Endoplasmic Reticulum-plasma Membrane Contact Research Articles

Rapid formation of ER‐PM contacts during early stages of phagocytosis

Membrane contact sites (MCS) are specialized structures where the endoplasmic reticulum (ER) and other organelle form come in close proximity (~10–30 nm). Functionally, these regions are thought to serve as hubs for cellular processes such as inter‐organellar exchange of lipids, calcium homeostasis, and the access of phosphatases to substrates. Consequently, the spatial‐temporal organization of these structures will impact numerous cellular functions. The formation of ER plasma membrane (PM) contact sites are controlled by a variety of membrane tether proteins as well as the morphology of the ER. We and others have found that the sub‐plasmalemmal cortical actin cytoskeleton, a mesh‐like network that occupies 100–200 nm below the PM, can also limit the formation of ER‐PM contact sites. Yet, how ER‐PM junctions are regulated in circumstances where cells have extensive cortical actin remodeling is not understood. An example of such cellular process involving dynamic cortical actin re‐arrangement is phagocytosis. Phagocytosis is an actin‐dependent processes used to internalize particulate material (≥0.5 μm) that serves both antimicrobial and homeostatic functions. The role of the ER in phagocytosis has been a matter of much controversy. Electron micrographs of macrophages undergoing phagocytosis revealed the presence of ER in extensive, close contact with the forming phagosome. It is speculated that the role for ER in this context is to form ER‐PM contact sites with the PM. My studies have revealed that during phagocytosis, the disassembly of F‐actin from the base of the phagocytic cup allows for the formation of new ER‐PM contact sites. ER‐PM contacts formed promptly and specifically where the polymerized actin has been cleared. As such I have found that the spatial occupancy of ER‐PM junction increased ~3 fold during phagocytosis. Finally, I identified PTP1B, a tyrosine phosphatase, as one of the ER‐PM contact proteins that may be functionally important during phagocytosis.Support or Funding InformationNSERC PGS‐DCIHR Project Grant

Rare earth elements induce cytoskeleton-dependent and PI4P-associated rearrangement of SYT1/SYT5 endoplasmic reticulum-plasma membrane contact site complexes in Arabidopsis.

In plant cells, environmental stressors promote changes in connectivity between the cortical endoplasmic reticulum (ER) and the plasma membrane (PM). Although this process is tightly regulated in space and time, the molecular signals and structural components mediating these changes in interorganelle communication are only starting to be characterized. In this report, we confirm the presence of a putative tethering complex containing the synaptotagmins 1 and 5 (SYT1 and SYT5) and the Ca2+- and lipid-binding protein 1 (CLB1/SYT7). This complex is enriched at ER-PM contact sites (EPCSs), has slow responses to changes in extracellular Ca2+, and displays severe cytoskeleton-dependent rearrangements in response to the trivalent lanthanum (La3+) and gadolinium (Gd3+) rare earth elements (REEs). Although REEs are generally used as non-selective cation channel blockers at the PM, here we show that the slow internalization of REEs into the cytosol underlies the activation of the Ca2+/calmodulin intracellular signaling, the accumulation of phosphatidylinositol-4-phosphate (PI4P) at the PM, and the cytoskeleton-dependent rearrangement of the SYT1/SYT5 EPCS complexes. We propose that the observed EPCS rearrangements act as a slow adaptive response to sustained stress conditions, and that this process involves the accumulation of stress-specific phosphoinositide species at the PM.

Structural and functional relationships between plasmodesmata and plant endoplasmic reticulum–plasma membrane contact sites consisting of three synaptotagmins

Synaptotagmin 1 (SYT1) has been recognised as a tethering factor of plant endoplasmic reticulum (ER)-plasma membrane (PM) contact sites (EPCSs) and partially localises to around plasmodesmata (PD). However, other components of EPCSs associated with SYT1 and functional links between the EPCSs and PD have not been identified. We explored interactors of SYT1 by immunoprecipitation and mass analysis. The dynamics, morphology and spatial arrangement of the ER in Arabidopsis mutants lacking the EPCS components were investigated using confocal microscopy and electron microscopy. PD permeability of EPCS mutants was assessed using a virus movement protein and free green fluorescent protein (GFP) as indicators. We identified two additional components of the EPCSs, SYT5 and SYT7, that interact with SYT1. The mutants of the three SYTs were defective in the anchoring of the ER to the PM. The ER near the PD entrance appeared to be weakly squeezed in the triple mutant compared with the wild-type. The triple mutant suppressed cell-to-cell movement of the virus movement protein, but not GFP diffusion. We revealed major additional components of EPCS associated with SYT1 and suggested that the EPCSs arranged around the PD squeeze the ER to regulate active transport via PD.

The endoplasmic reticulum-plasma membrane tethering protein Ist2 regulates endoplasmic reticulum stress response in <italic>Candida albicans</italic>

The endoplasmic reticulum-plasma membrane contact (ER-PM contact) is a novel membrane contact system in eukaryotic cells that mediates the close exchange of materials and messengers between ER and PM. However, the types and function of the ER-PM tethering proteins of the opportunistic pathogenic fungus Candida albicans remain poorly understood. In the present study, we found that Ist2 co-localizes with the ER-PM contact sites of C. albicans and that its deletion causes a marked decrease in the ER-PM contact sites as revealed by fluorescence observation. This indicates that Ist2 is a key ER-PM tethering protein of C. albicans . The measurement of growth curves, fluorescence detection of the unfolded protein response (UPR) reporting system, and the measurement of the cytosolic Ca2+ level revealed that the IST2 deletion strain ist2 Δ/Δ considerably increased in biomass, the UPR reporting gene PRB1 expression was attenuated, and the cytoplasmic Ca2+ levels under ER stress caused by tunicamycin decreased. In addition, the Ca2+ chelating agent EGTA could eliminate the difference between ist2 Δ/Δ and WT in ER stress tolerance. Therefore, Ist2 is an important ER-PM tethering protein in C. albicans and a negative regulator of ER stress responses, which mediates an increase in cytosolic Ca2+ level under ER stress, resulting in attenuated ER stress tolerance. The results of the present study facilitate our understanding of the structure and function of the fungal ER-PM contact and provide an important basis for the development of novel antifungal targets.

Remodeling of ER–plasma membrane contact sites but not STIM1 phosphorylation inhibits Ca2+ influx in mitosis

Store-operated Ca2+ entry (SOCE), mediated by the endoplasmic reticulum (ER) Ca2+ sensor stromal interaction molecule 1 (STIM1) and the plasma membrane (PM) channel Orai1, is inhibited during mitosis. STIM1 phosphorylation has been suggested to mediate this inhibition, but it is unclear whether additional pathways are involved. Here, we demonstrate using various approaches, including a nonphosphorylatable STIM1 knock-in mouse, that STIM1 phosphorylation is not required for SOCE inhibition in mitosis. Rather, multiple pathways converge to inhibit Ca2+ influx in mitosis. STIM1 interacts with the cochaperone BAG3 and localizes to autophagosomes in mitosis, and STIM1 protein levels are reduced. The density of ER-PM contact sites (CSs) is also dramatically reduced in mitosis, thus physically preventing STIM1 and Orai1 from interacting to activate SOCE. Our findings provide insights into ER-PM CS remodeling during mitosis and a mechanistic explanation of the inhibition of Ca2+ influx that is required for cell cycle progression.

Lipid transporter TMEM24/C2CD2L is a Ca2+-regulated component of ER–plasma membrane contacts in mammalian neurons

Close appositions between the endoplasmic reticulum (ER) and the plasma membrane (PM) are a general feature of all cells and are abundant in neurons. A function of these appositions is lipid transport between the two adjacent bilayers via tethering proteins that also contain lipid transport modules. However, little is known about the properties and dynamics of these proteins in neurons. Here we focused on TMEM24/C2CD2L, an ER-localized SMP domain containing phospholipid transporter expressed at high levels in the brain, previously shown to be a component of ER-PM contacts in pancreatic β-cells. TMEM24 is enriched in neurons versus glial cells and its levels increase in parallel with neuronal differentiation. It populates ER-PM contacts in resting neurons, but elevations of cytosolic Ca2+ mediated by experimental manipulations or spontaneous activity induce its transient redistribution throughout the entire ER. Dissociation of TMEM24 from the plasma membrane is mediated by phosphorylation of an array of sites in the C-terminal region of the protein. These sites are only partially conserved in C2CD2, the paralogue of TMEM24 primarily expressed in nonneuronal tissues, which correspondingly display a much lower sensitivity to Ca2+ elevations. ER-PM contacts in neurons are also sites where Kv2 (the major delayed rectifier K+ channels in brain) and other PM and ER ion channels are concentrated, raising the possibility of a regulatory feedback mechanism between neuronal excitability and lipid exchange between the ER and the PM.

Yeast-model-based study identified myosin- and calcium-dependent calmodulin signalling as a potential target for drug intervention in chorea-acanthocytosis.

ABSTRACTChorea-acanthocytosis (ChAc) is a rare neurodegenerative disease associated with mutations in the human VPS13A gene. The mechanism of ChAc pathogenesis is unclear. A simple yeast model was used to investigate the function of the single yeast VSP13 orthologue, Vps13. Vps13, like human VPS13A, is involved in vesicular protein transport, actin cytoskeleton organisation and phospholipid metabolism. A newly identified phenotype of the vps13Δ mutant, sodium dodecyl sulphate (SDS) hypersensitivity, was used to screen a yeast genomic library for multicopy suppressors. A fragment of the MYO3 gene, encoding Myo3-N (the N-terminal part of myosin, a protein involved in the actin cytoskeleton and in endocytosis), was isolated. Myo3-N protein contains a motor head domain and a linker. The linker contains IQ motifs that mediate the binding of calmodulin, a negative regulator of myosin function. Amino acid substitutions that disrupt the interaction of Myo3-N with calmodulin resulted in the loss of vps13Δ suppression. Production of Myo3-N downregulated the activity of calcineurin, a protein phosphatase regulated by calmodulin, and alleviated some defects in early endocytosis events. Importantly, ethylene glycol tetraacetic acid (EGTA), which sequesters calcium and thus downregulates calmodulin and calcineurin, was a potent suppressor of vps13Δ. We propose that Myo3-N acts by sequestering calmodulin, downregulating calcineurin and increasing activity of Myo3, which is involved in endocytosis and, together with Osh2/3 proteins, functions in endoplasmic reticulum-plasma membrane contact sites. These results show that defects associated with vps13Δ could be overcome, and point to a functional connection between Vps13 and calcium signalling as a possible target for chemical intervention in ChAc. Yeast ChAc models may uncover the underlying pathological mechanisms, and may also serve as a platform for drug testing.This article has an associated First Person interview with the first author of the paper.

Oxysterol-binding protein-related protein (ORP) 6 localizes to the ER and ER-plasma membrane contact sites and is involved in the turnover of PI4P in cerebellar granule neurons

Oxysterol-binding protein (OSBP)-related proteins (ORPs) are conserved lipid binding proteins found in organisms ranging from yeast to mammals. Recent findings have indicated that these proteins mainly localize to contact sites of 2 different membranous organelles. ORP6, a member of the ORP subfamily III, is one of the least studied ORPs. Using approaches in molecular cell biology, we attempted to study the characteristics of ORP6 and found that ORP6 is abundantly expressed in mouse cultured neurons. Deconvolution microscopy of cultured cerebellar granular cells revealed that ORP6 is localized to the endoplasmic reticulum (ER) and ER–plasma membrane (PM) contact sites, where it co-localized with extended synaptotagmin2 (E-Syt2), a well-known ER–PM contact site marker. E-Syt2 also co-localized with ORP3, another subfamily III member, and ORP5, a subfamily IV member. However, ORP5 does not distribute to the same ER-PM contact sites as subfamily III members. Also, the co-expression of ORP3 but not ORP5 altered the distribution of ORP6 into the processes of cerebellar neurons. Immunoprecipitation demonstrated binding between the intermediate region of ORP6 and ORP3 or ORP6 itself. Additionally, the localization of ORP6 in the PM decreased when co-expressed with the intermediate region of ORP6, in which the pleckstrin homology (PH) domain and OSBP-related ligand binding domain (ORD) are deleted. Over-expression of this intermediate region shifted the location of a phophtidylinositol-4-phosphate (PI4P) marker from the Golgi to the PM. Knockdown of ORP6 resulted in the same shift of the PI4P marker. Collectively, our data suggests that the recruitment of ORP6 to ER–PM contact sites is involved in the turnover of PI4P in cerebellar granular neurons.

Endoplasmic reticulum-plasma membrane contact sites integrate sterol and phospholipid regulation.

Tether proteins attach the endoplasmic reticulum (ER) to other cellular membranes, thereby creating contact sites that are proposed to form platforms for regulating lipid homeostasis and facilitating non-vesicular lipid exchange. Sterols are synthesized in the ER and transported by non-vesicular mechanisms to the plasma membrane (PM), where they represent almost half of all PM lipids and contribute critically to the barrier function of the PM. To determine whether contact sites are important for both sterol exchange between the ER and PM and intermembrane regulation of lipid metabolism, we generated Δ-super-tether (Δ-s-tether) yeast cells that lack six previously identified tethering proteins (yeast extended synatotagmin [E-Syt], vesicle-associated membrane protein [VAMP]-associated protein [VAP], and TMEM16-anoctamin homologues) as well as the presumptive tether Ice2. Despite the lack of ER-PM contacts in these cells, ER-PM sterol exchange is robust, indicating that the sterol transport machinery is either absent from or not uniquely located at contact sites. Unexpectedly, we found that the transport of exogenously supplied sterol to the ER occurs more slowly in Δ-s-tether cells than in wild-type (WT) cells. We pinpointed this defect to changes in sterol organization and transbilayer movement within the PM bilayer caused by phospholipid dysregulation, evinced by changes in the abundance and organization of PM lipids. Indeed, deletion of either OSH4, which encodes a sterol/phosphatidylinositol-4-phosphate (PI4P) exchange protein, or SAC1, which encodes a PI4P phosphatase, caused synthetic lethality in Δ-s-tether cells due to disruptions in redundant PI4P and phospholipid regulatory pathways. The growth defect of Δ-s-tether cells was rescued with an artificial "ER-PM staple," a tether assembled from unrelated non-yeast protein domains, indicating that endogenous tether proteins have nonspecific bridging functions. Finally, we discovered that sterols play a role in regulating ER-PM contact site formation. In sterol-depleted cells, levels of the yeast E-Syt tether Tcb3 were induced and ER-PM contact increased dramatically. These results support a model in which ER-PM contact sites provide a nexus for coordinating the complex interrelationship between sterols, sphingolipids, and phospholipids that maintain PM composition and integrity.

ORP5 and ORP8 bind phosphatidylinositol-4, 5-biphosphate (PtdIns(4,5)P2) and regulate its level at the plasma membrane

ORP5 and ORP8, members of the oxysterol-binding protein (OSBP)-related proteins (ORP) family, are endoplasmic reticulum membrane proteins implicated in lipid trafficking. ORP5 and ORP8 are reported to localize to endoplasmic reticulum–plasma membrane junctions via binding to phosphatidylinositol-4-phosphate (PtdIns(4)P), and act as a PtdIns(4)P/phosphatidylserine counter exchanger between the endoplasmic reticulum and plasma membrane. Here we provide evidence that the pleckstrin homology domain of ORP5/8 via PtdIns(4,5)P2, and not PtdIns(4)P binding mediates the recruitment of ORP5/8 to endoplasmic reticulum–plasma membrane contact sites. The OSBP-related domain of ORP8 can extract and transport multiple phosphoinositides in vitro, and knocking down both ORP5 and ORP8 in cells increases the plasma membrane level of PtdIns(4,5)P2 with little effect on PtdIns(4)P. Overall, our data show, for the first time, that phosphoinositides other than PtdIns(4)P can also serve as co-exchangers for the transport of cargo lipids by ORPs.

Autophagosomal membranes assemble at ER-plasma membrane contact sites

ABSTRACTThe biogenesis of autophagosome, the double membrane bound organelle related to macro-autophagy, is a complex event requiring numerous key-proteins and membrane remodeling events. Our recent findings identify the extended synaptotagmins, crucial tethers of Endoplasmic Reticulum-plasma membrane contact sites, as key-regulators of this molecular sequence.

Endoplasmic Reticulum-Plasma Membrane Contact Sites.

The endoplasmic reticulum (ER) has a broad localization throughout the cell and forms direct physical contacts with all other classes of membranous organelles, including the plasma membrane (PM). A number of protein tethers that mediate these contacts have been identified, and study of these protein tethers has revealed a multiplicity of roles in cell physiology, including regulation of intracellular Ca2+ dynamics and signaling as well as control of lipid traffic and homeostasis. In this review, we discuss the cross talk between the ER and the PM mediated by direct contacts. We review factors that tether the two membranes, their properties, and their dynamics in response to the functional state of the cell. We focus in particular on the role of ER-PM contacts in nonvesicular lipid transport between the two bilayers mediated by lipid transfer proteins.

The Rim101 pathway contributes to ER stress adaptation through sensing the state of plasma membrane.

Yeast cells sense alterations in the plasma membrane (PM) lipid asymmetry and external alkalization by the sensor protein Rim21, which functions in the Rim101 pathway. Rim101 signaling is initiated at the PM by the recruitment of the Rim101 signaling complex. The PM physically associates with the cortical endoplasmic reticulum (ER) to form ER-PM contact sites, where several signaling events, lipid exchange, and ion transport take place. In the present study, we investigated the spatial relationship between ER-PM contact sites and the sites of Rim101 signaling. Rim101 signaling mostly proceeds outside ER-PM contact sites in the PM and did not require intact ER-PM contact for its activation. Rather, the Rim101 pathway was constitutively activated by ER-PM contact site disruption, which is known to cause ER stress. ER stress induced by tunicamycin treatment activated the Rim101 pathway. Furthermore, the sensitivity of cells to tunicamycin without ER-PM contact was considerably elevated by the deletion of RIM21. These results suggest that the Rim101 pathway is important for the adaptation to ER stress by compensating for alterations in PM lipid asymmetry induced by ER stress.

Arabidopsis SYT1 maintains stability of cortical endoplasmic reticulum networks and VAP27-1-enriched endoplasmic reticulum-plasma membrane contact sites.

Arabidopsis synaptotagmin 1 (SYT1) is localized on the endoplasmic reticulum-plasma membrane (ER-PM) contact sites in leaf and root cells. The ER-PM localization of Arabidopsis SYT1 resembles that of the extended synaptotagmins (E-SYTs) in animal cells. In mammals, E-SYTs have been shown to regulate calcium signaling, lipid transfer, and endocytosis. Arabidopsis SYT1 was reported to be essential for maintaining cell integrity and virus movement. This study provides detailed insight into the subcellular localization of SYT1 and VAP27-1, another ER-PM-tethering protein. SYT1 and VAP27-1 were shown to be localized on distinct ER-PM contact sites. The VAP27-1-enriched ER-PM contact sites (V-EPCSs) were always in contact with the SYT1-enriched ER-PM contact sites (S-EPCSs). The V-EPCSs still existed in the leaf epidermal cells of the SYT1 null mutant; however, they were less stable than those in the wild type. The polygonal networks of cortical ER disassembled and the mobility of VAP27-1 protein on the ER-PM contact sites increased in leaf cells of the SYT1 null mutant. These results suggest that SYT1 is responsible for stabilizing the ER network and V-EPCSs.

ORP5/ORP8 localize to endoplasmic reticulum-mitochondria contacts and are involved in mitochondrial function.

The oxysterol-binding protein (OSBP)-related proteins ORP5 and ORP8 have been shown recently to transport phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) at ER-PM contact sites. PS is also transferred from the ER to mitochondria where it acts as precursor for mitochondrial PE synthesis. Here, we show that, in addition to ER-PM contact sites, ORP5 and ORP8 are also localized to ER-mitochondria contacts and interact with the outer mitochondrial membrane protein PTPIP51. A functional lipid transfer (ORD) domain was required for this localization. Interestingly, ORP5 and ORP8 depletion leads to defects in mitochondria morphology and respiratory function.

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

Membrane contact sites are structures where two organelles come close together to regulate flow of material and information between them. One typeof 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?

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.

Phosphatidylinositol and phosphatidic acid transport between the ER and plasma membrane during PLC activation requires the Nir2 protein.

Phospholipase C (PLC)-mediated hydrolysis of the limited pool of plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] requires replenishment from a larger pool of phosphatidylinositol (PtdIns) via sequential phosphorylation by PtdIns 4-kinases and phosphatidylinositol 4-phosphate (PtdIns4P) 5-kinases. Since PtdIns is synthesized in the endoplasmic reticulum (ER) and PtdIns(4,5)P2 is generated in the PM, it has been postulated that PtdIns transfer proteins (PITPs) provide the means for this lipid transfer function. Recent studies identified the large PITP protein, Nir2 as important for PtdIns transfer from the ER to the PM. It was also found that Nir2 was required for the transfer of phosphatidic acid (PtdOH) from the PM to the ER. In Nir2-depleted cells, activation of PLC leads to PtdOH accumulation in the PM and PtdIns synthesis becomes severely impaired. In quiescent cells, Nir2 is localized to the ER via interaction of its FFAT domain with ER-bound VAMP-associated proteins VAP-A and-B. After PLC activation, Nir2 also binds to the PM via interaction of its C-terminal domains with diacylglycerol (DAG) and PtdOH. Through these interactions, Nir2 functions in ER-PM contact zones. Mutations in VAP-B that have been identified in familial forms of amyotrophic lateral sclerosis (ALS or Lou-Gehrig's disease) cause aggregation of the VAP-B protein, which then impairs its binding to several proteins, including Nir2. These findings have shed new lights on the importance of non-vesicular lipid transfer of PtdIns and PtdOH in ER-PM contact zones with a possible link to a devastating human disease.

RdgBα reciprocally transfers PA and PI at ER-PM contact sites to maintain PI(4,5)P2 homoeostasis during phospholipase C signalling in Drosophila photoreceptors.

Phosphatidylinositol (PI) is the precursor lipid for the synthesis of PI 4,5-bisphosphate [PI(4,5)P2] at the plasma membrane (PM) and is sequentially phosphorylated by the lipid kinases, PI 4-kinase and phosphatidylinositol 4-phosphate (PI4P)-5-kinase. Receptor-mediated hydrolysis of PI(4,5)P2 takes place at the PM but PI resynthesis occurs at the endoplasmic reticulum (ER). Thus PI(4,5)P2 resynthesis requires the reciprocal transport of two key intermediates, phosphatidic acid (PA) and PI between the ER and the PM. PI transfer proteins (PITPs), defined by the presence of the PITP domain, can facilitate lipid transfer between membranes; the PITP domain comprises a hydrophobic cavity with dual specificity but accommodates a single phospholipid molecule. The class II PITP, retinal degeneration typeB (RdgB)α is a multi-domain protein and its PITP domain can bind and transfer PI and PA. In Drosophila photoreceptors, a well-defined G-protein-coupled phospholipase Cβ (PLCβ) signalling pathway, phototransduction defects resulting from loss of RdgBα can be rescued by expression of the PITP domain provided it is competent for both PI and PA transfer. We propose that RdgBα proteins maintain PI(4,5)P2 homoeostasis after PLC activation by facilitating the reciprocal transport of PA and PI at ER-PM membrane contact sites.

Examination of Sec22 Homodimer Formation and Role in SNARE-dependent Membrane Fusion

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein complexes play essential roles in catalyzing intracellular membrane fusion events although the assembly pathway and molecular arrangement of SNARE complexes in membrane fusion reactions are not well understood. Here we monitored interactions of the R-SNARE protein Sec22 through a cysteine scanning approach and detected efficient formation of cross-linked Sec22 homodimers in cellular membranes when cysteine residues were positioned in the SNARE motif or C terminus of the transmembrane domain. When specific Sec22 cysteine derivatives are present on both donor COPII vesicles and acceptor Golgi membranes, the formation of disulfide cross-links provide clear readouts on trans- and cis-SNARE arrangements during this fusion event. The Sec22 transmembrane domain was required for efficient homodimer formation and for membrane fusion suggesting a functional role for Sec22 homodimers. We propose that Sec22 homodimers promote assembly of higher-order SNARE complexes to catalyze membrane fusion. Sec22 is also reported to function in macroautophagy and in formation of endoplasmic reticulum-plasma membrane contact sites therefore homodimer assembly may regulate Sec22 activity across a range of cellular processes.