Bohm, M. Helfrich, S. Schulz, S. Schoch, Enzymes of the last steps of chlorophyll biosynthesis: modification of the substrate structure helps to understand the topology of the active centers, Biochemistry, 44 (2005) 10864-10872. [86] A. Mork-Jansson, A.K. Bue, D. Gargano, C. Furnes, V. Reisinger, J. Arnold, K. Kmiec, L.A. Eichacker, Lil3 Assembles with Proteins Regulating Chlorophyll Synthesis in Barley, PloS one, 10 (2015) e0133145.

Acknowledgements

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We thank Andrew Hitchcock for critical reading of the manuscript. The work was supported by the by the National Program of Sustainability I, ID: LO1416, and by the project P501/11/0377

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from the Czech Science Foundation.

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FIGURES

Figure 1. A hypothetical model of the HliA/B pair bound to the CP47 –PsbH subcomplex viewed from the stromal side perpendicularly to (A), or parallel with (B), the membrane plane. The putative Hlip pair binds 6 Chls and 2 -cars [23] and is attached to CP47 in the

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vicinity of the small subunit PsbH [38]. The Hlip pair can quench energy absorbed by CP47 via interaction of its Chls and/or -cars with the accessible Chls of CP47, namely Chl 650 (putative

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interaction designated by oval). The figure was made by Chimera software using oxygenevolving Photosystem II complex from Thermosynechococcus vulcanus (PDB ID: 3ARC ) [47].

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Figure 2. A working model built from current knowledge about photoprotection of the

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early steps of the PSII biogenesis by Hlips oligomers. A) The Chl-synthase enzyme (ChlG) is associated with translocon apparatus (SecYEG) via YidC insertase and photoprotected by a

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HliD/C oligomer. The newly synthesized D1 assembly modul (D1m) remains physically attached to the translocon and interacts with HliD/C, which effectively quenches all absorbed light energy. The Ycf39 protein binds HliD/C from the stromal site, however its function remains enigmatic. B) During the next step of PSII assembly the D2 module (D2m) associates with D1m, and the HliD/C oligomer is able to protect the resulting RCII*. C) The CP47 module (CP47m) is photoprotected by a different Hlips oligomer (HliA/B) and after attachment of CP47m to RCII* (D) the RC47 assembly intermediate containing HliA/B is released from the translocon machinery. Schemes represent views perpendicular to the membrane plane; individual circles in the PSII proteins designate transmembrane helixes.

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Figure 3. The effect of a C-terminal His-tag on the localization of the HliB protein in

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Synechocystis cells Mutant cells either lacking hliC and hliD genes (HliCD, left) or expressing C-terminally His6-tagged HliB instead of HliB (HliB-His, right) were exposed to illumination

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(500 µmol photon m-2 s-1) for 20 min and used for membrane isolation. Membranes were solubilized by n-dodecyl -D-maltoside and proteins separated by two-dimensional PAGE consisting of blue native PAGE in the 4-14% gel in the first dimension and 12-20% gel containing 7 M urea in the second dimension. The gel was stained by Sypro Orange (Sypro stain) and electroblotted to a PVDF membrane, which was probed by an antibody specific for HliA/B. The vertical line designates the presence of HliB in unassembled CP47 and the horizontal lines HliB-His in PSII-PSI supercomplexes. Designation: RCCS1, supercomplex of Photosystem I trimer and Photosystem II; PSI(3) and PSI(1), Photosystem I trimer and monomer, respectively; RCC(2) and RCC(1), dimeric and monomeric PSII core complex; RC47 the PSII core complex lacking CP43; U.P. unassembled proteins fraction.

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ACCEPTED MANUSCRIPT Highlights:

High-light-induced proteins are small proteins mostly located in Photosystem II

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Hlips bind chlorophyll and β-carotene and are related to plant LHCII antennae

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Hlips protect machinery for synthesis of chlorophyll-proteins

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Hlips quench energy absorbed by chlorophylls via transfer to S1 state of β-carotene

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Hlips are involved in reuse of chlorophyll after chlorophyll-protein degradation

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