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The analysis of myo-inositol phosphates (InsPs) and myo-inositol pyrophosphates (PP-InsPs) is a daunting challenge due to the large number of possible isomers, the absence of a chromophore, the high charge density, the low abundance, and the instability of the esters and anhydrides. Given their importance in biology, an analytical approach to follow and understand this complex signaling hub is desirable. Here, capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry (ESI-MS) is implemented to analyze complex mixtures of InsPs and PP-InsPs with high sensitivity. Stable isotope labeled (SIL) internal standards allow for matrix-independent quantitative assignment. The method is validated in wild-type and knockout mammalian cell lines and in model organisms. SIL-CE-ESI-MS enables the accurate monitoring of InsPs and PP-InsPs arising from compartmentalized cellular synthesis pathways, by feeding cells with either [13C6]-myo-inositol or [13C6]-D-glucose. In doing so, we provide evidence for the existence of unknown inositol synthesis pathways in mammals, highlighting the potential of this method to dissect inositol phosphate metabolism and signalling., Myo-Inositol phosphates (InsPs) and pyrophosphates (PP-InsPs) are important second messengers but their analysis remains challenging. Here, the authors develop a capillary electrophoresis-mass spectrometry method for the identification and quantitation of InsP and PP-InsP isomers in cells and tissues.

Most Gram-negative phytopathogenic bacteria inject type III effector (T3E) proteins into plant cells to manipulate signaling pathways to the pathogen’s benefit. In resistant plants, specialized immune receptors recognize single T3Es or their biochemical activities, thus halting pathogen ingress. However, molecular function and mode of recognition for most T3Es remains elusive. Here, we show that the Xanthomonas T3E XopH possesses phytase activity, i.e., dephosphorylates phytate (myo-inositol-hexakisphosphate, InsP6), the major phosphate storage compound in plants, which is also involved in pathogen defense. A combination of biochemical approaches, including a new NMR-based method to discriminate inositol polyphosphate enantiomers, identifies XopH as a naturally occurring 1-phytase that dephosphorylates InsP6 at C1. Infection of Nicotiana benthamiana and pepper by Xanthomonas results in a XopH-dependent conversion of InsP6 to InsP5. 1-phytase activity is required for XopH-mediated immunity of plants carrying the Bs7 resistance gene, and for induction of jasmonate- and ethylene-responsive genes in N. benthamiana., Plant pathogens translocate type III effector (T3E) proteins that may be recognized by plants to trigger immunity. Here, the authors show that the Xanthomonas T3E XopH possesses a novel 1-phytase activity that is required for XopH-mediated immunity of plants carrying the Bs7 resistance gene.

Diphosphorylated inositol polyphosphates, also referred to as inositol pyrophosphates, are important signaling molecules that regulate critical cellular activities in many eukaryotic organisms, such as membrane trafficking, telomere maintenance, ribosome biogenesis, and apoptosis. In mammals and fungi, two distinct classes of inositol phosphate kinases mediate biosynthesis of inositol pyrophosphates: Kcs1/IP6K- and Vip1/PPIP5K-like proteins. Here, we report that PPIP5K homologs are widely distributed in plants and that Arabidopsis thaliana VIH1 and VIH2 are functional PPIP5K enzymes. We show a specific induction of inositol pyrophosphate InsP8 by jasmonate and demonstrate that steady state and jasmonate-induced pools of InsP8 in Arabidopsis seedlings depend on VIH2. We identify a role of VIH2 in regulating jasmonate perception and plant defenses against herbivorous insects and necrotrophic fungi. In silico docking experiments and radioligand binding-based reconstitution assays show high-affinity binding of inositol pyrophosphates to the F-box protein COI1-JAZ jasmonate coreceptor complex and suggest that coincidence detection of jasmonate and InsP8 by COI1-JAZ is a critical component in jasmonate-regulated defenses.

Synthetic genetic array analyses identify powerful genetic interactions between a thermosensitive allele (sec14-1ts) of the structural gene for the major yeast phosphatidylinositol transfer protein (SEC14) and a structural gene deletion allele (tlg2Δ) for the Tlg2 target membrane-soluble N-ethylmaleimide-sensitive factor attachment protein receptor. The data further demonstrate Sec14 is required for proper trans-Golgi network (TGN)/endosomal dynamics in yeast. Paradoxically, combinatorial depletion of Sec14 and Tlg2 activities elicits trafficking defects from the endoplasmic reticulum, and these defects are accompanied by compromise of the unfolded protein response (UPR). UPR failure occurs downstream of Hac1 mRNA splicing, and it is further accompanied by defects in TOR signaling. The data link TGN/endosomal dynamics with ceramide homeostasis, UPR activity, and TOR signaling in yeast, and they identify the Sit4 protein phosphatase as a primary conduit through which ceramides link to the UPR. We suggest combinatorial Sec14/Tlg2 dysfunction evokes inappropriate turnover of complex sphingolipids in endosomes. One result of this turnover is potentiation of ceramide-activated phosphatase-mediated down-regulation of the UPR. These results provide new insight into Sec14 function, and they emphasize the TGN/endosomal system as a central hub for homeostatic regulation in eukaryotes.

Sec14p is the major phosphatidylinositol (PtdIns)/phosphatidylcholine (PtdCho) transfer protein in the budding yeast Saccharomyces cerevisiae and is the founding member of a large eukaryotic protein superfamily. This protein catalyzes the exchange of either PtdIns or PtdCho between membrane bilayers in vitro and this exchange reaction requires no external input of energy or of other protein cofactors. Despite the previous elucidation of the crystal structure of a detergent-bound form of Sec14p, the conformational changes that accompany the phospholipid-exchange reaction remain undefined. Moreover, a structural appreciation of how Sec14p or its homologs bind their various phospholipid substrates remains elusive. Here, the purification and crystallization of yeast Sfh1p, the protein most closely related to Sec14p, are reported. A combination of electrospray ionization mass-spectrometry and collision-induced decomposition mass-spectrometry methods indicate that recombinant Sfh1p loads predominantly with phosphatidylethanolamine. Unlike phospholipid-bound forms of Sec14p, this form of Sfh1p crystallizes readily in the absence of detergent. Sfh1p crystals diffract to 2.5 Å and belong to the orthorhombic primitive space group P212121, with unit-cell parameters a = 49.40, b = 71.55, c = 98.21 Å, α = β = γ = 90°. One Sfh1p molecule is present in the asymmetric unit (V M = 2.5 Å3 Da−1; V s = 50%). Crystallization of a phospholipid-bound Sec14p-like protein is a critical first step in obtaining the first high-resolution picture of how proteins of the Sec14p superfamily bind their phospholipid ligands. This information will significantly extend our current understanding of how Sec14p-like proteins catalyze phospholipid exchange.

Many eukaryotic proteins regulating phosphate (Pi) homeostasis contain SPX domains that are receptors for inositol pyrophosphates (PP-InsP), suggesting that PP-InsPs may regulate Pi homeostasis. Here we report that deletion of two diphosphoinositol pentakisphosphate kinases VIH1/2 impairs plant growth and leads to constitutive Pi starvation responses. Deletion of phosphate starvation response transcription factors partially rescues vih1 vih2 mutant phenotypes, placing diphosphoinositol pentakisphosphate kinases in plant Pi signal transduction cascades. VIH1/ 2 are bifunctional enzymes able to generate and break-down PP-InsPs. Mutations in the kinase active site lead to increased Pi levels and constitutive Pi starvation responses. ATP levels change significantly in different Pi growth conditions. ATP-Mg2+ concentrations shift the relative kinase and phosphatase activities of diphosphoinositol pentakisphosphate kinases in vitro. Pi inhibits the phosphatase activity of the enzyme. Thus, VIH1 and VIH2 relay changes in cellular ATP and Pi concentrations to changes in PP-InsP levels, allowing plants to maintain sufficient Pi levels.

Myo-Inositol (Ins)-derived molecules are involved in many cellular processes. Besides the role as compatible solute, Ins represents the building block for a variety of molecules, which function as signaling molecules. Cells utilize insoluble (lipid-bound) and soluble Ins derivatives for diverse signal transduction pathways, many of them essential for cell survival. In yeast and mammalian cells, Ins-derived signaling molecules are involved in many processes, such as phosphate starvation or insulin signaling. In plants first insights in the roles of this class of molecules are just emerging. In this work, I address the roles of Ins-derived signaling molecules during abiotic and biotic stress in Saccharomyces cerevisiae and Arabidopsis thaliana. In the presented manuscript (Johnen et al., in preparation) the role of Ins derivatives in overcoming aluminum (Al) toxicity, representing a major abiotic limitation for crop production worldwide, was investigated. We report that the ectopic expression of AtSFH5 and ScSFH1, encoding proteins, which belong to the ScSec14 lipid transfer protein family, mediates Al tolerance in the model organism yeast, likely independent from the uptake or compartmentalisation of Al. We show that through expression of ScSFH1 the negative charge of the plasma membrane (PM) is increased suggesting PM charge as an early toxicity target for Al. This idea is corroborated by yeast and plant genetics, as well as by cell biological analyses, showing that at physiological relevant Al concentrations proper membrane targeting of proteins is impaired and that an increase or a decrease of the PM charge led to higher or lower Al tolerance in yeast and plants, respectively. Based on a combination of cell biological, genetic and biochemical experiments, we propose a model in which AtSFH5 and ScSfh1 mediate in vivo phospholipid transfer, resulting in either increased levels or increased deprotonation state (and thus activity) of phosphatidyl inositol-4-phosphate (PtdIns(4)P) at the PM, thereby leading to an increase in negative PM charge and Al tolerance. Human fungal pathogens cause invasive fungal infections, which are associated with high morbidity and mortality rates. With only three classes of antifungal drugs in therapeutic use there is a lack of diversity of antifungal agents. In Pries, Nöcker, Khan, Johnen, Hong et al. (2018), we identified two chemotypes, the picolinamides and the benzamides, as specific ScSec14 inhibitors in S. cerevisiae, representing lead structures for the development of antifungal drugs. This finding is based on chemogenomic profiling in yeast and is confirmed by genetic and biochemical evidence. The presented co-crystal structure of ScSec14 bound to a compound belonging to the picolinamide chemotype represents the first structure of inhibitor-bound Sec14 and lays the groundwork for developing new antifungal drugs. Inositol pyrophosphates (PP-InsPs) are a specific class of Ins derivatives containing one or more high-energy diphosphate (or pyrophosphate) groups. In mammals and yeast these molecules are involved in a variety of cellular processes. In contrast to that, in plants insights in the biosynthesis or the roles of this class of molecules remained elusive at the onset of this work. In Laha et al. (2015) and Laha et al. (2016) we identified two proteins of A. thaliana, AtVIH1 and AtVIH2, as functional PPIP5K homologs catalyzing the biosynthesis of the PP- InsP InsP8 in planta. Based on a series of bioassays investigating the performance of herbivores and fungal pathogens on plant mutant lines defective inositol polyphosphate biosynthesis, we provide evidence that InsP8 plays a role in tuning the Jasmonic acid (JA)- dependent pathogen defense. Furthermore, it is presented that methyl-JA treatment led to an increase of InsP8 and did not have a major effect on other inositol polyphosphate species. Even though mutant lines with decreased InsP8 levels are more susceptible to plant pathogens, the levels of active JA species are increased compared to wild type plants. Based these findings, combined with Ask1-AtCOI1-AtJAZ reconstitution assays, yeast 2-hyrbid (Y2H) studies and in silico docking experiments, we propose that coincidence detection of active JA species and InsP8 by the SCF1AtCOI1-AtJAZ co-receptor complex regulates JA signaling and is thereby regulating JA-related plant defenses. Collectively, the presented data in this work highlight the importance of Ins-derived signaling molecules during abiotic and biotic stress in yeast and plants, and expand our knowledge of the roles of this exciting class of molecules

Sec14-like phosphatidylinositol/phosphatidylcholine (PtdIns/PtdCho) transfer proteins (PITPs) represent important regulatory components that integrate phospholipid metabolism and membrane trafficking in eukaryotes. Data derived from yeast studies suggest that this regulation results from a Sec14-mediated PtdIns/PtdCho exchange reaction that stimulates PtdIns kinase activity, thus facilitating the generation of phosphoinositides (PIPs) such as PtdIns(4)P and PtdIns(4,5)P2. The present work reports recent findings on the roles and functional mechanisms of yeast and plant Sec14-like PITPs. The Arabidopsis Sec14-nodulin AtSFH1, an important regulator of root hair formation, was used as a model to show that the nodulin domain represents a plasma membrane association module with high binding specificity towards PtdIns(4,5)P2. A Lys-rich C-terminal motif is necessary for PIP binding activity, which is amplified by homo-oligomerization of the nodulin domain. Both PIP association and homooligomerization are essential properties of AtSFH1 and mutants defective in PtdIns(4,5)P2 binding can be rescued by a translational fusion with a bona fide PtdIns(4,5)P2 binding domain. A model for the mode of action of AtSFH1 is proposed herein, and suggests that the physical linkage of the N-terminal Sec14 and the nodulin domains couples PIP synthesis and organization, so as to promote defined landmarks for PIP effectors that modulate developmental control of polarized membrane growth. A second approach used to gain further insights on AtSFH1 functions is also discussed. EMS-mutagenized plants that suppress the atsfh1-1-dependent short root hair henotype were isolated. By employing nextgeneration sequencing combined with deep candidate resequencing (dCARE), a mutation in the AtSTR1 sulfurtranferase encoding gene was identified. The preliminary results presented here suggest that cyanide detoxifying enzymes could represent additional cellular components that regulate root hair development, possibly in a pathway that is independent of AtSFH1. Finally, a directed evolution screen was performed in order to identify mutations that confer Sec14-like activities to the functionally inactive yeast Sec14 homolog Sfh1. Biochemical, biophysical and computational approaches helped to discover a network of transient interactions that propagates conformational energy from the lipid binding pocket to the ‘helical gating module’ that controls lipid access, thus enhancing the rates of phospholipid exchange and presentation in the mutant proteins. Taken together, the discoveries presented herein provide important details into the mechanisms regulating phospholipid exchange and PIP organization by Sec14-like PITPs, as well as comprehensive clues of how these fascinating proteins ultimately promote phosphoinositide homeostasis in eukaryotes.