The molecular basis of tRNA selectivity by human pseudouridine synthase 3.

Pseudouridine (Ψ), the isomer of uridine, is ubiquitously found in RNA, including tRNA, rRNA, and mRNA. Human pseudouridine synthase 3 (PUS3) catalyzes pseudouridylation of position 38/39 in tRNAs. However, the molecular mechanisms by which it recognizes its RNA targets and achieves site specificity remain elusive. Here, we determine single-particle cryo-EM structures of PUS3 in its apo form and bound to three tRNAs, showing how the symmetric PUS3 homodimer recognizes tRNAs and positions the target uridine next to its active site. Structure-guided and patient-derived mutations validate our structural findings in complementary biochemical assays. Furthermore, we deleted PUS1 and PUS3 in HEK293 cells and mapped transcriptome-wide Ψ sites by Pseudo-seq. Although PUS1-dependent sites were detectable in tRNA and mRNA, we found no evidence that human PUS3 modifies mRNAs. Our work provides the molecular basis for PUS3-mediated tRNA modification in humans and explains how its tRNA modification activity is linked to intellectual disabilities.

SugarPy facilitates the universal, discovery-driven analysis of intact glycopeptides.

MOTIVATION: Protein glycosylation is a complex post-translational modification with crucial cellular functions in all domains of life. Currently, large-scale glycoproteomics approaches rely on glycan database dependent algorithms and are thus unsuitable for discovery-driven analyses of glycoproteomes. RESULTS: Therefore, we devised SugarPy, a glycan database independent Python module, and validated it on the glycoproteome of human breast milk. We further demonstrated its applicability by analyzing glycoproteomes with uncommon glycans stemming from the green alga Chlamydomonas reinhardtii and the archaeon Haloferax volcanii. SugarPy also facilitated the novel characterization of glycoproteins from the red alga Cyanidioschyzon merolae. AVAILABILITY AND IMPLEMENTATION: The source code is freely available on GitHub (https://github.com/SugarPy/SugarPy), and its implementation in Python ensures support for all operating systems. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Improved Proteomics-Based Drug Mechanism-of-Action Studies Using 16-Plex Isobaric Mass Tags.

Multiplexed quantitative proteomics enabled complex workflows to study the mechanisms by which small molecule drugs interact with the proteome such as thermal proteome profiling (TPP) or multiplexed proteome dynamics profiling (mPDP). TPP measures changes in protein thermal stability in response to drug treatment and thus informs on direct targets and downstream regulation events, while the mPDP approach enables the discovery of regulated protein synthesis and degradation events caused by small molecules and other perturbations. The isobaric mass tags available for multiplexed proteomics have thus far limited the efficiency and sensitivity by which such experiments could be performed. Here we evaluate a recent generation of 16-plex isobaric mass tags and demonstrate the sensitive and time efficient identification of Staurosporine targets in HepG2 cell extracts by recording full thermal denaturation/aggregation profiles of vehicle and compound treated samples in a single mass spectrometry experiment. In 2D-TPP experiments, isothermal titration over seven concentrations per temperature enabled comprehensive selectivity profiling of Staurosporine with EC50 values for kinase targets tightly matching to the kinobeads gold standard assay. Finally, we demonstrate time and condition-based multiplexing of dynamic SILAC labeling experiments to delineate proteome-wide effects of the molecular glue Indisulam on synthesis and degradation rates.

Enhancing Open Modification Searches via a Combined Approach Facilitated by Ursgal.

The identification of peptide sequences and their post-translational modifications (PTMs) is a crucial step in the analysis of bottom-up proteomics data. The recent development of open modification search (OMS) engines allows virtually all PTMs to be searched for. This not only increases the number of spectra that can be matched to peptides but also greatly advances the understanding of the biological roles of PTMs through the identification, and the thereby facilitated quantification, of peptidoforms (peptide sequences and their potential PTMs). Whereas the benefits of combining results from multiple protein database search engines have been previously established, similar approaches for OMS results have been missing so far. Here we compare and combine results from three different OMS engines, demonstrating an increase in peptide spectrum matches of 8-18%. The unification of search results furthermore allows for the combined downstream processing of search results, including the mapping to potential PTMs. Finally, we test for the ability of OMS engines to identify glycosylated peptides. The implementation of these engines in the Python framework Ursgal facilitates the straightforward application of the OMS with unified parameters and results files, thereby enabling yet unmatched high-throughput, large-scale data analysis.

Nano LC-MS using capillary columns enables accurate quantification of modified ribonucleosides at low femtomol levels.

Post-transcriptional chemical modifications of (t)RNA molecules are crucial in fundamental biological processes, such as translation. Despite their biological importance and accumulating evidence linking them to various human diseases, technical challenges have limited their detection and accurate quantification. Here, we present a sensitive capillary nanoflow liquid chromatography mass spectrometry (nLC-MS) pipeline for quantitative high-resolution analysis of ribonucleoside modifications from complex biological samples. We evaluated two porous graphitic carbon (PGC) materials and one end-capped C18 reference material as stationary phases for reversed-phase separation. We found that these matrices have complementing retention and separation characteristics, including the capability to separate structural isomers. PGC and C18 matrices yielded excellent signal-to-noise ratios in nLC-MS while differing in the separation capability and sensitivity for various nucleosides. This emphasizes the need for tailored LC-MS setups for optimally detecting as many nucleoside modifications as possible. Detection ranges spanning up to six orders of magnitude enable the analysis of individual ribonucleosides down to femtomol concentrations. Furthermore, normalizing the obtained signal intensities to a stable isotope labeled spike-in enabled direct comparison of ribonucleoside levels between different samples. In conclusion, capillary columns coupled to nLC-MS constitute a powerful and sensitive tool for quantitative analysis of modified ribonucleosides in complex biological samples. This setup will be invaluable for further unraveling the intriguing and multifaceted biological roles of RNA modifications.

N-Glycoproteomic Characterization of Mannosidase and Xylosyltransferase Mutant Strains of Chlamydomonasreinhardtii.

At present, only little is known about the enzymatic machinery required for N-glycosylation in Chlamydomonas reinhardtii, leading to the formation of N-glycans harboring Xyl and methylated Man. This machinery possesses new enzymatic features, as C. reinhardtii N-glycans are independent of ß1,2-N-acetylglucosaminyltransferase I. Here we have performed comparative N-glycoproteomic analyses of insertional mutants of mannosidase 1A (IM Man1A ) and xylosyltransferase 1A (IM XylT1A ). The disruption of man1A affected methylation of Man and the addition of terminal Xyl. The absence of XylT1A led to shorter N-glycans compared to the wild type. The use of a IM Man1A xIM XylT1A double mutant revealed that the absence of Man1A suppressed the IM XylT1A phenotype, indicating that the increased N-glycan trimming is regulated by core ß1,2-Xyl and is dependent on Man1A activity. These data point toward an enzymatic cascade in the N-glycosylation pathway of C. reinhardtii with interlinked roles of Man1A and XylT1A. The results described herein represent the first step toward a functional characterization of the enzymatic N-glycosylation machinery in C. reinhardtii.

Temporal quantitative phosphoproteomics of ADP stimulation reveals novel central nodes in platelet activation and inhibition.

Adenosine diphosphate (ADP) enhances platelet activation by virtually any other stimulant to complete aggregation. It binds specifically to the G-protein-coupled membrane receptors P2Y1 and P2Y12, stimulating intracellular signaling cascades, leading to integrin αIIbß3 activation, a process antagonized by endothelial prostacyclin. P2Y12 inhibitors are among the most successful antiplatelet drugs, however, show remarkable variability in efficacy. We reasoned whether a more detailed molecular understanding of ADP-induced protein phosphorylation could identify (1) critical hubs in platelet signaling toward aggregation and (2) novel molecular targets for antiplatelet treatment strategies. We applied quantitative temporal phosphoproteomics to study ADP-mediated signaling at unprecedented molecular resolution. Furthermore, to mimic the antagonistic efficacy of endothelial-derived prostacyclin, we determined how Iloprost reverses ADP-mediated signaling events. We provide temporal profiles of 4797 phosphopeptides, 608 of which showed significant regulation. Regulated proteins are implicated in well-known activating functions such as degranulation and cytoskeletal reorganization, but also in less well-understood pathways, involving ubiquitin ligases and GTPase exchange factors/GTPase-activating proteins (GEF/GAP). Our data demonstrate that ADP-triggered phosphorylation occurs predominantly within the first 10 seconds, with many short rather than sustained changes. For a set of phosphorylation sites (eg, PDE3ASer312, CALDAG-GEFISer587, ENSASer109), we demonstrate an inverse regulation by ADP and Iloprost, suggesting that these are central modulators of platelet homeostasis. This study demonstrates an extensive spectrum of human platelet protein phosphorylation in response to ADP and Iloprost, which inversely overlap and represent major activating and inhibitory pathways.

pyQms enables universal and accurate quantification of mass spectrometry data.

Quantitative mass spectrometry (MS) is a key technique in many research areas (1), including proteomics, metabolomics, glycomics, and lipidomics. Because all of the corresponding molecules can be described by chemical formulas, universal quantification tools are highly desirable. Here, we present pyQms, an open-source software for accurate quantification of all types of molecules measurable by MS. pyQms uses isotope pattern matching that offers an accurate quality assessment of all quantifications and the ability to directly incorporate mass spectrometer accuracy. pyQms is, due to its universal design, applicable to every research field, labeling strategy, and acquisition technique. This opens ultimate flexibility for researchers to design experiments employing innovative and hitherto unexplored labeling strategies. Importantly, pyQms performs very well to accurately quantify partially labeled proteomes in large scale and high throughput, the most challenging task for a quantification algorithm.

An evolutionary approach uncovers a diverse response of tRNA 2-thiolation to elevated temperatures in yeast.

Chemical modifications of transfer RNA (tRNA) molecules are evolutionarily well conserved and critical for translation and tRNA structure. Little is known how these nucleoside modifications respond to physiological stress. Using mass spectrometry and complementary methods, we defined tRNA modification levels in six yeast species in response to elevated temperatures. We show that 2-thiolation of uridine at position 34 (s(2)U34) is impaired at temperatures exceeding 30°C in the commonly used Saccharomyces cerevisiae laboratory strains S288C and W303, and in Saccharomyces bayanus. Upon stress relief, thiolation levels recover and we find no evidence that modified tRNA or s(2)U34 nucleosides are actively removed. Our results suggest that loss of 2-thiolation follows accumulation of newly synthesized tRNA that lack s(2)U34 modification due to temperature sensitivity of the URM1 pathway in S. cerevisiae and S. bayanus. Furthermore, our analysis of the tRNA modification pattern in selected yeast species revealed two alternative phenotypes. Most strains moderately increase their tRNA modification levels in response to heat, possibly constituting a common adaptation to high temperatures. However, an overall reduction of nucleoside modifications was observed exclusively in S288C. This surprising finding emphasizes the importance of studies that utilize the power of evolutionary biology, and highlights the need for future systematic studies on tRNA modifications in additional model organisms.

Identification of methylated GnTI-dependent N-glycans in Botryococcus brauni.

In contrast to mammals and vascular plants, microalgae show a high diversity in the N-glycan structures of complex N-glycoproteins. Although homologues for ß1,2-N-acetylglucosaminyltransferase I (GnTI), a key enzyme in the formation of complex N-glycans, have been identified in several algal species, GnTI-dependent N-glycans have not been detected so far. We have performed an N-glycoproteomic analysis of the hydrocarbon oils accumulating green microalgae Botryococcus braunii. Thereby, the analysis of intact N-glycopeptides allowed the determination of N-glycan compositions. Furthermore, insights into the role of N-glycosylation in B. braunii were gained from functional annotation of the identified N-glycoproteins. In total, 517 unique N-glycosylated peptides have been identified, including intact N-glycopeptides that harbored N-acetylhexosamine (HexNAc) at the nonreducing end. Surprisingly, these GnTI-dependent N-glycans were also found to be modified with (di)methylated hexose. The identification of GnTI-dependent N-glycans in combination with N-glycan methylation in B. braunii revealed an uncommon type of N-glycan processing in this microalgae.

Phosphorylation of GENOMES UNCOUPLED 4 Alters Stimulation of Mg Chelatase Activity in Angiosperms.

GENOMES UNCOUPLED 4 (GUN4) is a positive regulator of light-dependent chlorophyll biosynthesis. GUN4 activates Mg chelatase (MgCh) that catalyzes the insertion of an Mg2+ ion into protoporphyrin IX. We show that Arabidopsis (Arabidopsis thaliana) GUN4 is phosphorylated at Ser 264 (S264), the penultimate amino acid residue at the C terminus. While GUN4 is preferentially phosphorylated in darkness, phosphorylation is reduced upon accumulation of Mg porphyrins. Expression of a phosphomimicking GUN4(S264D) results in an incomplete complementation of the white gun4-2 null mutant and a chlorotic phenotype comparable to gun4 knockdown mutants. Phosphorylated GUN4 has a reduced stimulatory effect on MgCh in vitro and in vivo but retains its protein stability and tetrapyrrole binding capacity. Analysis of GUN4 found in oxygenic photosynthetic organisms reveals the evolution of a C-terminal extension, which harbors the phosphorylation site of GUN4 expressed in angiosperms. Homologs of GUN4 from Synechocystis and Chlamydomonas lack the conserved phosphorylation site found in a C-terminal extension of angiosperm GUN4. Biochemical studies proved the importance of the C-terminal extension for MgCh stimulation and inactivation of GUN4 by phosphorylation in angiosperms. An additional mechanism regulating MgCh activity is proposed. In conjunction with the dark repression of 5-aminolevulinic acid synthesis, GUN4 phosphorylation minimizes the flow of intermediates into the Mg branch of the tetrapyrrole metabolic pathway for chlorophyll biosynthesis.

Ursgal, Universal Python Module Combining Common Bottom-Up Proteomics Tools for Large-Scale Analysis.

Proteomics data integration has become a broad field with a variety of programs offering innovative algorithms to analyze increasing amounts of data. Unfortunately, this software diversity leads to many problems as soon as the data is analyzed using more than one algorithm for the same task. Although it was shown that the combination of multiple peptide identification algorithms yields more robust results, it is only recently that unified approaches are emerging; however, workflows that, for example, aim to optimize search parameters or that employ cascaded style searches can only be made accessible if data analysis becomes not only unified but also and most importantly scriptable. Here we introduce Ursgal, a Python interface to many commonly used bottom-up proteomics tools and to additional auxiliary programs. Complex workflows can thus be composed using the Python scripting language using a few lines of code. Ursgal is easily extensible, and we have made several database search engines (X!Tandem, OMSSA, MS-GF+, Myrimatch, MS Amanda), statistical postprocessing algorithms (qvality, Percolator), and one algorithm that combines statistically postprocessed outputs from multiple search engines ("combined FDR") accessible as an interface in Python. Furthermore, we have implemented a new algorithm ("combined PEP") that combines multiple search engines employing elements of "combined FDR", PeptideShaker, and Bayes' theorem.

STATE TRANSITION7-Dependent Phosphorylation Is Modulated by Changing Environmental Conditions, and Its Absence Triggers Remodeling of Photosynthetic Protein Complexes.

In plants and algae, the serine/threonine kinase STN7/STT7, orthologous protein kinases in Chlamydomonas reinhardtii and Arabidopsis (Arabidopsis thaliana), respectively, is an important regulator in acclimation to changing light environments. In this work, we assessed STT7-dependent protein phosphorylation under high light in C. reinhardtii, known to fully induce the expression of light-harvesting complex stress-related protein3 (LHCSR3) and a nonphotochemical quenching mechanism, in relationship to anoxia where the activity of cyclic electron flow is stimulated. Our quantitative proteomics data revealed numerous unique STT7 protein substrates and STT7-dependent protein phosphorylation variations that were reliant on the environmental condition. These results indicate that STT7-dependent phosphorylation is modulated by the environment and point to an intricate chloroplast phosphorylation network responding in a highly sensitive and dynamic manner to environmental cues and alterations in kinase function. Functionally, the absence of the STT7 kinase triggered changes in protein expression and photoinhibition of photosystem I (PSI) and resulted in the remodeling of photosynthetic complexes. This remodeling initiated a pronounced association of LHCSR3 with PSI-light harvesting complex I (LHCI)-ferredoxin-NADPH oxidoreductase supercomplexes. Lack of STT7 kinase strongly diminished PSII-LHCII supercomplexes, while PSII core complex phosphorylation and accumulation were significantly enhanced. In conclusion, our study provides strong evidence that the regulation of protein phosphorylation is critical for driving successful acclimation to high light and anoxic growth environments and gives new insights into acclimation strategies to these environmental conditions.

The interplay of light and oxygen in the reactive oxygen stress response of Chlamydomonas reinhardtii dissected by quantitative mass spectrometry.

Light and oxygen are factors that are very much entangled in the reactive oxygen species (ROS) stress response network in plants, algae and cyanobacteria. The first obligatory step in understanding the ROS network is to separate these responses. In this study, a LC-MS/MS based quantitative proteomic approach was used to dissect the responses of Chlamydomonas reinhardtii to ROS, light and oxygen employing an interlinked experimental setup. Application of novel bioinformatics tools allow high quality retention time alignment to be performed on all LC-MS/MS runs increasing confidence in protein quantification, overall sequence coverage and coverage of all treatments measured. Finally advanced hierarchical clustering yielded 30 communities of co-regulated proteins permitting separation of ROS related effects from pure light effects (induction and repression). A community termed redox(II) was identified that shows additive effects of light and oxygen with light as the first obligatory step. Another community termed 4-down was identified that shows repression as an effect of light but only in the absence of oxygen indicating ROS regulation, for example, possibly via product feedback inhibition because no ROS damage is occurring. In summary the data demonstrate the importance of separating light, O2 and ROS responses to define marker genes for ROS responses. As revealed in this study, an excellent candidate is DHAR with strong ROS dependent induction profiles.

The Vac14-interaction network is linked to regulators of the endolysosomal and autophagic pathway.

The scaffold protein Vac14 acts in a complex with the lipid kinase PIKfyve and its counteracting phosphatase FIG4, regulating the interconversion of phosphatidylinositol-3-phosphate to phosphatidylinositol-3,5-bisphosphate. Dysfunctional Vac14 mutants, a deficiency of one of the Vac14 complex components, or inhibition of PIKfyve enzymatic activity results in the formation of large vacuoles in cells. How these vacuoles are generated and which processes are involved are only poorly understood. Here we show that ectopic overexpression of wild-type Vac14 as well as of the PIKfyve-binding deficient Vac14 L156R mutant causes vacuoles. Vac14-dependent vacuoles and PIKfyve inhibitor-dependent vacuoles resulted in elevated levels of late endosomal, lysosomal, and autophagy-associated proteins. However, only late endosomal marker proteins were bound to the membranes of these enlarged vacuoles. In order to decipher the linkage between the Vac14 complex and regulators of the endolysosomal pathway, a protein affinity approach combined with multidimensional protein identification technology was conducted, and novel molecular links were unraveled. We found and verified the interaction of Rab9 and the Rab7 GAP TBC1D15 with Vac14. The identified Rab-related interaction partners support the theory that the regulation of vesicular transport processes and phosphatidylinositol-modifying enzymes are tightly interconnected.

pyGCluster, a novel hierarchical clustering approach.

SUMMARY: pyGCluster is a clustering algorithm focusing on noise injection for subsequent cluster validation. The reproducibility of a large amount of clusters obtained with agglomerative hierarchical clustering is assessed. Furthermore, a multitude of different distance-linkage combinations are evaluated. Finally, highly reproducible clusters are meta-clustered into communities. Graphical illustration of the results as node and expression maps is implemented. AVAILABILITY AND IMPLEMENTATION: pyGCluster requires Python 2.7, it is freely available at http://pyGCluster.github.io and published under MIT license. Dependencies are NumPy, SciPy and optionally fastcluster and rpy2. CONTACT: christan@fufezan.net SUPPLEMENTARY INFORMATION: Supplementary data is available at Bioinformatics online and at http://pyGCluster.github.io.

Proton Gradient Regulation5-Like1-Mediated Cyclic Electron Flow Is Crucial for Acclimation to Anoxia and Complementary to Nonphotochemical Quenching in Stress Adaptation.

To investigate the functional importance of Proton Gradient Regulation5-Like1 (PGRL1) for photosynthetic performances in the moss Physcomitrella patens, we generated a pgrl1 knockout mutant. Functional analysis revealed diminished nonphotochemical quenching (NPQ) as well as decreased capacity for cyclic electron flow (CEF) in pgrl1. Under anoxia, where CEF is induced, quantitative proteomics evidenced severe down-regulation of photosystems but up-regulation of the chloroplast NADH dehydrogenase complex, plastocyanin, and Ca2+ sensors in the mutant, indicating that the absence of PGRL1 triggered a mechanism compensatory for diminished CEF. On the other hand, proteins required for NPQ, such as light-harvesting complex stress-related protein1 (LHCSR1), violaxanthin de-epoxidase, and PSII subunit S, remained stable. To further investigate the interrelation between CEF and NPQ, we generated a pgrl1 npq4 double mutant in the green alga Chlamydomonas reinhardtii lacking both PGRL1 and LHCSR3 expression. Phenotypic comparative analyses of this double mutant, together with the single knockout strains and with the P. patens pgrl1, demonstrated that PGRL1 is crucial for acclimation to high light and anoxia in both organisms. Moreover, the data generated for the C. reinhardtii double mutant clearly showed a complementary role of PGRL1 and LHCSR3 in managing high light stress response. We conclude that both proteins are needed for photoprotection and for survival under low oxygen, underpinning a tight link between CEF and NPQ in oxygenic photosynthesis. Given the complementarity of the energy-dependent component of NPQ (qE) and PGRL1-mediated CEF, we suggest that PGRL1 is a capacitor linked to the evolution of the PSII subunit S-dependent qE in terrestrial plants.

The metabolic status drives acclimation of iron deficiency responses in Chlamydomonas reinhardtii as revealed by proteomics based hierarchical clustering and reverse genetics.

Iron is a crucial cofactor in numerous redox-active proteins operating in bioenergetic pathways including respiration and photosynthesis. Cellular iron management is essential to sustain sufficient energy production and minimize oxidative stress. To produce energy for cell growth, the green alga Chlamydomonas reinhardtii possesses the metabolic flexibility to use light and/or carbon sources such as acetate. To investigate the interplay between the iron-deficiency response and growth requirements under distinct trophic conditions, we took a quantitative proteomics approach coupled to innovative hierarchical clustering using different "distance-linkage combinations" and random noise injection. Protein co-expression analyses of the combined data sets revealed insights into cellular responses governing acclimation to iron deprivation and regulation associated with photosynthesis dependent growth. Photoautotrophic growth requirements as well as the iron deficiency induced specific metabolic enzymes and stress related proteins, and yet differences in the set of induced enzymes, proteases, and redox-related polypeptides were evident, implying the establishment of distinct response networks under the different conditions. Moreover, our data clearly support the notion that the iron deficiency response includes a hierarchy for iron allocation within organelles in C. reinhardtii. Importantly, deletion of a bifunctional alcohol and acetaldehyde dehydrogenase (ADH1), which is induced under low iron based on the proteomic data, attenuates the remodeling of the photosynthetic machinery in response to iron deficiency, and at the same time stimulates expression of stress-related proteins such as NDA2, LHCSR3, and PGRL1. This finding provides evidence that the coordinated regulation of bioenergetics pathways and iron deficiency response is sensitive to the cellular and chloroplast metabolic and/or redox status, consistent with systems approach data.

Exploring the N-glycosylation pathway in Chlamydomonas reinhardtii unravels novel complex structures.

Chlamydomonas reinhardtii is a green unicellular eukaryotic model organism for studying relevant biological and biotechnological questions. The availability of genomic resources and the growing interest in C. reinhardtii as an emerging cell factory for the industrial production of biopharmaceuticals require an in-depth analysis of protein N-glycosylation in this organism. Accordingly, we used a comprehensive approach including genomic, glycomic, and glycoproteomic techniques to unravel the N-glycosylation pathway of C. reinhardtii. Using mass-spectrometry-based approaches, we found that both endogenous soluble and membrane-bound proteins carry predominantly oligomannosides ranging from Man-2 to Man-5. In addition, minor complex N-linked glycans were identified as being composed of partially 6-O-methylated Man-3 to Man-5 carrying one or two xylose residues. These findings were supported by results from a glycoproteomic approach that led to the identification of 86 glycoproteins. Here, a combination of in-source collision-induced dissodiation (CID) for glycan fragmentation followed by mass tag-triggered CID for peptide sequencing and PNGase F treatment of glycopeptides in the presence of (18)O-labeled water in conjunction with CID mass spectrometric analyses were employed. In conclusion, our data support the notion that the biosynthesis and maturation of N-linked glycans in the endoplasmic reticulum and Golgi apparatus occur via a GnT I-independent pathway yielding novel complex N-linked glycans that maturate differently from their counterparts in land plants.

Calcium-dependent regulation of cyclic photosynthetic electron transfer by a CAS, ANR1, and PGRL1 complex.

Cyclic photosynthetic electron flow (CEF) is crucial to photosynthesis because it participates in the control of chloroplast energy and redox metabolism, and it is particularly induced under adverse environmental conditions. Here we report that down-regulation of the chloroplast localized Ca(2+) sensor (CAS) protein by an RNAi approach in Chlamydomonas reinhardtii results in strong inhibition of CEF under anoxia. Importantly, this inhibition is rescued by an increase in the extracellular Ca(2+) concentration, inferring that CEF is Ca(2+)-dependent. Furthermore, we identified a protein, anaerobic response 1 (ANR1), that is also required for effective acclimation to anaerobiosis. Depletion of ANR1 by artificial microRNA expression mimics the CAS-depletion phenotype, and under anaerobic conditions the two proteins coexist within a large active photosystem I-cytochrome b(6)/f complex. Moreover, we provide evidence that CAS and ANR1 interact with each other as well as with PGR5-Like 1 (PGRL1) in vivo. Overall our data establish a Ca(2+)-dependent regulation of CEF via the combined function of ANR1, CAS, and PGRL1, associated with each other in a multiprotein complex.