Pex30-like proteins function as adaptors at distinct ER membrane contact sites

Membrane lipids and proteins synthesized in the ER are used for de novo assembly of organelles, such as lipid droplets and peroxisomes. After assembly, the growth of these organelles is supported by ER-derived lipids transferred at membrane contact sites (MCSs). How ER sites for organelle biogenesis and lipid transfer are established and regulated is unclear. Here, we investigate how the ER membrane protein Pex30 and its family members Pex28, Pex29, Pex31, and Pex32 target and function at multiple MCSs. We show that different Pex30 complexes function at distinct ER domains and MCSs. Pex30 targets ER–peroxisome MCSs when bound to Pex28 and Pex32, organizes the nuclear–vacuolar junction when bound to Pex29, and promotes the biogenesis of lipid droplets independently of other family members. Importantly, the reticulon homology domain (RHD) mediates the assembly of the various Pex30 complexes. Given the role of RHD in membrane shaping, our findings offer a mechanistic link between MCS and regulation of membrane curvature.     

Golgi apparatus: Homotypic fusion maintains biochemical gradients within the Golgi and improves the accuracy of protein maturation

Eukaryotic cells are compartmentalized into organelles which, although constantly exchanging proteins and lipids with their environment, maintain a relatively well-defined biochemical identity. How can such large heterogeneities of chemical composition between (and within) organelles be maintained if different organelles are in constant contact through mass transport? Generic nonlinearities in the transport processes, as would result from specific molecular interactions, can cause the spontaneous chemical differentiation of interacting organelles and compartments within organelles. For the Golgi apparatus, the role of which is to process an incoming flux of lipids and proteins, this spontaneous differentiation decreases inter-cisternal exchange and increases the protein transit time under conditions of high incoming flux, This mechanism enables the Golgi apparatus to spontaneously adjust the protein transit time to the amount of protein requiring processing, thereby improving the processing accuracy of even a limited amount of maturation enzymes.     

Staying in touch: the molecular era of organelle contact sites

Membrane contact sites (MCS) are close appositions between two organelles that facilitate both signaling and the passage of ions and lipids from one cellular compartment to another. Despite the fact that MCS have been observed for over 50 years now, we still know very little about the molecular machinery required to create them or their structure, function and regulation. In this review, we focus on the three best-characterized contact sites to date: the nucleus-vacuole junction and mitochondria-ER and plasma membrane-ER contact sites. In addition, we discuss principles arising from recent research and highlight several unanswered questions in the field.     

Short-range intracellular trafficking of small molecules across endoplasmic reticulum junctions

Intracellular trafficking is not mediated exclusively by vesicles. Additional, non-vesicular mechanisms transport material, in particular small molecules such as lipids and Ca(2+) ions, from one organelle to another. This transport occurs at narrow cytoplasmic gaps called membrane contact sites (MCSs), at which two organelles come into close apposition. Despite the conservation of these structures throughout evolution, little is known about this transport, largely because of a lack of knowledge of almost all molecular components of MCSs. Recently, this situation has started to change because the structural proteins that bridge an MCS are now known in a single case, and proteins implicated in lipid trafficking have been localized to MCSs. In the light of these advances, I hypothesize that the endoplasmic reticulum has a central role in the trafficking of lipids and ions by forming a network of MCSs with most other intracellular organelles.     

Piecing together the structural organisation of lipid exchange at membrane contact sites

Membrane contact sites (MCSs) are areas of close proximity between organelles, implicated in transport of small molecules and in organelle biogenesis. Lipid transfer proteins at MCSs facilitate the distribution of lipid species between organelle membranes. Such exchange processes rely on the apposition of two different membranes delimiting distinct compartments and a cytosolic intermembrane space. Maintaining organelle identity while transferring molecules therefore implies control over MCS architecture both on the ultrastructural and molecular levels. Factors including intermembrane distance, density of resident proteins, and contact surface area fine-tune MCS function. Furthermore, the structural arrangement of lipid transfer proteins and associated proteins underpins the molecular mechanisms of lipid fluxes at MCSs. Thus, the architecture of MCSs emerges as an essential aspect of their function.     

Structural and biochemical insights into lipid transport by VPS13 proteins

VPS13 proteins are proposed to function at contact sites between organelles as bridges for lipids to move directionally and in bulk between organellar membranes. VPS13s are anchored between membranes via interactions with receptors, including both peripheral and integral membrane proteins. Here we present the crystal structure of VPS13s adaptor binding domain (VAB) complexed with a Pro-X-Pro peptide recognition motif present in one such receptor, the integral membrane protein Mcp1p, and show biochemically that other Pro-X-Pro motifs bind the VAB in the same site. We further demonstrate that Mcp1p and another integral membrane protein that interacts directly with human VPS13A, XK, are scramblases. This finding supports an emerging paradigm of a partnership between bulk lipid transport proteins and scramblases. Scramblases can re-equilibrate lipids between membrane leaflets as lipids are removed from or inserted into the cytosolic leaflet of donor and acceptor organelles, respectively, in the course of protein-mediated transport.     

SMP domain proteins in membrane lipid dynamics

Synaptotagmin-like mitochondrial-lipid-binding (SMP) domain proteins are evolutionarily conserved family of proteins in eukaryotes that localize between the endoplasmic reticulum (ER) and either the plasma membrane (PM) or other organelles. They are involved in tethering of these membrane contact sites through interaction with other proteins and membrane lipids. Recent structural and biochemical studies have demonstrated that SMP domain proteins transport a wide variety of lipid species by the ability of the SMP domain to harbor lipids through its unique hydrophobic cavity. Growing evidence suggests that SMP domain proteins play critical roles in cell physiology by their actions at membrane contact sites. In this review, we summarize the functions of SMP domain proteins and their direct roles in lipid transport across different membrane compartments. We also discuss their physiological functions in organisms as well as “bypass” pathways that act in parallel with SMP domain proteins at membrane contact sites.     

Phosphoinositide transport and metabolism at membrane contact sites

Phosphoinositides are a family of signaling lipids that play a profound role in regulating protein function at the membrane-cytosol interface of all cellular membranes. Underscoring their importance, mutations or alterations in phosphoinositide metabolizing enzymes lead to host of developmental, neurodegenerative, and metabolic disorders that are devastating for human health. In addition to lipid enzymes, phosphoinositide metabolism is regulated and controlled at membrane contact sites (MCS). Regions of close opposition typically between the ER and other cellular membranes, MCS are non-vesicular lipid transport portals that engage in extensive communication to influence organelle homeostasis. This review focuses on lipid transport, specifically phosphoinositide lipid transport and metabolism at MCS.     

Phagosome‐endoplasmic reticulum contacts: Kissing and not running

The role of the endoplasmic reticulum (ER) in phagocytosis has been the subject of debate for over a decade. Proteomic determinations and dynamic microscopy of live cells led to conflicting conclusions. Recent insights into the existence of a variety of membrane contact sites (MCS) may help reconcile the seemingly disparate views. Specifically, earlier results can be rationalized considering that the ER forms specialized MCS with nascent and maturing phagosomes, without undergoing fusion. The composition and function of documented ER‐to‐phagosome contact sites is described. In addition, we speculate about the possible existence of additional phagosomal contact sites, based on available knowledge of interactions between the ER and other endocytic compartments. The interaction between phagosomes and the ER has been the subject of debate. Earlier observations that led to the suggestion that the ER fuses with the phagosomal membrane can now be explained in the light of recent evidence that intimate contacts form between the two organelles.     

Oxysterol binding proteins: in more than one place at one time?

Oxysterols are potent signalling lipids that directly bind liver X receptors (LXRs) and a subset of oxysterol binding protein (OSBP) related proteins (ORPs). It is relatively well established that the oxysterol-regulated function of LXRs is to control the expression of genes involved in reverse cholesterol transport, catabolism of cholesterol, and lipogenesis. In contrast, the mechanisms by which oxysterols and ORPs affect cellular lipid metabolism have remained poorly understood. In this review, we summarize the information available on function of the ORPs and compare the two families of proteins binding oxysterol to demonstrate the different responses that similar lipids can elicit within cells. The other focus is on the membrane targeting determinants and the protein interaction partners of ORPs, which provide interesting clues to the mode(s) of ORP action. Specifically, we suggest a model in which a general property of ORPs is to function at membrane contact sites, specialized zones of communication between two different organelles.     

Lipid transfer and signaling at organelle contact sites: the tip of the iceberg

Membrane contact sites (MCSs) are formed by the close apposition of membranes of two organelles. They are zones where signals and small molecules, such as lipids and calcium, are exchanged between intracellular compartments. The past few years have seen considerable progress in our understanding of how MCSs form and facilitate the exchange of lipids and signals. Here we summarize what has been learned about MCSs between the endoplamic reticulum (ER) and the plasma membrane, the ER and mitochondria, and the ER and endosomes or lysosomes. These findings suggest that we are just beginning to understand how MCSs form and function.     

Biogenesis of lipid droplets--how cells get fatter

Lipid droplets are discrete organelles present in most cell types and organisms including bacteria, yeast, plants, insects and animals. Long considered as passive storage deposits, recent cell biology, proteomic and lipidomic analysis show that lipid droplets are dynamic organelles involved in multiple cellular functions. They have a central function in lipid distribution to different membrane-bound organelles and serve not only as main reservoirs of neutral lipids such as triglycerides and cholesterol but in addition, contain structural proteins, proteins involved in lipid synthesis and transmembrane proteins. A detailed model for how transmembrane proteins such as SNARE proteins can exist in lipid droplets is proposed.     

Lipids at membrane contact sites: cell signaling and ion transport

Communication between organelles is essential to coordinate cellular functions and the cell's response to physiological and pathological stimuli. Organellar communication occurs at membrane contact sites (MCSs), where the endoplasmic reticulum (ER) membrane is tethered to cellular organelle membranes by specific tether proteins and where lipid transfer proteins and cell signaling proteins are located. MCSs have many cellular functions and are the sites of lipid and ion transfer between organelles and generation of second messengers. This review discusses several aspects of MCSs in the context of lipid transfer, formation of lipid domains, generation of Ca²⁺ and cAMP second messengers, and regulation of ion transporters by lipids.     

The seipin complex Fld1/Ldb16 stabilizes ER–lipid droplet contact sites

Lipid droplets (LDs) are storage organelles consisting of a neutral lipid core surrounded by a phospholipid monolayer and a set of LD-specific proteins. Most LD components are synthesized in the endoplasmic reticulum (ER), an organelle that is often physically connected with LDs. How LD identity is established while maintaining biochemical and physical connections with the ER is not known. Here, we show that the yeast seipin Fld1, in complex with the ER membrane protein Ldb16, prevents equilibration of ER and LD surface components by stabilizing the contact sites between the two organelles. In the absence of the Fld1/Ldb16 complex, assembly of LDs results in phospholipid packing defects leading to aberrant distribution of lipid-binding proteins and abnormal LDs. We propose that the Fld1/Ldb16 complex facilitates the establishment of LD identity by acting as a diffusion barrier at the ER–LD contact sites.     

Multiple C2 domain–containing transmembrane proteins promote lipid droplet biogenesis and growth at specialized endoplasmic reticulum subdomains

Lipid droplets (LDs) are neutral lipid-containing organelles enclosed in a single monolayer of phospholipids. LD formation begins with the accumulation of neutral lipids within the bilayer of the endoplasmic reticulum (ER) membrane. It is not known how the sites of formation of nascent LDs in the ER membrane are determined. Here we show that multiple C2 domain–containing transmembrane proteins, MCTP1 and MCTP2, are at sites of LD formation in specialized ER subdomains. We show that the transmembrane domain (TMD) of these proteins is similar to a reticulon homology domain. Like reticulons, these proteins tubulate the ER membrane and favor highly curved regions of the ER. Our data indicate that the MCTP TMDs promote LD biogenesis, increasing LD number. MCTPs colocalize with seipin, a protein involved in LD biogenesis, but form more stable microdomains in the ER. The MCTP C2 domains bind charged lipids and regulate LD size, likely by mediating ER–LD contact sites. Together, our data indicate that MCTPs form microdomains within ER tubules that regulate LD biogenesis, size, and ER–LD contacts. Interestingly, MCTP punctae colocalized with other organelles as well, suggesting that these proteins may play a general role in linking tubular ER to organelle contact sites.     

Biogenesis of lipid droplets – how cells get fatter

Abstract

Lipid droplets are discrete organelles present in most cell types and organisms including bacteria, yeast, plants, insects and animals. Long considered as passive storage deposits, recent cell biology, proteomic and lipidomic analysis show that lipid droplets are dynamic organelles involved in multiple cellular functions. They have a central function in lipid distribution to different membrane-bound organelles and serve not only as main reservoirs of neutral lipids such as triglycerides and cholesterol but in addition, contain structural proteins, proteins involved in lipid synthesis and transmembrane proteins. A detailed model for how transmembrane proteins such as SNARE proteins can exist in lipid droplets is proposed.     

Sar1, a Novel Regulator of ER-Mitochondrial Contact Sites

Endoplasmic reticulum (ER)—mitochondrial contact sites play a pivotal role in exchange of lipids and ions between the two organelles. How size and function of these contact sites are regulated remains elusive. Here we report a previously unanticipated, but conserved role of the small GTPase Sar1 in the regulation of ER-mitochondrial contact site size. Activated Sar1 introduces membrane curvature through its N-terminal amphiphatic helix at the ER-mitochondria interphase and thereby reducing contact size. Conversely, the S. cerevisiae N3-Sar1 mutant, in which curvature induction is decreased, caused an increase in ER-mitochondrial contacts. As a consequence, ER tubules are no longer able to mark the prospective scission site on mitochondria, thereby impairing mitochondrial dynamics. Consistently, blocking mitochondrial fusion partially rescued, whereas deletion of the dynamin-like protein enhanced the phenotype in the sar1D32G mutant. We conclude that Sar1 regulates the size of ER-mitochondria contact sites through its effects on membrane curvature.     

Lipid droplet biogenesis is spatially coordinated at ER–vacuole contacts under nutritional stress

Eukaryotic cells store lipids in cytosolic organelles known as lipid droplets (LDs). Lipid droplet bud from the endoplasmic reticulum (ER), and may be harvested by the vacuole for energy during prolonged periods of starvation. How cells spatially coordinate LD production is poorly understood. Here, we demonstrate that yeast ER–vacuole contact sites (NVJs) physically expand in response to metabolic stress, and serve as sites for LD production. NVJ tether Mdm1 demarcates sites of LD budding, and interacts with fatty acyl‐CoA synthases at the NVJ periphery. Artificially expanding the NVJ through over‐expressing Mdm1 is sufficient to drive NVJ‐associated LD production, whereas ablating the NVJ induces defects in fatty acid‐to‐triglyceride production. Collectively, our data suggest a tight metabolic link between nutritional stress and LD biogenesis that is spatially coordinated at ER–vacuole contact sites.     

Endoplasmic Reticulum Structure and Interconnections with Other Organelles

The endoplasmic reticulum (ER) is a large, continuous membrane-bound organelle comprised of functionally and structurally distinct domains including the nuclear envelope, peripheral tubular ER, peripheral cisternae, and numerous membrane contact sites at the plasma membrane, mitochondria, Golgi, endosomes, and peroxisomes. These domains are required for multiple cellular processes, including synthesis of proteins and lipids, calcium level regulation, and exchange of macromolecules with various organelles at ER-membrane contact sites. The ER maintains its unique overall structure regardless of dynamics or transfer at ER-organelle contacts. In this review, we describe the numerous factors that contribute to the structure of the ER.The endoplasmic reticulum (ER) is a dynamic organelle responsible for many cellular functions, including the synthesis of proteins and lipids, and regulation of intracellular calcium levels. This review focuses on the distinct and complex morphology of the ER. The structure of the ER is complex because of the numerous distinct domains that exist within one continuous membrane bilayer. These domains are shaped by interactions with the cytoskeleton, by proteins that stabilize membrane shape, and by a homotypic fusion machinery that allows the ER membrane to maintain its continuity and identity. The ER also contains domains that contact the plasma membrane (PM) and other organelles including the Golgi, endosomes, mitochondria, lipid droplets, and peroxisomes. ER contact sites with other organelles and the PM are both abundant and dispersed throughout the cytoplasm, suggesting that they too could influence the overall architecture of the ER. As we will discuss here, ER shape and distribution are regulated by many intrinsic and extrinsic forces.     

Inter-organelle lipid transfer: a channel model for Vps13 and chorein-N motif proteins

Membrane contact sites, where two organelles are in close proximity, are critical regulators of cellular membrane homeostasis, with roles in signaling, lipid metabolism, and ion dynamics. A growing catalog of specialized lipid transfer proteins carry out lipid exchange at these sites. Currently characterized eukaryotic lipid transport proteins are shuttles that typically extract a single lipid from the membrane of the donor organelle, solubilize it during transport through the cytosol, and deposit it in the acceptor organelle membrane. Here, we highlight the recently identified chorein_N family of lipid transporters, including the Vps13 proteins and the autophagy protein Atg2. These are elongated proteins that, distinct from previously characterized transport proteins, bind tens of lipids at once. They feature an extended channel, most likely lined with hydrophobic residues. We discuss the possibility that they are not shuttles but instead are bridges between membranes, with lipids traversing the cytosol via the hydrophobic channel.