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 H 2 O 2 , 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.
A considerably small fraction of approximately 60–100 proteins of all chloroplast proteins are encoded by the plastid genome. Many of these proteins are major subunits of complexes with central functions within plastids. In comparison with other subcellular compartments and bacteria, many steps of chloroplast protein biogenesis are not well understood. We report here on the first study of chloroplast-localised trigger factor. In bacteria, this molecular chaperone is known to associate with translating ribosomes to facilitate the folding of newly synthesized proteins. Chloroplast trigger factors of the unicellular green algae Chlamydomonas reinhardtii and the vascular land plant Arabidopsis thaliana were characterized by biophysical and structural methods and compared to the Escherichia coli isoform. We show that chloroplast trigger factor is mainly monomeric and displays only moderate stability against thermal unfolding even under mild heat-stress conditions. The global shape and conformation of these proteins were determined in solution by small-angle X-ray scattering and subsequent ab initio modelling. As observed for bacteria, plastidic trigger factors have a dragon-like structure, albeit with slightly altered domain arrangement and flexibility. This structural conservation despite low amino acid sequence homology illustrates a remarkable evolutionary robustness of chaperone conformations across various kingdoms of life.
In plant cells, chloroplast gene expression is predominantly controlled through post-transcriptional regulation. Such fine-tuning is vital for precisely orchestrating protein complex assembly as for the photosynthesis machinery and for quickly responding to environmental changes. While regulation of chloroplast protein synthesis is of central importance, little is known about the degree and nature of the regulatory network, mainly due to challenges associated with the specific isolation of transient ribosome interactors. Here, we established a ribosome affinity purification method, which enabled us to broadly uncover putative ribosome-associated proteins in chloroplasts. Endogenously tagging of a protein of the large or small subunit revealed not only interactors of the holo complex, but also preferential interactors of the two subunits. This includes known canonical regulatory proteins as well as several new proteins belonging to the categories of protein and RNA regulation, photosystem biogenesis, redox control and metabolism. The sensitivity of the here applied screen was validated for various transiently interacting proteins. We further provided evidence for the existence of a ribosome-associated Nα-acetyltransferase in chloroplasts and its ability to acetylate substrate proteins at their N-terminus. The broad set of ribosome interactors underscores the potential to regulate chloroplast gene expression on the level of protein synthesis.
Biochemical processes in chloroplasts are important for virtually all life forms. Tight regulation of protein homeostasis and the coordinated assembly of protein complexes, composed of both imported and locally synthesized subunits, are vital to plastid functionality. Protein biogenesis requires the action of cotranslationally acting molecular chaperones. One such chaperone is trigger factor (TF), which is known to cotranslationally bind most newly synthesized proteins in bacteria, thereby assisting their correct folding and maturation. However, how these processes are regulated in chloroplasts remains poorly understood. We report here functional investigation of chloroplast-localized TF (TIG1) in the green alga (Chlamydomonas reinhardtii) and the vascular land plant Arabidopsis (Arabidopsis thaliana). We show that chloroplastic TIG1 evolved as a specialized chaperone. Unlike other plastidic chaperones that are functionally interchangeable with their prokaryotic counterpart, TIG1 was not able to complement the broadly acting ortholog in Escherichia coli. Whereas general chaperone properties such as the prevention of aggregates or substrate recognition seems to be conserved between bacterial and plastidic TFs, plant TIG1s differed by associating with only a relatively small population of translating ribosomes. Furthermore, a reduction of plastidic TIG1 levels leads to deregulated protein biogenesis at the expense of increased translation, thereby disrupting the chloroplast energy household. This suggests a central role of TIG1 in protein biogenesis in the chloroplast.
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 non-lethal 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 become bottlenecks under heat but become optimized upon heat acclimation to sustain correct co-translational protein complex assembly.
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