Phaseolus vulgaris STP13.1 is an H+-coupled monosaccharide transporter, present in source leaves and seed coats, with higher substrate affinity at depolarized potentials.

Sugar transport proteins (STPs) are high-affinity H+-coupled hexose symporters. Recently, the contribution of STP13 to bacterial and fungal pathogen resistance across multiple plant species has garnered significant interest. Quantitative PCR analysis of source leaves, developing embryos, and seed coats of Phaseolus vulgaris L. (common bean) revealed that PvSTP13.1 was expressed in source leaves and seed coats throughout seed development. In contrast, PvSTP13.1 transcripts were detected at exceedingly low levels in developing embryos. To characterize the transport mechanism, PvSTP13.1 was expressed in Xenopus laevis oocytes, and inward-directed currents were analyzed using two-electrode voltage clamping. PvSTP13.1 was shown to function as an H+-coupled monosaccharide symporter exhibiting a unique high affinity for hexoses and aldopentoses at depolarized membrane potentials. Specifically, of the 31 assessed substrates, which included aldohexoses, deoxyhexoses, fructose, 3-O-methyl-D-glucose, aldopentoses, polyols, glycosides, disaccharides, trisaccharides, and glucuronic acid, PvSTP13.1 displayed the highest affinity (K 0.5) for glucose (43 µM), mannose (92 µM), galactose (145 µM), fructose (224 µM), xylose (1.0 mM), and fucose (3.7 mM) at pH 5.6 at a depolarized membrane potential of -40 mV. The results presented here suggest PvSTP13.1 contributes to retrieval of hexoses from the apoplasmic space in source leaves and coats of developing seeds.

Sugar loading of crop seeds - a partnership of phloem, plasmodesmal and membrane transport.

Sugar loading of developing seeds comprises a cohort of transport events that contribute to reproductive success and seed yield. Understanding these events is most advanced for grain crops (Brassicaceae, Fabaceae and Gramineae) and Arabidopsis. For these species, 75-80% of their final seed biomass is derived from phloem-imported sucrose. Sugar loading consecutively traverses three genomically distinct, and symplasmically isolated, seed domains: maternal pericarp/seed coat, filial endosperm and filial embryo. Sink status of each domain co-ordinately transitions from growth to storage. The latter is dominated by embryos (Brassicaceae and Fabaceae) or endosperms (Gramineae). Intradomain sugar transport occurs symplasmically through plasmodesmata. Interdomain sugar transport relies on plasma-membrane transporters operating in efflux (maternal and endosperm) or influx (endosperm and embryo) modes. Discussed is substantial progress made in identifying, and functionally evaluating, sugar symporters (STPs, SUTs or SUCs) and uniporters (SWEETs). These findings have underpinned a mechanistic understanding of seed loading. Less well researched are possible physical limitations imposed by hydraulic conductivities of differentiating protophloem and of subsequent plasmodesmal transport. The latter is coupled with sugar homeostasis within each domain mediated by sugar transporters. A similar conclusion is ascribed to fragmentary understanding of regulatory mechanisms integrating transport events with seed growth and storage.

Investigation of Protein-Lipid Interactions Using Native Mass Spectrometry.

Integral membrane proteins are embedded in biological membranes where various lipids modulate their structure and function. There exists a critical need to elucidate how these lipids participate in the physiological and pathological processes associated with the membrane protein dysfunction. Native mass spectrometry (MS), combined with ion mobility spectrometry (IM), is emerging as a powerful tool to probe membrane protein complexes and their interactions with ligands, lipids, and other small molecules. Unlike other biophysical approaches, native IM-MS can resolve individual ligand/lipid binding events. We have developed a novel method using native MS, coupled with a temperature-control apparatus, to determine the thermodynamic parameters of individual ligand or lipid binding events to proteins. This approach has been validated using several soluble protein-ligand systems wherein MS results are compared with those acquired from conventional biophysical techniques, such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). Using these principles, it is possible to elucidate the thermodynamics of individual lipid binding to integral membrane proteins. Herein, we use the ammonia channel (AmtB) from Escherichia coli as a model membrane protein. Remarkably, distinct thermodynamic signatures for AmtB binding to lipids with different headgroups and acyl chain configurations are observed. Additionally, using a mutant form of AmtB that abolishes a specific lipid binding site, distinct changes have been discovered in the thermodynamic signatures compared with the wild-type, implying that these signatures can identify key residues involved in specific lipid binding and potentially differentiate between specific lipid binding sites. This chapter provides procedures and findings associated with temperature-controlled native MS as a novel approach to interrogate membrane proteins and their interactions with lipids and other molecules.

Integrating Sugar Metabolism With Transport: Elevation of Endogenous Cell Wall Invertase Activity Up-Regulates SlHT2 and SlSWEET12c Expression for Early Fruit Development in Tomato.

Early fruit development is critical for determining crop yield. Cell wall invertase (CWIN) and sugar transporters both play important roles in carbon allocation and plant development. However, there is little information about the relationship between CWIN and those functionally related sugar transporters during fruit development. By using transgenic tomato with an elevated CWIN activity, we investigated how an increase in CWIN activity may regulate the expression of sugar transporter genes during fruit development. Our analyses indicate that CWIN activity may be under tight regulation by multiple regulators, including two invertase inhibitors (INVINHs) and one defective CWIN (deCWIN) in tomato ovaries prior to anthesis. Among the sugar transporters, expression of SlSWEET12c for sucrose efflux and SlHT2 for hexose uptake was enhanced by the elevated CWIN activity at 10 and 15 days after anthesis of tomato fruit development, respectively. The findings show that some specific sugars will eventually be exported transporters (SWEETs) and hexose transporters (HTs) respond to elevate CWIN activity probably to promote rapid fruit expansion when sucrose efflux from phloem and hexose uptake by parenchyma cell are in high demand. The analyses provide new leads for improving crop yield by manipulating CWIN-responsive sugar transporters, together with CWIN itself, to enhance fruit development and sugar accumulation.

A Sec14-like phosphatidylinositol transfer protein paralog defines a novel class of heme-binding proteins.

Yeast Sfh5 is an unusual member of the Sec14-like phosphatidylinositol transfer protein (PITP) family. Whereas PITPs are defined by their abilities to transfer phosphatidylinositol between membranes in vitro, and to stimulate phosphoinositide signaling in vivo, Sfh5 does not exhibit these activities. Rather, Sfh5 is a redox-active penta-coordinate high spin FeIII hemoprotein with an unusual heme-binding arrangement that involves a co-axial tyrosine/histidine coordination strategy and a complex electronic structure connecting the open shell iron d-orbitals with three aromatic ring systems. That Sfh5 is not a PITP is supported by demonstrations that heme is not a readily exchangeable ligand, and that phosphatidylinositol-exchange activity is resuscitated in heme binding-deficient Sfh5 mutants. The collective data identify Sfh5 as the prototype of a new class of fungal hemoproteins, and emphasize the versatility of the Sec14-fold as scaffold for translating the binding of chemically distinct ligands to the control of diverse sets of cellular activities.

Transfer cells: what regulates the development of their intricate wall labyrinths?

Transfer cells (TCs) support high nutrient rates into, or at symplasmic discontinuities within, the plant body. Their transport capacity is conferred by an amplified plasma membrane surface area, enriched in nutrient transporters, supported on an intricately invaginated wall labyrinth (WL). Thus, development of the WL is at the heart of TC function. Enquiry has shifted from describing WL architecture and formation to discovering mechanisms regulating WL assembly. Experimental systems used to examine these phenomena are critiqued. Considerable progress has been made in identifying master regulators that commit stem cells to a TC fate (e.g. the maize Myeloblastosis (MYB)-related R1-type transcription factor) and signals that induce differentiated cells to undergo trans-differentiation to a TC phenotype (e.g. sugar, auxin and ethylene). In addition, signals that provide positional information for assembly of the WL include apoplasmic hydrogen peroxide and cytosolic Ca2+ plumes. The former switches on, and specifies the intracellular site for WL construction, while the latter creates subdomains to direct assembly of WL invaginations. Less is known about macromolecule species and their spatial organization essential for WL assembly. Emerging evidence points to a dependency on methyl-esterified homogalacturonan accumulation, unique patterns of cellulose and callose deposition and spatial positioning of arabinogalactan proteins.

Enhanced Characterization of Membrane Protein Complexes by Ultraviolet Photodissociation Mass Spectrometry.

Development of chemical chaperones to solubilize membrane protein complexes in aqueous solutions has allowed for gas-phase analysis of their native-like assemblies, including rapid evaluation of stability and interacting partners. Characterization of protein primary sequence, however, has thus far been limited. Ultraviolet photodissociation (UVPD) generates a multitude of sequence ions for the E. coli ammonia channel (AmtB), provides improved localization of a possible post-translational modification of aquaporin Z (AqpZ), and surpasses previous reports of sequence coverage for mechanosensitive channel of large conductance (MscL). Variations in UVPD sequence ion abundance have been shown to correspond to structural changes induced upon some perturbation. Preliminary results are reported here for elucidating increased rigidity or flexibility of MscL when bound to various phospholipids.

Enzymes contributing to the hydrogen peroxide signal dynamics that regulate wall labyrinth formation in transfer cells.

Transfer cells are characterized by an amplified plasma membrane area supported on a wall labyrinth composed of a uniform wall layer (UWL) from which wall ingrowth (WI) papillae arise. Adaxial epidermal cells of developing Vicia faba cotyledons, when placed in culture, undergo a rapid (hours) trans-differentiation to a functional epidermal transfer cell (ETC) phenotype. The trans-differentiation event is controlled by a signalling cascade comprising auxin, ethylene, apoplasmic reactive oxygen species (apoROS), and cytosolic Ca2+. Apoplasmic hydrogen peroxide (apoH2O2) was confirmed as the apoROS regulating UWL and WI papillae formation. Informed by an ETC-specific transcriptome, a pharmacological approach identified a temporally changing cohort of H2O2 biosynthetic enzymes. The cohort contained a respiratory burst oxidase homologue, polyamine oxidase, copper amine oxidase, and a suite of class III peroxidases. Collectively these generated two consecutive bursts in apoH2O2 production. Spatial organization of biosynthetic/catabolic enzymes was deduced from responses to pharmacologically blocking their activities on the cellular and subcellular distribution of apoH2O2. The findings were consistent with catalase activity constraining the apoH2O2 signal to the outer periclinal wall of the ETCs. Strategic positioning of class III peroxidases in this outer domain shaped subcellular apoH2O2 signatures that differed during assembly of the UWL and WI papillae.

Probing Heterogeneous Lipid Interactions with Membrane Proteins Using Mass Spectrometry.

Native mass spectrometry (Native MS) enables the detection of intact membrane protein complexes in the gas phase. Membrane proteins are encapsulated in nonionic detergent micelles that protect them during transfer into the gas phase and preserves structure and noncovalent interactions. Herein, we describe methods to gently transfer membrane protein complexes bound to a mixture of heterogeneous lipid species into the gas phase. Through careful titrations, equilibrium dissociation constants can be directly determined to elucidate lipid interactions that induce positive, neutral, or negative allostery. These methods can lead to the identification of lipids that modulate membrane protein structure and function.

Generation of Charge-Reduced Ions of Membrane Protein Complexes for Native Ion Mobility Mass Spectrometry Studies.

Recent advances in native mass spectrometry (MS) have enabled the elucidation of how small molecule binding to membrane proteins modulates their structure and function. The protein-stabilizing osmolyte, trimethylamine oxide (TMAO), exhibits attractive properties for native MS studies. Here, we report significant charge reduction, nearly threefold, for three membrane protein complexes in the presence of this osmolyte without compromising mass spectral resolution. TMAO improves the ability to resolve individual lipid-binding events to the ammonia channel (AmtB) by over 200% compared to typical native conditions. The generation of ions with compact structure and access to a larger number of lipid-binding events through the incorporation of TMAO increases the utility of IM-MS for structural biology studies. Graphical Abstract.

Ethylene and hydrogen peroxide regulate formation of a sterol-enriched domain essential for wall labyrinth assembly in transfer cells.

Transfer cells (TCs) facilitate high rates of nutrient transport into, and within, the plant body. Their transport function is conferred by polarized wall ingrowth papillae, deposited upon a specialized uniform wall layer, that form a scaffold supporting an amplified area of plasma membrane enriched in nutrient transporters. We explored the question of whether lipid-enriched domains of the TC plasma membrane could serve as organizational platforms for proteins regulating the construction of the intricate TC wall labyrinth using developing Vicia faba cotyledons. When these cotyledons are placed in culture, their adaxial epidermal cells trans-differentiate to a TC phenotype regulated by auxin, ethylene, extracellular hydrogen peroxide (apoH2O2), and cytosolic Ca2+ ([Ca2+]cyt) arranged in series. Staining cultured cotyledons with the sterol-specific dye, Filipin III, detected a polarized sterol-enriched domain in the plasma membrane of their trans-differentiating epidermal transfer cells (ETCs). Ethylene activated sterol biosynthesis while extracellular apoH2O2 directed sterol-enriched vesicles to fuse with the outer periclinal region of the ETC plasma membrane. The sterol-enriched domain was essential for generating the [Ca2+]cyt signal and orchestrating construction of both the uniform wall layer and wall ingrowth papillae. A model is presented outlining how the sterol-enriched plasma membrane domain forms and functions to regulate wall labyrinth assembly.

Development and Evaluation of a Reverse-Entry Ion Source Orbitrap Mass Spectrometer.

As a step towards development of a high-resolution ion mobility mass spectrometer using the orbitrap mass analyzer platform, we describe herein a novel reverse-entry ion source (REIS) coupled to the higher-energy C-trap dissociation (HCD) cell of an orbitrap mass spectrometer with extended mass range. Development of the REIS is a first step in the development of a drift tube ion mobility-orbitrap MS. The REIS approach retains the functionality of the commercial instrument ion source which permits the uninterrupted use of the instrument during development as well as performance comparisons between the two ion sources. Ubiquitin (8.5 kDa) and lipid binding to the ammonia transport channel (AmtB, 126 kDa) protein complex were used as model soluble and membrane proteins, respectively, to evaluate the performance of the REIS instrument. Mass resolution obtained with the REIS is comparable to that obtained using the commercial ion source. The charge state distributions for ubiquitin and AmtB obtained on the REIS are in agreement with previous studies which suggests that the REIS-orbitrap EMR retains native structure in the gas phase. Graphical Abstract ᅟ.

Allostery revealed within lipid binding events to membrane proteins.

Membrane proteins interact with a myriad of lipid species in the biological membrane, leading to a bewildering number of possible protein-lipid assemblies. Despite this inherent complexity, the identification of specific protein-lipid interactions and the crucial role of lipids in the folding, structure, and function of membrane proteins is emerging from an increasing number of reports. Fundamental questions remain, however, regarding the ability of specific lipid binding events to membrane proteins to alter remote binding sites for lipids of a different type, a property referred to as allostery [Monod J, Wyman J, Changeux JP (1965) J Mol Biol 12:88-118]. Here, we use native mass spectrometry to determine the allosteric nature of heterogeneous lipid binding events to membrane proteins. We monitored individual lipid binding events to the ammonia channel (AmtB) from Escherichia coli, enabling determination of their equilibrium binding constants. We found that different lipid pairs display a range of allosteric modulation. In particular, the binding of phosphatidylethanolamine and cardiolipin-like molecules to AmtB exhibited the largest degree of allosteric modulation, inspiring us to determine the cocrystal structure of AmtB in this lipid environment. The 2.45-Å resolution structure reveals a cardiolipin-like molecule bound to each subunit of the trimeric complex. Mutation of a single residue in AmtB abolishes the positive allosteric modulation observed for binding phosphatidylethanolamine and cardiolipin-like molecules. Our results demonstrate that specific lipid-protein interactions can act as allosteric modulators for the binding of different lipid types to integral membrane proteins.

Protonation state of glutamate 73 regulates the formation of a specific dimeric association of mVDAC1.

The voltage-dependent anion channel (VDAC) is the most abundant protein in the outer mitochondrial membrane and constitutes the primary pathway for the exchange of ions and metabolites between the cytosol and the mitochondria. There is accumulating evidence supporting VDAC's role in mitochondrial metabolic regulation and apoptosis, where VDAC oligomerization has been implicated with these processes. Herein, we report a specific pH-dependent dimerization of murine VDAC1 (mVDAC1) identified by double electron-electron resonance and native mass spectrometry. Intermolecular distances on four singly spin-labeled mVDAC1 mutants were used to generate a model of the low-pH dimer, establishing the presence of residue E73 at the interface. This dimer arrangement is different from any oligomeric state previously described, and it forms as a steep function of pH with an apparent pKa of 7.4. Moreover, the monomer-dimer equilibrium affinity constant was determined using native MS, revealing a nearly eightfold enhancement in dimerization affinity at low pH. Mutation of E73 to either alanine or glutamine severely reduces oligomerization, demonstrating the role of protonated E73 in enhancing dimer formation. Based on these results, and the known importance of E73 in VDAC physiology, VDAC dimerization likely plays a significant role in mitochondrial metabolic regulation and apoptosis in response to cytosolic acidification during cellular stress.

Mechanisms of phloem unloading: shaped by cellular pathways, their conductances and sink function.

Phloem unloading represents a series of cell-to-cell transport steps transferring phloem-mobile constituents from phloem to sink tissues/organs to fuel their development or resource storage. Our analysis focuses on unloading of two major phloem-mobile constituents, sugars and water. Their unloading can occur across phloem plasma membranes (apoplasmic unloading), through plasmodesmata interconnecting phloem and sink cells (symplasmic unloading) or predominately symplasmically with an intervening post-phloem apoplasmic step. In planta studies of phloem unloading encounter substantial technical challenges in accessing phloem within a meshwork of vascular/ground tissues. Thus, current understanding of phloem-unloading mechanisms largely has been deduced from indirect experimental measures or modelling. Here we highlight recent advances in understanding phloem unloading mechanisms and identify where important knowledge gaps remain.

Transcript Profiling Identifies Gene Cohorts Controlled by Each Signal Regulating Trans-Differentiation of Epidermal Cells of Vicia faba Cotyledons to a Transfer Cell Phenotype.

Transfer cells (TCs) support high rates of membrane transport of nutrients conferred by a plasma membrane area amplified by lining a wall labyrinth comprised of an uniform wall layer (UWL) upon which intricate wall ingrowth (WI) papillae are deposited. A signal cascade of auxin, ethylene, extracellular hydrogen peroxide (H2O2) and cytosolic Ca2+ regulates wall labyrinth assembly. To identify gene cohorts regulated by each signal, a RNA- sequencing study was undertaken using Vicia faba cotyledons. When cotyledons are placed in culture, their adaxial epidermal cells spontaneously undergo trans-differentiation to epidermal TCs (ETCs). Expressed genes encoding proteins central to wall labyrinth formation (signaling, intracellular organization, cell wall) and TC function of nutrient transport were assembled. Transcriptional profiles identified 9,742 annotated ETC-specific differentially expressed genes (DEGs; Log2fold change > 1; FDR p ≤ 0.05) of which 1,371 belonged to signaling (50%), intracellular organization (27%), cell wall (15%) and nutrient transporters (9%) functional categories. Expression levels of 941 ETC-specific DEGs were found to be sensitive to the known signals regulating ETC trans-differentiation. Significantly, signals acting alone, or in various combinations, impacted similar numbers of ETC-specific DEGs across the four functional gene categories. Amongst the signals acting alone, H2O2 exerted most influence affecting expression levels of 56% of the ETC-specific DEGs followed by Ca2+ (21%), auxin (18%) and ethylene (5%). The dominance by H2O2 was evident across all functional categories, but became more attenuated once trans-differentiation transitioned into WI papillae formation. Amongst the eleven signal combinations, H2O2/Ca2+ elicited the greatest impact across all functional categories accounting for 20% of the ETC-specific DEG cohort. The relative influence of the other signals acting alone, or in various combinations, varied across the four functional categories and two phases of wall labyrinth construction. These transcriptome data provide a powerful information platform from which to examine signal transduction pathways and how these regulate expression of genes encoding proteins engaged in intracellular organization, cell wall construction and nutrient transport.

A Structurally Specialized Uniform Wall Layer is Essential for Constructing Wall Ingrowth Papillae in Transfer Cells.

Transfer cells are characterized by wall labyrinths with either a flange or reticulate architecture. A literature survey established that reticulate wall ingrowth papillae ubiquitously arise from a modified component of their wall labyrinth, termed the uniform wall layer; a structure absent from flange transfer cells. This finding sparked an investigation of the deposition characteristics and role of the uniform wall layer using a Vicia faba cotyledon culture system. On transfer of cotyledons to culture, their adaxial epidermal cells spontaneously trans-differentiate to a reticulate architecture comparable to their abaxial epidermal transfer cell counterparts formed in planta. Uniform wall layer construction commenced once adaxial epidermal cell expansion had ceased to overlay the original outer periclinal wall on its inner surface. In contrast to the dense ring-like lattice of cellulose microfibrils in the original primary wall, the uniform wall layer was characterized by a sparsely dispersed array of linear cellulose microfibrils. A re-modeled cortical microtubule array exerted no influence on uniform wall layer formation or on its cellulose microfibril organization. Surprisingly, formation of the uniform wall layer was not dependent upon depositing a cellulose scaffold. In contrast, uniform wall cellulose microfibrils were essential precursors for constructing wall ingrowth papillae. On converging to form wall ingrowth papillae, the cellulose microfibril diameters increased 3-fold. This event correlated with up-regulated differential, and transfer-cell specific, expression of VfCesA3B while transcript levels of other cellulose biosynthetic-related genes linked with primary wall construction were substantially down-regulated.

A Ca2+-dependent remodelled actin network directs vesicle trafficking to build wall ingrowth papillae in transfer cells.

The transport function of transfer cells is conferred by an enlarged plasma membrane area, enriched in nutrient transporters, that is supported on a scaffold of wall ingrowth (WI) papillae. Polarized plumes of elevated cytosolic Ca2+ define loci at which WI papillae form in developing adaxial epidermal transfer cells of Vicia faba cotyledons that are induced to trans-differentiate when the cotyledons are placed on culture medium. We evaluated the hypothesis that vesicle trafficking along a Ca2+-regulated remodelled actin network is the mechanism that underpins this outcome. Polarized to the outer periclinal cytoplasm, a Ca2+-dependent remodelling of long actin bundles into short, thin bundles was found to be essential for assembling WI papillae but not the underlying uniform wall layer. The remodelled actin network directed polarized vesicle trafficking to sites of WI papillae construction, and a pharmacological study indicated that both exo- and endocytosis contributed to assembly of the papillae. Potential candidates responsible for the Ca2+-dependent actin remodelling, along with those underpinning polarized exo- and endocyotosis, were identified in a transcriptome RNAseq database generated from the trans-differentiating epidermal cells. Of most significance, endocytosis was controlled by up-regulated expression of a dynamin-like isoform. How a cycle of localized exo- and endocytosis, regulated by Ca2+-dependent actin remodelling, assembles WI papillae is discussed.