Structure of a PSI–LHCI–Cyt B6f Supercomplex in Chlamydomonas Reinhardtii Promoting Cyclic Electron Flow Under Anaerobic Conditions

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Structure of a PSI–LHCI–cyt b6f supercomplex in Chlamydomonas reinhardtii promoting cyclic electron flow under anaerobic conditions

Janina Steinbecka,b, Ian L. Rossb, Rosalba Rothnagelb, Philipp Gäbeleina, Stefan Schulzea,1, Nichole Gilesc, Rubbiya Alib,2, Rohan Drysdaleb, Emma Siereckic, Yann Gambinc, Henning Stahlbergd, Yuichiro Takahashie, Michael Hipplera,3, and Ben Hankamerb,3

aInstitute of Plant Biology and Biotechnology, University of Münster, 48143 Münster, Germany; bInstitute for Molecular Bioscience, University of Queensland, St. Lucia, QLD 4072, Australia; cEuropean Molecular Biology Laboratory Single Molecule Science, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW 2052, Australia; dCenter for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, CH-4058 Basel, Switzerland; and eResearch Institute for Interdisciplinary Science, Okayama University, 700-8530 Okayama, Japan

Edited by Krishna K. Niyogi, Howard Hughes Medical Institute and University of California, Berkeley, CA, and approved August 23, 2018 (received for review June 13, 2018)

Photosynthetic linear electron flow (LEF) produces ATP and how these membrane protein complexes can contribute to both NADPH, while cyclic electron flow (CEF) exclusively drives photophos- functional modes. Extensive biochemical and biophysical analy- phorylation to supply extra ATP. The fine-tuning of linear and cyclic ses using the green alga Chlamydomonas reinhardtii suggest that electron transport levels allows photosynthetic organisms to balance efficient CEF depends on the formation of a CEF supercomplex – – light energy absorption with cellular energy requirements under consisting of PSI, cyt b6f, and subunits ferredoxin NADP oxidore- constantly changing light conditions. As LEF and CEF share many ductase (FNR), Proton Gradient Regulation-Like 1 (PGRL1), An- electron transfer components, a key question is how the same indi- aerobic Response 1 (ANR1), and Calcium Sensor (CAS). The CEF vidual structural units contribute to these two different functional supercomplex is proposed to enhance CEF over LEF when stromal electron carriers are reduced (excess NADPH) and ATP is limiting modes. Here, we report the structural identification of a photosystem I (7–10). However, structural evidence for this supercomplex in C. – – PLANT BIOLOGY (PSI) light harvesting complex I (LHCI) cytochrome (cyt) b6fsupercom- reinhardtii Chlamydomonas reinhardtii is lacking, probably due to its putative dynamic nature. plex isolated from the unicellular alga un- Here, under CEF-inducing anaerobic conditions, a sucrose der anaerobic conditions, which induces CEF. This provides strong density gradient (SDG) fraction with CEF activity (7, 8) was evidence for the model that enhanced CEF is induced by the formation isolated from C. reinhardtii, and a PSI–light harvesting complex I of CEF supercomplexes, when stromal electron carriers are reduced, to (LHCI)–cyt b6f-containing CEF supercomplex within it, was generate additional ATP. The additional identification of PSI–LHCI– structurally characterized. The physical association between LHCII complexes is consistent with recent findings that both CEF en- PSI–LHCI and cyt b6f was supported using single molecule hancement and state transitions are triggered by similar conditions, but can occur independently from each other. Single molecule fluores- Significance cence correlation spectroscopy indicates a physical association between cyt b f and fluorescent chlorophyll containing PSI–LHCI super- 6 To optimize photosynthetic performance and minimize pho- complexes. Single particle analysis identified top-view projections of tooxidative damage, photosynthetic organisms evolved to ef- the corresponding PSI–LHCI–cyt b f supercomplex. Based on molecular 6 ficiently balance light energy absorption and electron transport modeling and mass spectrometry analyses, we propose a model in with cellular energy requirements under constantly changing which dissociation of LHCA2 and LHCA9 from PSI supports the forma- light conditions. The regulation of linear electron flow (LEF) tion of this CEF supercomplex. This is supported by the finding that and cyclic electron flow (CEF) contributes to this fine-tuning. a Δlhca2 knockout mutant has constitutively enhanced CEF. Here we present a model of the formation and structural molecular organization of a CEF-performing photosystem I (PSI)–light cyclic electron flow | supercomplex | photosystem I | cytochrome b f | 6 harvesting complex I (LHCI)–cytochrome (cyt) b f supercomplex Chlamydomonas reinhardtii 6 from the green alga Chlamydomonas reinhardtii.Suchastruc- tural arrangement could modulate the distinct operation of LEF hotosynthesis captures solar energy and stores it in the form and CEF to optimize light energy utilization, despite the same Pof chemical energy, which is essential to support life on individual structural units contributing to these two different Earth. Photosynthetic electron transport operates in two modes: functional modes. linear (LEF) and cyclic electron flow (CEF). LEF yields ATP and NADPH, while CEF exclusively drives ATP production (1). Author contributions: J.S., I.L.R., M.H., and B.H. designed research; J.S., I.L.R., P.G., and Fine-tuning LEF and CEF maintains the ATP/NADPH equi- N.G. performed research; H.S. and Y.T. contributed new reagents/analytic tools; J.S., I.L.R., R.R., P.G., S.S., R.A., R.D., E.S., Y.G., M.H., and B.H. analyzed data; and J.S., I.L.R., M.H., and librium and efficient carbon assimilation (2, 3). CEF also plays B.H. wrote the paper. an important role in photoprotection (4, 5) as it maintains the The authors declare no conflict of interest. Δ necessary pH across the thylakoid membrane to allow energy- This article is a PNAS Direct Submission. dependent nonphotochemical quenching and to control the rate Published under the PNAS license. limiting step of LEF (6). The dynamic tuning between LEF and 1Present address: Department of Biology, University of Pennsylvania, Philadelphia, CEF is therefore essential for efficient photosynthesis. PA 19104. LEF involves in-series activity of photosystem II (PSII), cyto- 2Present address: Centre for Microscopy and Microanalysis, University of Queensland, chrome (cyt) b6f, and photosystem I (PSI), while CEF involves St. Lucia, QLD 4072, Australia. only PSI and cyt b6f. During CEF, electrons released by PSI are 3To whom correspondence may be addressed. Email: [email protected] or reinjected into the photosynthetic electron transport chain at the [email protected]. plastoquinone (PQ) pool or at the stromal side of the cyt b6f This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. complex. The fact that LEF and CEF share many electron 1073/pnas.1809973115/-/DCSupplemental. transfer components (e.g., PSI and cyt b6f) raises the question of

www.pnas.org/cgi/doi/10.1073/pnas.1809973115 PNAS Latest Articles | 1of6 Downloaded by guest on September 23, 2021 fluorescence (SMF) correlation spectroscopy. Immunoblot and A mass spectrometry (MS) analyses also clearly identified PSI, LHCI, cyt b6f as well as FNR, PGRL1, ANR1, and CAS. Their structural organization was characterized using crosslinking, MS, and single particle analysis (SPA). In Chlamydomonas PSI–LHCI, the LHCA2 and LHCA9 subunits are located at its PSAG/H side (11) similar to the recently resolved PSI structure of a red alga (12). Our CEF data suggest a dynamic dissociation/association model, in which LHCA2 and LHCA9 dissociate from PSI–LHCI enabling CEFsupercomplexformation. B Results

Identification of a PSI–LHCI–cyt b6f Supercomplex. CEF supercomplexes of C. reinhardtii were isolated from anaerobically cul- C tured cells (7, 8). Isolated thylakoid membranes were solubilized with n-dodecyl α-D-maltoside (α-DDM) and fractionated using SDG centrifugation (7, 8). Immunoblot analysis identified a high molecular weight SDG fraction containing the major CEF supercomplex components PSI and cyt b6f(Fig.1B and C). Previous Fig. 2. Cytochrome b6f is physically associated with chlorophyll fluorescent work had demonstrated CEF activities in the same SDG fractions, proteins in the CEF supercomplex sucrose density region revealed by SMF but it remained possible that this was due to (i) colocalization of cyt coincidence analysis. (A) Coincident events, i.e., the simultaneous bursts of green and red fluorescence, indicative of the physical association of DyLight b6f and PSI in small residual membrane patches (since α-DDM is a mild detergent) or (ii) comigration of separate cyt b fandPSI 488-labeled cyt f and chlorophyll fluorescent proteins, are most abundant in 6 the CEF supercomplex region of the SDG. The frequency of coincident events supercomplexes on the SDG (e.g., due to the presence of other relative to total fluorescent events recorded over a period of 60 s is plotted molecular partners such as LHCII trimers, ATPase dimers, or for selected fractions over the corresponding SDG. A false positive rate of NDH). We therefore used SMF correlation spectroscopy (SI Ap- 5% was applied to exclude the possible random excitation of two single pendix,Figs.S1–S3) to demonstrate a single molecule, physical in- fluorescent proteins as experimentally examined previously (13). (B) Pooled – SI teraction between cyt b6fandPSILHCI complexes (Fig. 2 and CEF supercomplex fractions from five SDGs of a cyt f His6-tag strain were Appendix,Fig.S3;seeSI Appendix for more details). Total fluo- concentrated, labeled with DyLight 488–trisNTA, and ultracentrifuged on a rescence profiles across the SDG fractions showed that the loca- subsequent SDG to enrich for potential CEF supercomplexes. (C) Immunoblot tions of cyt f (labeled with green DyLight 488), as well as PSI detection of cyt f, PSAC, and Psba D1 over the SDG fractions confirms that the highest frequency of coincident events correlates with the localization of these proteins in the high molecular weight CEF supercomplex region.

and PSII in the SDG correspond well with increases in green fluorescence (Fig. 1 A and C) and red chlorophyll fluorescence signals (Fig. 1 A and B), respectively. The fraction 19– 22 pool was repurified (Fig. 1 and SI Appendix, SI Materials and Methods) via a subsequent SDG (Fig. 2B) and measured by SMF correlation spectroscopy to discriminate between single (red or green) vs. coincident (red and green) fluorescent events (SI Appendix,Fig.S2B and C) corresponding to discrete PSI–LHCI and cyt b6f molecules vs. associated PSI– LHCI–cyt b6f supercomplexes (13, 14). The frequency of SMF coincident events (Fig. 2A) in the numbered fractions of the repurified complex gradient (Fig. 2B) clearly revealed that DyLight-tagged cyt b6f is physically associatedwithachloro- phyll fluorescent complex in subfractions 23–27, consistent with the location of cyt f and PsaC (Fig. 2C). Coincident events were substantially higher than in the control fractions measured and exceeded the number of coincident peaks of very high molecular weight fractions (e.g., fraction 14), in which broad SMF peaks were occasionally visible due to the presence of small membrane patches (SI Appendix,Fig.S3B). Importantly, these membrane patches contributing to false positive coincident events were not visible in fractions 23–27, reinforcing the conclusion that the observed coincidence is Fig. 1. Identification of the CEF supercomplex peak fraction in SDG by SMF due to genuine CEF supercomplexes. Although an interaction spectroscopy and Western blot analysis. (A) Fluorescence intensity screening of cyt f with PSII cannot be excluded based on these mea- of each SDG fraction (log scale) confirms good correlation of red chlorophyll surements alone, functional interaction between PSI–LHCI fluorescence with the location of the photosynthetic complexes in the SDG and cyt b6f has been reported (7, 8, 10), while a PSII–cyt b6f (B) and reveals two main peaks of green fluorescent DyLight 488–trisNTA supercomplex is unprecedented. Furthermore, immunoblot- labeled cyt f. As control for the green fluorescent signal, a SDG of an un- ting and electron microscopy (EM) confirmed that the con- labeled cyt f His-Tag strain was screened (displayed in gray). (B) SDG of an- centration of PSI in these fractions was approximately 10 aerobic α-DDM solubilized cyt f His-Tag thylakoids separated into 77 times that of PSII (see below). Collectively these results fractions. (C) Immunoblot detection of cyt f, the PSI subunit PSAC, and the – PSII subunit PsbA D1 show that the PSAC signal peaks with the higher mo- provide evidence for a physical association between PSI lecular weight green fluorescent peak signal of the DyLight 488–trisNTA LHCI and cyt b6f essential for the formation of CEF super- labeled cyt f at fraction 20. complexes in solution.

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Fig. 3. Structural characterization of potential PSI–LHCI–LHCII supercomplexes from C. reinhardtii by single particle electron microscopy. A–C show three of the projections from SI Appendix, Fig. S6, which have densities additional to those of the PSI–LHCI supercomplex. (D–F) These projection maps have been overlaid with the densities of the PSI core complex [yellow (17), Protein Data Bank (PDB) 4Y28] and the LHCA proteins (green, PDB 4Y28). A LHCII trimer

[magenta or cyan (42), PDB 1RWT] can be seen to fit well into these three densities. (G) Modeling a cyt b6f monomer [purple (19), PDB 1Q90] into the additional density next to PSI–LHCI supercomplex (A) shows a poor fit. (H) Overlay of the large PSI supercomplex projection map from Fig. 4A and a meshed density map of A.(I) Poor fit of two LHCII trimers (42) into the projection map from Fig. 4A next to the PSI–LHCI complex. This suggests that the additional

densities in A–C are likely LHCII trimers, while the new density in H and Fig. 4A best fits a cyt b6f dimer. (Scale bar: 5 nm.)

Structural Characterization of the CEF Supercomplex. Initial single A projection map of a PSI–LHCI-containing supercomplex particle analysis revealed heterogeneity within the PSI–LHCI– representing 50% of classes of SI Appendix, Fig. S7 (SI Appen- cyt b6f supercomplex SDG fraction (SI Appendix, Fig. S4). dix), is depicted in Fig. 4A. The molecular model overlay of PSI– Consequently, subsequent EM preparations employed an addi- LHCI (Fig. 4B, yellow and green, respectively) clearly identifies tional gentle affinity purification step using a His-tagged PSI– the PSI–LHCI supercomplex (17, 18) and an additional density LHCI (PSAA His-Tag) construct (15). To stabilize this super- adjacent to its PSAG–LHCA1 side that can accommodate a cyt complex, chemical protein crosslinking was performed during b6f dimer (Fig. 4B, purple, ref. 19; see also SI Appendix, Fig. S9). the solubilization step before affinity purification (Materials and Corresponding PSI–LHCI supercomplex controls are provided Methods). This enabled the identification of putative CEF super- in Fig. 4 C and D. Based on this, the first top view of the dynamic complexes by transmission electron microscopy (TEM) SPA. A CEF supercomplex of the green alga C. reinhardtii containing total number of 2,708 micrographs of negatively stained particles PSI, LHCI, and cyt b6f is proposed (Fig. 4B). were recorded and yielded a total of 526,519 projection images of Fig. 5 provides more detailed structural insights into factors protein complexes (SI Appendix,Fig.S4B). Using SPA with Relion proposed to control the formation of this CEF supercomplex. The (16), these particles were classified into 350 classes (SI Appendix, PSI dataset (SI Appendix,Fig.S6B) included a larger PSI–LHCI Fig. S4C). Parallel liquid chromatography (LC)-MS/MS analysis supercomplex (Fig. 5A) than is shown in Fig. 4C (see Fig. 5B, blue was conducted (SI Appendix,Fig.S8) and it identified contaminants shading) highlighting the dynamic nature of these supercomplexes. including mitochondrial NADH dehydrogenase (79,709 particles), The additional (Fig. 5B, unshaded) density on the PSAG side is mitochondrial ATPase (26,704 particles), and PSII–LHCII com- attributed to LHCA2, LHCA9, and PSAH (Fig. 5C) based on refs. plexes (14,664 particles), and the corresponding SPA classes were 11 and 18. These subunits appear to have dissociated in many identified (SI Appendix,Fig.S5) and removed from the dataset, isolated PSI–LHCI particles (Fig. 4 C and D) and are also absent yielding 160,819 potential PSI–LHCI-containing particles (SI Ap- in the CEF supercomplex (Fig. 4B). This suggests that PSAG and pendix A ,Fig.S6 ). A subset of 52,316 particles were identified as LHCA1 form an interface with the cyt b6fdimer(Fig.5C and D). top-view projections of single PSI–LHCI complexes (50 classes, see SI Appendix,Fig.S6B), as opposed to 12,179 top-view projections A Proposed Role for LHCA2 and LHCA9 in CEF Supercomplex Formation. that contained additional density adjacent to the PSI–LHCI com- The PSI–LHCI supercomplex projection map (Fig. 5A)fitswell plex (SI Appendix,Fig.S6C), potentially consisting of cyt b6for with the newly proposed model of Chlamydomonas PSI–LHCI LHCs. Next, tilt views and PSI–LHCI complexes lacking well- (11). According to crosslinking and interaction studies, Ozawa connected additional densities were eliminated from this dataset, et al. (11) concluded that LHCA2 and LHCA9 are not included in yielding 1,139 particles. These were classified into 16 classes (SI the two LHCI layers (Fig. 5C, Bottom, green), but are located at Appendix,Fig.S7) and analyzed (Fig. 3) to confirm the ability to the other side of the PSI core (Fig. 5C, Top, green) and associate distinguish between PSI–LHCI–cyt b6f supercomplexes (Fig. 4) and with PSAB and PSAH. This subunit assignment fits the additional PSI–LHCI–LHCII supercomplexes (Fig. 3 A–F,seeDiscussion). PSI density identified here (Fig. 5 B and C). This model therefore

Steinbeck et al. PNAS Latest Articles | 3of6 Downloaded by guest on September 23, 2021 cyt b6f dimer (Fig. 4B, purple). Third, these TEM data combined with functional data from LHCA2 knockouts yielded a model, which incorporates all of the above information and implies a role for LHCA2 and LHCA9 in the CEF supercomplex assembly process (Fig. 5 E and F). The assembly of the CEF supercomplex when stromal electron carriers are reduced to produce extra ATP (20, 21) places the PSI– LHCI supercomplex and the cyt b6f dimer in close proximity to favor CEF over LEF. As electron transport via plastocyanin and ferredoxin is thought to be diffusion limited (22), the regulation of the distances by structural alignment of the PSI–LHCI supercomplex with cyt b6f could control electron transfer kinetics, as seen via regulation of thylakoid stacking in vascular plants (23).

CEF Supercomplex Assembly and the Promotion of Cyclic Electron Flow. A surprising finding from the structural data (Fig. 5) was the absence of densities corresponding to LHCA2, LHCA9, and PSAH in the CEF supercomplex (Fig. 5 D vs. B). This led us to examine the functional consequence of LHCA2 knockout on CEF. Strikingly, the Δlhca2 knockout mutant (11) showed constitutively high CEF rates under aerobic conditions, that exceeded WT rates in anaerobic conditions (Fig. 5E). This suggests potential roles for LHCA2 and LHCA9, whose association to the PSI core is unstable in the absence of LHCA2 (11), during the CEF supercomplex assembly process: LHCA2 and LHCA9 could therefore block the CEF supercomplex formation, and Fig. 4. Structural characterization of a PSI–LHCI–cyt b6f supercomplex from their removal could expose a binding site for cyt b6f required to C. reinhardtii by single particle transmission electron microscopy. (A) Aver- drive CEF supercomplex assembly. This suggests a role for LHCs aged TEM projection map of a large supercomplex consisting of PSI and LHCI above and beyond their light harvesting and energy dissipation with an additional particle at its PSAG LHCA1 side (sum of 132 particles, functions. Notably, LHCA6 is important for NADP(H) de- representing 50% of classes in SI Appendix, Fig. S7).(B) Structural assign- hydrogenase–PSI supercomplex formation in Arabidopsis (24, 25), ment of this supercomplex based on fitting with the crystal structures of the independently showing the role of a LHCA polypeptide in

PSI–LHCI complex (17) and the cyt b6f complex (19) [Protein Data Bank (PDB) supercomplex formation. Closer analysis of a series of identified accession nos. 4Y28 and 1Q90, respectively]. The PSI core complex is shown in PSI–LHCI supercomplexes with additional putative bound LHCII yellow with LHCA proteins highlighted in green. The cyt b6f complex is trimers shows that other LHC complexes could potentially com- – shown in purple. (C) Averaged projection map of a PSI LHCI complex missing petitively block cyt b6f dimer binding (e.g., Fig. 3H, see below). PSAH at its core. (D) Structural assignment of the PSI–LHCI complex similar to B. Eight LHCA proteins were modeled into the double-layered LHCI belt State Transitions–CEF. Both state transitions and CEF enhance- according to ref. 11. Arrows indicate the position of PSAG (yellow) and two ment are triggered by similar conditions, for example, the redox LHCA1 subunits (green). (Scale bar: 5 nm.) state of the chloroplast, although these processes can occur independently of one another (8, 10). Hence, the presence of different PSI–LHCI-containing supercomplexes under anaer- differs from the one presented in ref. 18. Importantly, quantitative SI Appendix obic conditions is expected. To eliminate the possibility that MS analysis ( ,Fig.S8) confirmed that LHCA2 and the proposed CEF supercomplex could represent a PSI–LHCI– LHCA9 together with PSAH are most easily lost during purifi- LHCII complex, we modeled two LHCII trimers into this den- cation (SI Appendix,Fig.S8A), consistent with Fig. 5. Strikingly, a I Δlhca2 sity; these fitted poorly (Fig. 3 ). While the focus of this paper is knockout mutant showed enhanced cyclic electron flow on the CEF supercomplex, it is of note that we also identified compared with wild-type (WT) levels under anaerobic conditions; – – Δlhca2 three PSI LHCI LHCII complexes (Fig. 3) with an LHCII even under aerobic conditions, CEF rates in were already trimer-like density localized at the LHCI belt side of the complex. as high as for WT in anaerobic conditions (Fig. 5E). This suggests SI Ap- – These complexes represented the other 50% of classes in that the dissociation of LHCA2 and LHCA9 from the PSI LHCI pendix,Fig.S7. MS data confirmed that LHCII proteins were less supercomplex is important for the assembly of the CEF super- depleted by PSI His-Tag purification than PSII core subunits (SI F complex and the promotion of CEF (Fig. 5 ). Appendix,Fig.S8F), indicating that additional LHCIIs might in- deed be attached to PSI–LHCI. The supramolecular organization Discussion of these PSI–LHCI–LHCII supercomplexes differs substantially Structure of the Proposed CEF Supercomplex. In 2010, Iwai et al. (7) from other C. reinhardtii PSI–LHCI–LHCII supercomplexes al- presented biochemical evidence for the existence of a CEF ready described, in which two LHCII trimers and one LHCII supercomplex in the green alga Chlamydomonas. Here, this monomer were attached to the PSAH PSAL side of PSI opposite biochemical evidence is explained in structural terms. First, SMF the LHCI ring (26). In contrast, in the PSI–LHCI–LHCII com- spectroscopy supported the existence of discrete detergent sol- plexes identified here, an LHCII trimer localized at previously – ubilized supercomplexes containing both cyt b6f and PSI LHCI unreported positions at the outer LHCI belt side (Fig. 3 D–F), in solution (Figs. 1 and 2 and SI Appendix, Fig. S2); using MS, the which are similar to a recent structure described in Arabidopsis CEF supercomplex preparation was shown to contain FNR, thaliana where an LHCII trimer was associated with PSI at the PGRL1, ANR1, and CAS (SI Appendix, Fig. S8) which are im- side of LHCA2 and LHCA3 (27). portant for CEF (8, 9). Second, having identified the presence of The density assigned to the cyt b6f dimer at the PSAG– intact PSI–LHCI–cyt b6f supercomplexes (SI Appendix, Fig. S2), LHCA1 side of PSI–LHCI in the newly identified CEF super- over 500,000 TEM projection images of molecules in this prep- complex (Fig. 5) is located similarly to that observed in TEM SI Appendix – aration were analyzed by SPA ( , Figs. S4 S7). This images of detergent solubilized PSI–LHCI–cyt b6f complexes analysis yielded a top-view projection map of the proposed CEF from A. thaliana (27). In the Chlamydomonas CEF super- B supercomplex that is able to accommodate the PSI core (Fig. 4 , complex, the cyt b6f dimer interacts via its long side (Figs. 4B and yellow), its eight LHCI antenna proteins (Fig. 4B, green), and a 5D); in contrast in A. thaliana, the cyt b6f dimer is reported to

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1809973115 Steinbeck et al. Downloaded by guest on September 23, 2021 Fig. 5. Dissociation of LHCA2 and LHCA9 from the PSI–LHCI complex favors the association of PSI–LHCI– AB F

cyt b6f supercomplexes and enhances cyclic electron flow. (A) Averaged TEM projection map of a PSI–LHCI complex with an additional density at its core. (B) Overlay of the PSI–LHCI complex from A with Fig. 4C. (C) Structural assignment of the PSI–LHCI complex from A based on fitting with the crystal structures of the PSI–LHCI complex (17). The additional densities compared with the smaller PSI–LHCI complex (B) were modeled with PSAH (blue) and two additional CD LHCA proteins (green) at the PSI core according to ref. 11. (D) Overlay of the CEF supercomplex projection map from Fig. 4A with the PSI–LHCI complex from Fig. 4C. (Scale bar: 5 nm.) (E) Cyclic electron transfer rates of a Δlhca2 mutant compared with wild-type levels in aerobic and anaerobic conditions. Rates were measured in steady state upon a transi- − − tion from darkness to light with ∼130 μEm 2·s 1 light E intensity. To exclude contribution of PSII to the electron transfer, cultures were treated with 40 μM DCMU. Anaerobic conditions were achieved by ad- − dition of 100 mM glucose and 2 mg·mL 1 glucose oxidase. To alleviate PSI acceptor side limitation upon transition to anaerobiosis, anaerobic samples were kept in the darkness for 40 min and continu- ously illuminated for 2 min before the rate measurements (n = 6 biological replicates ± SD). Statistical analysis: one-way ANOVA followed by a Tukey test for pairwise comparison of the means (***P < 0.001;

*P < 0.05). (F) Structural model of CEF supercomplex formation upon dissociation of LHCA2, LHCA9, and PSAH from the PSI–LHCI complex. The PSI core is PLANT BIOLOGY

shown in yellow, LHCI proteins in bright green, cyt b6f in purple. Plastocyanin (cyt f in cyan, PSAF in orange) and ferredoxin (PSAD/E in red and cyt b6 in pink)- binding regions are indicated. All high-resolution components have been filtered to 20 Å to avoid overinterpretation.

− interact with PSI–LHCI via its short side (27). Since Chlamy- to 3–4 × 106 cells mL 1 and harvested by centrifugation (4,600 × g, 5 min, − domonas PSI is larger than plant PSI due to higher numbers of 25 °C), resuspending to 2 × 108 cells mL 1 in H1 buffer (25 mM Hepes-KOH pH 7.5, 5 mM MgCl2, 0.3 M sucrose). Anaerobic conditions were induced using 2 LHCI subunits forming a second LHCI belt and the potentially − − algae-specific positions of LHCA2 and LHCA9 with respect to mg·mL 1 glucose oxidase (Aspergillus niger) and 50 units·mL 1 catalase (bo- the PSI core, differences may exist in regulation of CEF assembly vine liver, Sigma-Aldrich) with 100 mM glucose in the dark for 60 min (10). formation in plants compared with Chlamydomonas. The binding Thylakoid and Photosynthetic Complex Isolation and DyLight 488–Tris–NTA Labeling of the cyt b6f dimer to the PSI–LHCI supercomplex positions the plastocyanin binding sites of cyt f (28, 29) and PSAF (30) close to of Photosynthetic Complexes. Thylakoids and photosynthetic complexes were one another, making the distance for electron transfer of plas- isolated according to refs. 37 and 38; see also SI Appendix, SI Materials and Methods.Cytf–His-Tag was labeled with DyLight 488– Tris–NTA before solubili- tocyanin between cyt b6fandPSI–LHCI at the luminal side rela- tively short. However, electron transfer partitioning between LEF zation and SDG-based purification (SI Appendix, SI Materials and Methods). and CEF is most likely regulated at the level of PQ reduction (31) Single Molecule Fluorescence Measurements. Single molecule spectroscopy (SI andthereforelocatedtowardthestromalside.Cytb6f dimer associated with its long side to PSI–LHCI would bring the more cen- Appendix,Fig.S2) was performed based on refs. 13 and 14 and SI Appendix, SI trally located components involved in the stromal electron transfer, Materials and Methods. Two excitation lasers (488 nm and 561 nm) focused in solution using a 40×/1.2 N.A. water immersion objective (Zeiss) simultaneously such as cyt b and Rieske subunits as well as the PSAA/PSAB re- 6 excite green (here: the DyLight 488-labeled cyt f protein, see SI Appendix, SI action center and ferredoxin binding sites PSAC, PSAD, and PSAE Materials and Methods for more details) and red (here: chlorophyll-containing (32, 33) into much closer proximity compared with a short side- proteins) fluorophores. Fluorescence was collected and separated using a 565-nm bound cyt b6f, with cyt f and PSAF still in close proximity. dichroic mirror; signal from DyLight 488-labeled cyt f protein was passed Building on these advances, the next challenge is to obtain a through a 525/20-nm band pass filter, while chlorophyll fluorescence was fil- physically stable and pure CEF supercomplex for future atomic tered by a 580-nm long pass filter. The fluorescence of the two channels was resolution structure determination to identify the position of recorded simultaneously in 1-ms time bins for 60 s. For single-molecule co- additional small proteins in the CEF supercomplex like FNR, incidence detection, the coincidence ratio was calculated as in ref. 13. PGRL1, ANR1, and CAS (7–9). These subunits were identified SI Appendix C in the CEF supercomplex SDG fraction ( , Fig. S8 ) Immunoblot Analysis. A fixed volume (20 μL) of representative fractions across and are expected to be attached to large supercomplexes (7–9) to the SDG gradient was analyzed by 4–12% Bis-Tris SDS/PAGE (Invitrogen). enable them to migrate to this high molecular density. We at- Proteins were transferred to PVDF Millipore membrane (Merck) with the tribute the low proportion of intact complexes on negative stain XCell Blot II Module (Invitrogen) and blocked with skimmed milk. The TEM grids to the labile nature of the supercomplex. A better membrane was incubated with antibodies against PSAC (1:1,000; Agrisera), understanding of the role of LHCA2 and LHCA9 in CEF assembly Psba D1 (1:10,000; Agrisera), and cyt f (1:5,000; Agrisera), and anti-rabbit IgG could lead to the production of physically stable supercomplexes, (HRP, 1:2,500; Sigma Aldrich) as the secondary antibody. Signal detection which would greatly assist in solving its atomic structure. was performed with ECL (Amersham GE Healthcare).

Materials and Methods Chemical Crosslinking. To crosslink proteins with disuccinimidyl suberate (DSS) Strains and Culture Conditions. The C. reinhardtii strains cyt f–His-Tag (19, 34), (Thermo Fisher), PSAA His-Tag thylakoids were resuspended in Hepes buffer PSAA–His-Tag (15), a Δlhca2 (35), and CC-4533 (WT) were used in this study. (50 M Hepes-KOH pH 8, 5 mM MgCl2)andfreshlydissolvedDSS(inDMSO, Cells were grown in tris acetate phosphate (TAP) media (36) (22 °C, 50 μM 20 mg/mL stock concentration) was added to the sample (final concentration, photons m−2·s−1, 120 rpm shaking). For thylakoid isolation, cells were grown 0.15 mg/mL) before solubilization of proteins. The crosslinking reaction was

Steinbeck et al. PNAS Latest Articles | 5of6 Downloaded by guest on September 23, 2021 performed for 30 min at room temperature in the dark with occasional in- Tecnai 12 TEM operated at 120 kV (FEI Company) connected to a Direct version and stopped by adding Tris buffer (final concentration, 15 mM pH 7.5, Electron LC-1100 lens-coupled 4k × 4k CCD camera (nominal magnification of 1 mM EDTA). 67,000×,2× pixel binning, 4.34 Å pixel size at specimen level). An initial 2D class average was calculated from manually picked particles to enable auto- Purification of His-Tagged Proteins. His-tagged proteins were purified by mated particle selection using RELION (16). A total of 526,519 particles were immobilized metal ion chromatography according to ref. 15 with the fol- collected from 2,708 digital micrographs (examples shown in SI Appendix,Fig. – – lowing modifications. PSI LHCI cyt b6f containing fractions from six SDGs S4 A and B, circular mask of 380 Å). Single particle images were analyzed with were pooled (∼4 mL) and loaded onto a 1-mL HiTrap HP column (GE RELION (16) (see SI Appendix, SI Materials and Methods for details). Healthcare) preequilibrated with 5 mM tricine-KOH pH 8, 0.02% α-DDM, 10 mM NaCl, 5 mM MgSO , 0.5 M sucrose, 2 mM imidazole. The column was 4 Spectroscopic Measurements. P700 absorption and electrochromic shift signal washed with two washing buffers, with increasing the imidazole concen- measurements were performed with a LED pump-probe JTS-10 spectro- trations (10 mM to 20 mM) and decreasing the sucrose concentration (0.5 M to 0 M, 10 mL each). Elution was performed by increasing the imidazole photometer (BioLogic) as described previously (10). Single turnover measure- concentration to 200 mM (4 elution fractions, 500 μL each). ments used a dye laser emitting at 640 nm, pumped by the second harmonic of a Minilite II Nd:YAG laser (Continuum). C. reinhardtii cells were harvested and μ · −1 Mass Spectrometry. For quantitative mass spectrometric analysis, the PSAA resuspended to a 20 g mL chlorophyll concentration [20 mM Hepes, pH 7.2, His-Tag strain was isotopically labeled with 14N and 15N. The pooled 15N 10% Ficoll (wt/vol)], and incubated (20 min, dark, constant shaking to avoid

labeled PSI–LHCI–cyt b6f fractions (before His-Tag purification) were mixed anaerobiosis). Anaerobic conditions were reached as described above. To 14 1:1 with the His-Tag purified eluate of the N labeled PSI–LHCI–cyt b6f eliminate contribution of linear electron flow, the PSII inhibitor 3-(3,4- sample. Duplicates were performed with a label swap. Samples were dichlorophenyl)-1,1-dimethylurea (DCMU, 40 μM) was added. Electron flow

digested with trypsin in a 0.5-mL Amicon Ultra ultrafiltration device (30-kDa rates were determined as the product of kox [P700red] (10). More details about cutoff; Millipore) (39) with minor modifications. Mass spectrometry was experimental procedures are listed in SI Appendix, SI Materials and Methods. performed according to ref. 38. Identification and quantification of peptide spectrum matches were conducted in the framework of Ursgal (40) and ACKNOWLEDGMENTS. We thank S. Hawat for help with preparation of MS/ using pyQms (41). See SI Appendix, SI Materials and Methods for details. MS samples and H. Nüsse and U. Keller for their kind permission and assis- tance to glow discharge grids at the EM facility of the Institute for Medical Transmission Electron Microscopy. A total of 5 μL of the PSAA–His-Tag purified and Biophysics (University of Münster, Germany). B.H. acknowledges support SDG fraction was applied to glow-discharged 400-mesh copper TEM grids from Australian Research Council Grants DP130100346 and DP160101018. coated with a thin continuous film of evaporated carbon and complexes M.H. acknowledges support from Deutsche Forschungsgemeinschaft (DFG) stained with 2% uranyl acetate (wt/vol). Single particles were imaged on a Grant HI 739/13-1.

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