Functional Interaction between Cytochrome P450 and UDP-Glucuronosyltransferase on the Endoplasmic Reticulum Membrane: One of Post-translational Factors Which Possibly Contributes to Their Inter-Individual Differences.

Cytochrome P450 (P450) and uridine 5'-diphosphate (UDP)-glucuronosyltransferase (UGT) catalyze oxidation and glucuronidation in drug metabolism, respectively. It is believed that P450 and UGT work separately because they perform distinct reactions and exhibit opposite membrane topologies on the endoplasmic reticulum (ER). However, given that some chemicals are sequentially metabolized by P450 and UGT, it is reasonable to consider that the enzymes may interact and work cooperatively. Previous research by our team detected protein-protein interactions between P450 and UGT by analyzing solubilized rat liver microsomes with P450-immobilized affinity column chromatography. Although P450 and UGT have been known to form homo- and hetero-oligomers, this is the first report indicating a P450-UGT association. Based on our previous study, we focused on the P450-UGT interaction and reported lines of evidence that the P450-UGT association is a functional protein-protein interaction that can alter the enzymatic capabilities, including enhancement or suppression of the activities of P450 and UGT, helping UGT to acquire novel regioselectivity, and inhibiting substrate binding to P450. Biochemical and molecular bioscientific approaches suggested that P450 and UGT interact with each other at their internal hydrophobic domains in the ER membrane. Furthermore, several in vivo studies have reported the presence of a functional P450-UGT association under physiological conditions. The P450-UGT interaction is expected to function as a novel post-translational factor for inter-individual differences in the drug-metabolizing enzymes.

Topology of phosphatidylserine synthase 1 in the endoplasmic reticulum membrane.

Phosphatidylserine (PS) synthase 1 (PSS1) of mammalian cells is a multiple membrane-spanning protein of the endoplasmic reticulum (ER) and regulated by inhibition with the product PS. Alanine-scanning mutagenesis of PSS1 has revealed eight amino acid residues as those crucial for its activity and six as those important for its regulation. Furthermore, three missense mutations in the human PSS1 gene, which lead to regulatory dysfunctions of PSS1 and are causative of Lenz-Majewski syndrome, have been identified. In this study, we investigated the membrane topology of PSS1 by means of epitope insertion and immunofluorescence. According to a 10-transmembrane segment model supported by topology analysis of PSS1, all the 8 amino acid residues crucial for the enzyme activity were localized to the luminal side of the lipid bilayer or the lumen of the ER, whereas all the 9 amino acid residues involved in the enzyme regulation were localized to the cytosol or the cytoplasmic side of the lipid bilayer of the ER. This localization of the functional amino acid residues suggests that PSS1 is regulated by inhibition with PS in the cytoplasmic leaflet of the ER membrane and synthesizes PS at the luminal leaflet.

Substrate and product complexes reveal mechanisms of Hedgehog acylation by HHAT.

Hedgehog proteins govern crucial developmental steps in animals and drive certain human cancers. Before they can function as signaling molecules, Hedgehog precursor proteins must undergo amino-terminal palmitoylation by Hedgehog acyltransferase (HHAT). We present cryo-electron microscopy structures of human HHAT in complex with its palmitoyl-coenzyme A substrate and of a product complex with a palmitoylated Hedgehog peptide at resolutions of 2.7 and 3.2 angstroms, respectively. The structures reveal how HHAT overcomes the challenges of bringing together substrates that have different physiochemical properties from opposite sides of the endoplasmic reticulum membrane within a membrane-embedded active site for catalysis. These principles are relevant to related enzymes that catalyze the acylation of Wnt and of the appetite-stimulating hormone ghrelin. The structural and mechanistic insights may advance the development of inhibitors for cancer.

Hexokinase 2 in Cancer: A Prima Donna Playing Multiple Characters.

Hexokinases are a family of ubiquitous exose-phosphorylating enzymes that prime glucose for intracellular utilization. Hexokinase 2 (HK2) is the most active isozyme of the family, mainly expressed in insulin-sensitive tissues. HK2 induction in most neoplastic cells contributes to their metabolic rewiring towards aerobic glycolysis, and its genetic ablation inhibits malignant growth in mouse models. HK2 can dock to mitochondria, where it performs additional functions in autophagy regulation and cell death inhibition that are independent of its enzymatic activity. The recent definition of HK2 localization to contact points between mitochondria and endoplasmic reticulum called Mitochondria Associated Membranes (MAMs) has unveiled a novel HK2 role in regulating intracellular Ca2+ fluxes. Here, we propose that HK2 localization in MAMs of tumor cells is key in sustaining neoplastic progression, as it acts as an intersection node between metabolic and survival pathways. Disrupting these functions by targeting HK2 subcellular localization can constitute a promising anti-tumor strategy.

Unraveling the complex enzymatic machinery making a key galactolipid in chloroplast membrane: a multiscale computer simulation.

Chloroplast membranes have a high content of the uncharged galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG). These galactolipids are essential for the biogenesis of plastids and functioning of the photosynthetic machinery. A monotopic glycosyltransferase, monogalactosyldiacylglycerol synthase synthesizes the bulk of MGDG. It is embedded in the outer leaflet of the inner envelope membrane of chloroplasts. The protein transfers a galactose residue from UDP-galactose to diacylglycerol (DAG); it needs anionic lipids such as phosphatidylglycerol (PG) to be active. The intricacy of the organization and the process of active complex assembly and synthesis have been investigated at the Coarse-Grained and All-Atom of computer simulation levels to cover large spatial and temporal scales. The following self-assembly process and catalytic events can be drawn; (1) in the membrane, in the absence of protein, there is a spontaneous formation of PG clusters to which DAG molecules associate, (2) a reorganization of the clusters occurs in the vicinity of the protein once inserted in the membrane, (3) an accompanying motion of the catalytic domain of the protein brings DAG in the proper position for the formation of the active complex MGD1/UDP-Gal/DAG/PG for which an atomistic model of interaction is proposed.

A kinase cascade on the yeast lysosomal vacuole regulates its membrane dynamics: conserved kinase Env7 is phosphorylated by casein kinase Yck3.

Membrane fusion/fission is a highly dynamic and conserved process that responds to intra- and extracellular signals. Whereas the molecular machineries involved in membrane fusion/fission have been dissected, regulation of membrane dynamics remains poorly understood. The lysosomal vacuole of budding yeast (Saccharomyces cerevisiae) has served as a seminal model in studies of membrane dynamics. We have previously established that yeast ENV7 encodes an ortholog of STK16-related kinases that localizes to the vacuolar membrane and downregulates vacuolar membrane fusion. Additionally, we have previously reported that Env7 phosphorylation in vivo depends on YCK3, a gene that encodes a vacuolar membrane casein kinase I (CKI) homolog that nonredundantly functions in fusion regulation. Here, we report that Env7 physically interacts with and is directly phosphorylated by Yck3. We also establish that Env7 vacuole fusion/fission regulation and vacuolar localization are mediated through its Yck3-dependent phosphorylation. Through extensive site-directed mutagenesis, we map phosphorylation to the Env7 C terminus and confirm that Ser-331 is a primary and preferred phosphorylation site. Phospho-deficient Env7 mutants were defective in negative regulation of membrane fusion, increasing the number of prominent vacuoles, whereas a phosphomimetic substitution at Ser-331 increased the number of fragmented vacuoles. Bioinformatics approaches confirmed that Env7 Ser-331 is within a motif that is highly conserved in STK16-related kinases and that it also anchors an SXXS CKI phosphorylation motif (328SRFS331). This study represents the first report on the regulatory mechanism of an STK16-related kinase. It also points to regulation of vacuolar membrane dynamics via a novel Yck3-Env7 kinase cascade.

Structure of the ER membrane complex, a transmembrane-domain insertase.

The endoplasmic reticulum (ER) membrane complex (EMC) cooperates with the Sec61 translocon to co-translationally insert a transmembrane helix (TMH) of many multi-pass integral membrane proteins into the ER membrane, and it is also responsible for inserting the TMH of some tail-anchored proteins1-3. How EMC accomplishes this feat has been unclear. Here we report the first, to our knowledge, cryo-electron microscopy structure of the eukaryotic EMC. We found that the Saccharomyces cerevisiae EMC contains eight subunits (Emc1-6, Emc7 and Emc10), has a large lumenal region and a smaller cytosolic region, and has a transmembrane region formed by Emc4, Emc5 and Emc6 plus the transmembrane domains of Emc1 and Emc3. We identified a five-TMH fold centred around Emc3 that resembles the prokaryotic YidC insertase and that delineates a largely hydrophilic client protein pocket. The transmembrane domain of Emc4 tilts away from the main transmembrane region of EMC and is partially mobile. Mutational studies demonstrated that the flexibility of Emc4 and the hydrophilicity of the client pocket are required for EMC function. The EMC structure reveals notable evolutionary conservation with the prokaryotic insertases4,5, suggests that eukaryotic TMH insertion involves a similar mechanism, and provides a framework for detailed understanding of membrane insertion for numerous eukaryotic integral membrane proteins and tail-anchored proteins.

Membrane Chaperoning of a Thylakoid Protease Whose Structural Stability Is Modified by the Protonmotive Force.

Protein folding is a complex cellular process often assisted by chaperones, but it can also be facilitated by interactions with lipids. Disulfide bond formation is a common mechanism to stabilize a protein. This can help maintain functionality amid changes in the biochemical milieu, including those relating to energy-transducing membranes. Plastidic Type I Signal Peptidase 1 (Plsp1) is an integral thylakoid membrane signal peptidase that requires an intramolecular disulfide bond for in vitro activity. We have investigated the interplay between disulfide bond formation, lipids, and pH in the folding and activity of Plsp1. By combining biochemical approaches with a genetic complementation assay using Arabidopsis thaliana plants, we provide evidence that interactions with lipids in the thylakoid membrane have reconstitutive chaperoning activity toward Plsp1. Further, the disulfide bridge appears to prevent an inhibitory conformational change resulting from proton motive force-mimicking pH conditions. Broader implications related to the folding of proteins in energy-transducing membranes are discussed.

TORC1 regulates vacuole membrane composition through ubiquitin- and ESCRT-dependent microautophagy.

Cellular adaptation in response to nutrient limitation requires the induction of autophagy and lysosome biogenesis for the efficient recycling of macromolecules. Here, we discovered that starvation and TORC1 inactivation not only lead to the up-regulation of autophagy and vacuole proteins involved in recycling but also result in the down-regulation of many vacuole membrane proteins to supply amino acids as part of a vacuole remodeling process. Down-regulation of vacuole membrane proteins is initiated by ubiquitination, which is accomplished by the coordination of multiple E3 ubiquitin ligases, including Rsp5, the Dsc complex, and a newly characterized E3 ligase, Pib1. The Dsc complex is negatively regulated by TORC1 through the Rim15-Ume6 signaling cascade. After ubiquitination, vacuole membrane proteins are sorted into the lumen for degradation by ESCRT-dependent microautophagy. Thus, our study uncovered a complex relationship between TORC1 inactivation and vacuole biogenesis.

Spatiotemporal control of phosphatidic acid signaling with optogenetic, engineered phospholipase Ds.

Phosphatidic acid (PA) is both a central phospholipid biosynthetic intermediate and a multifunctional lipid second messenger produced at several discrete subcellular locations. Organelle-specific PA pools are believed to play distinct physiological roles, but tools with high spatiotemporal control are lacking for unraveling these pleiotropic functions. Here, we present an approach to precisely generate PA on demand on specific organelle membranes. We exploited a microbial phospholipase D (PLD), which produces PA by phosphatidylcholine hydrolysis, and the CRY2-CIBN light-mediated heterodimerization system to create an optogenetic PLD (optoPLD). Directed evolution of PLD using yeast membrane display and IMPACT, a chemoenzymatic method for visualizing cellular PLD activity, yielded a panel of optoPLDs whose range of catalytic activities enables mimicry of endogenous, physiological PLD signaling. Finally, we applied optoPLD to elucidate that plasma membrane, but not intracellular, pools of PA can attenuate the oncogenic Hippo signaling pathway. OptoPLD represents a powerful and precise approach for revealing spatiotemporally defined physiological functions of PA.

De novo phosphatidylcholine synthesis is required for autophagosome membrane formation and maintenance during autophagy.

Macroautophagy/autophagy can enable cancer cells to withstand cellular stress and maintain bioenergetic homeostasis by sequestering cellular components into newly formed double-membrane vesicles destined for lysosomal degradation, potentially affecting the efficacy of anti-cancer treatments. Using 13C-labeled choline and 13C-magnetic resonance spectroscopy and western blotting, we show increased de novo choline phospholipid (ChoPL) production and activation of PCYT1A (phosphate cytidylyltransferase 1, choline, alpha), the rate-limiting enzyme of phosphatidylcholine (PtdCho) synthesis, during autophagy. We also discovered that the loss of PCYT1A activity results in compromised autophagosome formation and maintenance in autophagic cells. Direct tracing of ChoPLs with fluorescence and immunogold labeling imaging revealed the incorporation of newly synthesized ChoPLs into autophagosomal membranes, endoplasmic reticulum (ER) and mitochondria during anticancer drug-induced autophagy. Significant increase in the colocalization of fluorescence signals from the newly synthesized ChoPLs and mCherry-MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) was also found on autophagosomes accumulating in cells treated with autophagy-modulating compounds. Interestingly, cells undergoing active autophagy had an altered ChoPL profile, with longer and more unsaturated fatty acid/alcohol chains detected. Our data suggest that de novo synthesis may be required to increase autophagosomal ChoPL content and alter its composition, together with replacing phospholipids consumed from other organelles during autophagosome formation and turnover. This addiction to de novo ChoPL synthesis and the critical role of PCYT1A may lead to development of agents targeting autophagy-induced drug resistance. In addition, fluorescence imaging of choline phospholipids could provide a useful way to visualize autophagosomes in cells and tissues. ABBREVIATIONS: AKT: AKT serine/threonine kinase; BAX: BCL2 associated X, apoptosis regulator; BECN1: beclin 1; ChoPL: choline phospholipid; CHKA: choline kinase alpha; CHPT1: choline phosphotransferase 1; CTCF: corrected total cell fluorescence; CTP: cytidine-5'-triphosphate; DCA: dichloroacetate; DMEM: dulbeccos modified Eagles medium; DMSO: dimethyl sulfoxide; EDTA: ethylenediaminetetraacetic acid; ER: endoplasmic reticulum; GDPD5: glycerophosphodiester phosphodiesterase domain containing 5; GFP: green fluorescent protein; GPC: glycerophosphorylcholine; HBSS: hanks balances salt solution; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LPCAT1: lysophosphatidylcholine acyltransferase 1; LysoPtdCho: lysophosphatidylcholine; MRS: magnetic resonance spectroscopy; MTORC1: mechanistic target of rapamycin kinase complex 1; PCho: phosphocholine; PCYT: choline phosphate cytidylyltransferase; PLA2: phospholipase A2; PLB: phospholipase B; PLC: phospholipase C; PLD: phospholipase D; PCYT1A: phosphate cytidylyltransferase 1, choline, alpha; PI3K: phosphoinositide-3-kinase; pMAFs: pancreatic mouse adult fibroblasts; PNPLA6: patatin like phospholipase domain containing 6; Pro-Cho: propargylcholine; Pro-ChoPLs: propargylcholine phospholipids; PtdCho: phosphatidylcholine; PtdEth: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; RPS6: ribosomal protein S6; SCD: stearoyl-CoA desaturase; SEM: standard error of the mean; SM: sphingomyelin; SMPD1/SMase: sphingomyelin phosphodiesterase 1, acid lysosomal; SGMS: sphingomyelin synthase; WT: wild-type.

The role of xanthophylls in the supramolecular organization of the photosynthetic complex LHCII in lipid membranes studied by high-resolution imaging and nanospectroscopy.

The xanthophyll cycle is a regulatory mechanism operating in the photosynthetic apparatus of plants. It consists of the conversion of the xanthophyll pigment violaxanthin to zeaxanthin, and vice versa, in response to light intensity. According to the current understanding, one of the modes of regulatory activity of the cycle is associated with the influence on a molecular organization of pigment-protein complexes. In the present work, we analyzed the effect of violaxanthin and zeaxanthin on the molecular organization of the LHCII complex, in the environment of membranes formed with chloroplast lipids. Nanoscale imaging based on atomic force microscopy (AFM) showed that the presence of exogenous xanthophylls promotes the formation of the protein supramolecular structures. Nanoscale infrared (IR) absorption analysis based on AFM-IR nanospectroscopy suggests that zeaxanthin promotes the formation of LHCII supramolecular structures by forming inter-molecular ß-structures. Meanwhile, the molecules of violaxanthin act as "molecular spacers" preventing self-aggregation of the protein, potentially leading to uncontrolled dissipation of excitation energy in the complex. This latter mechanism was demonstrated with the application of fluorescence lifetime imaging microscopy. The intensity-averaged chlorophyll a fluorescence lifetime determined in the LHCII samples without exogenous xanthophylls at the level of 0.72 ns was longer in the samples containing exogenous violaxanthin (2.14 ns), but shorter under the presence of zeaxanthin (0.49 ns) thus suggesting a role of this xanthophyll in promotion of the formation of structures characterized by effective excitation quenching. This mechanism can be considered as a representation of the overall photoprotective activity of the xanthophyll cycle.

Src in endosomal membranes promotes exosome secretion and tumor progression.

c-Src is a membrane-associated tyrosine kinase that has key roles in the signaling transduction that controls cell growth, adhesion, and migration. In the early stage of carcinogenesis, c-Src is activated under the plasma membrane and transduces oncogenic signals. Here we show that c-Src localized to the endosomal membrane has unique functions in c-Src-transformed cells. Our results indicate that activated c-Src in the endosomal membrane promoted the secretion of exosomes, in which c-Src was encapsulated. In addition, the ESCRT-interacting molecule, Alix was identified as a c-Src-interacting protein in exosomes. We revealed that the interaction between the SH3 domain of c-Src and the proline-rich region of Alix activates ESCRT-mediated intra-luminal vesicle (ILV) formation, resulting in the upregulation of exosome secretion in c-Src-transformed cells. We observed also a correlation between malignant phenotypes and Alix-dependent aberrant exosome secretion in Src-upregulated cancer cells. Collectively, our findings provide a unique mechanism for the upregulation of exosomes in cancer cells, as well as new insights into the significance of exosome secretion in cancer progression.

Tripping the light fantastic in membrane redox biology: linking dynamic structures to function in ER electron transfer chains.

How the dynamics of proteins assist catalysis is a contemporary issue in enzymology. In particular, this holds true for membrane-bound enzymes, where multiple structural, spectroscopic and biochemical approaches are needed to build up a comprehensive picture of how dynamics influence enzyme reaction cycles. Of note are the recent studies of cytochrome P450 reductases (CPR)-P450 (CYP) endoplasmic reticulum redox chains, showing the relationship between dynamics and electron flow through flavin and haem redox centres and the impact this has on monooxygenation chemistry. These studies have led to deeper understanding of mechanisms of electron flow, including the timing and control of electron delivery to protein-bound cofactors needed to facilitate CYP-catalysed reactions. Individual and multiple component systems have been used to capture biochemical behaviour and these have led to the emergence of more integrated models of catalysis. Crucially, the effects of membrane environment and composition on reaction cycle chemistry have also been probed, including effects on coenzyme binding/release, thermodynamic control of electron transfer, conformational coupling between partner proteins and vectorial versus 'off pathway' electron flow. Here, we review these studies and discuss evidence for the emergence of dynamic structural models of electron flow along human microsomal CPR-P450 redox chains.

Fluorescence in blue light (FLU) is involved in inactivation and localization of glutamyl-tRNA reductase during light exposure.

Fluorescent in blue light (FLU) is a negative regulator involved in dark repression of 5-aminolevulinic acid (ALA) synthesis and interacts with glutamyl-tRNA reductase (GluTR), the rate-limiting enzyme of tetrapyrrole biosynthesis. In this study, we investigated FLU's regulatory function in light-exposed FLU-overexpressing (FLUOE) Arabidopsis lines and under fluctuating light intensities in wild-type (WT) and flu seedlings. FLUOE lines suppress ALA synthesis in the light, resulting in reduced chlorophyll content, but more strongly in low and high light than in medium growth light. This situation indicates that FLU's impact on chlorophyll biosynthesis depends on light intensity. FLU overexpressors contain strongly increased amounts of mainly membrane-associated GluTR. These findings correlate with FLU-dependent localization of GluTR to plastidic membranes and concomitant inhibition, such that only the soluble GluTR fraction is active. The overaccumulation of membrane-associated GluTR indicates that FLU binding enhances GluTR stability. Interestingly, under fluctuating light, the leaves of flu mutants contain less chlorophyll compared with WT and become necrotic. We propose that FLU is basically required for fine-tuned ALA synthesis. FLU not only mediates dark repression of ALA synthesis, but functions also to control balanced ALA synthesis under variable light intensities to ensure the adequate supply of chlorophyll.

The ER membrane protein complex promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis.

The eukaryotic endoplasmic reticulum (ER) membrane contains essential complexes that oversee protein biogenesis and lipid metabolism, impacting nearly all aspects of cell physiology. The ER membrane protein complex (EMC) is a newly described transmembrane domain (TMD) insertase linked with various phenotypes, but whose clients and cellular responsibilities remain incompletely understood. We report that EMC deficiency limits the cellular boundaries defining cholesterol tolerance, reflected by diminished viability with limiting or excessive extracellular cholesterol. Lipidomic and proteomic analyses revealed defective biogenesis and concomitant loss of the TMD-containing ER-resident enzymes sterol-O-acyltransferase 1 (SOAT1) and squalene synthase (SQS, also known as FDFT1), which serve strategic roles in the adaptation of cells to changes in cholesterol availability. Insertion of the weakly hydrophobic tail-anchor (TA) of SQS into the ER membrane by the EMC ensures sufficient flux through the sterol biosynthetic pathway while biogenesis of polytopic SOAT1 promoted by the EMC provides cells with the ability to store free cholesterol as inert cholesteryl esters. By facilitating insertion of TMDs that permit essential mammalian sterol-regulating enzymes to mature accurately, the EMC is an important biogenic determinant of cellular robustness to fluctuations in cholesterol availability.This article has an associated First Person interview with the first author of the paper.

Lysosomes in nutrient signalling: A focus on pancreatic ß-cells.

Regulated insulin secretion from pancreatic ß-cells is a major process maintaining glucose homeostasis in mammals. Enhancing insulin release in response to chronic nutrient overload and obesity-related insulin resistance (pre-diabetes) requires several adaptive cellular mechanisms maintaining ß-cell health under such stresses. Once these mechanisms are overwhelmed, ß-cell failure occurs leading to full-blown Type 2 Diabetes (T2D). Nutrient-dependent macroautophagy represents one such adaptive mechanism in ß-cells. While macroautophagy levels are high and protective in ß-cells in pre-diabetes, they decrease at later stages contributing to ß-cell failure. However, mechanisms compromising macroautophagy in ß-cells remain poorly understood. In this review, we discuss how recently discovered signalling cascades that emanate from the limiting membrane of lysosomes contribute to changes in macroautophagy flux in physiology and disease. In particular, these mechanisms are put into context with ß-cell function highlighting most recently described links between nutrient-dependent lysosomal signalling pathways and insulin secretion. Understanding these mechanisms in response to metabolic stress might pave the way for development of more tailored treatment strategies aimed at preserving ß-cell health.

Atlastins remodel the endoplasmic reticulum for selective autophagy.

Specific receptors are required for the autophagic degradation of endoplasmic reticulum (ER), known as ER-phagy. However, little is known about how the ER is remodeled and separated for packaging into autophagosomes. We developed two ER-phagy-specific reporter systems and found that Atlastins are key positive effectors and also targets of ER-phagy. Atlastins are ER-resident GTPases involved in ER membrane morphology, and Atlastin-depleted cells have decreased ER-phagy under starvation conditions. Atlastin's role in ER-phagy requires a functional GTPase domain and proper ER localization, both of which are also involved in ER architecture. The three Atlastin family members functionally compensate for one another during ER-phagy and may form heteromeric complexes with one another. We further find that Atlastins act downstream of the FAM134B ER-phagy receptor, such that depletion of Atlastins represses ER-autophagy induced by the overexpression of FAM134B. We propose that during ER-phagy, Atlastins remodel ER membrane to separate pieces of FAM134B-marked ER for efficient autophagosomal engulfment.

Ten Prominent Host Proteases in Plant-Pathogen Interactions.

Proteases are enzymes integral to the plant immune system. Multiple aspects of defence are regulated by proteases, including the hypersensitive response, pathogen recognition, priming and peptide hormone release. These processes are regulated by unrelated proteases residing at different subcellular locations. In this review, we discuss 10 prominent plant proteases contributing to the plant immune system, highlighting the diversity of roles they perform in plant defence.

The role of endomembrane-localized VHA-c in plant growth.

In plant cells, the vacuolar-type H+-ATPase (V-ATPase), a large multis`ubunit endomembrane proton pump, plays an important role in acidification of subcellular organelles, pH and ion homeostasis, and endocytic and secretory trafficking. V-ATPase subunit c (VHA-c) is essential for V-ATPase assembly, and is directly responsible for binding and transmembrane transport of protons. In previous studies, we identified a PutVHA-c gene from Puccinellia tenuiflora, and investigated its function in plant growth. Subcellular localization revealed that PutVHA-c is mainly localized in endosomal compartments. Overexpression of PutVHA-c enhanced V-ATPase activity and promoted plant growth in transgenic Arabidopsis. Furthermore, the activity of V-ATPase affected intracellular transport of the Golgi-derived endosomes. Our results showed that endomembrane localized-VHA-c contributes to plant growth by influencing V-ATPase-dependent endosomal trafficking. Here, we discuss these recent findings and speculate on the VHA-c mediated molecular mechanisms involved in plant growth, providing a better understanding of the functions of VHA-c and V-ATPase.