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Abstract: The generation of novel subsets of phosphorylation sites is needed to complement the present Arabidopsis plasma membrane phosphoprotein repertoire, where several families of proteins are under-represented. In this work, different combinations of chromatographic steps were first compared for capacity to resolve model phosphopeptides before characterisation from PSD fragments in MALDI MS/MS. Nearly half of the phosphorylation sites detected in the Arabidopsis plasmalemma using the optimised procedure were novel, and two-thirds of protein accessions identified secondary active transporters. These included phosphate/H+ symporters, ammonium and nitrate transporters, different alkali cation exchangers, a urea/H+ symporter, a glucose transporter, a purine permease, and peptide transporters. There has been previous functional evidence for phosphorylation of only a minority of these, the regulation of others having been essentially investigated at the transcriptional level. The demonstration of active phosphorylation sites in such a diverse set of secondary transporter families suggests that this regulation level plays a major role in the response of plants to nutrient availability. [Copyright &y& Elsevier]

Tsetse flies (Glossina spp.) are major vectors of African trypanosomes, causing either Human or Animal African Trypanosomiasis (HAT or AAT). Several approaches have been developed to control the disease, among which is the anti‐vector Sterile Insect Technique. Another approach to anti‐vector strategies could consist of controlling the fly's vector competence through hitherto unidentified regulatory factors (genes, proteins, biological pathways, etc.). The present work aims to evaluate the protein abundance in the midgut of wild tsetse flies (Glossina palpalis palpalis) naturally infected by Trypanosoma congolense s.l. Infected and non‐infected flies were sampled in two HAT/AAT foci in Southern Cameroon. After dissection, the proteomes from the guts of parasite‐infected flies were compared to that of uninfected flies to identify quantitative and/or qualitative changes associated with infection. Among the proteins with increased abundance were fructose‐1,6‐biphosphatase, membrane trafficking proteins, death proteins (or apoptosis proteins) and SERPINs (inhibitor of serine proteases, enzymes considered as trypanosome virulence factors) that displayed the highest increased abundance. The present study, together with previous proteomic and transcriptomic studies on the secretome of trypanosomes from tsetse fly gut extracts, provides data to be explored in further investigations on, for example, mammal host immunisation or on fly vector competence modification via para‐transgenic approaches. [ABSTRACT FROM AUTHOR]

The hydraulic conductivity of plant roots ( Lpr) is determined in large part by the activity of aquaporins. Mechanisms occurring at the post-translational level, in particular phosphorylation of aquaporins of the plasma membrane intrinsic protein 2 ( PIP2) subfamily, are thought to be of critical importance for regulating root water transport. However, knowledge of protein kinases and phosphatases acting on aquaporin function is still scarce. In the present work, we investigated the Lpr of knockout A rabidopsis plants for four Ca2+-dependent protein kinases. cpk7 plants showed a 30% increase in Lpr because of a higher aquaporin activity. A quantitative proteomic analysis of wild-type and cpk7 plants revealed that PIP gene expression and PIP protein quantity were not correlated and that CPK7 has no effect on PIP2 phosphorylation. In contrast, CPK7 exerts a negative control on the cellular abundance of PIP1s, which likely accounts for the higher Lpr of cpk7. In addition, this study revealed that the cellular amount of a few additional proteins including membrane transporters is controlled by CPK7. The overall work provides evidence for CPK7-dependent stability of specific membrane proteins. [ABSTRACT FROM AUTHOR]

Osmotic stress can be detrimental to plants, whose survival relies heavily on proteomic plasticity. Protein ubiquitination is a central post-translational modification in osmotic-mediated stress. In this study, we used the K-Ɛ-GG antibody enrichment method integrated with high-resolution mass spectrometry to compile a list of 719 ubiquitinated lysine (K-Ub) residues from 450 Arabidopsis root membrane proteins (58% of which are transmembrane proteins), thereby adding to the database of ubiquitinated substrates in plants. Although no ubiquitin (Ub) motifs could be identified, the presence of acidic residues close to K-Ub was revealed. Our ubiquitinome analysis pointed to a broad role of ubiquitination in the internalization and sorting of cargo proteins. Moreover, the simultaneous proteome and ubiquitinome quantification showed that ubiquitination is mostly not involved in membrane protein degradation in response to short osmotic treatment but that it is putatively involved in protein internalization, as described for the aquaporin PIP2;1. Our in silico analysis of ubiquitinated proteins shows that two E2 Ub-conjugating enzymes, UBC32 and UBC34, putatively target membrane proteins under osmotic stress. Finally, we revealed a positive role for UBC32 and UBC34 in primary root growth under osmotic stress. [ABSTRACT FROM AUTHOR]

Abstract: The study of phosphoproteome on a global scale represents one of the challenges in the post-genomic era. Here, we propose an integrated procedure starting from the crude protein extract, that consists of sequential purification steps, and ending up in the identification of phosphorylation sites. This involves (i) an enrichment in phosphoproteins with a commercially available chromatography matrix, (ii) a 2-D gel analysis of the enriched fraction followed by the selective staining with the phosphospecific fluorescent dye Pro-Q Diamond, (iii) a phosphopeptide capture, from the tryptic lysate of 2-D spots, using IMAC micro-columns. In the end, the identification of the phosphoproteins and their corresponding phosphorylation sites were achieved by MALDI-TOF-TOF spectrometry. The method was applied to contrasting samples prepared from cell suspension cultures of Arabidopsis thaliana and roots of Medicago truncatula. The results obtained, demonstrated the robustness of the combination of two enrichment stages, sequentially at the protein and at the peptide levels, to analyse phosphoproteins in plants. [Copyright &y& Elsevier]

Iron-sulfur (Fe-S) clusters are one of the most ancient and ubiquitous prosthetic groups, and they are required by a variety of proteins involved in important metabolic processes. Apicomplexan parasites have inherited different plastidic and mitochondrial Fe-S clusters biosynthesis pathways through endosymbiosis. We have investigated the relative contributions of these pathways to the fitness of Toxoplasma gondii, an apicomplexan parasite causing disease in humans, by generating specific mutants. Phenotypic analysis and quantitative proteomics allowed us to highlight notable differences in these mutants. Both Fe-S cluster synthesis pathways are necessary for optimal parasite growth in vitro, but their disruption leads to markedly different fates: impairment of the plastidic pathway leads to a loss of the organelle and to parasite death, while disruption of the mitochondrial pathway trigger differentiation into a stress resistance stage. This highlights that otherwise similar biochemical pathways hosted by different sub-cellular compartments can have very different contributions to the biology of the parasites, which is something to consider when exploring novel strategies for therapeutic intervention. Author summary: Toxoplasma gondii is a ubiquitous unicellular parasite that harbours two organelles of endosymbiotic origin: the mitochondrion, and a relict plastid named the apicoplast. Each one of these organelles contains its own machinery for synthesizing iron-sulfur clusters, which are important protein co-factors. In this study, we show that interfering with either the mitochondrial or the plastidic iron-sulfur cluster synthesizing machinery has a profound impact on parasite growth. However, while disrupting the plastidic pathway led to an irreversible loss of the organelle and subsequent death of the parasite, disrupting the mitochondrial pathway led to conversion of the parasites into a stress resistance form. We used a comparative quantitative proteomic analysis of the mutants, combined with experimental validation, to provide mechanistic clues into these different phenotypic outcomes. Although the consequences of disrupting each pathway were manifold, our data highlighted potential changes at the metabolic level. For instance, the plastidic iron-sulfur cluster synthesis pathway may be important for maintaining the lipid homeostasis of the parasites, while the mitochondrial pathway is likely crucial for maintaining their respiratory capacity. Interestingly, we have discovered that other mutants severely impacted for mitochondrial function, in particular the respiratory chain, are able to survive and initiate conversion to the stress resistance form. This illustrates a different capacity for T. gondii to adapt for survival in response to distinct metabolic dysregulations. [ABSTRACT FROM AUTHOR]

Summary: Iron (Fe) is a major micronutrient and is required for plant growth and development. Nongrass species have evolved a reduction‐based strategy to solubilize and take up Fe. The secretion of Fe‐mobilizing coumarins (e.g. fraxetin, esculetin and sideretin) by plant roots plays an important role in this process. Although the biochemical mechanisms leading to their biosynthesis have been well described, very little is known about their cellular and subcellular localization or their mobility within plant tissues.Spectral imaging was used to monitor, in Arabidopsis thaliana, the in planta localization of Fe‐mobilizing coumarins and scopolin. Molecular, genetic and biochemical approaches were also used to investigate the dynamics of coumarin accumulation in roots.These approaches showed that root hairs play a major role in scopoletin secretion, whereas fraxetin and esculetin secretion occurs through all epidermis cells. The findings of this study also showed that the transport of coumarins from the cortex to the rhizosphere relies on the PDR9 transporter under Fe‐deficient conditions. Additional experiments support the idea that coumarins move throughout the plant body via the xylem sap and that several plant species can take up coumarins present in the surrounding media.Altogether, the data presented here demonstrate that coumarin storage and accumulation in roots is a highly complex and dynamic process. [ABSTRACT FROM AUTHOR]

Summary: In Arabidopsis thaliana, NRT2.1 codes for a main component of the root nitrate high‐affinity transport system. Previous studies revealed that post‐translational regulation of NRT2.1 plays an important role in the control of root nitrate uptake and that one mechanism could correspond to NRT2.1 C‐terminus processing.To further investigate this hypothesis, we produced transgenic plants with truncated forms of NRT2.1. This revealed an essential sequence for NRT2.1 activity, located between the residues 494 and 513.Using a phospho‐proteomic approach, we found that this sequence contains one phosphorylation site, at serine 501, which can inactivate NRT2.1 function when mimicking the constitutive phosphorylation of this residue in transgenic plants. This phenotype could neither be explained by changes in abundance of NRT2.1 and NAR2.1, a partner protein of NRT2.1, nor by a lack of interaction between these two proteins.Finally, the relative level of serine 501 phosphorylation was found to be increased by ammonium nitrate in wild‐type plants, leading to the inactivation of NRT2.1 and to a decrease in high affinity nitrate transport into roots. Altogether, these observations reveal a new and essential mechanism for the regulation of NRT2.1 activity. See also the Commentary on this article by Rahikainen & Kangasjärvi (2020), 228: 802–804. [ABSTRACT FROM AUTHOR]

In contrast to desiccation-tolerant 'orthodox' seeds, so-called 'intermediate' seeds cannot survive complete drying and are short-lived. All species of the genus Coffea produce intermediate seeds, but they show a considerable variability in seed desiccation tolerance (DT), which may help to decipher the molecular basis of seed DT in plants. We performed a comparative transcriptome analysis of developing seeds in three coffee species with contrasting desiccation tolerance. Seeds of all species shared a major transcriptional switch during late maturation that governs a general slow-down of metabolism. However, numerous key stress-related genes, including those coding for the late embryogenesis abundant protein EM6 and the osmosensitive calcium channel ERD4, were up-regulated during DT acquisition in the two species with high seed DT, C. arabica and C. eugenioides. By contrast, we detected up-regulation of numerous genes involved in the metabolism, transport, and perception of auxin in C. canephora seeds with low DT. Moreover, species with high DT showed a stronger down-regulation of the mitochondrial machinery dedicated to the tricarboxylic acid cycle and oxidative phosphorylation. Accordingly, respiration measurements during seed dehydration demonstrated that intermediate seeds with the highest DT are better prepared to cease respiration and avoid oxidative stresses. [ABSTRACT FROM AUTHOR]

Antigen Ki-67 is a nuclear protein expressed in proliferating mammalian cells. It is widely used in cancer histopathology but its functions remain unclear. Here, we show that Ki-67 controls heterochromatin organisation. Altering Ki-67 expression levels did not significantly affect cell proliferation in vivo. Ki-67 mutant mice developed normally and cells lacking Ki-67 proliferated efficiently. Conversely, upregulation of Ki-67 expression in differentiated tissues did not prevent cell cycle arrest. Ki-67 interactors included proteins involved in nucleolar processes and chromatin regulators. Ki-67 depletion disrupted nucleologenesis but did not inhibit pre-rRNA processing. In contrast, it altered gene expression. Ki-67 silencing also had wide-ranging effects on chromatin organisation, disrupting heterochromatin compaction and long-range genomic interactions. Trimethylation of histone H3K9 and H4K20 was relocalised within the nucleus. Finally, overexpression of human or Xenopus Ki-67 induced ectopic heterochromatin formation. Altogether, our results suggest that Ki-67 expression in proliferating cells spatially organises heterochromatin, thereby controlling gene expression. [ABSTRACT FROM AUTHOR]

Nutritional symbiotic interactions require the housing of large numbers of microbial symbionts, which produce essential compounds for the growth of the host. In the legume-rhizobium nitrogen-fixing symbiosis, thousands of rhizobium microsymbionts, called bacteroids, are confined intracellularly within highly specialized symbiotic host cells. In Inverted Repeat-Lacking Clade (IRLC) legumes such as Medicago spp., the bacteroids are kept under control by an arsenal of nodulespecific cysteine-rich (NCR) peptides, which induce the bacteria in an irreversible, strongly elongated, and polyploid state. Here, we show that in Aeschynomene spp. legumes belonging to the more ancient Dalbergioid lineage, bacteroids are elongated or spherical depending on the Aeschynomene spp. and that these bacteroids are terminally differentiated and polyploid, similar to bacteroids in IRLC legumes. Transcriptome, in situ hybridization, and proteome analyses demonstrated that the symbiotic cells in the Aeschynomene spp. nodules produce a large diversity of NCR-like peptides, which are transported to the bacteroids. Blocking NCR transport by RNA interference-mediated inactivation of the secretory pathway inhibits bacteroid differentiation. Together, our results support the view that bacteroid differentiation in the Dalbergioid clade, which likely evolved independently from the bacteroid differentiation in the IRLC clade, is based on very similar mechanisms used by IRLC legumes. [ABSTRACT FROM AUTHOR]

The aim of this study was to assess whether endosperm-specific carotenoid biosynthesis influenced core metabolic processes in maize embryo and endosperm and how global seed metabolism adapted to this expanded biosynthetic capacity. Although enhancement of carotenoid biosynthesis was targeted to the endosperm of maize kernels, a concurrent up-regulation of sterol and fatty acid biosynthesis in the embryo was measured. Targeted terpenoid analysis, and non-targeted metabolomic, proteomic, and transcriptomic profiling revealed changes especially in carbohydrate metabolism in the transgenic line. In-depth analysis of the data, including changes of metabolite pools and increased enzyme and transcript concentrations, gave a first insight into the metabolic variation precipitated by the higher upstream metabolite demand by the extended biosynthesis capacities for terpenoids and fatty acids. An integrative model is put forward to explain the metabolic regulation for the increased provision of terpenoid and fatty acid precursors, particularly glyceraldehyde 3-phosphate and pyruvate or acetyl-CoA from imported fructose and glucose. The model was supported by higher activities of fructokinase, glucose 6-phosphate isomerase, and fructose 1,6-bisphosphate aldolase indicating a higher flux through the glycolytic pathway. Although pyruvate and acetyl-CoA utilization was higher in the engineered line, pyruvate kinase activity was lower. A sufficient provision of both metabolites may be supported by a by-pass in a reaction sequence involving phosphoenolpyruvate carboxylase, malate dehydrogenase, and malic enzyme. [ABSTRACT FROM AUTHOR]

During its life cycle, the protozoan pathogen L eishmania donovani is exposed to contrasting environments inside insect vector and vertebrate host, to which the parasite must adapt for extra- and intracellular survival. Combining null mutant analysis with phosphorylation site-specific mutagenesis and functional complementation we genetically tested the requirement of the L . donovani chaperone cyclophilin 40 ( LdCyP40) for infection. Targeted replacement of LdCyP40 had no effect on parasite viability, axenic amastigote differentiation, and resistance to various forms of environmental stress in culture, suggesting important functional redundancy to other parasite chaperones. However, ultrastructural analyses and video microscopy of cyp40−/− promastigotes uncovered important defects in cell shape, organization of the subpellicular tubulin network and motility at stationary growth phase. More importantly, cyp40−/− parasites were unable to establish intracellular infection in murine macrophages and were eliminated during the first 24 h post infection. Surprisingly, cyp40−/− infectivity was restored in complemented parasites expressing a CyP40 mutant of the unique S274 phosphorylation site. Together our data reveal non-redundant CyP40 functions in parasite cytoskeletal remodelling relevant for the development of infectious parasites in vitro independent of its phosphorylation status, and provide a framework for the genetic analysis of L eishmania-specific phosphorylation sites and their role in regulating parasite protein function. [ABSTRACT FROM AUTHOR]

The water status of plant leaves depends on the efficiency of the water supply, from the vasculature to inner tissues. This process is under hormonal and environmental regulation and involves aquaporin water channels. In Arabidopsis thaliana , the rosette hydraulic conductivity (K  ros) is higher in darkness than it is during the day. Knockout plants showed that three plasma membrane intrinsic proteins (PIPs) sharing expression in veins (PIP1;2, PIP2;1, and PIP2;6) contribute to rosette water transport, and PIP2;1 can fully account for K  ros responsiveness to darkness. Directed expression of PIP2;1 in veins of a pip2;1 mutant was sufficient to restore K  ros. In addition, a positive correlation, in both wild-type and PIP2;1 -overexpressing plants, was found between K  ros and the osmotic water permeability of protoplasts from the veins but not from the mesophyll. Thus, living cells in veins form a major hydraulic resistance in leaves. Quantitative proteomic analyses showed that light-dependent regulation of K  ros is linked to diphosphorylation of PIP2;1 at Ser-280 and Ser-283. Expression in pip2;1 of phosphomimetic and phosphorylation-deficient forms of PIP2;1 demonstrated that phosphorylation at these two sites is necessary for K  ros enhancement under darkness. These findings establish how regulation of a single aquaporin isoform in leaf veins critically determines leaf hydraulics. [ABSTRACT FROM AUTHOR]

Summary: Accurate chromosome segregation relies upon a mitotic checkpoint that monitors kinetochore attachment toward opposite spindle poles before enabling chromosome disjunction []. The MPS1/TTK protein kinase is a core component of the mitotic checkpoint that lies upstream of MAD2 and BubR1 both at the kinetochore and in the cytoplasm []. To gain insight into the mechanisms underlying the regulation of MPS1 kinase, we undertook the identification of Xenopus MPS1 phosphorylation sites by mass spectrometry. We mapped several phosphorylation sites onto MPS1 and we show that phosphorylation of S283 in the noncatalytic region of MPS1 is required for full kinase activity. This phosphorylation potentiates MPS1 catalytic efficiency without impairing its affinity for the substrates. By using Xenopus egg extracts depleted of endogenous MPS1 and reconstituted with single point mutants, we show that phosphorylation of S283 is essential to activate the mitotic checkpoint. This phosphorylation does not regulate the localization of MPS1 to the kinetochore but is required for the recruitment of MAD1/MAD2, demonstrating its role at the kinetochore. Constitutive phosphorylation of S283 lowers the number of kinetochores required to hold the checkpoint, which suggests that CDK-dependent phosphorylation of MPS1 is essential to sustain the mitotic checkpoint when few kinetochores remain unattached. [Copyright &y& Elsevier]

Background: Genome-wide statistics established that long intrinsically disordered regions (over 30 residues) are predicted in a large part of proteins in all eukaryotes, with a higher ratio in trans-membrane proteins. At functional level, such unstructured and flexible regions were suggested for years to favour phosphorylation events. In plants, despite increasing evidence of the regulation of transport and signalling processes by phosphorylation events, only few data are available without specific information regarding plasma membrane proteins, especially at proteome scale. Results: Using a dedicated phosphoproteomic workflow, 75 novel and unambiguous phosphorylation sites were identified in Arabidopsis plasma membrane. Bioinformatics analysis showed that this new dataset concerned mostly integral proteins involved in key functions of the plasma membrane (such as transport and signal transduction, including protein phosphorylation). It thus expanded by 15% the directory of phosphosites previously characterized in signalling and transport proteins. Unexpectedly, 66% of phosphorylation sites were predicted to be located outside long intrinsically disordered regions. This result was further corroborated by analysis of publicly available data for the plasma membrane. Conclusions: The new phosphoproteomics data presented here, with published datasets and functional annotation, suggest a previously unexpected topology of phosphorylation in the plant plasma membrane proteins. The significance of these new insights into the so far overlooked properties of the plant plasma membrane phosphoproteome and the long disordered regions is discussed. [ABSTRACT FROM AUTHOR]

Leishmania is exposed to a sudden increase in environmental temperature during the infectious cycle that triggers stage differentiation and adapts the parasite phenotype to intracellular survival in the mammalian host. The absence of classical promoter-dependent mechanisms of gene regulation and constitutive expression of most of the heat-shock proteins (HSPs) in these human pathogens raise important unresolved questions as to regulation of the heat-shock response and stage-specific functions of Leishmania HSPs. Here we used a gel-based quantitative approach to assess the Leishmania donovani phosphoproteome and revealed that 38% of the proteins showed significant stage-specific differences, with a strong focus of amastigote-specific phosphoproteins on chaperone function. We identified Sill/HOP-containing chaperone complexes that interact with ribosomal client proteins in an amastigote-specific manner. Genetic analysis of STI1/HOP phosphorylation sites in conditional sti1-/- null mutant parasites revealed two phosphoserine residues essential for parasite viability. Phosphorylation of the major Leishmania chaperones at the pathogenic stage suggests that these proteins may be promising drug targets via inhibition of their respective protein kinases. [ABSTRACT FROM AUTHOR]

Besides the direct effects of radiations, indirect effects are observed within the surrounding non-irradiated area; irradiated cells relay stress signals in this close proximity, inducing the so-called radiation-induced bystander effect. These signals received by neighboring unirradiated cells induce specific responses similar with those of direct irradiated cells. To understand the cellular response of bystander cells, we performed a 2D gel-based proteomic study of the chondrocytes receiving the conditioned medium of low-dose irradiated chondrosarcoma cells. The conditioned medium was directly analyzed by mass spectrometry in order to identify candidate bystander factors involved in the signal transmission. The proteomic analysis of the bystander chondrocytes highlighted 20 proteins spots that were significantly modified at low dose, implicating several cellular mechanisms, such as oxidative stress responses, cellular motility, and exosomes pathways. In addition, the secretomic analysis revealed that the abundance of 40 proteins in the conditioned medium of 0.1 Gy irradiated chondrosarcoma cells was significantly modified, as compared with the conditioned medium of non-irradiated cells. A large cluster of proteins involved in stress granules and several proteins involved in the cellular response to DNA damage stimuli were increased in the 0.1 Gy condition. Several of these candidates and cellular mechanisms were confirmed by functional analysis, such as 8-oxodG quantification, western blot, and wound-healing migration tests. Taken together, these results shed new lights on the complexity of the radiation-induced bystander effects and the large variety of the cellular and molecular mechanisms involved, including the identification of a new potential actor, namely the stress granules. [ABSTRACT FROM AUTHOR]