Characterization of AtDGK2 in relation to Contact Sites

Contact Sites are conserved cellular regions where two membranes of different organelles are very close but not merged. Contact sites between the endoplasmic reticulum (ER) and the plasma membrane (ER-PM CS) play important roles in metabolic functions. We have identified AtDGK2 (Diacylglycerol kinase 2) as an interactor of SYT1 (Synaptotagmin1), which is a protein located at ER-PM CS. DGKs phosphorylate diacylglycerol to produce phosphatidic acid, both important signalling molecules. Arabidopsis thaliana has seven AtDGKs, but only AtDGK1 and AtDGK2 present an ER transmembrane domain, the rest are cytoplasmic. We have analysed the subcellular localization and functions of these two proteins.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. BIO2017-82609-

Undercovering the molecular mechanisms of lipid signalling at ER-PM contact sites in tomato (Solanum lycopersicum) under abiotic stress conditions

Abiotic stresses cause large reductions in crop production. Therefore, is important to understand how plants respond in order to develop varieties with increased resistance. Lipid-transport proteins (LTP) are emerging as key players of lipid signaling in response to numerous stresses. Specifically, SYT1, a protein first identified by its role in abiotic stress tolerance, is now recognized as an endoplasmic reticulum-plasma membrane contact site tether capable. Our recent data support that SYT1 in involved on non-vesicular lipid-transport of diacyl glycerol (DAG) through its SMP domain. This data together with the interaction of SYT1 with a diacyl glycerol kinase (DGK) suggest a lipid signaling pathway where the product of phospholipase C, diacylglycerol, might be simultaneously translocated from the plasma membrane to the endoplasmic-reticulum by SYT1 and phosphorylated to phosphatidic acid by DGK at the plasma membrane. Using in vitro biochemical approaches we are investigating the affinity of specific lipid species transported by SYT1 using lipid-competition assays, where a fluorescent lipid competes for SYT1 binding-pocket with different lipid species. Using bioinformatic we are obtaining insight into the lipid signal pathway involving PHOSPHOLIPASE C (PLC), DIACYLGLYCEROL KINASE (DGK) and SYNAPTOTAGMIN1 (SYT1) in tomatoUniversidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

NTMC2T5: lipid transfer proteins at ER-chloroplast contact sites involved in plant stress.

Chloroplasts are the site of fatty acid synthesis in plants; however, these fatty acids are assembled into glycerolipids at the ER. Later on, some of these ER-assembled glycerolipids will be transferred back to the chloroplasts to be further modified and to form part of the chloroplastic membranes. Previous reports have shown that under some abiotic stresses, these plastid membranes suffer a large lipid remodelling and new precursors massively need to be transported from the ER to the chloroplast or vice versa. It has been suggested that the newly synthetized ER lipids are delivered to chloroplast via a non-vesicular pathway, likely through lipid transport proteins (LTP). These LTP would be localized in membrane contact sites (MCS). Some LTP at MCS contain particular domains, as the synaptotagmin-like mitochondrial lipid-binding (SMP) domain. We have studied the occurrence of SMP proteins in A. thaliana and S. lycopersicum. By using transient expression in N. benthamiana leaves and confocal microscopy, we have identified the NTMC2T5 family with two homologs in A. thaliana and only one in S. lycopersicum. They are anchored to the chloroplast outer membrane, and they interact in trans with the ER (ER-chloroplast MCS). We have observed that clustering of chloroplasts around the nucleus occurred when we overexpressed these proteins and Arabidopsis double knock-out mutant for these proteins showed less chloroplasts attached to nuclei at control conditions. And, we have investigated the NTMC2T5 protein domains involved in this clustering. Moreover, our analysis has demonstrated that Arabidopsis simple mutants show lower germination rates in media supplemented with NaCl and lower rates of expanded cotyledons in media supplemented with ABA. We have also performed biotinylation-based proximity labelling proteomics experiments in order to identify interactors of these proteins. Finally, we have performed lipidomic analysis to understand the role of these proteins.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

SYT6: a newly identified protein involved in ER - trans-Golgi network Membrane Contact Sites

Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.SYT6 is a newly identified lipid transport protein from ER - trans-Golgi network Membrane Contact Sites. Our results show that: • SYT6 contacts trans-Golgi network vesicles through its coiled-coil domain. • SYT6 can efectively respond to Ca2+ using its terminal C2C domain. • SYT6-C2 domains preferentially bind to negatively charged membranes (with PI4P and PS) in presence of Ca2+.This research has been funded by AEI (PID2021-127649OB-I00 and PGC-2018-098789-B-I00) and FEDER-Junta de Andalucía (UMA18-FEDERJA-154). The attendance to this meeting was supported by Plan Propio de Investigación, Transferencia y Divulgación Científica de la Universidad de Málaga, Campus de Excelencia Internacional Andalucía Tech

Role of Arabidopsis DGK1 and DGK2 in cold stress

There are regions, present in all eukaryotic cells, where the membranes of two different organelles are very close (10-30nm), but without fusion, due to proteins which act as tether. These regions are named Membrane Contact Sites (MCS), as for example those formed by endoplasmic reticulum (ER) and plasma membrane (PM). ER-PM CS play important roles in communication between membranes, lipid homeostasis and Ca2+ influx. When a plant is challenge with a stress, phospholipase C is activated at PM producing diacylglycerol (DAG) and inositol phosphates from the hydrolysis of PIP(4,5)P2 or PI4P. DAG is then phosphorylated by diacylglycerol kinases (DGKs) producing phosphatidic acid (PA). Both, DAG and PA, are molecules involved in signalling (Arisz et al., 2009). The genome of Arabidopsis thaliana contain 7 genes encoding DGKs. Most of them are predicted to be cytosolic, with only DGK1 and DGK2 are anchored to the ER due a transmembrane domain. Using different approaches (Co-Immunoprecipitation, confocal microscopy, FRET, Tap-tag…), our group has uncover, that AtDGK1 and AtDGK2 (Diacylglycerol kinase 1, AT5G07920 and Diacylglycerol kinase 2, At5g63770) form a complex with the lipid transport protein located at ER-PM CS known as Synaptotagmin1 (SYT1, At2g20990) (Pérez-Sancho et al., 2015). SYT1 is able to bind preferentially DAG (Ruiz-Lopez et al., 2021), which support the idea that SYT1, DGK1, and DGK2 function coordinated to regulate the levels of DAG at the PM. DGK1 and DGK2 are induced upon low temperatures and found that dgk2 mutants show reduced root growth in low temperature and have reduced freezing tolerance. Our studies suggest that DGK1 and DGK2 act in concert with SYT1 to regulate the production of DAG and PA at ER-PM CS and highlight the importance of these proteins for the correct response to stress.The authors acknowledge the support by: (1) Ministerio de Ciencia e Innovación BIO2017-82609-R, PGC2018-098789-B-100; (2) Ministerio de Economía, Industria y Competitividad (BES-2015-071256, RyC-2013-12699). Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

How are DGK1 and DGK2 involved in membrene contact sites?

Eukaryotic cells have regions of interaction between two organelles where some proteins, which act as tether, bring both membranes closer (10-30 nm) without fusion, named membrane contact sites (MCS). Two organelles that can form MCS are endoplasmic reticulum (ER) and plasma membrane (PM). ER-PM CS play important metabolic functions such as communication between both membranes, lipid homeostasis and Ca2+ influx. Our group has identified that AtDGK1 and AtDGK2 (Diacylglycerol kinase 1, AT5G07920 and Diacylglycerol kinase 2, At5g63770) form a complex with a well-known protein located at ER-PM CS, Synaptotagmin1 (SYT1, At2g20990). Upon perception of stress, phospholipase C (PLC) is activated at the plasma membrane to hydrolyse PIP(4,5)P2 or PI4P in order to generate DAG and inositol phosphates. Diacylglycerol (DAG) is phosphorylated by diacylglycerol kinases (DGKs) to produce phosphatidic acid (PA). DAG and PA are important cell signalling molecules. There are seven DGKs encoded in Arabidopsis thaliana genome, but only DGK1 and DGK2 have a transmembrane domain that anchors them to the endoplasmic reticulum, the rest are cytoplasmic. DGK1 and DGK2 appear to play a role in stress response as both are induced by exposure to low temperatures and wounding. Also, we found that dgk2 knockout mutant produces lower resistance to freezing. Using confocal microscopy, we have analysed the subcellular localization of these two proteins and investigated their interaction with SYT1 and between them using FRET and co-immunoprecipitation studies. Additionally, we report that the mutation of DGK1 is lethal in homozygosity. Our studies suggest that DGK1 and DGK2 act in concert with SYT1 to regulate the production of PA at ER-PM CS and highlight the importance of these proteins for the correct response to stress tolerance.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxylase kinase isoenzymes play an important role in the filling and quality of Arabidopsis thaliana seed

Three plant-type phosphoenolpyruvate carboxylase (PPC1 to PPC3) and two phosphoenolpyruvate carboxylase kinase (PPCKs: PPCK1 and 2) genes are present in the Arabidopsis thaliana genome. In seeds, all PPC genes were found to be expressed. Examination of individual ppc mutants showed little reduction of PEPC protein and global activity, with the notable exception of PPC2 which represent the most abundant PEPC in dry seeds. Ppc mutants exhibited moderately lower seed parameters (weight, area, yield, germination kinetics) than wild type. In contrast, ppck1-had much altered (decreased) yield. At the molecular level, ppc3-was found to be significantly deficient in global seed nitrogen (nitrate, amino-acids, and soluble protein pools). Also, N-deficiency was much more marked in ppck1-, which exhibited a tremendous loss of 95% and 90% in nitrate and proteins, respectively. The line ppck2-had accumulated amino-acids but lower levels of soluble proteins. Regarding carboxylic acid pools, Krebs cycle intermediates were found to be diminished in all mutants; this was accompanied by a consistent decrease in ATP. Lipids were stable in ppc mutants, however ppck1-seeds accumulated more lipids while ppck2-seeds showed high level of polyunsaturated fatty acid oleic and linolenic (omega 3). Altogether, the results indicate that the complete PEPC and PPCK family are needed for normal C/N metabolism ratio, growth, development, yield and quality of the seed.Ministerio de Economía y Competitividad AGL2012-35708, AGL2016-75413-PJunta de Andalucía P12-FQM-48

SMP-CONTAINING PROTEINS AT MEMBRANE CONTACT SITES: SUBCELLULAR LOCALIZATION AND CHARACTERIZATION.

Membrane contact sites (MCS) are microdomains where two membranes of two different organelles are in close apposition, but they do not fuse. MCS are essential for non-vesicular transport of lipids. This lipid transport is mediated by several families of proteins which all of them contain a lipid transport domain, as the synaptotagmin-like mitochondrial lipid-binding (SMP) domain. The most studied SMP protein is Arabidopsis SYT1 which is known to be involved in tolerance to multiple abiotic stresses. Later studies in other SMP proteins of the same family have shown that SYT1 and homologous such as SYT3 or SYT5 gave similar results. However, little information is available about the role other SMP proteins in plants. We have studied the occurrence of additional SMP proteins in A. thaliana and S. lycopersicum. In order to identify these proteins, SMP sequences from human and yeast were used to identify their remote orthologues in A. thaliana and S. lycopersicum, allowing the identification of several putative encoding SMP domains. We have found that some of the identified proteins are exclusive of plants as they do not have direct orthologs in yeast nor human. Transient expression in N. benthamiana leaves followed by confocal microscopy was used to study the subcellular localization of these proteins. Our results show that some of these proteins are localized at ER-Golgi contact sites and two other proteins at ER-Chloroplast sites. Finally, to determine whether these proteins are involved in abiotic stress tolerance, we have analysed the root growth and seed germination rates of Arabidopsis mutants for these genes under different conditions. Some of these mutants have shown different germination rates in media supplemented with NaCl and different rates of expanded cotyledons in media supplemented with ABA. These results suggest that some these proteins may be implicated in abiotic stress signalling through an ABA-dependent pathway.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. This work is supported by grants from: Ministerio de Ciencia, Innovación y Universidades (grant PGC2018-098789-B-I00), UMA-FEDER (grant UMA18-FEDERJA-154) and Ministerio de Ciencia e Innovación (BIO2017-82609-R)

NTMC2T5 protein family: newly identified ER-chloroplast contact site proteins involved in abiotic stress.

Plants are sessile organisms and therefore they have perfected a complex molecular signalling network to detect and respond to the different environmental stresses such as high temperatures, salinity, or drought. In plants, fatty acid synthesis takes place at chloroplasts, and they are assembled into glycerolipids and sphingolipids at the endoplasmic reticulum (ER). Then, the newly synthetized lipids in the ER are delivered to chloroplast via a non-vesicular pathway, likely through lipid transport proteins (LTP). These LTP would be localized in ER-chloroplast membrane contact sites (MCS). Synaptotagmin-like mitochondrial-lipid-binding (SMP) domain proteins are evolutionarily conserved LTP in eukaryotes that localize at MCS. They are involved in tethering of these MCS through interaction with other proteins/membrane lipids and in transferring of glycerolipids between these two membranes. We have studied the occurrence of SMP proteins in A. thaliana and S. lycopersicum by searching remote orthologs of human E-Syt1 (SMP protein). By using transient expression in N. benthamiana leaves and confocal microscopy, we have identified the NTMC2T5 family with two homologs in A. thaliana and only one in S. lycopersicum that are anchored to the chloroplast outer membrane and are interacting with the ER (at ER-chloroplast MCS). Our preliminary data have unequivocally demonstrated that NTMC2T5 proteins are anchored to the chloroplast, and they bind in trans the ER. Additionally, it is predicted that these proteins contain a SMP domain which is a lipid-transfer domain, indicating that these proteins could be responsible for some of the lipid transferring events at ER-chloroplast MCS that are still unknown. Our preliminary phenotypic analyses have shown that these proteins are involved in salt tolerance. Finally, we have observed that clustering of chloroplasts around the nucleus occurred when we overexpressed these proteins in Nicotiana benthamiana leaves.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech