Session 1: Structure, mechanisms and organisation of protein complexes in thylakoids
Dynamic lateral heterogeneity of plant thylakoid protein complexes
Dr Jan Anderson, University of Auckland, NZ
The concept that in photosynthesis two photosystems cooperate in series, immortalised in Hill and Bendall’s (1960) Z scheme, was still a black box defining neither structural nor the molecular organization of the photosystems within thylakoids. Digitonin fragmentation of isolated chloroplasts, followed by differential centrifugation, yielded a heavier granal fraction enriched in PSII which bound a third “enigmatic” cytochrome b-559, and a lighter PSI fraction, proving that indeed there were two photosystems. The differentiation of the continuous thylakoid membrane network into grana and stroma thylakoids is a morphological reflection of the non-random distribution of PSII and PSI and ATP synthase, which became known as lateral heterogeneity. Long-term acclimation of sun versus shade plants modulates the composition and function and membrane appression of thylakoids. Significantly, highly dynamic rapid grana-stacking/ destacking, with reversible macro-organization of PSII/LHCII supercomplex arrays within grana optimise photosynthetic function in vivo over the entire range of irradiance; this still poses a grana conundrum.
Wah Soon Chow, Australian National University, Australia
Electron cryo-tomography of membrane protein complexes in mitochondria and chloroplasts
Professor Werner Kühlbrandt, Max-Planck-Institute of Biophysics, Germany
Electron crystallography of two-dimensional (2D) crystals makes it possible to examine membrane proteins in the quasi-native environment of a lipid bilayer at high to moderately high resolution. Recently, we have used electron crystallography to investigate functionally important conformational changes in membrane transport proteins such as the sodium/proton antiporters NhaA and NhaP, or the structure of channelrhodopsin.
“Single particle” cryo-EM is well suited to study the structure of large macromolecular assemblies in the 3.2 to 20Å resolution range. A recent example is our 19Å map of a mitochondrial respiratory chain supercomplex consisting of one copy of complex 1, two copies of complex III, and one of complex IV. The fit of the x-ray structures to our map indicates short pathways for efficient electron shuttling between complex I and III by ubiquinol, and between complex III and IV by cytochrome c.
Electron cryo-tomography can visualize large protein complexes in their cellular context at 30-50Å resolution, bridging the gap between protein crystallography and light microscopy. Cryo-ET is particularly suitable for studying biological membranes and large membrane protein complexes in situ. Cryo-ET of chloroplast thylakoids revealed the ATP synthase in flat stromal membranes and grana end membranes. We also could localize photosystem-II dimers in stacked and unstacked grana membranes. Together with the high-resolution structure of LHC-II, this enabled us to build a molecular model of membrane interaction in chloroplast grana.
In mitochondria of 6 different species we studied (2 mammals, 3 fungi, 1 plant), we found long rows of ATP synthase dimers along the tightly curved cristae ridges, whereas it is always monomeric and confined to flat membrane regions in chloroplasts. The proton pumps of the mitochondrial respiratory chain seemed to be confined to the flat membrane regions on either side of the dimer rows. This highly conserved arrangement appears to be a fundamental feature of all mitochondria from healthy cells, and suggests a fundamental role of the mitochondrial cristae as proton traps for efficient ATP synthesis. Interestingly, the inner membrane of mitochondria from the aging model organism Podospora anserina, a filamentous fungus with a fixed life span of ~20 days, undergoes a dramatic change as the cells age, and the dimer rows break up.
Goswami P, Paulino C, Hizlan D, Vonck J, Yildiz Ö, Kühlbrandt W (2010) Structure of the archaeal Na+/H+ antiporter NhaP1 and functional role of transmembrane helix 1. EMBO J. 30, 439-449.
Strauss M, Hofhaus G, Schröder R R, Kühlbrandt W (2008) Dimer ribbons of ATP synthase shape the inner mitochondrial membrane. EMBO J 27, 1154-1160.
Gipson P, Mills D, Wouts R, Grininger M, Vonck J, Kühlbrandt W (2010):
Direct structural insight into the substrate-shuttling mechanism of yeast fatty acid synthase by electron cryomicroscopy.
Proc Natl Acad Sci USA107, 9164-9169
Kühlbrandt W, Wang DN, Fujiyoshi Y (1994):
Atomic model of plant light-harvesting complex by electron crystallography. Nature367, 614-621.
Appel M, Hizlan D, Vinothkumar KR, Ziegler C and Kühlbrandt W (2009):
Conformations of NhaA, the Na+/H+ exchanger from Escherichia coli, in the pH-activated and ion-translocating states.
J Mol Biol388, 659-672.
Strauss M, Hofhaus G, Schröder RR, Kühlbrandt W (2008):
Dimer ribbons of ATP synthase shape the inner mitochondrial membrane.
EMBO J27, 1154-1160.
Daum B, Nicastro D, Austin II J, McIntosh JR, Kühlbrandt W (2010):
Arrangement of photosystem-II and ATP synthase in chloroplast membranes of spinach and pea.
Plant Cell22, 1299-1312.
How ATP synthase works
Professor Sir John Walker FMedSci FRS, MRC-Mitochondrial Biology Unit, UK
The ATP synthase found in chloroplasts has many features in common with the ATP synthases found in eubacteria and mitochondria. Their overall architectures are similar, and they all consist of two rotary motors linked by a stator and a flexible rotor. When rotation of the membrane bound rotor is driven by proton motive force, the direction of rotation ensures that ATP is made from ADP and phosphate in the globular catalytic domain. When ATP serves as the source of energy and is hydrolysed in the catalytic domain, the rotor turns in the opposite sense and protons are pumped outwards through the membrane domain, and away from the catalytic domain. The lecture will describe the common features of their catalytic mechanisms. However, the ATP synthase from chloroplasts, eubacteria and mitochondria differ in several key features, in their mechanisms of regulation and most fundamentally in the energy cost that is paid to make an ATP molecule. The most efficient ATP synthase is found in the mitochondria from multicellular animals. The ATP synthases in unicellular organisms, and chloroplasts, pay various higher costs that seem to reflect the supply of available energy.
Lipid functions in cytochrome bc complexes; an odd event in evolution
Professor William Cramer, Purdue University, USA
Lipid binding sites and properties were compared in the hetero-oligomeric cytochrome b6f and bc1 complexes that function in photosynthetic and respiratory membrane energy transduction. Seven lipid binding sites in the cyanobacterial b6f complex overlap three natural sites in the Chlamydomonasreinhardtii algal complex and four sites in the yeast mitochondrial bc1 complex. Inferences of lipid binding sites and functions are supported by sequence, interatomic distance, and B-factor information on interacting lipid groups and coordinating amino acid residues. Lipid functions in the b6f complex include the consequence of substitution of the eighth (‘H’) trans-membrane helix present in the mitochondrial cytochrome b subunit by a lipid and chlorin ring in b6f. The question of the function of this lipid substitution is of interest. The quinol oxidation site is on the p- (lumenal) side of the ‘H’ helix or the substituted lipid. Oxidation of PQH2 is coupled to the n (stromal)-side activation of an LHC kinase. It is suggested that the presence of the lipid may enable the trans-membrane signaling that activates the kinase. (Support from NIH GM-038323).
S S Hasan, Purdue University, USA
E Yamashita, Osaka University, Japan
Photosystem II structure and function: successes and challenges.
Professor James Barber FRS, Imperial College London, UK
It was the work of Jan Anderson, together with Keith Boardman (1) that showed it was possible to physically separate Photosystem I (PSI) from Photosysten II (PSII) and later it was Jan Anderson (2) who realised the importance of this work in terms of the fluid-mosaic model as applied to the thylakoid membrane. Since then there has been a steady progress in the development of biochemical procedures to isolate both PSII and PSI for physical, biochemical, molecular biological and structural studies, culminating in their crystallization and structural determination at atomic resolution. There are crystal structures for PSII and PSI isolated from cyanobacteria (3,4,5,6) and for PSI from higher plants (7). In the case of higher plants, the biochemical procedures developed have built on the recognition that PSII and PSI are lateral separated between granal and stromal regions as proposed by Jan Anderson and Bertil Andersson (8)
The structure of the cyanobacterial PSII has now been resolved to 1.9A (5). It is a dimer having a molecular mass of 700 kDa and the crystal structures have provided organisational details of the 19/20 subunits (16/17 intrinsic and 3 extrinsic) which make up each monomer and revealed information about the position and protein environments of the cofactors involved in the absorption of light, charge separation and water splitting. This level of detail has yet to be elucidated for higher plant PSII especially important because there are a number of significant differences between PSII of cyanobacteria and that of higher plants including differences in core subunits composition (both intrinsic and extrinsic) and light harvesting systems (Chla/Chlb binding proteins in plants and phycobilisomes in cyanobacteria). Progress towards this end will be presented.
1. Boardman, N K & Anderson, J M. 1964 Isolation from spinach chloroplasts of particles containing different proportions of chlorophyll a and chlorophyll b and their possible role in the light reactions of photosynthesis. Nature 203, 166-167.
2. Anderson, J M. 1975 The molecular organization of chloroplast thylakoids. Biochim Biophys Acta 416, 191-235.
3. Ferreira, K N, Iverson, T M, Maghlaoui, K, Barber J & Iwata, S. 2004 Architecture of the photosynthetic oxygen evolving center. Science 303, 1831–1838.
4. Loll, B, Kern, J, Saenger, W, Zouni, A & Biesiadka, J. 2005 Towards complete cofactor arrangement in the 3.0 Å resolution structure of photosystem II. Nature 438, 1040–1044.
5 Umena, Y, Kawakami, K, Shen, J R & Kamiya, N. 2011 Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 Å. Nature 473, 55–60.
6. Jordan, P, Fromme, P, Witt, H T, Klukas, O, Saenger, W & Krauß, N. 2001 Three-dimensional structure of cyanobacterial photosystem I at 2.5 Å resolution. Nature 411, 909–917.
7. Amunts, A, Drory, O & Nelson, N. 2007 The structure of a plant photosystem I supercomplex at 3.4 Å resolution. Nature 447, 58–63.
8. Andersson, B & Anderson, J M. 1980 Lateral heterogeneity in the distribution of chlorophyll protein complexes of the thylakoid membranes of spinach chloroplasts. Biochim Biophys Acta 593, 427–440.
Structure, function, evolution and utilization of photosystem I
Professor Nathan Nelson, Tel Aviv University, Israel
Abstract not yet available.
Structure of the dimeric RC-LH1 complex from Rhodobaca bororiensis studied by electron microscopy
Professor Egbert Boekema, Groningen University, The Netherlands
Electron microscopy and single particle averaging are important tools to investigate structures of large membrane proteins. For instance, supercomplexes of plant thylakoid membranes and inner mitochondrial membranes are a popular topic. In this contribution, we performed an investigation on isolated RC-LH1 complexes of Rhodobaca with the aim of establishing the peripheral antenna conformation, and in particular the structural role of PufX. Rhodobaca is an alkaliphilic purple nonsulfur bacterium found in African Rift valley soda lakes [Milford 2000]. Projection maps of dimeric complexes were obtained at 13 Å resolution and show the positions of the α-helices of 2x14 LH1 units subunits. They are organized in two half-rings of 2 x 13 units. In addition, there are two units in the interface of the two halves of the dimer. Between the interface and the two half rings are two openings on each side. Next to the openings there are additional densities present, considered to be occupied by in total 4 PufX molecules. The model differs from previously proposed configurations for other purple bacteria, in which the LH1 ribbon is continuous, but with two openings at the end and in which the dimeric RC-LH1 complex contains only two PufX molecules.
Milford, A D, Achenbach, L A, Jung, D O and Madigan, M T (2000) Rhodobaca bogoriensis gen nov and sp nov, an alkaliphilic purple nonsulfur bacterium from African Rift Valley soda lakes. Arch Microbiol 174, 18-27
Dmitry A Semchonok, University of Groningen, The Netherlands
Colette Jungas, CEA, DSV, IBEB, Laboratoire de Biologie Cellulaire and CNRS, UMR Biologie Végétale et Microbiologie Environnementales/Université Aix-Marseille, France