Stromal Side Of Thylakoid Membrane Research Articles

The Unique Light-Harvesting System of the Algal Phycobilisome: Structure, Assembly Components, and Functions.

The phycobilisome (PBS) is the major light-harvesting apparatus in cyanobacteria and red algae. It is a large multi-subunit protein complex of several megadaltons that is found on the stromal side of thylakoid membranes in orderly arrays. Chromophore lyases catalyse the thioether bond between apoproteins and phycobilins of PBSs. Depending on the species, composition, spatial assembly, and, especially, the functional tuning of different phycobiliproteins mediated by linker proteins, PBSs can absorb light between 450 and 650 nm, making them efficient and versatile light-harvesting systems. However, basic research and technological innovations are needed, not only to understand their role in photosynthesis but also to realise the potential applications of PBSs. Crucial components including phycobiliproteins, phycobilins, and lyases together make the PBS an efficient light-harvesting system, and these provide a scheme to explore the heterologous synthesis of PBS. Focusing on these topics, this review describes the essential components needed for PBS assembly, the functional basis of PBS photosynthesis, and the applications of phycobiliproteins. Moreover, key technical challenges for heterologous biosynthesis of phycobiliproteins in chassis cells are discussed.

In vivo regulation of thylakoid proton motive force in immature leaves.

In chloroplast, proton motive force (pmf) is critical for ATP synthesis and photoprotection. To prevent photoinhibition of photosynthetic apparatus, proton gradient (ΔpH) across the thylakoid membranes needs to be built up to minimize the production of reactive oxygen species (ROS) in thylakoid membranes. However, the regulation of thylakoid pmf in immature leaves is little known. In this study, we compared photosynthetic electron sinks, P700 redox state, non-photochemical quenching (NPQ), and electrochromic shift (ECS) signal in immature and mature leaves of a cultivar of Camellia. The immature leaves displayed lower linear electron flow and cyclic electron flow, but higher levels of NPQ and P700 oxidation ratio under high light. Meanwhile, we found that pmf and ΔpH were higher in the immature leaves. Furthermore, the immature leaves showed significantly lower thylakoid proton conductivity than mature leaves. These results strongly indicated that immature leaves can build up enough ΔpH by modulating proton efflux from the lumenal side to the stromal side of thylakoid membranes, which is essential to prevent photoinhibition via thermal energy dissipation and photosynthetic control of electron transfer. This study highlights that the activity of chloroplast ATP synthase is a key safety valve for photoprotection in immature leaves.

The contribution of individual domains of chloroplast protein AtDeg2 to its chaperone and proteolytic activities

The thylakoid protease AtDeg2 is a non-ATP hydrolyzing chloroplast protease/chaperone peripherally connected with stromal side of thylakoid membrane. Its linear structure consists of protease domain and two PDZ domains. To unveil the significance of individual domains, chaperone and proteolytic activities of AtDeg2, its mutated recombinant versions have been developed and their ability to suppress protein aggregation and resolubilization of protein aggregates as well as the ability to degrade substrate protein was examined in vitro. Our work reveals for the first time that AtDeg2 is able not only to suppress aggregation of denatured proteins, but to resolubilize existing protein aggregates as well. We show that PDZ2 domain contributes significantly to both chaperone and protease activities of AtDeg2, whereas PDZ1 is required for chaperone but superfluous for proteolytic activity. Protease domain – but not S-268 in its catalytic site – contributes to chaperone activities of AtDeg2. These results show an entirely new function of AtDeg2 chaperone/protease (i.e., disaggregation of protein aggregates) and allow to identify structural motifs required for “old” and new functions of AtDeg2.

TEF30 Interacts with Photosystem II Monomers and Is Involved in the Repair of Photodamaged Photosystem II in Chlamydomonas reinhardtii.

The remarkable capability of photosystem II (PSII) to oxidize water comes along with its vulnerability to oxidative damage. Accordingly, organisms harboring PSII have developed strategies to protect PSII from oxidative damage and to repair damaged PSII. Here, we report on the characterization of the THYLAKOID ENRICHED FRACTION30 (TEF30) protein in Chlamydomonas reinhardtii, which is conserved in the green lineage and induced by high light. Fractionation studies revealed that TEF30 is associated with the stromal side of thylakoid membranes. By using blue native/Deriphat-polyacrylamide gel electrophoresis, sucrose density gradients, and isolated PSII particles, we found TEF30 to quantitatively interact with monomeric PSII complexes. Electron microscopy images revealed significantly reduced thylakoid membrane stacking in TEF30-underexpressing cells when compared with control cells. Biophysical and immunological data point to an impaired PSII repair cycle in TEF30-underexpressing cells and a reduced ability to form PSII supercomplexes after high-light exposure. Taken together, our data suggest potential roles for TEF30 in facilitating the incorporation of a new D1 protein and/or the reintegration of CP43 into repaired PSII monomers, protecting repaired PSII monomers from undergoing repeated repair cycles or facilitating the migration of repaired PSII monomers back to stacked regions for supercomplex reassembly.

AtDeg2 – a chloroplast protein with dual protease/chaperone activity

Chloroplast protease AtDeg2 (an ATP-independent serine endopeptidase) is cytosolically synthesized as a precursor, which is imported into the chloroplast stroma and deprived of its transit peptide. Then the mature protein undergoes routing to its functional location at the stromal side of thylakoid membrane. In its linear structure AtDeg2 molecule contains the protease domain with catalytic triad (HDS) and two PDZ domains (PDZ1 and PDZ2). In vivo AtDeg2 most probably exists as a supposedly inactive haxamer, which may change its oligomeric stage to form active 12-mer, or 24-mer. AtDeg2 has recently been demonstrated to exhibit dual protease/chaperone function. This review is focused on the current awareness with regard to AtDeg2 structure and functional significance.

Interaction of a plant 14-3-3 protein with the signal peptide of a thylakoid-targeted chloroplast precursor protein and the presence of 14-3-3 isoforms in the chloroplast stroma.

The 14-3-3 proteins are acidic, dimeric proteins that have been implicated in many eukaryotic cellular processes because of direct protein association with enzymes and other metabolic and regulatory proteins. 14-3-3 proteins are largely considered to be cytoplasmic, but a search for proteins that specifically interact with a plant 14-3-3 resulted in the isolation of a nuclear-encoded, thylakoid-targeted chloroplast precursor, the full-length Arabidopsis photosystem I N-subunit At pPSI-N (P.C. Sehnke, R.J. Ferl ¿1995 Plant Physiol 109: 1126). Using precursor truncations in the two-hybrid system, it was determined that the leader sequence is the site of PSI-N that associates with 14-3-3. This suggested the novel possibility that 14-3-3 would be found within chloroplasts. Immuno-electron microscopy of leaf tissue and western analysis of chloroplast fractions with monoclonal anti-14-3-3 antibodies localized 14-3-3 proteins to the chloroplast stroma and the stromal side of thylakoid membranes. Using peptide-generated, isoform-specific antibodies, GF14nu, GF14epsilon, GF14mu, and GF14upsilon were shown to be present in the chloroplast stromal extract. These isoforms represent two distinct phylogenetic 14-3-3 groupings. These data suggest a novel interorganellar role for these phylogenetically distinct 14-3-3 proteins.