Synaptotagmin-like Mitochondrial Lipid-binding Protein Domain Research Articles

Insights into membrane association of the SMP domain of extended synaptotagmin

The Synaptotagmin-like Mitochondrial-lipid-binding Protein (SMP) domain is a newly identified lipid transfer module present in proteins that regulate lipid homeostasis at membrane contact sites (MCSs). However, how the SMP domain associates with the membrane to extract and unload lipids is unclear. Here, we performed in vitro DNA brick-assisted lipid transfer assays and in silico molecular dynamics simulations to investigate the molecular basis of the membrane association by the SMP domain of extended synaptotagmin (E-Syt), which tethers the tubular endoplasmic reticulum (ER) to the plasma membrane (PM). We demonstrate that the SMP domain uses its tip region to recognize the extremely curved subdomain of tubular ER and the acidic-lipid-enriched PM for highly efficient lipid transfer. Supporting these findings, disruption of these mechanisms results in a defect in autophagosome biogenesis contributed by E-Syt. Our results suggest a model that provides a coherent picture of the action of the SMP domain at MCSs.

Reconstitution and biochemical studies of extended synaptotagmin-mediated lipid transport.

Extended synaptotagmins (E-Syts) are a family of lipid transfer proteins (LTPs) located at the endoplasmic reticulum (ER)-plasma membrane (PM) contact sites in eukaryotic cells. They possess a conserved synaptotagmin-like mitochondrial-lipid-binding protein (SMP) domain and two to five C2 domains. While the membrane tethering function of E-Syts has been well studied in diverse species, recent studies revealed that the mammalian E-Syt1 and its yeast homolog tricalbin 3 (Tcb3) could transport lipids between the opposed membrane. Mechanical studies suggested SYT1 transfers lipids fundamentally through the SMP domain, but the lipid transport requires the regulation of C2 domain-mediated membrane tethering. In addition, both E-Syt1 and Tcb3 are Ca2+-modulated LTPs, which sense and interact with Ca2+ through the C2 domains. This chapter describes the in vitro reconstitution and biochemical assays for studying the functions and mechanisms of E-Syts, by expressing and purifying recombinant proteins, preparing reconstitution systems, and developing assays for membrane tethering and lipid transport.

Calcium-dependent and -independent lipid transfer mediated by tricalbins in yeast

Membrane contact sites (MCSs) formed between the endoplasmic reticulum (ER) and the plasma membrane (PM) provide a platform for nonvesicular lipid exchange. The ER-anchored tricalbins (Tcb1, Tcb2, and Tcb3) are critical tethering factors at ER–PM MCSs in yeast. Tricalbins possess a synaptotagmin-like mitochondrial-lipid-binding protein (SMP) domain and multiple Ca2+-binding C2 domains. Although tricalbins have been suggested to be involved in lipid exchange at the ER–PM MCSs, it remains unclear whether they directly mediate lipid transport. Here, using in vitro lipid transfer assays, we discovered that tricalbins are capable of transferring phospholipids between membranes. Unexpectedly, while its lipid transfer activity was markedly elevated by Ca2+, Tcb3 constitutively transferred lipids even in the absence of Ca2+. The stimulatory activity of Ca2+ on Tcb3 required intact Ca2+-binding sites on both the C2C and C2D domains of Tcb3, while Ca2+-independent lipid transport was mediated by the SMP domain that transferred lipids via direct interactions with phosphatidylserine and other negatively charged lipid molecules. These findings establish tricalbins as lipid transfer proteins, and reveal Ca2+-dependent and -independent lipid transfer activities mediated by these tricalbins, providing new insights into their mechanism in maintaining PM integrity at ER–PM MCSs.

Structural Studies of a Novel Extended Synaptotagmin with Only Two C2 Domains from <i>Trypanosoma Brucei</i>

Extended synaptotagmins (E-Syts) are a group of evolutionarily conserved proteins localizing at membrane contact sites between the endoplasmic reticulum (ER) and the plasma membrane, where they mediate inter-membrane lipid transfer and control plasma membrane lipid homeostasis. There are three E-Syts in mammals, which all contain an N-terminal transmembrane (TM) hairpin for the association with the ER membrane, and a central synaptotagmin-like mitochondrial-lipid binding protein (SMP) domain as a carrier for phospholipids. Additionally, mammalian E-Syt1 and E-Syt2/E-Syt3 respectively have five and three C2 domains at their C-terminus, which mediate their interaction with the plasma membrane. Here we report a novel E-Syt from the protist parasite Trypanosoma brucei, TbE-Syt. Despite also having the TM hairpin and the SMP domain, TbE-Syt contains only two C2 domains, which makes it the shortest E-Syt currently identified. We determined a 1.5 A resolution crystal structure of the TbE-Syt C2B domain, which showed that it binds two Ca2+ ions. Our mutagenesis studies demonstrated that TbE-Syt-C2B binds lipids via both Ca2+- and PI(4,5)P2-dependent means. Our bioinformatics analyses showed that TbE-Syt-C2A lacks the Ca2+-binding site found in C2B but may still interact with lipids via a basic surface patch. Furthermore, in contrast to the rigid V-shaped arrangement of the C2A and C2B domains in human E-Syt2, our analysis suggests that the two C2 domains in TbE-Syt are connected by a long flexible linker. We propose a working model for how TbE-Syt might tether the ER membrane and the plasma membrane to transfer lipids between the two organelles.

Determining the Lipid-Binding Specificity of SMP Domains: An ERMES Subunit as a Case Study.

Membrane contact sites between the endoplasmic reticulum (ER) and mitochondria function as a central hub for the exchange of phospholipids and calcium. The yeast Endoplasmic Reticulum-Mitochondrion Encounter Structure (ERMES) complex is composed of five subunits that tether the ER and mitochondria. Three ERMES subunits (i.e., Mdm12, Mmm1, and Mdm34) contain the synaptotagmin-like mitochondrial lipid-binding protein (SMP) domain. The SMP domain belongs to the tubular lipid-binding protein (TULIP) superfamily, which consists of ubiquitous lipid scavenging and transfer proteins. Herein, we describe the methods for expression and purification of recombinant Mdm12, a bona fide SMP-containing protein, together with the subsequent identification of its bound phospholipids by high-performance thin-layer chromatography (HPTLC) and the characterization of its lipid exchange and transfer functions using lipid displacement and liposome flotation in vitro assays with liposomes as model biological membranes. These methods can be applied to the study and characterization of novel lipid-binding and lipid-transfer proteins.

ER-mitochondria tethering by PDZD8 regulates Ca2+ dynamics in mammalian neurons.

Interfaces between organelles are emerging as critical platforms for many biological responses in eukaryotic cells. In yeast, the ERMES complex is an endoplasmic reticulum (ER)-mitochondria tether composed of four proteins, three of which contain a SMP (synaptotagmin-like mitochondrial-lipid binding protein) domain. No functional ortholog for any ERMES protein has been identified in metazoans. Here, we identified PDZD8 as an ER protein present at ER-mitochondria contacts. The SMP domain of PDZD8 is functionally orthologous to the SMP domain found in yeast Mmm1. PDZD8 was necessary for the formation of ER-mitochondria contacts in mammalian cells. In neurons, PDZD8 was required for calcium ion (Ca2+) uptake by mitochondria after synaptically induced Ca2+-release from ER and thereby regulated cytoplasmic Ca2+ dynamics. Thus, PDZD8 represents a critical ER-mitochondria tethering protein in metazoans. We suggest that ER-mitochondria coupling is involved in the regulation of dendritic Ca2+ dynamics in mammalian neurons.

In silico analysis of coevolution among ERMES proteins, Pex11, and Lam6.

In eukaryotic cells, communication and dynamic interactions among different organelles are important for maintaining cellular homeostasis. The endoplasmic reticulum (ER) mitochondria encounter structure (ERMES) complex establishes membrane contact sites between ER and mitochondria and is essential for phospholipid transport, protein import, and mitochondrial dynamics and inheritance. In this work, in silico analyses were used to probe the intramolecular interactions in ERMES proteins and the interactions that support the ERMES complex. Based on mutual information (MI), sites of intramolecular coevolution are predicted in the core proteins Mmm1, Mdm10, Mdm12, Mdm34, the peroxisomal protein Pex11, and cytoplasmic Lam6; these sites are linked to structural features of the proteins. Intermolecular coevolution is predicted among the synaptotagmin-like mitochondrial lipid-binding protein (SMP) domains of Mmm1, Mdm12, and Mdm34. Segments of Pex11 and Lam6 also share MI with the SMP domains of Mmm1 and Mdm12 and with the N terminus of Mdm34, implicating Mdm34 as part of a hub for interactions between ERMES and other complexes. In contrast, evidence of limited intermolecular coevolution involving the outer membrane protein Mdm10 was detected only with Mmm1 and Pex11. The results support models for the organization of these interacting proteins and suggest roles for Pex11 and Lam6 in regulating complex formation.

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

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 Ca(2+), 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 Ca(2+) In this work, we show that extended synaptotagmins (E-Syts), poorly understood membrane proteins at endoplasmic reticulum-plasma membrane contact sites, are Ca(2+)-dependent LTPs. Using both recombinant and endogenous mammalian proteins, we discovered that E-Syts transfer glycerophospholipids between membrane bilayers in the presence of Ca(2+) 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 Ca(2+)-bound C2 domains. Strikingly, the Ca(2+)-regulated lipid transfer activity of E-Syts was fully recapitulated when the SMP domain was fused to the cytosolic domain of synaptotagmin-1, the Ca(2+)sensor in synaptic vesicle fusion, indicating that a common mechanism of membrane tethering governs the Ca(2+)regulation of lipid transfer and vesicle fusion. Finally, we showed that microsomal vesicles isolated from mammalian cells contained robust Ca(2+)-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 Ca(2+)regulation.

Extended‐Synaptotagmins as Lipid Transporters at ER‐PM Contact Sites

Growing evidence suggests that close appositions between the endoplasmic reticulum (ER) and other membranes, including appositions with the plasma membrane (PM), mediate exchange of lipids between these bilayers. The mechanisms of such exchange, which allows lipid transfer independently of vesicular transport, remain poorly understood. The presence of a synaptotagmin‐like mitochondrial‐lipid‐binding protein (SMP) domain, a proposed lipid‐binding module, in several proteins localized at membrane contact sites raised the possibility that such domains may be implicated in lipid transport. SMP‐containing proteins include components of the ERMES complex, an ER–mitochondrial tether, and the extended synaptotagmins, which are ER–PM tethers. We present at 2.44 Å resolution the crystal structure of a fragment of human extended synaptotagmin 2 (E‐SYT2), including an SMP domain and two adjacent C2 domains. The SMP domain has a β‐barrel structure like protein modules in the tubular‐lipid‐binding (TULIP) superfamily. It dimerizes to form an approximately 90‐Å‐long cylinder traversed by a channel lined entirely with hydrophobic residues, with the two C2A–C2B fragments forming arched structures flexibly linked to the SMP domain. Importantly, structural analysis complemented by mass spectrometry revealed the presence of glycerophospholipids in the E‐SYT2 SMP channel, indicating a direct role for E‐SYTs in lipid transport. These findings provide strong evidence for a role of SMP‐domain‐containing proteins in the control of lipid transfer at membrane contact sites and have broad implications beyond the field of ER‐to‐PM appositions.Note that this abstract is very similar to that in our initial report in Nature, 2014, 510:552–555.Support or Funding InformationThis work was supported by the National Institutes of Health (GM080616 to K.M.R., R37NS36251 and DK082700 to P.D.C.). M.R.W. and F.T. are funded by grants from the National University of Singapore via the Life Sciences Institute (LSI) and the Biochemical Research Council (BMRC‐SERC Diag‐034). C.M.S. was recipient of an National Science Foundation Graduate Research Fellowship (DGE‐1122492). Y.S. was supported by the Uehara Memorial Foundation and the Japanese Society for Promotion of Science.NIH GM808

Conserved SMP domains of the ERMES complex bind phospholipids and mediate tether assembly

Membrane contact sites (MCS) between organelles are proposed as nexuses for the exchange of lipids, small molecules, and other signals crucial to cellular function and homeostasis. Various protein complexes, such as the endoplasmic reticulum-mitochondrial encounter structure (ERMES), function as dynamic molecular tethers between organelles. Here, we report the reconstitution and characterization of subcomplexes formed by the cytoplasm-exposed synaptotagmin-like mitochondrial lipid-binding protein (SMP) domains present in three of the five ERMES subunits--the soluble protein Mdm12, the endoplasmic reticulum (ER)-resident membrane protein Mmm1, and the mitochondrial membrane protein Mdm34. SMP domains are conserved lipid-binding domains found exclusively in proteins at MCS. We show that the SMP domains of Mdm12 and Mmm1 associate into a tight heterotetramer with equimolecular stoichiometry. Our 17-Å-resolution EM structure of the complex reveals an elongated crescent-shaped particle in which two Mdm12 subunits occupy symmetric but distal positions at the opposite ends of a central ER-anchored Mmm1 homodimer. Rigid body fitting of homology models of these SMP domains in the density maps reveals a distinctive extended tubular structure likely traversed by a hydrophobic tunnel. Furthermore, these two SMP domains bind phospholipids and display a strong preference for phosphatidylcholines, a class of phospholipids whose exchange between the ER and mitochondria is essential. Last, we show that the three SMP-containing ERMES subunits form a ternary complex in which Mdm12 bridges Mmm1 to Mdm34. Our findings highlight roles for SMP domains in ERMES assembly and phospholipid binding and suggest a structure-based mechanism for the facilitated transport of phospholipids between organelles.

Protein sorting to intracellular compartments.

The organization of eukaryotic cells depends on correct targeting of proteins and lipids to the organelles at which they function, but how organelle identity is maintained in the face of a constant flux of material is a major problem for the cell. New mechanisms addressing this question were revealed at the Minisymposium on “Membrane Traffic: Dynamic and Regulation.” Starting with the endoplasmic reticulum (ER), Silvere Pagant (Miller lab) showed that some plasma membrane (PM) proteins require multiple signals to drive capture into COPII vesicles. One signal comes from the cargo protein itself, and the second comes signal from a cargo receptor; both signals simultaneously engage the cargo adaptor for the COPII coat, Sec24. Prasanna Satpute-Krishnan (Lippincott-Schwartz and Hegde labs), recipient of the 2014 Merton Bernfield Award, presented her work on a novel quality-control mechanism for glycosylphosphatidylinositol (GPI)-anchored proteins called RESET (for rapid ER stress-induced export). ER stress causes receptor-mediated exit of GPI-anchored proteins from the ER through the secretory pathway to the PM, from which they are endocytosed and degraded in lysosomes. Only misfolded GPI-anchored proteins bind tightly to the cargo receptor, Tmp21, providing a new paradigm in ER stress clearance. Moving from the ER to the Golgi, Mie Wong (Munro lab) presented an elegant system to test the function of a family of diverse Golgi-localized coiled-coil proteins. When different golgins were ectopically attached to mitochondria, specific classes of vesicles were redirected to mitochondria, clearly demonstrating a vesicle capture role for these proteins. A possible mechanism of lipid transfer from the ER to the PM was revealed by a structural study presented by Chris Schauder (Reinisch and de Camilli groups). The synaptotagmin-like mitochondrial-lipid binding protein (SMP) domain of the ER–PM tether E-Syt2 forms a long hydrophobic channel that can harbor multiple phospholipid molecules. Two SMP domains form a stacked dimer to double the length of the channel. Continuing the theme of lipid composition in organelle identity, Cathy Jackson presented her group's results showing how amphipathic helices (AHs) of distinct chemistries specifically target different organelles through recognition of organelle lipid composition. Regulation of a lipid-binding AH of the Arf guanine nucleotide exchange factor (GEF) GBF1 mediates its localization to either the Golgi or lipid droplets. Looking at a related Arf GEF, yeast Sec7, Brian Richardson (Fromme lab) reported the first crystal structure of a noncatalytic region of these proteins. The N-terminal region is made up of α-helices forming a series of armadillo folds that form an extended platform. This structure provides insight into how this region is involved in dimerization and protein interactions. New vesicle tethering and targeting mechanisms were reported, with Chris Stroupe presenting how the homotypic fusion and protein sorting (HOPS)/class C Vps tethering complex connects specific membrane compartments. This complex binds to a Rab GTPase in one membrane and directly to a second, highly curved membrane through a curvature-sensing amphipathic lipid packing sensor (ALPS) motif. Margaret Heider (Munson lab) provided the most comprehensive view to date of the architecture of the exocyst complex, a multisubunit tether that targets secretory vesicles to the PM. The session ended with a talk by Christine Insinna-Kettenhofen (Westlake group) reporting on a new mechanism for targeting of vesicles to cilia mediated by the epidermal growth factor receptor substrate 15 homology domain–containing (EHD) proteins. These proteins are localized to vesicles, where they cooperate with the Rab8-Rab11 cascade and soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) to regulate morphological changes to the vesicle membrane important for fusion.

Structure of a lipid-bound extended synaptotagmin indicates a role in lipid transfer.

Growing evidence suggests that close appositions between the endoplasmic reticulum (ER) and other membranes, including appositions with the plasma membrane (PM), mediate exchange of lipids between the two bilayers. The mechanisms of such exchange, which allows lipid transfer independently of vesicular transport, remain poorly understood. The presence of an SMP (synaptotagmin-like-mitochondrial-lipid binding protein) domain, a proposed lipid binding module, in several proteins localized at membrane contact sites raised the possibility that such domains may be implicated in lipid transport1,2. SMP-containing proteins include components of the ERMES complex, an ER-mitochondrial tether3, and the Extended-Synaptotagmins/tricalbins, which are ER-PM tethers4-6. Here we present at 2.44 Å resolution the crystal structure of a fragment of Extended-Synaptotagmin 2 (E-Syt2), including an SMP domain and two adjacent C2 domains. The SMP domain has a beta-barrel structure like protein modules in the TULIP superfamily. It dimerizes to form a ~90 Å long cylinder traversed by a channel lined entirely with hydrophobic residues, with the two C2A-C2B fragments forming arched structures flexibly linked to the SMP domain. Importantly, structural analysis complemented by mass spectrometry revealed the presence of glycerophospholipids in the E-Syt2 SMP channel, indicating a direct role for E-Syts in lipid transport. These findings provide strong evidence for a role of SMP domain containing proteins in the control of lipid transfer at membrane contact sites and have broad implication beyond the field of ER to PM appositions.

A conserved membrane-binding domain targets proteins to organelle contact sites

Membrane contact sites (MCSs), where the membranes of two organelles are closely apposed, are regions where small molecules such as lipids or calcium are exchanged between organelles. We have identified a conserved membrane-binding domain found exclusively in proteins at MCSs in Saccharomyces cerevisiae. The synaptotagmin-like-mitochondrial-lipid binding protein (SMP) domain is conserved across species. We show that all seven proteins that contain this domain in yeast localize to one of three MCSs. Human proteins with SMP domains also localize to MCSs when expressed in yeast. The SMP domain binds membranes and is necessary for protein targeting to MCSs. Proteins containing this domain could be involved in lipid metabolism. This is the first protein domain found exclusively in proteins at MCSs.