Cryo-EM structure of the polyphosphate polymerase VTC reveals coupling of polymer synthesis to membrane transit.

The eukaryotic vacuolar transporter chaperone (VTC) complex acts as a polyphosphate (polyP) polymerase that synthesizes polyP from adenosine triphosphate (ATP) and translocates polyP across the vacuolar membrane to maintain an intracellular phosphate (Pi ) homeostasis. To discover how the VTC complex performs its function, we determined a cryo-electron microscopy structure of an endogenous VTC complex (Vtc4/Vtc3/Vtc1) purified from Saccharomyces cerevisiae at 3.1 Å resolution. The structure reveals a heteropentameric architecture of one Vtc4, one Vtc3, and three Vtc1 subunits. The transmembrane region forms a polyP-selective channel, likely adopting a resting state conformation, in which a latch-like, horizontal helix of Vtc4 limits the entrance. The catalytic Vtc4 central domain is located on top of the pseudo-symmetric polyP channel, creating a strongly electropositive pathway for nascent polyP that can couple synthesis to translocation. The SPX domain of the catalytic Vtc4 subunit positively regulates polyP synthesis by the VTC complex. The noncatalytic Vtc3 regulates VTC through a phosphorylatable loop. Our findings, along with the functional data, allow us to propose a mechanism of polyP channel gating and VTC complex activation.

The ascorbate biosynthesis pathway in plants is known, but there is a way to go with understanding control and functions.

Ascorbate (vitamin C) is one of the most abundant primary metabolites in plants. Its complex chemistry enables it to function as an antioxidant, as a free radical scavenger, and as a reductant for iron and copper. Ascorbate biosynthesis occurs via the mannose/l-galactose pathway in green plants, and the evidence for this pathway being the major route is reviewed. Ascorbate accumulation is leaves is responsive to light, reflecting various roles in photoprotection. GDP-l-galactose phosphorylase (GGP) is the first dedicated step in the pathway and is important in controlling ascorbate synthesis. Its expression is determined by a combination of transcription and translation. Translation is controlled by an upstream open reading frame (uORF) which blocks translation of the main GGP-coding sequence, possibly in an ascorbate-dependent manner. GGP associates with a PAS-LOV protein, inhibiting its activity, and dissociation is induced by blue light. While low ascorbate mutants are susceptible to oxidative stress, they grow nearly normally. In contrast, mutants lacking ascorbate do not grow unless rescued by supplementation. Further research should investigate possible basal functions of ascorbate in severely deficient plants involving prevention of iron overoxidation in 2-oxoglutarate-dependent dioxygenases and iron mobilization during seed development and germination.

Single-cell visualization and quantification of trace metals in Chlamydomonas lysosome-related organelles.

The acidocalcisome is an acidic organelle in the cytosol of eukaryotes, defined by its low pH and high calcium and polyphosphate content. It is visualized as an electron-dense object by transmission electron microscopy (TEM) or described with mass spectrometry (MS)-based imaging techniques or multimodal X-ray fluorescence microscopy (XFM) based on its unique elemental composition. Compared with MS-based imaging techniques, XFM offers the additional advantage of absolute quantification of trace metal content, since sectioning of the cell is not required and metabolic states can be preserved rapidly by either vitrification or chemical fixation. We employed XFM in Chlamydomonas reinhardtii to determine single-cell and organelle trace metal quotas within algal cells in situations of trace metal overaccumulation (Fe and Cu). We found up to 70% of the cellular Cu and 80% of Fe sequestered in acidocalcisomes in these conditions and identified two distinct populations of acidocalcisomes, defined by their unique trace elemental makeup. We utilized the vtc1 mutant, defective in polyphosphate synthesis and failing to accumulate Ca, to show that Fe sequestration is not dependent on either. Finally, quantitation of the Fe and Cu contents of individual cells and compartments via XFM, over a range of cellular metal quotas created by nutritional and genetic perturbations, indicated excellent correlation with bulk data from corresponding cell cultures, establishing a framework to distinguish the nutritional status of single cells.

Fire-driven vegetation type conversion in Southern California.

One consequence of global change causing widespread concern is the possibility of ecosystem conversions from one type to another. A classic example of this is vegetation type conversion (VTC) from native woody shrublands to invasive annual grasslands in the biodiversity hotspot of Southern California. Although the significance of this problem is well recognized, understanding where, how much, and why this change is occurring remains elusive owing to differences in results from studies conducted using different methods, spatial extents, and scales. Disagreement has arisen particularly over the relative importance of short-interval fires in driving these changes. Chronosequence approaches that use space for time to estimate changes have produced different results than studies of changes at a site over time. Here we calculated the percentage woody and herbaceous cover across Southern California using air photos from ~1950 to 2019. We assessed the extent of woody cover change and the relative importance of fire history, topography, soil moisture, and distance to human infrastructure in explaining change across a hierarchy of spatial extents and regions. We found substantial net decline in woody cover and expansion of herbaceous vegetation across all regions, but the most dramatic changes occurred in the northern interior and southern coastal areas. Variables related to frequent, short-interval fire were consistently top ranked as the explanation for shrub to grassland type conversion, but low soil moisture and topographic complexity were also strong correlates. Despite the consistent importance of fire, there was substantial geographical variation in the relative importance of drivers, and these differences resulted in different mapped predictions of VTC. This geographical variation is important to recognize for management decision-making and, in addition to differences in methodological design, may also partly explain differences in previous study results. The overwhelming importance of short-interval fire has management implications. It suggests that actions should be directed away from imposing fires to preventing fires. Prevention can be controlled through management actions that limit ignitions, fire spread, and the damage sustained in areas that do burn. This study also demonstrates significant potential for changing fire regimes to drive large-scale, abrupt ecological change.

Model systems for studying polyphosphate biology: a focus on microorganisms.

Polyphosphates (polyP) are polymers of inorganic phosphates joined by high-energy bonds to form long chains. These chains are present in all forms of life but were once disregarded as 'molecular fossils'. PolyP has gained attention in recent years following new links to diverse biological roles ranging from energy storage to cell signaling. PolyP research in humans and other higher eukaryotes is limited by a lack of suitable tools and awaits the identification of enzymatic players that would enable more comprehensive studies. Therefore, many of the most important insights have come from single-cell model systems. Here, we review determinants of polyP metabolism, regulation, and function in major microbial systems, including bacteria, fungi, protozoa, and algae. We highlight key similarities and differences that may aid in our understanding of how polyP impacts cell physiology at a molecular level.

Ascorbic acid modulation by ABI4 transcriptional repression of VTC2 in the salt tolerance of Arabidopsis.

BACKGROUND: Abscisic acid (ABA) plays an important role in plant abiotic stress responses, and ABA INSENSITIVE 4 (ABI4) is a pivotal transcription factor in the ABA signaling pathway. In Arabidopsis, ABI4 negatively regulates salt tolerance; however, the mechanism through which ABI4 regulates plant salt tolerance is poorly understood. Our previous study showed that ABI4 directly binds to the promoter of the VITAMIN C DEFECTIVE 2 (VTC2) gene, inhibiting the transcription of VTC2 and ascorbic acid (AsA) biosynthesis. RESULTS: In the present study, we found that treatment with exogenous AsA could alleviate salt stress sensitivity of ABI4-overexpressing transgenic plants. The decreased AsA content and increased reactive oxygen species (ROS) levels in ABI4-overexpressing seedlings under salt treatment indicated that AsA-promoted ROS scavenging was related to ABI4-mediated salt tolerance. Gene expression analysis showed that ABI4 was induced at the early stage of salt stress, giving rise to reduced VTC2 expression. Accordingly, the abundance of the VTC2 protein decreased under the same salt stress conditions, and was absent in the ABI4 loss-of-function mutants, suggesting that the transcriptional inhibition of ABI4 on VTC2 resulted in the attenuation of VTC2 function. In addition, other encoding genes in the AsA biosynthesis and recycling pathways showed different responses to salt stress, demonstrating that AsA homeostasis is complicated under salinity stress. CONCLUSIONS: This study elucidates the negative modulation of ABI4 in salt stress tolerance through the regulation of AsA biosynthesis and ROS accumulation in plants.

Induction of S-nitrosoglutathione reductase protects root growth from ammonium toxicity by regulating potassium homeostasis in Arabidopsis and rice.

Ammonium (NH4+) is toxic to root growth in most plants already at moderate levels of supply, but mechanisms of root growth tolerance to NH4+ remain poorly understood. Here, we report that high levels of NH4+ induce nitric oxide (NO) accumulation, while inhibiting potassium (K+) acquisition via SNO1 (sensitive to nitric oxide 1)/SOS4 (salt overly sensitive 4), leading to the arrest of primary root growth. High levels of NH4+ also stimulated the accumulation of GSNOR (S-nitrosoglutathione reductase) in roots. GSNOR overexpression improved root tolerance to NH4+. Loss of GSNOR further induced NO accumulation, increased SNO1/SOS4 activity, and reduced K+ levels in root tissue, enhancing root growth sensitivity to NH4+. Moreover, the GSNOR-like gene, OsGSNOR, is also required for NH4+ tolerance in rice. Immunoblotting showed that the NH4+-induced GSNOR protein accumulation was abolished in the VTC1- (vitamin C1) defective mutant vtc1-1, which is hypersensititive to NH4+ toxicity. GSNOR overexpression enhanced vtc1-1 root tolerance to NH4+. Our findings suggest that induction of GSNOR increases NH4+ tolerance in Arabidopsis roots by counteracting NO-mediated suppression of tissue K+, which depends on VTC1 function.

Enzymes of Polyphosphate Metabolism in Yeast: Properties, Functions, Practical Significance.

Inorganic polyphosphates (polyP) are the linear polymers of orthophosphoric acid varying in the number of phosphate residues linked by the energy-rich phosphoanhydride bonds. PolyP is an essential component in living cells. Knowledge of polyP metabolizing enzymes in eukaryotes is necessary for understanding molecular mechanisms of polyP metabolism in humans and development of new approaches for treating bone and cardiovascular diseases associated with impaired mineral phosphorus metabolism. Yeast cells represent a rational experimental model for this research due to availability of the methods for studying phosphorus metabolism and construction of knockout mutants and strains overexpressing target proteins. Multicomponent system of polyP metabolism in Saccharomyces cerevisiae cells is presented in this review discussing properties, functioning, and practical significance of the enzymes involved in the synthesis and degradation of this important metabolite.

Objects seen as scenes: Neural circuitry for attending whole or parts.

Depending on our goals, we pay attention to the global shape of an object or to the local shape of its parts, since it's difficult to do both at once. This typically effortless process can be impaired in disease. However, it is not clear which cortical regions carry the information needed to constrain shape processing to a chosen global/local level. Here, novel stimuli were used to dissociate functional MRI responses to global and local shapes. This allowed identification of cortical regions containing information about level (independent from shape). Crucially, these regions overlapped part of the cortical network implicated in scene processing. As expected, shape information (independent of level) was mainly located in category-selective areas specialized for object- and face-processing. Regions with the same informational profile were strongly linked (as measured by functional connectivity), but were weak when the profiles diverged. Specifically, in the ventral-temporal-cortex (VTC) regions favoring level and shape were consistently separated by the mid-fusiform sulcus (MFS). These regions also had limited crosstalk despite their spatial proximity, thus defining two functional pathways within VTC. We hypothesize that object hierarchical level is processed by neural circuitry that also analyses spatial layout in scenes, contributing to the control of the spatial-scale used for shape recognition. Use of level information tolerant to shape changes could guide whole/part attentional selection but facilitate illusory shape/level conjunctions under impoverished vision.

From underlying chemistry to therapeutic potential: open questions in the new field of lysine polyphosphorylation.

Polyphosphorylation is a newly described non-enzymatic post-translational modification wherein long chains of inorganic phosphates are attached to lysine residues. The first targets of polyphosphorylation identified were S. cerevisiae proteins Nsr1 and Top1. Building on this theme, we recently exploited functional genomics tools in yeast to identify 15 new targets, including a conserved network of nucleolar proteins implicated in ribosome biogenesis. We also described the polyphosphorylation of six human proteins, suggesting that this unique post-translational modification could be conserved throughout eukaryotes. The study of polyphosphorylation seems poised to uncover novel modes of protein regulation in pathways spanning diverse biological processes. In this review, we establish a framework for future work by outlining critical questions related to the biochemistry of polyphosphorylation, its therapeutic potential, and everything in between.

Cardiac Computed Tomography (CT) Evaluation of Valvular Heart Disease in Transcatheter Interventions.

PURPOSE OF REVIEW: To establish the actual role of CT in the growing field of transcatheter valve interventions (THV). RECENT FINDINGS: The development of empirical CT measurements, which are linked with outcomes. CT is a reliable technique for assessing risk and planning transcatheter valve interventions for mitral and aortic valves. Pulmonic and tricuspid valve assessment with CT imaging is still in the early stages but there is room for development.

Two New Cytoarchitectonic Areas on the Human Mid-Fusiform Gyrus.

Areas of the fusiform gyrus (FG) within human ventral temporal cortex (VTC) process high-level visual information associated with faces, limbs, words, and places. Since classical cytoarchitectonic maps do not adequately reflect the functional and structural heterogeneity of the VTC, we studied the cytoarchitectonic segregation in a region, which is rostral to the recently identified cytoarchitectonic areas FG1 and FG2. Using an observer-independent and statistically testable parcellation method, we identify 2 new areas, FG3 and FG4, in 10 human postmortem brains on the mid-FG. The mid-fusiform sulcus reliably identifies the cytoarchitectonic transition between FG3 and FG4. We registered these cytoarchitectonic areas to the common reference space of the single-subject Montreal Neurological Institute (MNI) template and generated probability maps, which reflect the intersubject variability of both areas. Future studies can relate in vivo neuroimaging data with these microscopically defined cortical areas to functional parcellations. We discuss these results in the context of both large-scale functional maps and fine-scale functional clusters that have been identified within the human VTC. We propose that our observer-independent cytoarchitectonic parcellation of the FG better explains the functional heterogeneity of the FG compared with the homogeneity of classic cytoarchitectonic maps.

Regulation of ascorbate biosynthesis in green algae has evolved to enable rapid stress-induced response via the VTC2 gene encoding GDP-l-galactose phosphorylase.

Ascorbate (vitamin C) plays essential roles in stress resistance, development, signaling, hormone biosynthesis and regulation of gene expression; however, little is known about its biosynthesis in algae. In order to provide experimental proof for the operation of the Smirnoff-Wheeler pathway described for higher plants and to gain more information on the regulation of ascorbate biosynthesis in Chlamydomonas reinhardtii, we targeted the VTC2 gene encoding GDP-l-galactose phosphorylase using artificial microRNAs. Ascorbate concentrations in VTC2 amiRNA lines were reduced to 10% showing that GDP-l-galactose phosphorylase plays a pivotal role in ascorbate biosynthesis. The VTC2 amiRNA lines also grow more slowly, have lower chlorophyll content, and are more susceptible to stress than the control strains. We also demonstrate that: expression of the VTC2 gene is rapidly induced by H2 O2 and 1 O2 resulting in a manifold increase in ascorbate content; in contrast to plants, there is no circadian regulation of ascorbate biosynthesis; photosynthesis is not required per se for ascorbate biosynthesis; and Chlamydomonas VTC2 lacks negative feedback regulation by ascorbate in the physiological concentration range. Our work demonstrates that ascorbate biosynthesis is also highly regulated in Chlamydomonas albeit via mechanisms distinct from those previously described in land plants.

D27E mutation of VTC1 impairs the interaction with CSN5B and enhances ascorbic acid biosynthesis and seedling growth in Arabidopsis.

Our previous investigation revealed that GDP-Man pyrophosphorylase (VTC1), a vital ascorbic acid (AsA) biosynthesis enzyme, could be degraded through interaction with the photomorphogenic factor COP9 signalosome subunit 5B (CSN5B) in the darkness, demonstrating the posttranscriptional regulation of light signal in AsA production. Here, we further report that a point mutation in D27E of VTC1 disables the interaction with CSN5B, resulting in enhancement of AsA biosynthesis and seedling growth in Arabidopsis thaliana. To identify the interaction sites with CSN5B, we first predicted the key amino acids in VTC1 via bioinformatics analysis. And then we biochemically and genetically demonstrated that the 27th Asp was the amino acid that influenced the interaction of VTC1 with CSN5B in plants. Moreover, transgenic lines overexpressing the site-specific mutagenesis from D27 (Asp) into E27 (Glu) in VTC1 showed enhanced AsA accumulation and reduced H2O2 content in Arabidopsis seedlings, compared with the lines overexpressing the mutation from D27 into N27 (Asn) in VTC1. In addition, this regulation of VTC1 D27E mutation promoted seedling growth. Together, our data reveal that the 27th amino acid of VTC1 confers a key regulation in the interaction with CSN5B and AsA biosynthesis, as well as in Arabidopsis seedling growth.

Coupled synthesis and translocation restrains polyphosphate to acidocalcisome-like vacuoles and prevents its toxicity.

Eukaryotes contain inorganic polyphosphate (polyP) and acidocalcisomes, which sequester polyP and store amino acids and divalent cations. Why polyP is sequestered in dedicated organelles is not known. We show that polyP produced in the cytosol of yeast becomes toxic. Reconstitution of polyP translocation with purified vacuoles, the acidocalcisomes of yeast, shows that cytosolic polyP cannot be imported, whereas polyP produced by the vacuolar transporter chaperone (VTC) complex, an endogenous vacuolar polyP polymerase, is efficiently imported and does not interfere with growth. PolyP synthesis and import require an electrochemical gradient, probably as a driving force for polyP translocation. VTC exposes its catalytic domain to the cytosol and carries nine vacuolar transmembrane domains. Mutations in the VTC transmembrane regions, which are likely to constitute the translocation channel, block not only polyP translocation but also synthesis. Given that they are far from the cytosolic catalytic domain of VTC, this suggests that the VTC complex obligatorily couples synthesis of polyP to its import in order to avoid toxic intermediates in the cytosol. Sequestration of otherwise toxic polyP might be one reason for the existence of acidocalcisomes in eukaryotes.

Enzymes of yeast polyphosphate metabolism: structure, enzymology and biological roles.

Inorganic polyphosphate (polyP) is found in all living organisms. The known polyP functions in eukaryotes range from osmoregulation and virulence in parasitic protozoa to modulating blood coagulation, inflammation, bone mineralization and cellular signalling in mammals. However mechanisms of regulation and even the identity of involved proteins in many cases remain obscure. Most of the insights obtained so far stem from studies in the yeast Saccharomyces cerevisiae. Here, we provide a short overview of the properties and functions of known yeast polyP metabolism enzymes and discuss future directions for polyP research.

Ascorbate efflux as a new strategy for iron reduction and transport in plants.

Iron (Fe) is essential for virtually all living organisms. The identification of the chemical forms of iron (the speciation) circulating in and between cells is crucial to further understand the mechanisms of iron delivery to its final targets. Here we analyzed how iron is transported to the seeds by the chemical identification of iron complexes that are delivered to embryos, followed by the biochemical characterization of the transport of these complexes by the embryo, using the pea (Pisum sativum) as a model species. We have found that iron circulates as ferric complexes with citrate and malate (Fe(III)3Cit2Mal2, Fe(III)3Cit3Mal1, Fe(III)Cit2). Because dicotyledonous plants only transport ferrous iron, we checked whether embryos were capable of reducing iron of these complexes. Indeed, embryos did express a constitutively high ferric reduction activity. Surprisingly, iron(III) reduction is not catalyzed by the expected membrane-bound ferric reductase. Instead, embryos efflux high amounts of ascorbate that chemically reduce iron(III) from citrate-malate complexes. In vitro transport experiments on isolated embryos using radiolabeled (55)Fe demonstrated that this ascorbate-mediated reduction is an obligatory step for the uptake of iron(II). Moreover, the ascorbate efflux activity was also measured in Arabidopsis embryos, suggesting that this new iron transport system may be generic to dicotyledonous plants. Finally, in embryos of the ascorbate-deficient mutants vtc2-4, vtc5-1, and vtc5-2, the reducing activity and the iron concentration were reduced significantly. Taken together, our results identified a new iron transport mechanism in plants that could play a major role to control iron loading in seeds.

Ascorbate deficiency influences the leaf cell wall glycoproteome in Arabidopsis thaliana.

The cell wall forms the first line of interaction between the plant and the external environment. Based on the observation that ascorbate-deficient vtc mutants of Arabidopsis thaliana have increased cell wall peroxidase activity, the cell wall glycoproteome of vtc2-2 was investigated. Glycoproteins were purified from fully expanded leaves by Concanavalin A affinity chromatography and analysed by liquid chromatography quadrupole time-of-flight mass spectrometry. This procedure identified 63 proteins with predicted glycosylation sites and cell wall localization. Of these, 11 proteins were differentially expressed between vtc2-2 and wild type. In particular, PRX33/34 were identified as contributing to increased peroxidase activity in response to ascorbate deficiency. This is the same peroxidase previously shown to contribute to hydrogen peroxide generation and pathogen resistance. Three fasciclin-like arabinogalactan proteins (FLA1, 2 and 8) had lower abundance in vtc2-2. Inspection of published microarray data shows that these also have lower gene expression in vtc1 and vtc2-1 and are decreased in expression by pathogen challenge and oxidative stresses. Ascorbate deficiency therefore impacts expression of cell wall proteins involved in pathogen responses and these presumably contribute to the increased resistance of vtc mutants to biotrophic pathogens.

Institutionally based videoconferencing.

The delivery of psychiatric care via video-teleconferencing (VTC) technology is thought to have reached a tipping point. As a medical speciality with relatively few material or technical requirements for service delivery, psychiatry has been one of the earliest to embrace the possibility of providing evaluations and treatment at a distance. Such technical infrastructure as is necessary can often be found in the institutions already in existence. It was natural therefore that institutionally based telepsychiatry would lay the foundation for the development of the field. In this article we review the history and development of institutional VTC in a wide variety of clinically supervised settings such as hospitals, outpatient clinics, and forensic settings. We cite evidence supporting institutionally sponsored use and expand on key takeaways for the development and expansion of videoconferencing in these settings. We also speculate on the future direction and development of psychiatric care provided by these arrangements.

Stx5 is a novel interactor of VLDL-R to affect its intracellular trafficking and processing.

We identified syntaxin 5 (Stx5), a protein involved in intracellular vesicle trafficking, as a novel interaction partner of the very low density lipoprotein (VLDL)-receptor (VLDL-R), a member of the LDL-receptor family. In addition, we investigated the effect of Stx5 on VLDL-R maturation, trafficking and processing. Here, we demonstrated mutual association of both proteins using several in vitro approaches. Furthermore, we detected a special maturation phenotype of VLDL-R resulting from Stx5 overexpression. We found that Stx5 prevented advanced Golgi-maturation of VLDL-R, but did not cause accumulation of the immature protein in ER, ER to Golgi compartments, or cis-Golgi ribbon, the main expression sites of Stx5. Rather more, abundantly present Stx5 was capable of translocating ER-/N-glycosylated VLDL-R to the plasma membrane, and thus was insensitive to BFA treatment and low temperature. Furthermore, abundant presence of Stx5 significantly interfered with VLDL-R reaching the trans-Golgi network. Based on our findings, we postulate that Stx5 can directly bind to the C-terminal domain of VLDL-R, thereby influencing the receptor's glycosylation, trafficking and processing characteristics. Resulting from that, we further suggest that Stx5 might play a role in modulating VLDL-R physiology by participating in an abrasively described or completely novel Golgi-bypass pathway.