Association of the Arabidopsis oleoyl Δ12-desaturase FAD2 with pre-cis-Golgi stacks at endoplasmic reticulum-Golgi-exit sites.

The unsaturation of phospholipids influences the function of membranes. In Arabidopsis thaliana, the oleoyl Δ12-desaturase FAD2 converts oleic (18:1Δ9 ) to linoleic acid (18:2Δ9,12 ) and influences phospholipid unsaturation in different cellular membranes. Despite its importance, the precise localization of Arabidopsis FAD2 has not been unambiguously described. As FAD2 is thought to modify phospholipid-associated fatty acids at the endoplasmic reticulum (ER), from where unsaturates are distributed to other cellular sites, we hypothesized that FAD2 locates to ER subdomains enabling trafficking of lipid intermediates through the secretory pathway. Fluorescent FAD2 fusions used to test this hypothesis were first assessed for functionality by heterologous expression in yeast (Saccharomyces cerevisiae), and in planta by Arabidopsis fad2 mutant rescue upon ectopic expression from an intrinsic FAD2 promoter fragment. Light sheet fluorescence, laser scanning confocal or spinning disc microscopy of roots, leaves, or mesophyll protoplasts showed the functional fluorescence-tagged FAD2 variants in flattened donut-shaped structures of ~0.5-1 µm diameter, in a pattern not resembling mere ER association. High-resolution imaging of coexpressed organellar markers showed fluorescence-tagged FAD2 in a ring-shaped pattern surrounding ER-proximal Golgi particles, colocalizing with pre-cis-Golgi markers. This localization required the unusual C-terminal retention signal of FAD2, and deletion or substitutions in this protein region resulted in relaxed distribution and diffuse association with the ER. The distinct association of FAD2 with pre-cis-Golgi stacks in Arabidopsis root and leaf tissue is consistent with a contribution of FAD2 to membrane lipid homeostasis through the secretory pathway, as verified by an increased plasma membrane liquid phase order in the fad2 mutant.

The pollen-specific class VIII-myosin ATM2 from Arabidopsis thaliana associates with the plasma membrane through a polybasic region binding anionic phospholipids.

Pollen tubes display polarized tip-growth and are a model to study the coordination of vesicular trafficking and cytoskeletal control. The molecular details of how dynamic actin filaments associate with the plasma membrane are currently unclear. In Arabidopsis thaliana, plasma membrane attachment of actin filaments may be mediated by four myosins representing the plant-specific myosin-subclass VIII, which localize to the plasma membrane and display only minor motor-activity. Here we explore the mode of membrane attachment of the pollen-expressed class VIII-myosins ATM2 and VIII-B through interaction with anionic membrane phospholipids. A fluorescent mCherry-ATM2-fusion decorated plasma membrane-peripheral actin filaments when expressed in tobacco pollen tubes, consistent with a role of class VIII-myosins at the membrane-cytoskeleton interface. As recombinant proteins, class VIII-myosins are prone to aggregation and to proteolysis, creating a challenge for their biochemical characterization. We describe a purification scheme for guanidinium chloride (GdmCl)-denatured recombinant proteins, followed by a renaturation protocol to obtain pure, soluble protein fragments of ATM2 and VIII-B. The fragments represent the C-terminal tail and coiled-coil-regions and lack the N-terminal actin-binding regions, IQ or motor domains. Based on lipid-overlays and liposome-sedimentation assays, the fragments of ATM2 and VIII-B bind anionic phospholipids. Small polybasic regions at the extreme C-termini were sufficient for lipid-binding of the respective protein fragments. When expressed in tobacco pollen tubes, a fluorescence-tagged variant of ATM2 lacking its lipid-binding region displayed substantially reduced plasma membrane association. The data indicate that class VIII-myosins may facilitate actin-plasma membrane attachment through interaction with anionic phospholipids, mediated by polybasic C-terminal lipid-binding domains.

Regulators regulated: Different layers of control for plasma membrane phosphoinositides in plants.

The membranes of plant cells serve diverse physiological roles, which are defined largely by the localized and dynamic recruitment of proteins. Signaling lipids, such as phosphoinositides, can aid protein recruitment to the plasma membrane via specific recognition of their head groups and influence vesicular trafficking, cytoskeletal dynamics and other processes, with ramifications for plant tissue architecture and development. Phosphoinositide abundance is dynamically regulated. Recent advances indicate various levels of control during development or upon environmental triggers, including transcriptional or posttranslational regulation of enzymes balancing biogenesis and degradation, or the nano-organization of membranes into self-organizing physiologically distinct microenvironments. As patterns of interlinked mechanisms emerge, the horizons of what we do not understand become more and more defined.

LC-MS Based Draft Map of the Arabidopsis thaliana Nuclear Proteome and Protein Import in Pattern Triggered Immunity.

Despite its central role as the ark of genetic information and gene expression the plant nucleus is surprisingly understudied. We isolated nuclei from the Arabidopsis thaliana dark grown cell culture left untreated and treated with flg22 and nlp20, two elicitors of pattern triggered immunity (PTI) in plants, respectively. An liquid chromatography mass spectrometry (LC-MS) based discovery proteomics approach was used to measure the nuclear proteome fractions. An enrichment score based on the relative abundance of cytoplasmic, mitochondrial and Golgi markers in the nuclear protein fraction allowed us to curate the nuclear proteome producing high quality catalogs of around 3,000 nuclear proteins under untreated and both PTI conditions. The measurements also covered low abundant proteins including more than 100 transcription factors and transcriptional co-activators. We disclose a list of several hundred potentially dual targeted proteins including proteins not yet found before for further study. Protein import into the nucleus in plant immunity is known. Here we sought to gain a broader impression of this phenomenon employing our proteomics data and found 157 and 73 proteins to possibly be imported into the nucleus upon stimulus with flg22 and nlp20, respectively. Furthermore, the abundance of 93 proteins changed significantly in the nucleus following elicitation of immunity. These results suggest promiscuous ribosome assembly and a role of prohibitins and cytochrome C in the nucleus in PTI.

Plasma membrane nano-organization specifies phosphoinositide effects on Rho-GTPases and actin dynamics in tobacco pollen tubes.

Pollen tube growth requires coordination of cytoskeletal dynamics and apical secretion. The regulatory phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is enriched in the subapical plasma membrane of pollen tubes of Arabidopsis thaliana and tobacco (Nicotiana tabacum) and can influence both actin dynamics and secretion. How alternative PtdIns(4,5)P2 effects are specified is unclear. In tobacco pollen tubes, spinning disc microscopy (SD) reveals dual distribution of a fluorescent PtdIns(4,5)P2-reporter in dynamic plasma membrane nanodomains vs. apparent diffuse membrane labeling, consistent with spatially distinct coexisting pools of PtdIns(4,5)P2. Several PI4P 5-kinases (PIP5Ks) can generate PtdIns(4,5)P2 in pollen tubes. Despite localizing to one membrane region, the PIP5Ks AtPIP5K2-EYFP and NtPIP5K6-EYFP display distinctive overexpression effects on cell morphologies, respectively related to altered actin dynamics or membrane trafficking. When analyzed by SD, AtPIP5K2-EYFP associated with nanodomains, whereas NtPIP5K6-EYFP localized diffusely. Chimeric AtPIP5K2-EYFP and NtPIP5K6-EYFP variants with reciprocally swapped membrane-associating domains evoked reciprocally shifted effects on cell morphology upon overexpression. Overall, active PI4P 5-kinase variants stabilized actin when targeted to nanodomains, suggesting a role of nanodomain-associated PtdIns(4,5)P2 in actin regulation. This notion is further supported by interaction and proximity of nanodomain-associated AtPIP5K2 with the Rho-GTPase NtRac5, and by its functional interplay with elements of Rho of plants signaling. Plasma membrane nano-organization may thus aid the specification of PtdIns(4,5)P2 functions to coordinate cytoskeletal dynamics and secretion.

Analysis of Phosphoinositides from Complex Plant Samples by Solid-Phase Adsorption Chromatography and Subsequent Quantification via Thin-Layer and Gas Chromatography.

The determination of phosphoinositide molecular species in plant material is challenging because of their low abundance concurrent with a very high abundance of other membrane lipids, such as plastidial glycolipids. Phosphoinositides harbor an inositol headgroup which carries one or more phosphate groups at different positions of the inositol, linked to diacylglycerol via a phosphodiester. Thus, a further analytical challenge is to distinguish the different inositol-phosphate headgroups as well as the fatty acids of the diacylglycerol backbone. The method presented in this chapter expands on previous protocols for phosphoinositide analysis by employing chromatographic enrichment of phospholipids and their separation from other, more abundant lipid classes, before analysis. Lipids extracted from plant material are first separated by solid-phase adsorption chromatography into fractions containing neutral lipids, glycolipids, or phospholipids. Lipids from the phospholipid fraction are then separated by thin-layer chromatography (TLC) according to their characteristic head groups, and the individual phosphatidylinositol-monophosphates and phosphatidylinositol-bisphosphates are isolated. Finally, the fatty acids associated with each isolated phosphatidylinositol-monophosphate or phosphatidylinositol-bisphosphate are analyzed in a quantitative fashion using gas chromatography (GC). The analysis of phosphoinositides by this combination of methods provides a cost-efficient and reliable alternative to lipidomics approaches requiring more extensive instrumentation.

A PAMP-triggered MAPK cascade inhibits phosphatidylinositol 4,5-bisphosphate production by PIP5K6 in Arabidopsis thaliana.

The phosphoinositide kinase PIP5K6 has recently been identified as a target for the mitogen-activated protein kinase (MAPK) MPK6. Phosphorylation of PIP5K6 inhibited the production of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2 ), impacting membrane trafficking and cell expansion in pollen tubes. Here, we analyzed whether MPK6 regulated PIP5K6 in vegetative Arabidopsis cells in response to the pathogen-associated molecular pattern (PAMP) flg22. Promoter-ß-glucuronidase analyses and quantitative real-time reverse transcription polymerase chain reaction data show PIP5K6 expressed throughout Arabidopsis tissues. Upon flg22 treatment of transgenic protoplasts, the PIP5K6 protein was phosphorylated, and this modification was reduced for a PIP5K6 variant lacking MPK6-targeted residues, or in protoplasts from mpk6 mutants. Upon flg22 treatment of Arabidopsis plants, phosphoinositide levels mildly decreased and a fluorescent reporter for PtdIns(4,5)P2 displayed reduced plasma membrane association, contrasting with phosphoinositide increases reported for abiotic stress responses. Flg22 treatment and chemical induction of the upstream MAPK kinase, MKK5, decreased phosphatidylinositol 4-phosphate 5-kinase activity in mesophyll protoplasts, indicating that the flg22-activated MAPK cascade limited PtdIns(4,5)P2 production. PIP5K6 expression or PIP5K6 protein abundance changed only marginally upon flg22 treatment, consistent with post-translational control of PIP5K6 activity. PtdIns(4,5)P2 -dependent endocytosis of FM 4-64, PIN2 and the NADPH-oxidase RbohD were reduced upon flg22 treatment or MKK5 induction. Reduced RbohD-endocytosis was correlated with enhanced ROS production. We conclude that MPK6-mediated phosphorylation of PIP5K6 limits the production of a functional PtdIns(4,5)P2 pool upon PAMP perception.

A dual role for cell plate-associated PI4Kß in endocytosis and phragmoplast dynamics during plant somatic cytokinesis.

Plant cytokinesis involves membrane trafficking and cytoskeletal rearrangements. Here, we report that the phosphoinositide kinases PI4Kß1 and PI4Kß2 integrate these processes in Arabidopsis thaliana (Arabidopsis) roots. Cytokinetic defects of an Arabidopsis pi4kß1 pi4kß2 double mutant are accompanied by defects in membrane trafficking. Specifically, we show that trafficking of the proteins KNOLLE and PIN2 at the cell plate, clathrin recruitment, and endocytosis is impaired in pi4kß1 pi4kß2 double mutants, accompanied by unfused vesicles at the nascent cell plate and around cell wall stubs. Interestingly, pi4kß1 pi4kß2 plants also display ectopic overstabilization of phragmoplast microtubules, which guide membrane trafficking at the cell plate. The overstabilization of phragmoplasts in the double mutant coincides with mislocalization of the microtubule-associated protein 65-3 (MAP65-3), which cross-links microtubules and is a downstream target for inhibition by the MAP kinase MPK4. Based on similar cytokinetic defects of the pi4kß1 pi4kß2 and mpk4-2 mutants and genetic and physical interaction of PI4Kß1 and MPK4, we propose that PI4Kß and MPK4 influence localization and activity of MAP65-3, respectively, acting synergistically to control phragmoplast dynamics.

Anillin facilitates septin assembly to prevent pathological outfoldings of central nervous system myelin.

Myelin serves as an axonal insulator that facilitates rapid nerve conduction along axons. By transmission electron microscopy, a healthy myelin sheath comprises compacted membrane layers spiraling around the cross-sectioned axon. Previously we identified the assembly of septin filaments in the innermost non-compacted myelin layer as one of the latest steps of myelin maturation in the central nervous system (CNS) (Patzig et al., 2016). Here we show that loss of the cytoskeletal adaptor protein anillin (ANLN) from oligodendrocytes disrupts myelin septin assembly, thereby causing the emergence of pathological myelin outfoldings. Since myelin outfoldings are a poorly understood hallmark of myelin disease and brain aging we assessed axon/myelin-units in Anln-mutant mice by focused ion beam-scanning electron microscopy (FIB-SEM); myelin outfoldings were three-dimensionally reconstructed as large sheets of multiple compact membrane layers. We suggest that anillin-dependent assembly of septin filaments scaffolds mature myelin sheaths, facilitating rapid nerve conduction in the healthy CNS.

Inhibition of phospholipases influences the metabolism of wound-induced benzylisoquinoline alkaloids in Papaver somniferum L.

Benzylisoquinoline alkaloids (BIAs) are important secondary plant metabolites and include medicinally relevant drugs, such as morphine or codeine. As the de novo synthesis of BIA backbones is (still) unfeasible, to date the opium poppy plant Papaver somniferum L. represents the main source of BIAs. The formation of BIAs is induced in poppy plants by stress conditions, such as wounding or salt treatment; however, the details about regulatory processes controlling BIA formation in opium poppy are not well studied. Environmental stresses, such as wounding or salinization, are transduced in plants by phospholipid-based signaling pathways, which involve different classes of phospholipases. Here we investigate whether pharmacological inhibition of phospholipase A2 (PLA2, inhibited by aristolochic acid (AA)) or phospholipase D (PLD; inhibited by 5-fluoro-2-indolyl des-chlorohalopemide (FIPI)) in poppy plants influences wound-induced BIA accumulation and the expression of key biosynthetic genes. We show that inhibition of PLA2 results in increased morphinan biosynthesis concomitant with reduced production of BIAs of the papaverine branch, whereas inhibition of PLD results in increased production of BIAs of the noscapine branch. The data suggest that phospholipid-dependent signaling pathways contribute to the activation of morphine biosynthesis at the expense of the production of other BIAs in poppy plants. A better understanding of the effectors and the principles of regulation of alkaloid biosynthesis might be the basis for the future genetic modification of opium poppy to optimize BIA production.

Proteasome inhibitor bortezomib enhances the effect of standard chemotherapy in small cell lung cancer.

Small cell lung cancer (SCLC) is an aggressive cancer showing a very poor prognosis because of metastasis formation at an early stage and acquisition of chemoresistance. One key driver of chemoresistance is the transcription factor Forkhead box protein M1 (FOXM1) that regulates cell cycle proliferation, maintenance of genomic stability, DNA damage response, and cell differentiation in numerous tumor entities. In this study we investigated the role of FOXM1 in SCLC progression and analyzed the effect of FOXM1 inhibition using two proteasome inhibitors, bortezomib and siomycin A. FOXM1 was strongly expressed in patient-derived SCLC samples (n=123) and its nuclear localization was associated with the proliferation marker Ki-67. Both proteasome inhibitors successfully inhibited FOXM1 expression leading to a significantly reduced proliferation and a decreased mitotic rate along with cell cycle arrest and apoptosis induction. These effects were further enhanced by addition of bortezomib to standard chemotherapy. Treatment of mice bearing chemoresistant SCLC xenografts with bortezomib reduced the mean bioluminescence signal of tumors by 54%. Similarly, treatment with cisplatin as a standard chemotherapy reduced the mean bioluminescence signal of tumors by 58%. However, in combination with standard chemotherapy bortezomib further reduced the mean bioluminescence signal by 93% (p=0.0258). In conclusion, we demonstrate the effect of bortezomib in inhibiting FOXM1 expression and thus in sensitizing resistant SCLC cells to standard chemotherapy. Thus, addition of bortezomib to standard chemotherapy might potently improve SCLC therapy, particularly in an extensive cancer stage.

MAPKs Influence Pollen Tube Growth by Controlling the Formation of Phosphatidylinositol 4,5-Bisphosphate in an Apical Plasma Membrane Domain.

An apical plasma membrane domain enriched in the regulatory phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is critical for polar tip growth of pollen tubes. How the biosynthesis of PtdIns(4,5)P2 by phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) is controlled by upstream signaling is currently unknown. The pollen-expressed PI4P 5-kinase PIP5K6 is required for clathrin-mediated endocytosis and polar tip growth in pollen tubes. Here, we identify PIP5K6 as a target of the pollen-expressed mitogen-activated protein kinase MPK6 and characterize the regulatory effects. Based on an untargeted mass spectrometry approach, phosphorylation of purified recombinant PIP5K6 by pollen tube extracts could be attributed to MPK6. Recombinant MPK6 phosphorylated residues T590 and T597 in the variable insert of the catalytic domain of PIP5K6, and this modification inhibited PIP5K6 activity in vitro. PIP5K6 interacted with MPK6 in yeast two-hybrid tests, immuno-pull-down assays, and by bimolecular fluorescence complementation at the apical plasma membrane of pollen tubes. In vivo, MPK6 expression resulted in reduced plasma membrane association of a fluorescent PtdIns(4,5)P2 reporter and decreased endocytosis without impairing membrane association of PIP5K6. Effects of PIP5K6 expression on pollen tube growth and cell morphology were attenuated by coexpression of MPK6 in a phosphosite-dependent manner. Our data indicate that MPK6 controls PtdIns(4,5)P2 production and membrane trafficking in pollen tubes, possibly contributing to directional growth.

WRKY43 regulates polyunsaturated fatty acid content and seed germination under unfavourable growth conditions.

Seed germination and postgerminative growth of Arabidopsis thaliana and various other plant species are arrested in response to unfavourable environmental conditions by signalling events involving the phytohormone abscisic acid (ABA). In this study, we showed that loss of the seed-specific WRKY DNA-BINDING PROTEIN 43 (WRKY43) conferred increased tolerance towards high salt, high osmolarity and low temperature during seed germination in Arabidopsis. The wrky43 loss of function lines displayed increased inhibition of seed germination in response to exogenous ABA; whereas lines overexpressing WRKY43 were more tolerant towards exogenous ABA. Biochemical analysis of fatty acid composition revealed that loss of WRKY43 increased polyunsaturated fatty acid content in seeds, particularly 18:2Δ9,12 and 18:3Δ9,12,15 in triacylglycerols and phospholipids, indicating an important physiological effect on fatty acid desaturation with ramifications for the tolerance of plants to cold and osmotic stress and possibly, for oilseed engineering. Molecular analyses showed that ABA-induced regulation of FUSCA3, ZAT10 and seed storage proteins were absent in the wrky43 mutant. In summary, WRKY43 encodes for a novel positive regulator of ABA-dependent gene regulation and as a potent modulator of fatty acid desaturation and seed filling, which results in increased tolerance to abiotic stress.

Arabidopsis phosphatidylinositol 4-phosphate 5-kinase 2 contains a functional nuclear localization sequence and interacts with alpha-importins.

The Arabidopsis phosphoinositide kinase PIP5K2 has been implicated in the control of membrane trafficking and is important for development and growth. In addition to cytosolic functions of phosphoinositides, a nuclear phosphoinositide system has been proposed, but evidence for nuclear phosphoinositides in plants is limited. Fluorescence-tagged variants of PIP5K2 reside in the nucleus of Arabidopsis root meristem cells, in addition to reported plasma membrane localization. Here we report on the interaction of PIP5K2 with alpha-importins and characterize its nuclear localization sequences (NLSs). The PIP5K2 sequence contains four putative NLSs (NLSa-NLSd) and only a PIP5K2 fragment containing NLSs is imported into nuclei of onion epidermis cells upon transient expression. PIP5K2 interacts physically with alpha-importin isoforms in cytosolic split-ubiquitin-based yeast two-hybrid tests, in dot-blot experiments and in immuno-pull-downs. A 27-amino-acid fragment of PIP5K2 containing NLSc is necessary and sufficient to mediate the nuclear import of a large cargo fusion consisting of two mCherry markers fused to RubisCO large subunit. Substitution of basic residues in NLSc results in reduced import of PIP5K2 or other cargoes into plant nuclei. The data suggest that PIP5K2 is subject to active, alpha-importin-mediated nuclear import, consistent with a nuclear role for PIP5K2 in addition to its reported cytosolic functions. The detection of both substrate and product of PIP5K2 in plant nuclei according to reporter fluorescence and immunofluorescence further supports the notion of a nuclear phosphoinositide system in plants. Variants of PIP5K2 with reduced nuclear residence might serve as tools for the future functional study of plant nuclear phosphoinositides.

Guilt by Association: A Phenotype-Based View of the Plant Phosphoinositide Network.

Eukaryotic membranes contain small amounts of phospholipids that have regulatory effects on the physiological functions of cells, tissues, and organs. Phosphoinositides (PIs)-the phosphorylated derivatives of phosphatidylinositol-are one example of such regulatory lipids. Although PIs were described in plants decades ago, their contribution to the regulation of physiological processes in plants is not well understood. In the past few years, evidence has emerged that PIs are essential for plant function and development. Recently reported phenotypes associated with the perturbation of different PIs suggest that some subgroups of PIs influence specific processes. Although the molecular targets of PI-dependent regulation in plants are largely unknown, the effects of perturbed PI metabolism can be used to propose regulatory modules that involve particular downstream targets of PI regulation. This review summarizes phenotypes associated with the perturbation of the plant PI network to categorize functions and suggest possible downstream targets of plant PI regulation.

Phospholipid composition and a polybasic motif determine D6 PROTEIN KINASE polar association with the plasma membrane and tropic responses.

Polar transport of the phytohormone auxin through PIN-FORMED (PIN) auxin efflux carriers is essential for the spatiotemporal control of plant development. The Arabidopsis thaliana serine/threonine kinase D6 PROTEIN KINASE (D6PK) is polarly localized at the plasma membrane of many cells where it colocalizes with PINs and activates PIN-mediated auxin efflux. Here, we show that the association of D6PK with the basal plasma membrane and PINs is dependent on the phospholipid composition of the plasma membrane as well as on the phosphatidylinositol phosphate 5-kinases PIP5K1 and PIP5K2 in epidermis cells of the primary root. We further show that D6PK directly binds polyacidic phospholipids through a polybasic lysine-rich motif in the middle domain of the kinase. The lysine-rich motif is required for proper PIN3 phosphorylation and for auxin transport-dependent tropic growth. Polybasic motifs are also present at a conserved position in other D6PK-related kinases and required for membrane and phospholipid binding. Thus, phospholipid-dependent recruitment to membranes through polybasic motifs might not only be required for D6PK-mediated auxin transport but also other processes regulated by these, as yet, functionally uncharacterized kinases.

Identification of a secretory phospholipase A2 from Papaver somniferum L. that transforms membrane phospholipids.

The full-length sequence of a new secretory phospholipase A2 was identified in opium poppy seedlings (Papaver somniferum L.). The cDNA of poppy phospholipase A2, denoted as pspla2, encodes a protein of 159 amino acids with a 31 amino acid long signal peptide at the N-terminus. PsPLA2 contains a PLA2 signature domain (PA2c), including the Ca(2+)-binding loop (YGKYCGxxxxGC) and the catalytic site motif (DACCxxHDxC) with the conserved catalytic histidine and the calcium-coordinating aspartate residues. The aspartate of the His/Asp dyad playing an important role in animal sPLA2 catalysis is substituted by a serine residue. Furthermore, the PsPLA2 sequence contains 12 conserved cysteine residues to form 6 structural disulfide bonds. The calculated molecular weight of the mature PsPLA2 is 14.0 kDa. Based on the primary structure PsPLA2 belongs to the XIB group of PLA2s. Untagged recombinant PsPLA2 obtained by expression in Escherichia coli, renaturation from inclusion bodies and purification by cation-exchange chromatography was characterized in vitro. The pH optimum for activity of PsPLA2 was found to be pH 7, when using mixed micelles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and Triton X-100. PsPLA2 specifically cleaves fatty acids from the sn-2 position of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and shows a pronounced preference for PC over phosphatidyl ethanolamine, -glycerol and -inositol. The active recombinant enzyme was tested in vitro against natural phospholipids isolated from poppy plants and preferably released the unsaturated fatty acids, linoleic acid and linolenic acid, from the naturally occurring mixture of substrate lipids.

Mitochondrial uncouplers inhibit clathrin-mediated endocytosis largely through cytoplasmic acidification.

ATP production requires the establishment of an electrochemical proton gradient across the inner mitochondrial membrane. Mitochondrial uncouplers dissipate this proton gradient and disrupt numerous cellular processes, including vesicular trafficking, mainly through energy depletion. Here we show that Endosidin9 (ES9), a novel mitochondrial uncoupler, is a potent inhibitor of clathrin-mediated endocytosis (CME) in different systems and that ES9 induces inhibition of CME not because of its effect on cellular ATP, but rather due to its protonophore activity that leads to cytoplasm acidification. We show that the known tyrosine kinase inhibitor tyrphostinA23, which is routinely used to block CME, displays similar properties, thus questioning its use as a specific inhibitor of cargo recognition by the AP-2 adaptor complex via tyrosine motif-based endocytosis signals. Furthermore, we show that cytoplasm acidification dramatically affects the dynamics and recruitment of clathrin and associated adaptors, and leads to reduction of phosphatidylinositol 4,5-biphosphate from the plasma membrane.

Synthesis of oleyl oleate wax esters in Arabidopsis thaliana and Camelina sativa seed oil.

Seed oil composed of wax esters with long-chain monoenoic acyl moieties represents a high-value commodity for industry. Such plant-derived sperm oil-like liquid wax esters are biodegradable and can have excellent properties for lubrication. In addition, wax ester oil may represent a superior substrate for biodiesel production. In this study, we demonstrate that the low-input oil seed crop Camelina sativa can serve as a biotechnological platform for environmentally benign wax ester production. Two biosynthetic steps catalysed by a fatty alcohol-forming acyl-CoA reductase (FAR) and a wax ester synthase (WS) are sufficient to achieve wax ester accumulation from acyl-CoA substrates. To produce plant-derived sperm oil-like liquid wax esters, the WS from Mus musculus (MmWS) or Simmondsia chinensis (ScWS) were expressed in combination with the FAR from Mus musculus (MmFAR1) or Marinobacter aquaeolei (MaFAR) in seeds of Arabidopsis thaliana and Camelina sativa. The three analysed enzyme combinations Oleo3:mCherry:MmFAR1∆c/Oleo3:EYFP:MmWS, Oleo3:mCherry:MmFAR1∆c/ScWS and MaFAR/ScWS showed differences in the wax ester molecular species profiles and overall biosynthetic performance. By expressing MaFAR/ScWS in Arabidopsis or Camelina up to 59% or 21% of the seed oil TAGs were replaced by wax esters, respectively. This combination also yielded wax ester molecular species with highest content of monounsaturated acyl moieties. Expression of the enzyme combinations in the Arabidopsis fae1 fad2 mutant background high in oleic acid resulted in wax ester accumulation enriched in oleyl oleate (18:1/18:1 > 60%), suggesting that similar values may be obtained with a Camelina high oleic acid line.