Competition co-immunoprecipitation reveals the interactors of the chloroplast CPN60 chaperonin machinery.

The functionality of all metabolic processes in chloroplasts depends on a balanced integration of nuclear- and chloroplast-encoded polypeptides into the plastid's proteome. The chloroplast chaperonin machinery is an essential player in chloroplast protein folding under ambient and stressful conditions, with a more intricate structure and subunit composition compared to the orthologous GroEL/ES chaperonin of Escherichia coli. However, its exact role in chloroplasts remains obscure, mainly because of very limited knowledge about the interactors. We employed the competition immunoprecipitation method for the identification of the chaperonin's interactors in Chlamydomonas reinhardtii. Co-immunoprecipitation of the target complex in the presence of increasing amounts of isotope-labelled competitor epitope and subsequent mass spectrometry analysis specifically allowed to distinguish true interactors from unspecifically co-precipitated proteins. Besides known substrates such as RbcL and the expected complex partners, we revealed numerous new interactors with high confidence. Proteins that qualify as putative substrate proteins differ from bulk chloroplast proteins by a higher content of beta-sheets, lower alpha-helical conformation and increased aggregation propensity. Immunoprecipitations targeted against a subunit of the co-chaperonin lid revealed the ClpP protease as a specific partner complex, pointing to a close collaboration of these machineries to maintain protein homeostasis in the chloroplast.

One-helix protein 2 is not required for the synthesis of photosystem II subunit D1 in Chlamydomonas.

In land plants and cyanobacteria, co-translational association of chlorophyll (Chl) to the nascent D1 polypeptide, a reaction center protein of photosystem II (PSII), requires a Chl binding complex consisting of a short-chain dehydrogenase (high chlorophyll fluorescence 244 [HCF244]/uncharacterized protein 39 [Ycf39]) and one-helix proteins (OHP1 and OHP2 in chloroplasts) of the light-harvesting antenna complex superfamily. Here, we show that an ohp2 mutant of the green alga Chlamydomonas (Chlamydomonas reinhardtii) fails to accumulate core PSII subunits, in particular D1 (encoded by the psbA mRNA). Extragenic suppressors arose at high frequency, suggesting the existence of another route for Chl association to PSII. The ohp2 mutant was complemented by the Arabidopsis (Arabidopsis thaliana) ortholog. In contrast to land plants, where psbA translation is prevented in the absence of OHP2, ribosome profiling experiments showed that the Chlamydomonas mutant translates the psbA transcript over its full length. Pulse labeling suggested that D1 is degraded during or immediately after translation. The translation of other PSII subunits was affected by assembly-controlled translational regulation. Proteomics showed that HCF244, a translation factor which associates with and is stabilized by OHP2 in land plants, still partly accumulates in the Chlamydomonas ohp2 mutant, explaining the persistence of psbA translation. Several Chl biosynthesis enzymes overaccumulate in the mutant membranes. Partial inactivation of a D1-degrading protease restored a low level of PSII activity in an ohp2 background, but not photoautotrophy. Taken together, our data suggest that OHP2 is not required for psbA translation in Chlamydomonas, but is necessary for D1 stabilization.

Structural features of chloroplast trigger factor determined at 2.6†Šresolution.

The folding of newly synthesized polypeptides requires the coordinated action of molecular chaperones. Prokaryotic cells and the chloroplasts of plant cells possess the ribosome-associated chaperone trigger factor, which binds nascent polypeptides at their exit stage from the ribosomal tunnel. The structure of bacterial trigger factor has been well characterized and it has a dragon-shaped conformation, with flexible domains responsible for ribosome binding, peptidyl-prolyl cis-trans isomerization (PPIase) activity and substrate protein binding. Chloroplast trigger-factor sequences have diversified from those of their bacterial orthologs and their molecular mechanism in plant organelles has been little investigated to date. Here, the crystal structure of the plastidic trigger factor from the green alga Chlamydomonas reinhardtii is presented at 2.6 Šresolution. Due to the high intramolecular flexibility of the protein, diffraction to this resolution was only achieved using a protein that lacked the N-terminal ribosome-binding domain. The eukaryotic trigger factor from C. reinhardtii exhibits a comparable dragon-shaped conformation to its bacterial counterpart. However, the C-terminal chaperone domain displays distinct charge distributions, with altered positioning of the helical arms and a specifically altered charge distribution along the surface responsible for substrate binding. While the PPIase domain shows a highly conserved structure compared with other PPIases, its rather weak activity and an unusual orientation towards the C-terminal domain points to specific adaptations of eukaryotic trigger factor for function in chloroplasts.

Fast and global reorganization of the chloroplast protein biogenesis network during heat acclimation.

Photosynthesis is a central determinant of plant biomass production, but its homeostasis is increasingly challenged by heat. Little is known about the sensitive regulatory principles involved in heat acclimation that underly the biogenesis and repair of chloroplast-encoded core subunits of photosynthetic complexes. Employing time-resolved ribosome and transcript profiling together with selective ribosome proteomics, we systematically deciphered these processes in chloroplasts of Chlamydomonas reinhardtii. We revealed protein biosynthesis and altered translation elongation as central processes for heat acclimation and showed that these principles are conserved between the alga and the flowering plant Nicotiana tabacum. Short-term heat exposure resulted in specific translational repression of chlorophyll a-containing core antenna proteins of photosystems I and II. Furthermore, translocation of ribosome nascent chain complexes to thylakoid membranes was affected, as reflected by the increased accumulation of stromal cpSRP54-bound ribosomes. The successful recovery of synthesizing these proteins under prolonged acclimation of nonlethal heat conditions was associated with specific changes of the co-translational protein interaction network, including increased ribosome association of chlorophyll biogenesis enzymes and acclimation factors responsible for complex assembly. We hypothesize that co-translational cofactor binding and targeting might be bottlenecks under heat but become optimized upon heat acclimation to sustain correct co-translational protein complex assembly.

Systems approaches identify the consequences of monosomy in somatic human cells.

Chromosome loss that results in monosomy is detrimental to viability, yet it is frequently observed in cancers. How cancers survive with monosomy is unknown. Using p53-deficient monosomic cell lines, we find that chromosome loss impairs proliferation and genomic stability. Transcriptome and proteome analysis demonstrates reduced expression of genes encoded on the monosomes, which is partially compensated in some cases. Monosomy also induces global changes in gene expression. Pathway enrichment analysis reveals that genes involved in ribosome biogenesis and translation are downregulated in all monosomic cells analyzed. Consistently, monosomies display defects in protein synthesis and ribosome assembly. We further show that monosomies are incompatible with p53 expression, likely due to defects in ribosome biogenesis. Accordingly, impaired ribosome biogenesis and p53 inactivation are associated with monosomy in cancer. Our systematic study of monosomy in human cells explains why monosomy is so detrimental and reveals the importance of p53 for monosomy occurrence in cancer.

Acclimation in plants - the Green Hub consortium.

Acclimation is the capacity to adapt to environmental changes within the lifetime of an individual. This ability allows plants to cope with the continuous variation in ambient conditions to which they are exposed as sessile organisms. Because environmental changes and extremes are becoming even more pronounced due to the current period of climate change, enhancing the efficacy of plant acclimation is a promising strategy for mitigating the consequences of global warming on crop yields. At the cellular level, the chloroplast plays a central role in many acclimation responses, acting both as a sensor of environmental change and as a target of cellular acclimation responses. In this Perspective article, we outline the activities of the Green Hub consortium funded by the German Science Foundation. The main aim of this research collaboration is to understand and strategically modify the cellular networks that mediate plant acclimation to adverse environments, employing Arabidopsis, tobacco (Nicotiana tabacum) and Chlamydomonas as model organisms. These efforts will contribute to 'smart breeding' methods designed to create crop plants with improved acclimation properties. To this end, the model oilseed crop Camelina sativa is being used to test modulators of acclimation for their potential to enhance crop yield under adverse environmental conditions. Here we highlight the current state of research on the role of gene expression, metabolism and signalling in acclimation, with a focus on chloroplast-related processes. In addition, further approaches to uncovering acclimation mechanisms derived from systems and computational biology, as well as adaptive laboratory evolution with photosynthetic microbes, are highlighted.

Co-Translational Protein Folding and Sorting in Chloroplasts.

Cells depend on the continuous renewal of their proteome composition during the cell cycle and in order to replace aberrant proteins or to react to changing environmental conditions. In higher eukaryotes, protein synthesis is achieved by up to five million ribosomes per cell. With the fast kinetics of translation, the large number of newly made proteins generates a substantial burden for protein homeostasis and requires a highly orchestrated cascade of factors promoting folding, sorting and final maturation. Several of the involved factors directly bind to translating ribosomes for the early processing of emerging nascent polypeptides and the translocation of ribosome nascent chain complexes to target membranes. In plant cells, protein synthesis also occurs in chloroplasts serving the expression of a relatively small set of 60-100 protein-coding genes. However, most of these proteins, together with nucleus-derived subunits, form central complexes majorly involved in the essential processes of photosynthetic light reaction, carbon fixation, metabolism and gene expression. Biogenesis of these heterogenic complexes adds an additional level of complexity for protein biogenesis. In this review, we summarize the current knowledge about co-translationally binding factors in chloroplasts and discuss their role in protein folding and ribosome translocation to thylakoid membranes.

The conserved theme of ribosome hibernation: from bacteria to chloroplasts of plants.

Cells are highly adaptive systems that respond and adapt to changing environmental conditions such as temperature fluctuations or altered nutrient availability. Such acclimation processes involve reprogramming of the cellular gene expression profile, tuning of protein synthesis, remodeling of metabolic pathways and morphological changes of the cell shape. Nutrient starvation can lead to limited energy supply and consequently, remodeling of protein synthesis is one of the key steps of regulation since the translation of the genetic code into functional polypeptides may consume up to 40% of a cell's energy during proliferation. In eukaryotic cells, downregulation of protein synthesis during stress is mainly mediated by modification of the translation initiation factors. Prokaryotic cells suppress protein synthesis by the active formation of dimeric so-called 'hibernating' 100S ribosome complexes. Such a transition involves a number of proteins which are found in various forms in prokaryotes but also in chloroplasts of plants. Here, we review the current understanding of these hibernation factors and elaborate conserved principles which are shared between species.

Commonalities and differences of chloroplast translation in a green alga and land plants.

Chloroplast gene expression is a fascinating and highly regulated process, which was mainly studied on specific genes in a few model organisms including the unicellular green alga Chlamydomonas (Chlamydomonas reinhardtii) and the embryophyte (land) plants tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana). However, a direct plastid genome-wide interspecies comparison of chloroplast gene expression that includes translation was missing. We adapted a targeted chloroplast ribosome profiling approach to quantitatively compare RNA abundance and translation output between Chlamydomonas, tobacco and Arabidopsis. The re-analysis of established chloroplast mutants confirmed the capability of the approach by detecting known as well as previously undetected translation defects (including the potential photosystem II assembly-dependent regulation of PsbH). Systematic comparison of the algal and land plant wild-type gene expression showed that, for most genes, the steady-state translation output is highly conserved among the three species, while the levels of transcript accumulation are more distinct. Whereas in Chlamydomonas transcript accumulation and translation output are closely balanced, this correlation is less obvious in embryophytes, indicating more pronounced translational regulation. Altogether, this suggests that green algae and land plants evolved different strategies to achieve conserved levels of protein synthesis.

ATP-dependent molecular chaperones in plastids--More complex than expected.

Plastids are a class of essential plant cell organelles comprising photosynthetic chloroplasts of green tissues, starch-storing amyloplasts of roots and tubers or the colorful pigment-storing chromoplasts of petals and fruits. They express a few genes encoded on their organellar genome, called plastome, but import most of their proteins from the cytosol. The import into plastids, the folding of freshly-translated or imported proteins, the degradation or renaturation of denatured and entangled proteins, and the quality-control of newly folded proteins all require the action of molecular chaperones. Members of all four major families of ATP-dependent molecular chaperones (chaperonin/Cpn60, Hsp70, Hsp90 and Hsp100 families) have been identified in plastids from unicellular algae to higher plants. This review aims not only at giving an overview of the most current insights into the general and conserved functions of these plastid chaperones, but also into their specific plastid functions. Given that chloroplasts harbor an extreme environment that cycles between reduced and oxidized states, that has to deal with reactive oxygen species and is highly reactive to environmental and developmental signals, it can be presumed that plastid chaperones have evolved a plethora of specific functions some of which are just about to be discovered. Here, the most urgent questions that remain unsolved are discussed, and guidance for future research on plastid chaperones is given. This article is part of a Special Issue entitled: Chloroplast Biogenesis.

The ribosome as a hub for protein quality control.

Cells face a constant challenge as they produce new proteins. The newly synthesized polypeptides must be folded properly to avoid aggregation. If proteins do misfold, they must be cleared to maintain a functional and healthy proteome. Recent work is revealing the complex mechanisms that work cotranslationally to ensure protein quality control during biogenesis at the ribosome. Indeed, the ribosome is emerging as a central hub in coordinating these processes, particularly in sensing the nature of the nascent protein chain, recruiting protein folding and translocation components, and integrating mRNA and nascent chain quality control. The tiered and complementary nature of these decision-making processes confers robustness and fidelity to protein homeostasis during protein synthesis.

The cotranslational function of ribosome-associated Hsp70 in eukaryotic protein homeostasis.

In eukaryotic cells a molecular chaperone network associates with translating ribosomes, assisting the maturation of emerging nascent polypeptides. Hsp70 is perhaps the major eukaryotic ribosome-associated chaperone and the first reported to bind cotranslationally to nascent chains. However, little is known about the underlying principles and function of this interaction. Here, we use a sensitive and global approach to define the cotranslational substrate specificity of the yeast Hsp70 SSB. We find that SSB binds to a subset of nascent polypeptides whose intrinsic properties and slow translation rates hinder efficient cotranslational folding. The SSB-ribosome cycle and substrate recognition is modulated by its ribosome-bound cochaperone, RAC. Deletion of SSB leads to widespread aggregation of newly synthesized polypeptides. Thus, cotranslationally acting Hsp70 meets the challenge of folding the eukaryotic proteome by stabilizing its longer, more slowly translated, and aggregation-prone nascent polypeptides.

The chloroplast HSP70B-CDJ2-CGE1 chaperones catalyse assembly and disassembly of VIPP1 oligomers in Chlamydomonas.

The vesicle-inducing protein in plastids (VIPP1) is essential for the biogenesis of thylakoid membranes in cyanobacteria and plants. VIPP1 and its bacterial ancestor PspA form large homo-oligomeric rings of >1 MDa. We recently demonstrated that VIPP1 interacts with the chloroplast J-domain co-chaperone CDJ2 and its chaperone partner HSP70B, and hypothesized that the chaperones might be involved in the assembly and/or disassembly of VIPP1 oligomers. To test this hypothesis, we analysed the composition of VIPP1/chaperone complexes in Chlamydomonas reinhardtii cell extracts and monitored effects of the chaperones on VIPP1 assembly states in vitro. We found that CGE1, the chloroplast GrpE homologue, is also part of complexes with HSP70B, CDJ2 and VIPP1. We observed that CDJ2-VIPP1 accumulated as low- and high-molecular-weight complexes in ATP-depleted cell extracts, but as intermediate-size complexes in extracts supplemented with ATP. This was consistent with a role for the chaperones in VIPP1 assembly and disassembly. Using purified proteins, we could demonstrate that the chaperones indeed facilitated both the assembly and disassembly of VIPP1 oligomers. Electron microscopy studies revealed that, in contrast to PspA, VIPP1 rings assembled into rod-shaped supercomplexes that were morphologically similar to microtubule-like structures observed earlier in various plastid types. VIPP1 rods, too, were disassembled by the chaperones, and chaperone-mediated rod disassembly also occurred when VIPP1 lacked an approximately 30-aa C-terminal extension present in VIPP1 homologues but absent in PspA. By regulating the assembly state of VIPP1, the chloroplast HSP70 chaperone system may play an important role in the maintenance/biogenesis of thylakoid membranes.

J-domain protein CDJ2 and HSP70B are a plastidic chaperone pair that interacts with vesicle-inducing protein in plastids 1.

J-domain cochaperones confer functional specificity to their heat shock protein (HSP)70 partner by recruiting it to specific substrate proteins. To gain insight into the functions of plastidic HSP70s, we searched in Chlamydomonas databases for expressed sequence tags that potentially encode chloroplast-targeted J-domain cochaperones. Two such cDNAs were found: the encoded J-domain proteins were named chloroplast DnaJ homolog 1 and 2 (CDJ1 and CDJ2). CDJ2 was shown to interact with a approximately 28-kDa protein that by mass spectrometry was identified as the vesicle-inducing protein in plastids 1 (VIPP1). In fractionation experiments, CDJ2 was detected almost exclusively in the stroma, whereas VIPP1 was found in low-density membranes, thylakoids, and in the stroma. Coimmunoprecipitation and mass spectrometry analyses identified stromal HSP70B as the major protein interacting with soluble VIPP1, and, as confirmed by cross-linking data, as chaperone partner of CDJ2. In blue native-PAGE of soluble cell extracts, CDJ2 and VIPP1 comigrated in complexes of >>669, approximately 150, and perhaps approximately 300 kDa. Our data suggest that CDJ2, presumably via coiled-coil interactions, binds to VIPP1 and presents it to HSP70B in the ATP state. Our findings and the previously reported requirement of VIPP1 for the biogenesis of thylakoid membranes point to a role for the HSP70B/CDJ2 chaperone pair in this process.