Lack of the light-harvesting complex CP24 affects the structure and function of the grana membranes of higher plant chloroplasts

The photosystem II (PSII) light-harvesting antenna in higher plants contains a number of highly conserved gene products whose function is unknown. Arabidopsis thaliana plants depleted of one of these, the CP24 light-harvesting complex, have been analyzed. CP24-deficient plants showed a decrease in light-limited photosynthetic rate and growth, but the pigment and protein content of the thylakoid membranes were otherwise almost unchanged. However, there was a major change in the macroorganization of PSII within these membranes; electron microscopy and image analysis revealed the complete absence of the C(2)S(2)M(2) light-harvesting complex II (LHCII)/PSII supercomplex predominant in wild-type plants. Instead, only C(2)S(2) supercomplexes, which are deficient in the LHCIIb M-trimers, were found. Spectroscopic analysis confirmed the disruption of the wild-type macroorganization of PSII. It was found that the functions of the PSII antenna were disturbed: connectivity between PSII centers was reduced, and maximum photochemical yield was lowered; rapidly reversible nonphotochemical quenching was inhibited; and the state transitions were altered kinetically. CP24 is therefore an important factor in determining the structure and function of the PSII light-harvesting antenna, providing the linker for association of the M-trimer into the PSII complex, allowing a specific macroorganization that is necessary both for maximum quantum efficiency and for photoprotective dissipation of excess excitation energy.     

Structural determination of the photosystem II core complex from spinach

A photosystem II core complex was purified with high yield from spinach by solubilization with beta-dodecylmaltoside. The complex consisted of polypeptides with molecular mass 47, 43, 34, 31, 9 and 4 kDa and some minor components, as detected by silver-staining of polyacrylamide gels. There was no indication for the chlorophyll-a/b-binding, light-harvesting complex polypeptides. The core complex revealed electron-transfer activity (1,5-diphenylcarbazide----2,6-dichloroindophenol) of about 30 mumol reduced 2,6-dichloroindophenol/mg chlorophyll/h. The structural integrity was analyzed by electron microscopy. The detergent-solubilized protein complex has the shape of a triangular disk with a maximum diameter of 13 nm and a maximum height of 6.8 nm. The shape of this core complex differs considerably from that of cyanobacterial photosystem II membrane fragments, which are elongated particles. The structural differences between both the complexes of higher plants and cyanobacteria are discussed with special emphasis on their association with the antenna apparatus in the photosynthetic membranes.     

A structural investigation of complex I and I + III2 supercomplex from Zea mays at 11-13 Å resolution: Assignment of the carbonic anhydrase domain and evidence for structural heterogeneity within complex I

The projection structures of complex I and the I + III₂ supercomplex from the C₄ plant Zea mays were determined by electron microscopy and single particle image analysis to a resolution of up to 11 Å. Maize complex I has a typical L-shape. Additionally, it has a large hydrophilic extra-domain attached to the centre of the membrane arm on its matrix-exposed side, which previously was described for Arabidopsis and which was reported to include carbonic anhydrase subunits. A comparison with the X-ray structure of homotrimeric γ-carbonic anhydrase from the archaebacterium Methanosarcina thermophila indicates that this domain is also composed of a trimer. Mass spectrometry analyses allowed to identify two different carbonic anhydrase isoforms, suggesting that the γ-carbonic anhydrase domain of maize complex I most likely is a heterotrimer. Statistical analysis indicates that the maize complex I structure is heterogeneous: a less-abundant “type II” particle has a 15 Å shorter membrane arm and an additional small protrusion on the intermembrane-side of the membrane arm if compared to the more abundant “type I” particle. The I + III₂ supercomplex was found to be a rigid structure which did not break down into subcomplexes at the interface between the hydrophilic and the hydrophobic arms of complex I. The complex I moiety of the supercomplex appears to be only of “type I”. This would mean that the “type II” particles are not involved in the supercomplex formation and, hence, could have a different physiological role.     

Lactococcus lactis YfiA is necessary and sufficient for ribosome dimerization

Dimerization and inactivation of ribosomes in Escherichia coli is a two‐step process that involves the binding of ribosome modulation factor (RMF) and hibernation promotion factor (HPF). Lactococcus lactis MG1363 expresses a protein, YfiA^Ll, which associates with ribosomes in the stationary phase of growth and is responsible for dimerization of ribosomes. We show that full‐length YfiA^Ll is necessary and sufficient for ribosome dimerization in L. lactis but also functions heterologously in vitro with E. coli ribosomes. Deletion of the yfiA gene has no effect on the growth rate but diminishes the survival of L. lactis under energy‐starving conditions. The N‐terminal domain of YfiA^Ll is homologous to HPF from E. coli, whereas the C‐terminal domain has no counterpart in E. coli. By assembling ribosome dimers in vitro, we could dissect the roles of the N‐ and C‐terminal domains of YfiA^Ll. It is concluded that the dimerization and inactivation of ribosomes in L. lactis and E. coli differ in several cellular and molecular aspects. In addition, two‐dimensional maps of dimeric ribosomes from L. lactis obtained by single particle electron microscopy show a marked structural difference in monomer association in comparison to the ribosome dimers in E. coli.     

The 5 kDa Protein NdhP Is Essential for Stable NDH-1L Assembly in Thermosynechococcus elongatus

The cyanobacterial NADPH:plastoquinone oxidoreductase complex (NDH-1), that is related to Complex I of eubacteria and mitochondria, plays a pivotal role in respiration as well as in cyclic electron transfer (CET) around PSI and is involved in a unique carbon concentration mechanism (CCM). Despite many achievements in the past, the complex protein composition and the specific function of many subunits of the different NDH-1 species remain elusive. We have recently discovered in a NDH-1 preparation from Thermosynechococcus elongatus two novel single transmembrane peptides (NdhP, NdhQ) with molecular weights below 5 kDa. Here we show that NdhP is a unique component of the ∼450 kDa NDH-1L complex, that is involved in respiration and CET at high CO₂ concentration, and not detectable in the NDH-1MS and NDH-1MS'' complexes that play a role in carbon concentration. C-terminal fusion of NdhP with his-tagged superfolder GFP and the subsequent analysis of the purified complex by electron microscopy and single particle averaging revealed its localization in the NDH-1L specific distal unit of the NDH-1 complex, that is formed by the subunits NdhD1 and NdhF1. Moreover, NdhP is essential for NDH-1L formation, as this type of NDH-1 was not detectable in a ΔndhP::Km mutant.     

Fine structure of granal thylakoid membrane organization using cryo electron tomography

The architecture of grana membranes from spinach chloroplasts was studied by cryo electron tomography. Tomographic reconstructions of ice-embedded isolated grana stacks enabled to resolve features of photosystem II (PSII) in the native membrane and to assign the absolute orientation of individual membranes of granal thylakoid discs. Averaging of 3D sub-volumes containing PSII complexes provided a 3D structure of the PSII complex at 40 ? resolution. Comparison with a recently proposed pseudo-atomic model of the PSII supercomplex revealed the presence of unknown protein densities right on top of 4 light harvesting complex II (LHCII) trimers at the lumenal side of the membrane. The positions of individual dimeric PSII cores within an entire membrane layer indicates that about 23% supercomplexes must be of smaller size than full C(2)S(2)M(2) supercomplexes, to avoid overlap.     

Structural characterization of NDH-1 complexes of Thermosynechococcus elongatus by single particle electron microscopy

The structure of the multifunctional NAD(P)H dehydrogenase type 1 (NDH-1) complexes from cyanobacteria was investigated by growing the wild type and specific ndh His-tag mutants of Thermosynechococcus elongatus BP-1 under different CO(2) conditions, followed by an electron microscopy (EM) analysis of their purified membrane protein complexes. Single particle averaging showed that the complete NDH-1 complex (NDH-1L) is L-shaped, with a relatively short hydrophilic arm. Two smaller complexes were observed, differing only at the tip of the membrane-embedded arm. The smallest one is considered to be similar to NDH-1M, lacking the NdhD1 and NdhF1 subunits. The other fragment, named NDH-1I, is intermediate between NDH-1L and NDH-1M and only lacks a mass compatible with the size of the NdhF1 subunit. Both smaller complexes were observed under low- and high-CO(2) growth conditions, but were much more abundant under the latter conditions. EM characterization of cyanobacterial NDH-1 further showed small numbers of NDH-1 complexes with additional masses. One type of particle has a much longer peripheral arm, similar to the one of NADH: ubiquinone oxidoreductase (complex I) in E. coli and other organisms. This indicates that Thermosynechococcus elongatus must have protein(s) which are structurally homologous to the E. coli NuoE, -F, and -G subunits. Another low-abundance type of particle (NDH-1U) has a second labile hydrophilic arm at the tip of the membrane-embedded arm. This U-shaped particle has not been observed before by EM in a NDH-I preparation.     

Structure and function of mitochondrial supercomplexes

The five complexes (complexes I–V) of the oxidative phosphorylation (OXPHOS) system of mitochondria can be extracted in the form of active supercomplexes. Single-particle electron microscopy has provided 2D and 3D data describing the interaction between complexes I and III, among I, III and IV and in a dimeric form of complex V, between two ATP synthase monomers. The stable interactions are called supercomplexes which also form higher-ordered oligomers. Cryo-electron tomography provides new insights on how these supercomplexes are arranged within intact mitochondria. The structure and function of OXPHOS supercomplexes are discussed.