scholarly journals The structure and flexibility analysis of the Arabidopsis Synaptotagmin 1 reveal the basis of its regulation at membrane contact sites

2021 ◽  
Author(s):  
Juan Luis Benavente ◽  
Dritan Siliqi ◽  
Lourdes Infantes ◽  
Laura Lagartera ◽  
Alberto Mills ◽  
...  
Keyword(s):  
Cell Function ◽  
Lipid Extraction ◽  
Membrane Lipid ◽  
Lipid Binding ◽  
Lipid Transfer ◽  
C2 Domains ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Cellular Environment ◽  
Synaptotagmin 1

Cell function requires the maintenance of membrane lipid homeostasis as changes in cellular environment unbalance this equilibrium. The non-vesicular lipid transfer at endoplasmic reticulum (ER) and plasma membrane (PM) contact sites (CS) is central to restore it. Extended synaptotagmins (E-Syts) are ER proteins that play a central role in this process as they act as molecular tethers with PM and as lipid transfer proteins between these organelles. E-Syts are constitutively anchored to the ER through an N-terminal hydrophobic segment and bind to the PM via C-terminal C2 domains. In plants, synaptotagmins (SYTs) are orthologous of E-Syts and regulate the ER-PM communication by the activity of their two C2 domains in response to abiotic stresses. We have combined macromolecular crystallography, small-angle X-ray scattering, structural bioinformatics and biochemical data to analyze the regulation of plant synaptotagmin 1 (SYT1). Our data show that the binding of SYT1 to the PM is regulated by the interaction of the first C2 domain through a Ca2+-dependent lipid binding site and by a site for phosphorylated forms of phosphatidylinositol in such a way that two different molecular signals are integrated in response to stress. In addition, our data show that SYT1 is highly flexible by virtue of up to three hinge points, including one that connects the two C2 domains. This feature provides conformational freedom to SYT1 to define a large and complementary interaction surface with the PM. This structural plasticity, in turn, may facilitate lipid extraction, protein loading and subsequent transfer between PM and ER.

scholarly journals The structure and flexibility analysis of the Arabidopsis synaptotagmin 1 reveal the basis of its regulation at membrane contact sites

2021 ◽  
Vol 4 (10) ◽  
pp. e202101152
Author(s):  
Juan L Benavente ◽  
Dritan Siliqi ◽  
Lourdes Infantes ◽  
Laura Lagartera ◽  
Alberto Mills ◽  
...  
Keyword(s):  
Lipid Extraction ◽  
Membrane Lipid ◽  
Lipid Binding ◽  
Variable Number ◽  
Lipid Transfer ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Synaptotagmin 1 ◽  
Hinge Points ◽  
A Site

Non-vesicular lipid transfer at ER and plasma membrane (PM) contact sites (CS) is crucial for the maintenance of membrane lipid homeostasis. Extended synaptotagmins (E-Syts) play a central role in this process as they act as molecular tethers of ER and PM and as lipid transfer proteins between these organelles. E-Syts are proteins constitutively anchored to the ER through an N-terminal hydrophobic segment and bind the PM via a variable number of C-terminal C2 domains. Synaptotagmins (SYTs) are the plant orthologous of E-Syts and regulate the ER–PM communication in response to abiotic stress. Combining different structural and biochemical techniques, we demonstrate that the binding of SYT1 to lipids occurs through a Ca2+-dependent lipid-binding site and by a site for phosphorylated forms of phosphatidylinositol, thus integrating two different molecular signals in response to stress. In addition, we show that SYT1 displays three highly flexible hinge points that provide conformational freedom to facilitate lipid extraction, protein loading, and subsequent transfer between PM and ER.

scholarly journals Lipid transfer proteins do their thing anchored at membrane contact sites… but what is their thing?

2016 ◽  
Vol 44 (2) ◽  
pp. 517-527 ◽  
Author(s):  
Louise H. Wong ◽  
Tim P. Levine
Keyword(s):  
Lipid Binding ◽  
Lipid Transfer ◽  
Transmembrane Helices ◽  
Lipid Transfer Proteins ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Multiple Contacts ◽  
Membrane Contact ◽  
Organelle Communication ◽  
Lipid Binding Proteins

Membrane contact sites are structures where two organelles come close together to regulate flow of material and information between them. One type of inter-organelle communication is lipid exchange, which must occur for membrane maintenance and in response to environmental and cellular stimuli. Soluble lipid transfer proteins have been extensively studied, but additional families of transfer proteins have been identified that are anchored into membranes by transmembrane helices so that they cannot diffuse through the cytosol to deliver lipids. If such proteins target membrane contact sites they may be major players in lipid metabolism. The eukaryotic family of so-called Lipid transfer proteins Anchored at Membrane contact sites (LAMs) all contain both a sterol-specific lipid transfer domain in the StARkin superfamily (related to StART/Bet_v1), and one or more transmembrane helices anchoring them in the endoplasmic reticulum (ER), making them interesting subjects for study in relation to sterol metabolism. They target a variety of membrane contact sites, including newly described contacts between organelles that were already known to make contact by other means. Lam1–4p target punctate ER–plasma membrane contacts. Lam5p and Lam6p target multiple contacts including a new category: vacuolar non-NVJ cytoplasmic ER (VancE) contacts. These developments confirm previous observations on tubular lipid-binding proteins (TULIPs) that established the importance of membrane anchored proteins for lipid traffic. However, the question remaining to be solved is the most difficult of all: are LAMs transporters, or alternately are they regulators that affect traffic more indirectly?

scholarly journals Three-dimensional architecture of extended synaptotagmin-mediated endoplasmic reticulum–plasma membrane contact sites

2015 ◽  
Vol 112 (16) ◽  
pp. E2004-E2013 ◽  
Author(s):  
Rubén Fernández-Busnadiego ◽  
Yasunori Saheki ◽  
Pietro De Camilli
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Mammalian Cells ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Lipid Binding ◽  
Dense Layer ◽  
Lipid Transfer ◽  
C2 Domains ◽  
Membrane Contact Sites

The close apposition between the endoplasmic reticulum (ER) and the plasma membrane (PM) plays important roles in Ca2+ homeostasis, signaling, and lipid metabolism. The extended synaptotagmins (E-Syts; tricalbins in yeast) are ER-anchored proteins that mediate the tethering of the ER to the PM and are thought to mediate lipid transfer between the two membranes. E-Syt cytoplasmic domains comprise a synaptotagmin-like mitochondrial-lipid–binding protein (SMP) domain followed by five C2 domains in E-Syt1 and three C2 domains in E-Syt2/3. Here, we used cryo-electron tomography to study the 3D architecture of E-Syt–mediated ER–PM contacts at molecular resolution. In vitrified frozen-hydrated mammalian cells overexpressing individual E-Syts, in which E-Syt–dependent contacts were by far the predominant contacts, ER–PM distance (19–22 nm) correlated with the amino acid length of the cytosolic region of E-Syts (i.e., the number of C2 domains). Elevation of cytosolic Ca2+ shortened the ER–PM distance at E-Syt1–dependent contacts sites. E-Syt–mediated contacts displayed a characteristic electron-dense layer between the ER and the PM. These features were strikingly different from those observed in cells exposed to conditions that induce contacts mediated by the stromal interaction molecule 1 (STIM1) and the Ca2+ channel Orai1 as well as store operated Ca2+ entry. In these cells the gap between the ER and the PM was spanned by filamentous structures perpendicular to the membranes. Our results define specific ultrastructural features of E-Syt–dependent ER–PM contacts and reveal their structural plasticity, which may impact on the cross-talk between the ER and the PM and the functions of E-Syts in lipid transport between the two bilayers.

scholarly journals Extended synaptotagmins are Ca2+-dependent lipid transfer proteins at membrane contact sites

2016 ◽  
Vol 113 (16) ◽  
pp. 4362-4367 ◽  
Author(s):  
Haijia Yu ◽  
Yinghui Liu ◽  
Daniel R. Gulbranson ◽  
Alex Paine ◽  
Shailendra S. Rathore ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Protein Transport ◽  
Vesicle Fusion ◽  
Lipid Binding ◽  
Common Mechanism ◽  
Lipid Transfer ◽  
Lipid Transfer Proteins ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact

Organelles are in constant communication with each other through exchange of proteins (mediated by trafficking vesicles) and lipids [mediated by both trafficking vesicles and lipid transfer proteins (LTPs)]. It has long been known that vesicle trafficking can be tightly regulated by the second messenger Ca2+, allowing membrane protein transport to be adjusted according to physiological demands. However, it remains unclear whether LTP-mediated lipid transport can also be regulated by Ca2+. In this work, we show that extended synaptotagmins (E-Syts), poorly understood membrane proteins at endoplasmic reticulum–plasma membrane contact sites, are Ca2+-dependent LTPs. Using both recombinant and endogenous mammalian proteins, we discovered that E-Syts transfer glycerophospholipids between membrane bilayers in the presence of Ca2+. E-Syts use their lipid-accommodating synaptotagmin-like mitochondrial lipid binding protein (SMP) domains to transfer lipids. However, the SMP domains themselves cannot transport lipids unless the two membranes are tightly tethered by Ca2+-bound C2 domains. Strikingly, the Ca2+-regulated lipid transfer activity of E-Syts was fully recapitulated when the SMP domain was fused to the cytosolic domain of synaptotagmin-1, the Ca2+ sensor in synaptic vesicle fusion, indicating that a common mechanism of membrane tethering governs the Ca2+ regulation of lipid transfer and vesicle fusion. Finally, we showed that microsomal vesicles isolated from mammalian cells contained robust Ca2+-dependent lipid transfer activities, which were mediated by E-Syts. These findings established E-Syts as a novel class of LTPs and showed that LTP-mediated lipid trafficking, like vesicular transport, can be subject to tight Ca2+ regulation.

scholarly journals Mdm1/Snx13 is a novel ER–endolysosomal interorganelle tethering protein

2015 ◽  
Vol 210 (4) ◽  
pp. 541-551 ◽  
Author(s):  
W. Mike Henne ◽  
Lu Zhu ◽  
Zsolt Balogi ◽  
Christopher Stefan ◽  
Jeffrey A. Pleiss ◽  
...  
Keyword(s):  
Neurological Disease ◽  
Lipid Binding ◽  
Sphingolipid Metabolism ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Effector Genes ◽  
Px Domain ◽  
In Trans ◽  
Membrane Contact ◽  
Lysosome Membrane

Although endolysosomal trafficking is well defined, how it is regulated and coordinates with cellular metabolism is unclear. To identify genes governing endolysosomal dynamics, we conducted a global fluorescence-based screen to reveal endomembrane effector genes. Screening implicated Phox (PX) domain–containing protein Mdm1 in endomembrane dynamics. Surprisingly, we demonstrate that Mdm1 is a novel interorganelle tethering protein that localizes to endoplasmic reticulum (ER)–vacuole/lysosome membrane contact sites (MCSs). We show that Mdm1 is ER anchored and contacts the vacuole surface in trans via its lipid-binding PX domain. Strikingly, overexpression of Mdm1 induced ER–vacuole hypertethering, underscoring its role as an interorganelle tether. We also show that Mdm1 and its paralogue Ydr179w-a (named Nvj3 in this study) localize to ER–vacuole MCSs independently of established tether Nvj1. Finally, we find that Mdm1 truncations analogous to neurological disease–associated SNX14 alleles fail to tether the ER and vacuole and perturb sphingolipid metabolism. Our work suggests that human Mdm1 homologues may play previously unappreciated roles in interorganelle communication and lipid metabolism.

scholarly journals The Hob Proteins: Putative, Novel Lipid Transfer Proteins at ER-PM Contact Sites

Contact ◽  
2021 ◽  
Vol 4 ◽  
pp. 251525642110523
Author(s):  
Sarah D. Neuman ◽  
Amy T. Cavanagh ◽  
Arash Bashirullah
Keyword(s):  
Structural Models ◽  
Model Systems ◽  
Lipid Transfer ◽  
Lipid Transfer Proteins ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Bona Fide ◽  
Membrane Contact ◽  
Mutant Cells ◽  
Critical Process

Nonvesicular transfer of lipids at membrane contact sites (MCS) has recently emerged as a critical process for cellular function. Lipid transfer proteins (LTPs) mediate this unique transport mechanism, and although several LTPs are known, the cellular complement of these proteins continues to expand. Our recent work has revealed the highly conserved but poorly characterized Hobbit/Hob proteins as novel, putative LTPs at endoplasmic reticulum-plasma membrane (ER-PM) contact sites. Using both S. cerevisiae and D. melanogaster model systems, we demonstrated that the Hob proteins localize to ER-PM contact sites via an N-terminal ER membrane anchor and conserved C-terminal sequences. These conserved C-terminal sequences bind to phosphoinositides (PIPs), and the distribution of PIPs is disrupted in hobbit mutant cells. Recently released structural models of the Hob proteins exhibit remarkable similarity to other bona fide LTPs, like VPS13A and ATG2, that function at MCS. Hobbit is required for viability in Drosophila, suggesting that the Hob proteins are essential genes that may mediate lipid transfer at MCS.

scholarly journals Reconstitution of autophagosome nucleation defines Atg9 vesicles as seeds for membrane formation

Science ◽  
2020 ◽  
Vol 369 (6508) ◽  
pp. eaaz7714 ◽  
Author(s):  
Justyna Sawa-Makarska ◽  
Verena Baumann ◽  
Nicolas Coudevylle ◽  
Sören von Bülow ◽  
Veronika Nogellova ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
De Novo ◽  
Lipid Transfer ◽  
Related Protein ◽  
Membrane Formation ◽  
Selective Autophagy ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact ◽  
Phosphatidylinositol 3

Autophagosomes form de novo in a manner that is incompletely understood. Particularly enigmatic are autophagy-related protein 9 (Atg9)–containing vesicles that are required for autophagy machinery assembly but do not supply the bulk of the autophagosomal membrane. In this study, we reconstituted autophagosome nucleation using recombinant components from yeast. We found that Atg9 proteoliposomes first recruited the phosphatidylinositol 3-phosphate kinase complex, followed by Atg21, the Atg2-Atg18 lipid transfer complex, and the E3-like Atg12–Atg5-Atg16 complex, which promoted Atg8 lipidation. Furthermore, we found that Atg2 could transfer lipids for Atg8 lipidation. In selective autophagy, these reactions could potentially be coupled to the cargo via the Atg19-Atg11-Atg9 interactions. We thus propose that Atg9 vesicles form seeds that establish membrane contact sites to initiate lipid transfer from compartments such as the endoplasmic reticulum.

Chloroplast lipid synthesis and lipid trafficking through ER–plastid membrane contact sites

2012 ◽  
Vol 40 (2) ◽  
pp. 457-463 ◽  
Author(s):  
Zhen Wang ◽  
Christoph Benning
Keyword(s):  
Fatty Acids ◽  
Current Knowledge ◽  
Lipid Transfer ◽  
Photosynthetic Membrane ◽  
Lipid Trafficking ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact ◽  
Envelope Membranes ◽  
Thylakoid Lipids

Plant chloroplasts contain an intricate photosynthetic membrane system, the thylakoids, and are surrounded by two envelope membranes at which thylakoid lipids are assembled. The glycoglycerolipids mono- and digalactosyldiacylglycerol, and sulfoquinovosyldiacylglycerol as well as phosphatidylglycerol, are present in thylakoid membranes, giving them a unique composition. Fatty acids are synthesized in the chloroplast and are either directly assembled into thylakoid lipids at the envelope membranes or exported to the ER (endoplasmic reticulum) for extraplastidic lipid assembly. A fraction of lipid precursors is reimported into the chloroplast for the synthesis of thylakoid lipids. Thus polar lipid assembly in plants requires tight co-ordination between the chloroplast and the ER and necessitates inter-organelle lipid trafficking. In the present paper, we discuss the current knowledge of the export of fatty acids from the chloroplast and the import of chloroplast lipid precursors assembled at the ER. Direct membrane contact sites between the ER and the chloroplast outer envelopes are discussed as possible conduits for lipid transfer.

scholarly journals Nanoscale architecture of a VAP-A-OSBP tethering complex at membrane contact sites

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Eugenio de la Mora ◽  
Manuela Dezi ◽  
Aurélie Di Cicco ◽  
Joëlle Bigay ◽  
Romain Gautier ◽  
...  
Keyword(s):  
Lipid Transfer Protein ◽  
Structural Information ◽  
Transmembrane Protein ◽  
Lipid Transfer ◽  
Oxysterol Binding Protein ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact ◽  
Golgi Network

AbstractMembrane contact sites (MCS) are subcellular regions where two organelles appose their membranes to exchange small molecules, including lipids. Structural information on how proteins form MCS is scarce. We designed an in vitro MCS with two membranes and a pair of tethering proteins suitable for cryo-tomography analysis. It includes VAP-A, an ER transmembrane protein interacting with a myriad of cytosolic proteins, and oxysterol-binding protein (OSBP), a lipid transfer protein that transports cholesterol from the ER to the trans Golgi network. We show that VAP-A is a highly flexible protein, allowing formation of MCS of variable intermembrane distance. The tethering part of OSBP contains a central, dimeric, and helical T-shape region. We propose that the molecular flexibility of VAP-A enables the recruitment of partners of different sizes within MCS of adjustable thickness, whereas the T geometry of the OSBP dimer facilitates the movement of the two lipid-transfer domains between membranes.

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