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Members of the Oxa1/YidC/Alb3 protein family are involved in the insertion, folding, and assembly of membrane proteins in mitochondria, bacteria, and chloroplasts. The thylakoid membrane protein Alb3 mediates the chloroplast signal recognition particle (cpSRP)-dependent posttranslational insertion of nuclear-encoded light harvesting chlorophyll a/b-binding proteins and participates in the biogenesis of plastid-encoded subunits of the photosynthetic complexes. These subunits are cotranslationally inserted into the thylakoid membrane, yet very little is known about the molecular mechanisms underlying docking of the ribosome-nascent chain complexes to the chloroplast SecY/Alb3 insertion machinery. Here, we show that nanodisc-embedded Alb3 interacts with ribosomes, while the homolog Alb4, also located in the thylakoid membrane, shows no ribosome binding. Alb3 contacts the ribosome with its C-terminal region and at least one additional binding site within its hydrophobic core region. Within the C-terminal region, two conserved motifs (motifs III and IV) are cooperatively required to enable the ribosome contact. Furthermore, our data suggest that the negatively charged C-terminus of the ribosomal subunit uL4c is involved in Alb3 binding. Phylogenetic analyses of uL4 demonstrate that this region newly evolved in the green lineage during the transition from aquatic to terrestrial life. [ABSTRACT FROM AUTHOR]

Members of the Oxa1/YidC/Alb3 protein family are involved in the insertion, folding, and assembly of membrane proteins in mitochondria, bacteria, and chloroplasts. The thylakoid membrane protein Alb3 mediates the chloroplast signal recognition particle (cpSRP)-dependent posttranslational insertion of nuclear-encoded light harvesting chlorophyll a/b-binding proteins and participates in the biogenesis of plastid-encoded subunits of the photosynthetic complexes. These subunits are cotranslationally inserted into the thylakoid membrane, yet very little is known about the molecular mechanisms underlying docking of the ribosome-nascent chain complexes to the chloroplast SecY/Alb3 insertion machinery. Here, we show that nanodisc-embedded Alb3 interacts with ribosomes, while the homolog Alb4, also located in the thylakoid membrane, shows no ribosome binding. Alb3 contacts the ribosome with its C-terminal region and at least one additional binding site within its hydrophobic core region. Within the C-terminal region, two conserved motifs (motifs III and IV) are cooperatively required to enable the ribosome contact. Furthermore, our data suggest that the negatively charged C-terminus of the ribosomal subunit uL4c is involved in Alb3 binding. Phylogenetic analyses of uL4 demonstrate that this region newly evolved in the green lineage during the transition from aquatic to terrestrial life.

VIPP proteins aid thylakoid biogenesis and membrane maintenance in cyanobacteria, algae, and plants. Some members of the Chlorophyceae contain two VIPP paralogs termed VIPP1 and VIPP2, which originate from an early gene duplication event during the evolution of green algae. VIPP2 is barely expressed under nonstress conditions but accumulates in cells exposed to high light intensities or H2O2, during recovery from heat stress, and in mutants with defective integration (alb3.1) or translocation (secA) of thylakoid membrane proteins. Recombinant VIPP2 forms rod‐like structures in vitro and shows a strong affinity for phosphatidylinositol phosphate. Under stress conditions, >70% of VIPP2 is present in membrane fractions and localizes to chloroplast membranes. A vipp2 knock‐out mutant displays no growth phenotypes and no defects in the biogenesis or repair of photosystem II. However, after exposure to high light intensities, the vipp2 mutant accumulates less HSP22E/F and more LHCSR3 protein and transcript. This suggests that VIPP2 modulates a retrograde signal for the expression of nuclear genes HSP22E/F and LHCSR3. Immunoprecipitation of VIPP2 from solubilized cells and membrane‐enriched fractions revealed major interactions with VIPP1 and minor interactions with HSP22E/F. Our data support a distinct role of VIPP2 in sensing and coping with chloroplast membrane stress. The VIPP2 protein from Chlamydomonas reinhardtii localizes predominantly to chloroplast membranes under stresses inducing protein mistargeting and misfolding. VIPP2 interacts with VIPP1 as major partner and with HSP22E/F as minor partner and modulates a retrograde signal for HSP22E/F gene expression in high light. [ABSTRACT FROM AUTHOR]

Light is the key for life on earth. Every photoautotrophic organism depends on the absorption of photons from the visible spectrum of solar radiation. Exposure to light enables foliar photosynthetic reactions, which function in the transformation from sun light into biochemical energy. The essential pigments for light absorption are chlorophyll and bacteriochlorophyll and the appropriate spatio-temporal synthesis and supply of these pigments is essential for the performance of their light-dependent function. The control of chlorophyll synthesis should prevent the accumulation of metabolic intermediates, which would lead to unwanted production of reactive oxygen species. In the worst-case scenario, these reactive oxygen species trigger programmed cell death. Hence, the tight control of the entire chlorophyll-synthesizing pathway is indispensable to ensure a fast and precise response on changing environmental conditions. While transcription and translation of genes involved in tetrapyrrole biosynthesis are expected to be responsible for long-term control, the posttranslational modification of proteins adds another layer of control, which is responsible for short-term modulations of protein expression and function. The rapidly increasing number of studies on posttranslational control of enzymes in tetrapyrrole biosynthesis identified already several interesting mechanisms and new factors, which contribute to the control of the metabolic flow of tetrapyrroles. [ABSTRACT FROM AUTHOR]

DnaK3, a highly conserved cyanobacterial chaperone of the Hsp70 family, binds to cyanobacterial thylakoid membranes, and an involvement of DnaK3 in the biogenesis of thylakoid membranes has been suggested. As shown here, light triggers synthesis of DnaK3 in the cyanobacterium Synechocystis sp. PCC 6803, which links DnaK3 to the biogenesis of thylakoid membranes and to photosynthetic processes. In a DnaK3 depleted strain, the photosystem content is reduced and the photosystem II activity is impaired, whereas photosystem I is regular active. An impact of DnaK3 on the activity of other thylakoid membrane complexes involved in electron transfer is indicated. In conclusion, DnaK3 is a versatile chaperone required for biogenesis and/or maintenance of thylakoid membrane-localized protein complexes involved in electron transfer reactions. As mentioned above, Hsp70 proteins are involved in photoprotection and repair of PS II in chloroplasts.

The photosynthetic light reaction takes place within the thylakoid membrane system in cyanobacteria and chloroplasts. Besides its global importance, the biogenesis, maintenance and dynamics of this membrane system are still a mystery. In the last two decades, strong evidence supported the idea that these processes involve IM30, the inner membrane-associated protein of 30 kDa , a protein also known as the vesicle-inducing protein in plastids 1 (Vipp1). Even though we just only begin to understand the precise physiological function of this protein, it is clear that interaction of IM30 with membranes is crucial for biogenesis of thylakoid membranes. Here we summarize and discuss forces guiding IM30-membrane interactions, as the membrane properties as well as the oligomeric state of IM30 appear to affect proper interaction of IM30 with membrane surfaces. Interaction of IM30 with membranes results in an altered membrane structure and can finally trigger fusion of adjacent membranes, when Mg 2 + is present. Based on recent results, we finally present a model summarizing individual steps involved in IM30-mediated membrane fusion. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider. [ABSTRACT FROM AUTHOR]

The IM30 (inner membrane-associated protein of 30 kDa), also known as the Vipp1 (vesicle-inducing protein in plastids 1), has a crucial role in thylakoid membrane biogenesis and maintenance. Recent results suggest that the protein binds peripherally to membranes containing negatively charged lipids. However, although IM30 monomers interact and assemble into large oligomeric ring complexes with different numbers of monomers, it is still an open question whether ring formation is crucial for membrane interaction. Here we show that binding of IM30 rings to negatively charged phosphatidylglycerol membrane surfaces results in a higher ordered membrane state, both in the head group and in the inner core region of the lipid bilayer. Furthermore, by using gold nanorods covered with phosphatidylglycerol layers and single particle spectroscopy, we show that not only IM30 rings but also lower oligomeric IM30 structures interact with membranes, although with higher affinity. Thus, ring formation is not crucial for, and even counteracts, membrane interaction of IM30. [ABSTRACT FROM AUTHOR]

As a key feature in oxygenic photosynthesis, thylakoid membranes play an essential role in the physiology of plants, algae, and cyanobacteria. Despite their importance in the process of oxygenic photosynthesis, their biogenesis has remained a mystery to the present day. A decade ago, vesicle-inducing protein in plastids 1 (Vipp1) was described to be involved in thylakoid membrane formation in chloroplasts and cyanobacteria. Most follow-up studies clearly linked Vipp1 to membranes and Vipp1 interactions as well as the defects observed after Vipp1 depletion in chloroplasts and cyanobacteria indicate that Vipp1 directly binds to membranes, locally stabilizes bilayer structures, and thereby retains membrane integrity. Here current knowledge about the structure and function of Vipp1 is summarized with a special focus on its relationship to the bacterial phage shock protein A (PspA), as both proteins share a common origin and appear to have retained many similarities in structure and function. [ABSTRACT FROM PUBLISHER]

To advance our knowledge of the model cyanobacterium Synechocystis sp. PCC 6803 we investigated the three-dimensional organization of the cytoplasm using standard transmission electron microscopy and electron tomography. Electron tomography allows a resolution of ~5 nm in all three dimensions, superior to the resolution of most traditional electron microscopy, which is often limited in part by the thickness of the section (70 nm). The thylakoid membrane pairs formed layered sheets that followed the periphery of the cell and converged at various sites near the cytoplasmic membrane. At some of these sites, the margins of thylakoid membranes associated closely along the external surface of rod-like structures termed thylakoid centers, which sometimes traversed nearly the entire periphery of the cell. The thylakoid membranes surrounded the central cytoplasm that contained inclusions such as ribosomes and carboxysomes. Lipid bodies were dispersed throughout the peripheral cytoplasm and often juxtaposed with cytoplasmic and thylakoid membranes suggesting involvement in thylakoid maintenance or biogenesis. Ribosomes were numerous and mainly located throughout the central cytoplasm with some associated with thylakoid and cytoplasmic membranes. Some ribosomes were attached along internal unit-membrane-like sheets located in the central cytoplasm and appeared to be continuous with existing thylakoid membranes. These results present a detailed analysis of the structure of Synechocystis sp. PCC 6803 using high-resolution bioimaging techniques and will allow future evaluation and comparison with gene-deletion mutants. [ABSTRACT FROM AUTHOR]

Dynamin, a GTP-binding protein found in rat brain, plays a role in endocytosis. Suborganellar fractionation studies of Arabidopsis leaf tissue revealed that a dynamin-like protein, ADL1, is localized in the thylakoid membranes of chloroplasts. This notion was supported further by in vivo targeting experiments using an ADL1—green fluorescent fusion protein and immunogold labeling with the anti-ADL1 antibody. Transgenic plants harboring various deletion mutant genes of ADL1 had a yellow leaf phenotype where the cells had very few chloroplasts. In addition, the remaining chloroplasts appeared morphologically not fully developed. The detailed structure of the chloroplasts revealed by electron microscopy showed a greatly reduced amount of thylakoid membranes. Also, the level of thylakoid membrane proteins such as the light-harvesting complex II and CP29 was greatly reduced in these transgenic plants. When we examined the expression of the ADL1 deletion mutant genes, these genes were highly expressed at the transcriptional level. However, the mutant ADL1s were not detectable at the protein level by Western blot analysis. Moreover, the endogenous ADL1 protein level was greatly reduced in these transgenic plants, probably due to a post-transcriptional silencing effect of the transgenes. We propose, therefore, that ADLl is involved in the biogenesis of thylakoid membranes. [ABSTRACT FROM AUTHOR]

A concise overview on the current knowledge of the proteolytic activities in chloroplasts is presented, with an emphasis on the proteolytic events associated with thylavoid membranes. The D1 reaction centre protein of photosystem II undergoes rapid light-dependent turnover and chlorophyll a/b-binding proteins are effectively degraded upon acclimation of plants to higher irradiances. Insights into the partially characterized proteolytic systems in each case will be presented, but the proteases involved still remain unknown. It can be envisaged, however, that the proteolysis is probably an as highly regulated phenomenon as the various steps during biosynthesis of the photosynthetic multiprotein complexes. From the protease point of view, more progress has recently been made in characterization of processing proteases involved m protein import into chloroplasts and in C-terminal processing of the Dl protein. Moreover, there are an increasing number of proteases in chloroplasts which have been discovered and identified as bacterial homologues. These include a Clp-type protease, a homologue of the bacterial protease FtsH and the cyanobacterial PcrA protease, all of which have a specific location in the chloroplast but their definite physiological substrates are still missing. Attempts are made to bring together the recent progress in the identification of proteases and characterisation of proteolytic events in chloroplasts. [ABSTRACT FROM AUTHOR]