Microsomal Membrane Fractions Research Articles

Characterization of a PIP Binding Site in the N-Terminal Domain of V-ATPase a4 and Its Role in Plasma Membrane Association

Vacuolar ATPases (V-ATPases) are multi-subunit ATP-dependent proton pumps necessary for cellular functions, including pH regulation and membrane fusion. The evidence suggests that the V-ATPase a-subunit's interaction with the membrane signaling lipid phosphatidylinositol (PIPs) regulates the recruitment of V-ATPase complexes to specific membranes. We generated a homology model of the N-terminal domain of the human a4 isoform (a4NT) using Phyre2.0 and propose a lipid binding domain within the distal lobe of the a4NT. We identified a basic motif, K234IKK237, critical for interaction with phosphoinositides (PIP), and found similar basic residue motifs in all four mammalian and both yeast a-isoforms. We tested PIP binding of wildtype and mutant a4NT in vitro. In protein lipid overlay assays, the double mutation K234A/K237A and the autosomal recessive distal renal tubular-causing mutation K237del reduced both PIP binding and association with liposomes enriched with PI(4,5)P2, a PIP enriched within plasma membranes. Circular dichroism spectra of the mutant protein were comparable to wildtype, indicating that mutations affected lipid binding, not protein structure. When expressed in HEK293, wildtype a4NT localized to the plasma membrane in fluorescence microscopy and co-purified with the microsomal membrane fraction in cellular fractionation experiments. a4NT mutants showed reduced membrane association and decreased plasma membrane localization. Depletion of PI(4,5)P2 by ionomycin caused reduced membrane association of the WT a4NT protein. Our data suggest that information contained within the soluble a4NT is sufficient for membrane association and that PI(4,5)P2 binding capacity is involved in a4 V-ATPase plasma membrane retention.

A Rapid and Universal Workflow for Label-Free-Quantitation-Based Proteomic and Phosphoproteomic Studies in Cereals.

Proteomics and phosphoproteomics are robust tools to analyze dynamics of post-transcriptional processes during growth and development. A variety of experimental methods and workflows have been published, but most of them were developed for model plants and have not been adapted to high-throughput platforms. Here, we describe an experimental workflow for proteome and phosphoproteome studies tailored to cereal crop tissues. The workflow consists of two parallel parts that are suitable for analyzing protein/phosphoprotein from total proteins and the microsomal membrane fraction. We present phosphoproteomic data regarding quantification coverage and analytical reproducibility for example preparations from maize root and shoot, wheat leaf, and a microsomal protein preparation from maize leaf. To enable users to adjust for tissue specific requirements, we provide two different methods of protein clean-up: traditional ethanol precipitation (PC) and a recently developed technology termed single-pot, solid-phase-enhanced sample preparation (SP3). Both the PC and SP3 methods are effective in the removal of unwanted substances in total protein crude extracts. In addition, two different methods of phosphopeptide enrichment are presented: a TiO2 -based method and Fe(III)-NTA cartridges on a robotized platform. Although the overall number of phosphopeptides is stable across protein clean-up and phosphopeptide enrichment methods, there are differences in the preferred phosphopeptides in each enrichment method. The preferred protocol depends on laboratory capabilities and research objective. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Total protein crude extraction Basic Protocol 2: Total protein clean-up with ethanol precipitation Alternate Protocol 1: Total protein clean-up with SP3 method Basic Protocol 3: Microsomal fraction protein extraction Basic Protocol 4: Protein concentration determination by Bradford assay Basic Protocol 5: In-solution digestion with trypsin Basic Protocol 6: Phosphopeptide enrichment with TiO2 Alternate Protocol 2: Phosphopeptide enrichment with Fe(III)-NTA cartridges Basic Protocol 7: Peptide desalting with C18 material Basic Protocol 8: LC-MS/MS analysis of (phospho)peptides and spectrum matching.

Active Transport of Lignin Precursors into Membrane Vesicles from Lignifying Tissues of Bamboo.

Lignin is the second most abundant natural polymer on Earth and is a major cell wall component in vascular plants. Lignin biosynthesis has three stages: biosynthesis, transport, and polymerization of its precursors. However, there is limited knowledge on lignin precursor transport, especially in monocots. In the present study, we aimed to elucidate the transport mode of lignin monomers in the lignifying tissues of bamboo (Phyllostachys pubescens). The growth manners and lignification processes of bamboo shoots were elucidated, which enabled us to obtain the lignifying tissues reproducibly. Microsomal membrane fractions were prepared from tissues undergoing vigorous lignification to analyze the transport activities of lignin precursors in order to show the ATP-dependent transport of coniferin and p-glucocoumaryl alcohol. The transport activities for both precursors depend on vacuolar type H+-ATPase and a H+ gradient across the membrane, suggesting that the electrochemical potential is the driving force of the transport of both substrates. These findings are similar to the transport properties of these lignin precursors in the differentiating xylem of poplar and Japanese cypress. Our findings suggest that transport of coniferin and p-glucocoumaryl alcohol is mediated by secondary active transporters energized partly by the vacuolar type H+-ATPase, which is common in lignifying tissues. The loading of these lignin precursors into endomembrane compartments may contribute to lignification in vascular plants.

A cell-free assay implicates a role of sphingomyelin and cholesterol in STING phosphorylation

Stimulator of interferon genes (STING) is essential for the type I interferon response induced by microbial DNA from virus or self-DNA from mitochondria/nuclei. In response to emergence of such DNAs in the cytosol, STING translocates from the endoplasmic reticulum to the Golgi, and activates TANK-binding kinase 1 (TBK1) at the trans-Golgi network (TGN). Activated TBK1 then phosphorylates STING at Ser365, generating an interferon regulatory factor 3-docking site on STING. How this reaction proceeds specifically at the TGN remains poorly understood. Here we report a cell-free reaction in which endogenous STING is phosphorylated by TBK1. The reaction utilizes microsomal membrane fraction prepared from TBK1-knockout cells and recombinant TBK1. We observed agonist-, TBK1-, “ER-to-Golgi” traffic-, and palmitoylation-dependent phosphorylation of STING at Ser365, mirroring the nature of STING phosphorylation in vivo. Treating the microsomal membrane fraction with sphingomyelinase or methyl-β-cyclodextrin, an agent to extract cholesterol from membranes, suppressed the phosphorylation of STING by TBK1. Given the enrichment of sphingomyelin and cholesterol in the TGN, these results may provide the molecular basis underlying the specific phosphorylation reaction of STING at the TGN.

Hepatic Synaptotagmin 1 is involved in the remodelling of liver plasma- membrane lipid composition and gene expression in male Apoe-deficient mice consuming a Western diet

Background and aims.The molecular mechanisms by which the liver develops steatotic disease still remain unclear. Previous studies using nutritional and genetic models of hepatic steatosis in mice showed that liver synaptotagmin 1 (Syt1) expression was associated with lipid droplet area. Hepatic Syt1 overexpression was used as a tool to explore its effect on hepatic and plasma lipids. Methods and resultsTo find out a cause-effect, hepatic mouse Syt1 mRNA was cloned into a vector driving hepatocyte-specific expression and administered by hydrodynamic injection to male Apoe-deficient mice fed on a Western diet, the latter as a model of rapid spontaneous steatosis development. Hepatic microsomal, large vesicle, lysosomal and plasma membrane fractions were enriched in SYT1 protein following gene overexpression. In these conditions, very low density lipoprotein esterified cholesterol increased. Likewise, the transgene caused an alteration in lipid droplet surface and a positive correlation between Syt1 expression and hepatic total cholesterol content. A lipidomic approach evidenced a decrease in lysophosphatidylcholine, phosphatidylcholine and triglycerides in isolated plasma membrane fraction. Expressions of genes involved in biosynthesis of bile acids, fatty acid metabolism, lipoprotein dynamics and vesicular transport were modified by the increased SYT1 expression. ConclusionsThese results indicate that this protein is involved in hepatic management of lipids and in the regulation of genes involved in lipid metabolism.

Affinity Labeling and Purification of Plant Chitin-Binding LysM Receptor with Chitin Octasaccharide Derivatives.

Lysin motif (LysM) is a carbohydrate-binding modules found in all kingdoms. LysM binds to N-acetylglucosamine-containing molecules such as peptidoglycan, chitin, Nod factor, and Myc factor and is found in peptidoglycan hydrolases, chitinases, and plant pathogen effectors and plant receptor/co-receptor for defense and symbiosis signaling. This chapter describes the synthesis of a nonradioactive chitin ligand, biotinylated chitin octasaccharide, (GlcNAc)8-Bio, and its application for the detection and characterization of chitin-binding LysM receptor CEBiP in the microsomal membrane fraction of rice suspension-cultured cells by affinity labeling. We also describe the purification of CEBiP from the plasma membrane of the rice cells by affinity chromatography with the synthesized (GlcNAc)8-APEA-CH-Sepharose as an affinity matrix.

Blood and Gill Carbonic Anhydrase in the Context of a Chondrichthyan Model of CO2 Excretion.

Pacific spiny dogfish (Squalus suckleyi) have been widely used as a representative species for chondrichthyan CO2 excretion. Pacific spiny dogfish have a slower red blood cell (RBC) carbonic anhydrase (CA) isoform than teleost fishes, extracellular CA activity, no endogenous plasma CA inhibitor, and plasma-accessible CA IV at the gills. Thus, both the RBC and plasma compartments contribute to bicarbonate ion () dehydration at the gills for CO2 excretion in contrast to teleost fishes, in which dehydration is restricted to RBCs. We compared CA activity levels, subcellular localization, and presence of plasma CA inhibitors in the blood and gills of 13 chondrichthyans to examine the hypothesis that the dogfish model of CO2 excretion applies broadly to chondrichthyans. In general, blood samples from the 12 other chondrichthyans examined had lower RBC CA activity than teleosts, some extracellular CA activity, and no endogenous plasma CA inhibitor. While type IV-like membrane-associated CA was found in the gills in all four of the chondrichthyans examined, S. suckleyi had three times more CA activity ( μmol CO2 min-1 mg protein-1) in the microsomal (membrane) fraction of gills than the other three. In addition, unexpected variation in CA characteristics was observed between chondrichthyan species. Thus, in general, it appears that the pattern of CA distribution in fishes can be generally categorized as either chondrichthyan or teleost models. However, further studies should examine the functional significance of the within-chondrichthyan differences we observed and investigate whether CO2 excretion patterns exist along a continuum or in discrete groups.

Periapical lesions decrease Akt serine phosphorylation and plasma membrane GLUT4 content in rat skeletal muscle.

Periapical lesion (PL) promotes insulin resistance; however, the mechanisms underlying this alteration are not fully understood. Therefore, in this study, we aimed to evaluate the Akt serine phosphorylation status and GLUT4 expression levels in the gastrocnemius muscle (GM) of rats with PL. Male Wistar rats (n = 42) were distributed equally into control (CN) and PL groups. The pulpal tissue of the PL group rats was exposed to the oral environment for 30days. Thereafter, glucose and insulin levels were assessed, followed by homeostasis model assessment of insulin resistance (HOMA-IR). The Akt serine phosphorylation and GLUT4 levels of microsomal (M) and plasma membrane (PM) fractions were evaluated by western blotting and analyzed statistically. Compared to CN group rats, PL group rats had lower insulin sensitivity (as observed by HOMA-IR), lower Akt serine phosphorylation status after insulin stimulus, and lower GLUT4 levels in the PM fraction. However, the M fraction in the PL group did not differ significantly from that of the CN group. PL decreases insulin sensitivity, Akt phosphorylation, and PM GLUT4 content. The present study indicates that preventing endodontic disease can thwart insulin resistance.

Novel adipocyte aminopeptidases are selectively upregulated by insulin in healthy and obese rats.

The lack of a complete assembly of the sensitivity of subcellular aminopeptidase (AP) activities to insulin in different pathophysiological conditions has hampered the complete view of the adipocyte metabolic pathways and its implications in these conditions. Here we investigated the influence of insulin on basic AP (APB), neutral puromycin-sensitive AP (PSA), and neutral puromycin-insensitive AP (APM) in high and low density microsomal and plasma membrane fractions from adipocytes of healthy and obese rats. Catalytic activities of these enzymes were fluorometrically monitoring in these fractions with or without insulin stimulus. Canonical traffic such as insulin-regulated AP was not detected for these novel adipocyte APs in healthy and obese rats. However, insulin increased APM in low density microsomal and plasma membrane fractions from healthy rats, APB in high density microsomal fraction from obese rats and PSA in plasma membrane fraction from healthy rats. A new concept of intracellular compartment-dependent upregulation of AP enzyme activities by insulin emerges from these data. This relatively selective regulation has pathophysiological significance, since these enzymes are well known to act as catalysts and receptor of peptides directly related to energy metabolism. Overall, the regulation of each one of these enzyme activities reflects certain dysfunction in obese individuals.

Adipocyte aminopeptidases in obesity and fasting

This study checked the existence of a diverse array of aminopeptidase (AP) enzymes in high (HDM) and low (LDM) density microsomal and plasma membrane (MF) fractions from adipocytes of control, monosodium glutamate obese and food deprived rats. Gene expression was detected for ArgAP, AspAP, MetAP, and two AlaAP (APM and PSA). APM and PSA had the highest catalytic efficiency, whereas AspAP the highest affinity. Subcellular distribution of AP activities depended on metabolic status. Comparing catalytic levels, AspAP in HDM, LDM and MF was absent in obese and control under food deprivation; PSA in LDM was 3.5-times higher in obese than in normally fed control and control and obese under food deprivation; MetAP in MF was 4.5-times higher in obese than in food deprived obese. Data show new AP enzymes genetically expressed in subcellular compartments of adipocytes, three of them with altered catalytic levels that respond to whole-body energetic demands.

Comparative analysis of Mg-dependent and Mg-independent HCO3(-) ATPases.

The comparative analysis between two enzymes, Mg-dependent and Mg-independent HCO3(-) ATPases, were studied in synaptosomal and microsomal membrane fractions of albino rat brain, using the method of kinetic analysis of the multi-sited enzyme systems. Therefore, it can be inferred that Mg-dependent HCO3(-) ATPase belongs to the group of "P-type" transporting ATPases. Mg-independent HCO3(-) ATPase with its kinetic properties may be attributed to the group of "Ecto" ATPases.

Heterodimeric capping protein from Arabidopsis is a membrane-associated, actin-binding protein.

The actin cytoskeleton is a major regulator of cell morphogenesis and responses to biotic and abiotic stimuli. The organization and activities of the cytoskeleton are choreographed by hundreds of accessory proteins. Many actin-binding proteins are thought to be stimulus-response regulators that bind to signaling phospholipids and change their activity upon lipid binding. Whether these proteins associate with and/or are regulated by signaling lipids in plant cells remains poorly understood. Heterodimeric capping protein (CP) is a conserved and ubiquitous regulator of actin dynamics. It binds to the barbed end of filaments with high affinity and modulates filament assembly and disassembly reactions in vitro. Direct interaction of CP with phospholipids, including phosphatidic acid, results in uncapping of filament ends in vitro. Live-cell imaging and reverse-genetic analyses of cp mutants in Arabidopsis (Arabidopsis thaliana) recently provided compelling support for a model in which CP activity is negatively regulated by phosphatidic acid in vivo. Here, we used complementary biochemical, subcellular fractionation, and immunofluorescence microscopy approaches to elucidate CP-membrane association. We found that CP is moderately abundant in Arabidopsis tissues and present in a microsomal membrane fraction. Sucrose density gradient separation and immunoblotting with known compartment markers were used to demonstrate that CP is enriched on membrane-bound organelles such as the endoplasmic reticulum and Golgi. This association could facilitate cross talk between the actin cytoskeleton and a wide spectrum of essential cellular functions such as organelle motility and signal transduction.

Tissue and Cell Membrane Lipid Composition in Rats on Adaptation to Highland Conditions

The effects of the physical factors of highland altitudes (3200 m) on the lipid composition of tissues and cell membranes were studied in rats. Adaptation of the animals to highland conditions was accompanied by changes in tissue phospholipid composition. Adaptive phospholipid re-composition was seen in the lungs, brain, liver, and skeletal muscle, and in the microsomal membrane fractions isolated from them, with increases in the quantities of phosphatidylinositol and phosphatidic acid. Adaptation at low temperature (+10°C) led to more significant changes in lipid peroxidation and phospholipid composition in tissues and membranes than adaptation in thermally neutral conditions (+30°C). Modification of tissue and cell membrane lipid composition in rats in highland conditions appeared to increase the body’s adaptive potential – the animals showed a tendency to increases in physical work capacity and resistance to hypoxia.

Squeezing at Entrance of Proton Transport Pathway in Proton-translocating Pyrophosphatase upon Substrate Binding

Homodimeric proton-translocating pyrophosphatase (H(+)-PPase; EC 3.6.1.1) is indispensable for many organisms in maintaining organellar pH homeostasis. This unique proton pump couples the hydrolysis of PPi to proton translocation across the membrane. H(+)-PPase consists of 14-16 relatively hydrophobic transmembrane domains presumably for proton translocation and hydrophilic loops primarily embedding a catalytic site. Several highly conserved polar residues located at or near the entrance of the transport pathway in H(+)-PPase are essential for proton pumping activity. In this investigation single molecule FRET was employed to dissect the action at the pathway entrance in homodimeric Clostridium tetani H(+)-PPase upon ligand binding. The presence of the substrate analog, imidodiphosphate mediated two sites at the pathway entrance moving toward each other. Moreover, single molecule FRET analyses after the mutation at the first proton-carrying residue (Arg-169) demonstrated that conformational changes at the entrance are conceivably essential for the initial step of H(+)-PPase proton translocation. A working model is accordingly proposed to illustrate the squeeze at the entrance of the transport pathway in H(+)-PPase upon substrate binding.

Reliability and Robustness of Simultaneous Absolute Quantification of Drug Transporters, Cytochrome P450 Enzymes, and Udp-Glucuronosyltransferases in Human Liver Tissue by Multiplexed MRM/Selected Reaction Monitoring Mode Tandem Mass Spectrometry with Nano-Liquid Chromatography

Mass spectrometry (MS)-based multiplexed multiple reaction monitoring quantification of proteins has recently evolved as a versatile tool for accurate, absolute quantification of proteins. The purpose of this study was to examine the validity of the present method with regard to standard bioanalytical criteria for drug transporters, cytochrome P450 (CYP) enzymes and uridine 5′-diphospho-glucuronosyltransferases (UGTs). Membrane preparations from human liver tissue were used for target protein quantification. As a result, the determination coefficients (r2) of all targets were greater than 0.986. In the absence of matrix, inaccuracy values (expressed as % deviation) were −8.1% to 20.3%, whereas imprecision values (expressed as % coefficient of variation) were within 15.9%. In the presence of matrix, which consisted of digested plasma membrane fraction for transporters and digested microsomal membrane fraction for CYP enzymes and UGTs, respectively, the inaccuracy was −15.3%–8.1%, and the imprecision were within 18.9%. Sufficient sample stability of membrane fraction was shown for three freeze–thaw cycles, 32 days at −20°C, and in processed samples for 7 days at 10°C. In conclusion, this study demonstrated, for the first time, that the MS-based assay with nano-liquid chromatography provides adequate reliability and robustness for the quantification of selected drug transporters, P450 enzymes and UGTs. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:4037–4043, 2011

The Endoplasmic Reticulum (ER)-associated Degradation System Regulates Aggregation and Degradation of Mutant Neuroserpin

Familial encephalopathy with neuroserpin inclusion bodies is a neurodegenerative disorder characterized by the accumulation of neuroserpin polymers in the endoplasmic reticulum (ER) of cortical and subcortical neurons in the CNS because of neuroserpin point mutations. ER-associated degradation (ERAD) is involved in mutant neuroserpin degradation. In this study, we demonstrate that two ER-associated E3 ligases, Hrd1 and gp78, are involved in the ubiquitination and degradation of mutant neuroserpin. Overexpression of Hrd1 and gp78 decreases the mutant neuroserpin protein level, whereas Hrd1 and gp78 knockdown increases mutant neuroserpin stability. Moreover, ERAD impairment by mutant valosin-containing protein increases the mutant neuroserpin protein level and aggregate formation. Thus, these findings identify mutant neuroserpin as an ERAD target and show that Hrd1 and gp78 mediate mutant neuroserpin turnover through the ERAD pathway.

Development of Filtration-Based Time-Resolved Fluorescence Assay for the High-Throughput Screening of Urotensin II Receptor Antagonist

The time-resolved fluorescence (TRF) receptor binding assay has many advantages over the traditional radioligand binding assay in terms of sensitivity and reproducibility for the screening of receptor ligands. The TRF-based urotensin receptor (UT) binding assay with an automatic vacuum filtration system was developed and evaluated for the high-throughput screening of UT receptor antagonists. For this assay development, the human recombinant urotensin II (UII) was modified by labeling europium at its N-terminal position (Eu-UII) and used as a fluorescent tracer. The microsomal membrane fraction of UT receptor was prepared from HEK293 cells stably expressing the human UT receptor. The 50% inhibitory concentration (IC(50)) values of UII from competition binding assays with Eu-UII were 2.76 nM, which is very similar to that of fluorescence polarization (FP)-based UT receptor binding experiment (2.18 nM). Comparing with the FP-based receptor binding assay for UII (Z' factor, 0.36), the current TRF assay presented improved Z' factor (0.76) with a relatively higher signal-to-background ratio (1.5 and 2.1, respectively). The known high-affinity UT receptor antagonists, palosuran and SB657510, exhibited IC(50) values of 23.6 and 73.4 nM, respectively, which were consistent with the IC(50) values from FP-based receptor binding assay (30.6 and 78.7 nM, respectively). These results suggest that our filtration-based TRF UT receptor binding assay can achieve the desired sensitivity with higher reproducibility to adapt for the high-throughput screening of compound libraries.

A nodulin/glutamine synthetase-like fusion protein is implicated in the regulation of root morphogenesis and in signalling triggered by flagellin

The nodulin/glutamine synthetase-like protein (NodGS) that we identified proteomically in Arabidopsis thaliana is a fusion protein composed of an N-terminal amidohydrolase domain that shares homology with nodulins and a C-terminal domain of prokaryotic glutamine synthetase type I. The protein is homologous to the FluG protein, a morphogenetic factor in fungi. Although genes encoding NodGS homologues are present in many plant genomes, their products have not yet been characterized. The Arabidopsis NodGS was present in an oligomeric form of ~700-kDa, mainly in the cytosol, and to a lesser extent in the microsomal membrane fraction. The oligomeric NodGS was incorporated into large heterogeneous protein complexes >700 kDa and partially co-immunoprecipitated with γ-tubulin. In situ and in vivo microscopic analyses revealed a NodGS signal in the cytoplasm, with endomembranes, particularly in the perinuclear area. NodGS had no detectable glutamine synthetase activity. Downregulation of NodGS by RNAi resulted in plants with a short main root, reduced meristematic activity and disrupted development of the root cap. Y2H analysis and publicly available microarray data indicated a role for NodGS in biotic stress signalling. We found that flagellin enhanced the expression of the NodGS protein, which was then preferentially localized in the nuclear periphery. Our results point to a role for NodGS in root morphogenesis and microbial elicitation. These data might help in understanding the family of NodGS/FluG-like fusion genes that are widespread in prokaryotes, fungi and plants.

Proteomic Analysis of Extracellular ATP-Regulated Proteins Identifies ATP Synthase β-Subunit as a Novel Plant Cell Death Regulator

Extracellular ATP is an important signal molecule required to cue plant growth and developmental programs, interactions with other organisms, and responses to environmental stimuli. The molecular targets mediating the physiological effects of extracellular ATP in plants have not yet been identified. We developed a well characterized experimental system that depletes Arabidopsis cell suspension culture extracellular ATP via treatment with the cell death-inducing mycotoxin fumonisin B1. This provided a platform for protein profile comparison between extracellular ATP-depleted cells and fumonisin B1-treated cells replenished with exogenous ATP, thus enabling the identification of proteins regulated by extracellular ATP signaling. Using two-dimensional difference in-gel electrophoresis and matrix-assisted laser desorption-time of flight MS analysis of microsomal membrane and total soluble protein fractions, we identified 26 distinct proteins whose gene expression is controlled by the level of extracellular ATP. An additional 48 proteins that responded to fumonisin B1 were unaffected by extracellular ATP levels, confirming that this mycotoxin has physiological effects on Arabidopsis that are independent of its ability to trigger extracellular ATP depletion. Molecular chaperones, cellular redox control enzymes, glycolytic enzymes, and components of the cellular protein degradation machinery were among the extracellular ATP-responsive proteins. A major category of proteins highly regulated by extracellular ATP were components of ATP metabolism enzymes. We selected one of these, the mitochondrial ATP synthase β-subunit, for further analysis using reverse genetics. Plants in which the gene for this protein was knocked out by insertion of a transfer-DNA sequence became resistant to fumonisin B1-induced cell death. Therefore, in addition to its function in mitochondrial oxidative phosphorylation, our study defines a new role for ATP synthase β-subunit as a pro-cell death protein. More significantly, this protein is a novel target for extracellular ATP in its function as a key negative regulator of plant cell death.

Arabidopsis Stromal-derived Factor2 (SDF2) Is a Crucial Target of the Unfolded Protein Response in the Endoplasmic Reticulum

Stresses increasing the load of unfolded proteins that enter the endoplasmic reticulum (ER) trigger a protective response termed the unfolded protein response (UPR). Stromal cell-derived factor2 (SDF2)-type proteins are highly conserved throughout the plant and animal kingdoms. In this study we have characterized AtSDF2 as crucial component of the UPR in Arabidopsis thaliana. Using a combination of biochemical and cell biological methods, we demonstrate that SDF2 is induced in response to ER stress conditions causing the accumulation of unfolded proteins. Transgenic reporter plants confirmed induction of SDF2 during ER stress. Under normal growth conditions SDF2 is highly expressed in fast growing, differentiating cells and meristematic tissues. The increased production of SDF2 due to ER stress and in tissues that require enhanced protein biosynthesis and secretion, and its association with the ER membrane qualifies SDF2 as a downstream target of the UPR. Determination of the SDF2 three-dimensional crystal structure at 1.95 A resolution revealed the typical beta-trefoil fold with potential carbohydrate binding sites. Hence, SDF2 might be involved in the quality control of glycoproteins. Arabidopsis sdf2 mutants display strong defects and morphological phenotypes during seedling development specifically under ER stress conditions, thus establishing that SDF2-type proteins play a key role in the UPR.