Gram-negative bacteria are surrounded by inner and outer membranes whose glycerophospholipid building blocks are synthesized in the inner membrane and redistributed to the outer membrane by poorly understood mechanisms. In eukaryotes, bridge-like lipid transfer proteins (BLTPs) transfer glycerophospholipids between membranes to support membrane homeostasis and biogenesis. Bacterial proteins in the AsmA family are related to BLTPs in structure and are proposed also to function in lipid transfer. Here we adapted a well-established in vitro lipid transfer assay to provide biochemical evidence that AsmA family proteins, TamB and YhdP, have the ability to transfer glycerophospholipids between membranes, supporting similar functions for AsmA proteins and BLTPs.
Membrane contact sites are organized by protein assemblies that physically couple organelles and coordinate lipid metabolism, yet the structural principles that enable lipid exchange across these junctions remain poorly defined. At the nuclear-vacuolar junction (NVJ) in budding yeast, the tethering protein Mdm1 and its binding partner Nvj3 form a complex that regulates lipid metabolic pathways, but the structural features underlying their interaction have not been resolved. Here, we use AlphaFold-based complex prediction and comparative structural analysis to define the organization of Nvj3-Mdm1 complex assembly. We identify a high-confidence heterodimer in which conserved PXA and PXC domains generate an extended tunnel spanning both proteins. Tunnel analysis predicts a core hydrophobic conduit traversing the Nvj3-Mdm1 interface, consistent with a lipid-compatible architecture. Evolutionary conservation is enriched at the Nvj3-Mdm1 interface. The predicted conduit shares geometric and physicochemical properties with bridge-like lipid transfer proteins, including Atg2, Fmp27, and Hob2, suggesting that heteromeric tether assemblies may contribute directly to inter-organelle lipid transfer. Notably, this conduit is predicted to arise from a heteromeric α-helical assembly rather than the β-sheet-rich architecture characteristic of canonical bridge-like lipid transfer proteins. Comparative phylogenetic analyses showed that Nvj3 and Mdm1 share broadly congruent evolutionary patterns across Saccharomycetes, consistent with their conserved functional association. Together, these findings define Nvj3 as a structural partner of Mdm1 and support a conduit-based model of lipid transfer at the NVJ.
Membrane contact sites (MCSs) are dynamic subcellular compartments formed between organelles that coordinate diverse aspects of cellular communication, including signaling, metabolism, and membrane organization. Tools capable of monitoring and controlling the spatially localized and dynamic properties of MCSs are needed to dissect their regulatory mechanisms and physiological roles. Recent advances in protein engineering have begun to address this need. Proximity-based reporters, chemogenetic approaches, and optogenetic systems have been developed to enable the visualization, interrogation, and manipulation of inter-organelle contacts with improved spatial and temporal precision. This minireview highlights key developments in these molecular toolkits and their representative applications in studying MCS biology. These approaches provide new insights into organelle crosstalk and may inform future therapeutic strategies targeting MCSs.
Subcellular targeting and functionality of plant sucrose transporters (SUTs) are affected at the post-translational level by protein-protein interactions. A systematic screening for SUT-interacting proteins identified a novel plasmodesma (PD)-localized membrane protein belonging to the HVA22 family of stress-induced ER proteins. It carries three transmembrane domains and three zinc-finger domains, interacts with all three sucrose transporters from potato, and co-localizes with PD callose and PD marker proteins. Detailed analyses of transgenic potato plants with decreased expression of this PD protein displaying phenotypic alterations regarding trichome length, leaf expansion, root length, flowering time and tuberization helped to determine its physiological function. These effects are partially graft-transmissible indicating the participation of phloem-mobile signals. Changes in the levels of callose in RNAi plants suggested effects on PD permeability. Co-infiltration experiments revealed enhanced mobility of sucrose transporter—GFP fusion proteins via plasmodesmata in the presence of the HVA22-like protein. The analysis of transgenic plants further suggests that HVA22 protein is a general regulator of PD permeability in potato. Taken together, the HVA22-like protein is an ER protein localized close to phloem plasmodesmata and enhances mobility of GFP proteins of different sizes.
Mitochondria and peroxisomes have long been recognized as interconnected. More than half a century ago it was observed that both types of cell organelles exhibit defects in peroxisome biogenesis disorders. Remarkably, until today, the molecular basis of this connection remains elusive. This Short Review aims to highlight some of the functional links between peroxisomes and mitochondria, and how genetic defects in peroxisomes may impact mitochondria.
Many Gram-negative bacterial pathogens deploy type III effector proteins (T3Es) to manipulate host cellular processes and suppress immune responses. Increasing evidence suggests that certain T3Es mimic eukaryotic FFAT (two phenylalanines in an acidic tract) motifs, enabling interaction with vesicle-associated membrane protein (VAMP)-associated proteins (VAPs). These interactions likely help pathogens target and exploit host membrane contact sites. However, the significance and distribution of FFAT mimicry across different bacterial pathogens remain poorly understood, which is crucial to uncovering its role in pathogenic strategies. In this study, we analyzed the T3E repertoire of the model plant pathogenic bacterium Pseudomonas syringae pv. tomato (Pst) DC3000 to identify potential FFAT motifs. Our preliminary data reveal that HopN1, a Pst T3E belonging to the YopT/AvrPphB family of cysteine proteases, contains at least one functional FFAT motif. Yeast two-hybrid and in planta co-immunoprecipitation assays confirmed that HopN1 interacts with plant VAP proteins. This interaction suggests that VAP binding may facilitate its localization to specific membrane compartments. Furthermore, HopN1 was shown to interact with a plant RHO-GTPase, hinting at a functional parallel to YopT in mammals. Our findings demonstrate that HopN1 interacts with VAP12 and a plant RHO-GTPase, suggesting a potential role in membrane-associated processes. However, whether HopN1 actively exploits VAP proteins for subcellular localization remains to be determined. While FFAT motif mimicry may contribute to effector targeting in plant-pathogenic bacteria, further studies are required to establish its functional significance in HopN1 virulence.
Membrane contact sites (MCSs) are microdomains that exchange ions and lipids between the membranes of two organelles. They facilitate the exchange of metabolites and act as a site for intracellular communication through material transport. Because of the important physiological significance of MCSs in localizing the exchange of substances and metabolic regulation, they are considered to play an important role in cell biology. Understanding MCS structure is essential for analyzing how substances move to and from each organelle. Several methods have been developed to analyze MCS function, with electron microscopy (EM) being the predominant technique when structural detail is needed. In this review, we summarize the ultrastructure of MCSs and how EM can be used to determine their role in cell biology.
VAMP-associated proteins (VAPs) are highly conserved, endoplasmic reticulum (ER)-resident receptors that tether the ER to various membrane compartments in eukaryotic cells. Each VAP contains a transmembrane helix at its extreme C-terminus and a conserved N-terminal major sperm protein (MSP) domain that mediates various cytosolic interactions via both protein and lipid binding. Here, I question the fundamental difference between protein- and lipid-based associations in VAP-driven membrane contact site (MCS) formation and function – could the lipid affinity of VAPs be an overlooked factor in MCS dynamic regulation?
Execution of all cellular functions depends on a healthy proteome, whose maintenance requires multimodal oversight. Roughly a third of human proteins reside in membranes and thus present unique topological challenges with respect to biogenesis and degradation. To meet these challenges, eukaryotes have evolved organellar pathways of protein folding and quality control. Most transmembrane proteins originate in the endoplasmic reticulum (ER), where they are subject to surveillance and, if necessary, removal through either ER-associated proteasomal degradation (cytosolic pathway) or selective autophagy (ER-phagy; organellar pathway). In the latter case, ER cargoes are shuttled to (endo)lysosomes – the same organelles that degrade cell surface molecules via endocytosis. Here, we provide an overview of dynamic coordination between the ER and endolysosomes, with a focus on their engagement in specialized physical interfaces termed membrane contact sites (MCSs). We cover how cross-compartmental integration through MCSs allows biosynthetic and proteolytic organelles to fine-tune each other's membrane composition, organization, and dynamics and facilitates recovery from proteotoxic stress. Along the way, we highlight recent developments and open questions at the crossroads between organelle biology and protein quality control and cast them against the backdrop of factor-specific diseases associated with perturbed membrane homeostasis.
Alzheimer's disease (AD) is the most common neurodegenerative disorder of the elderly and no cure is currently available, as the mechanisms leading to neuronal damage and cognitive impairments remain elusive. In the last years, accumulating evidence highlighted early perturbations of the communication between mitochondria and endoplasmic reticulum (ER) in AD models. In this short review, we summarize recent findings linking alterations of ER-mitochondria coupling with typical AD hallmarks.
Eukaryotic cells are subdivided into specialised organelle compartments, each with unique physiological environments and functions. Interaction and cross-talk between organelles is inherent to Eukaryotic life, and each organelle is physically interconnected to their surrounding subcellular components including the cytoskeleton and adjacent membrane compartments. In animals and yeast, the mechanisms of organelle interaction have been well characterised and are known to have fundamental importance to life. In contrast, we are only beginning to understand the mechanisms and functions of such interactions in plants. The discovery and ongoing characterisation of the NETWORKED (NET) protein family of plant actin-membrane adaptors has greatly advanced our understanding of the mechanisms of organelle-cytoskeletal interaction. Furthermore, unfolding investigation into the NET proteins has revealed their binding partner, VAMP-ASSOCIATED PROTEIN-27 (VAP27), to be a regulator of organelle tethering and interaction with previously unknown, specialised roles in plants. Research on NET and VAP27 proteins has rapidly increased our knowledge of the mechanisms regulating membrane interaction in plants, their functions in regulating cell structure and organisation, as well as their importance to plant growth, development and stress-response. Here, we discuss the discovery and characterisation of the NET and VAP27 proteins, their regulation of organelle interaction and their functions in plants.
Pex23 proteins are a family of fungal endoplasmic reticulum proteins. Hansenula polymorpha contains four members, two of which, Pex24 and Pex32, function in endoplasmic reticulum-peroxisome membrane contact sites. In the absence of the other two members, Pex23 and Pex29, mitochondria are fragmented and lipid droplet numbers are reduced. We here show that in Δpex23 and Δpex29 cells an increased portion of the lipid droplet protein Erg6 (C24-methyltransferase), an enzyme involved in ergosterol biosynthesis, localizes to mitochondria. Erg6 relocalization and the reduction in lipid droplet numbers are suppressed by an artificial endoplasmic reticulum-mitochondrion tether protein. Sterol measurements showed that the presence of Erg6 at mitochondria did not cause major changes in the overall sterol composition. Our findings suggest that Pex23 and Pex29 play a role in endoplasmic reticulum-mitochondrion contact sites which prevent mitochondrial mislocalization of Erg6.
Sites of close apposition between organelles, known as membrane contact sites (MCSs), are critical regulators of organelle function. Mitochondria form elaborate reticular networks that perform essential metabolic and signaling functions. Many mitochondrial functions are regulated by MCSs formed between mitochondria and other organelles. In this review, we aim to bring attention to an understudied, but physiologically important, MCS between mitochondria and the plasma membrane (PM). We first describe the molecular mechanism of mitochondria-PM tethering in budding yeast and discuss its role in regulating multiple biological processes, including mitochondrial dynamics and lipid metabolism. Next, we discuss the evidence for mitochondria-PM tethering in higher eukaryotes, with a specific emphasis on mitochondria-PM contacts in retinal cells, and speculate on their functions. Finally, we discuss unanswered questions to guide future research into the function of mitochondria-PM contact sites.
Alterations of endoplasmic reticulum (ER)-mitochondrial interaction have been associated with different pathological conditions, including neurodegenerative diseases, characterized by dysregulation of protein homeostasis. However, little is known about how enhanced ER-mitochondrial tethering affects cellular proteostatic machinery. Here, we transiently overexpressed synthetic ER-mitochondrial linkers (EMLs), stabilizing the ER-mitochondrial distance at ≤5 nm (denominated as 5 nm-EML) and ∼10 nm (10 nm-EML), in HeLa cells. No alterations were found in cell growth, although metabolic activity and total ATP were significantly reduced. In EML-expressing cells, global protein synthesis was significantly reduced, accompanied by a reduction of total PERK and eIF2α protein levels, but increased p-eIF2α. Unfolded protein response (UPR) markers ATF4 and ATF6 were upregulated, suggesting that enhanced ER-mitochondrial tethering deranges protein synthesis and induces a low-grade ER stress/UPR. To further investigate ER-mitochondrial tethering-induced protein dyshomeostasis, we performed shotgun mass spectrometry proteomics followed by bioinformatic analysis. Analysis of highly changed proteins and the most significantly overrepresented gene ontology (GO) terms revealed that ≤5 nm tethering preferentially affected the expression of proteins involved in RNA processing and splicing and proteasomal protein degradation, while ∼10 nm tethering preferentially affected protein translation. Both EMLs affected expression of proteins involved in mitochondrial bioenergetics and metabolism, defense against oxidative stress, ER protein homeostasis, signaling and secretion. Finally, lipidomic analysis suggests that 5 nm-EML and 10 nm-EML differentially affect lipid homeostasis. Altogether, our results suggest that enhanced ER-mitochondrial tethering leads to a profound remodeling of cellular protein homeostasis, which may play a key role in pathogenesis of Alzheimer's and other neurodegenerative diseases.
Detection of mitochondria-ER contacts (MERCs) from diffraction limited confocal images commonly uses fluorescence colocalization analysis of mitochondria and endoplasmic reticulum (ER) as well as split fluorescent probes, such as the split-GFP-based contact site sensor (SPLICS). However, inter-organelle distances (∼10–60 nm) for MERCs are lower than the 200–250 nm diffraction limited resolution obtained by standard confocal microscopy. Super-resolution microscopy of 3D volume analysis provides a two-fold resolution improvement (∼120 nm XY; 250 nm Z), which remains unable to resolve MERCs. MCS-DETECT, a membrane contact site (MCS) detection algorithm faithfully detects elongated ribosome-studded riboMERCs when applied to 3D STED super-resolution image volumes. Here, we expressed the SPLICS L reporter in HeLa cells co-transfected with the ER reporter RFP-KDEL and label fixed cells with antibodies to RFP and the mitochondrial protein TOM20. MCS-DETECT analysis of 3D STED volumes was compared to contacts determined by co-occurrence colocalization analysis of mitochondria and ER or the SPLICS L probe. Percent mitochondria coverage by MCS-DETECT derived contacts was significantly smaller than those obtained for colocalization analysis or SPLICS L , and more closely matched contact site metrics obtained by 3D electron microscopy. Further, STED analysis localized a subset of the SPLICS L label to mitochondria with some SPLICS L puncta observed to be completely enveloped by mitochondria in 3D views. These data suggest that MCS-DETECT reports on a limited set of MERCs that more closely corresponds to those observed by EM.
Mitochondria and lysosomes regulate a multitude of biological processes that are essential for the maintenance of nutrient and metabolic homeostasis and overall cell viability. Recent evidence reveals that these pivotal organelles, similarly to others previously studied, communicate through specialized membrane contact sites (MCSs), hereafter referred to as mitochondria-lysosome contacts (or MLCs), which promote their dynamic interaction without involving membrane fusion. Signal integration through MLCs is implicated in key processes, including mitochondrial fission and dynamics, and the exchange of calcium, cholesterol, and amino acids. Impairments in the formation and function of MLCs are increasingly associated with age-related diseases, specifically neurodegenerative disorders and lysosomal storage diseases. However, MLCs may play roles in other pathological contexts where lysosomes and mitochondria are crucial. In this review, we introduce the methodologies used to study MLCs and discuss known molecular players and key factors involved in their regulation in mammalian cells. We also argue other potential regulatory mechanisms depending on the acidic lysosomal pH and their impact on MLC's function. Finally, we explore the emerging implications of dysfunctional mitochondria-lysosome interactions in disease, highlighting their potential as therapeutic targets in cancer.
Coenzyme Q (CoQ) is an essential redox-active lipid that plays a major role in the electron transport chain, driving mitochondrial ATP synthesis. In Saccharomyces cerevisiae (yeast), CoQ biosynthesis occurs exclusively in the mitochondrial matrix via a large protein-lipid complex, the CoQ synthome, comprised of CoQ itself, late-stage CoQ-intermediates, and the polypeptides Coq3-Coq9 and Coq11. Coq11 is suggested to act as a negative modulator of CoQ synthome assembly and CoQ synthesis, as its deletion enhances Coq polypeptide content, produces an enlarged CoQ synthome, and restores respiration in mutants lacking the CoQ chaperone polypeptide, Coq10. The CoQ synthome resides in specific niches within the inner mitochondrial membrane, termed CoQ domains, that are often located adjacent to the endoplasmic reticulum-mitochondria encounter structure (ERMES). Loss of ERMES destabilizes the CoQ synthome and renders CoQ biosynthesis less efficient. Here we show that deletion of COQ11 suppresses the respiratory deficient phenotype of select ERMES mutants, results in repair and reorganization of the CoQ synthome, and enhances mitochondrial CoQ domains. Given that ER-mitochondrial contact sites coordinate CoQ biosynthesis, we used a Split-MAM (Mitochondrial Associated Membrane) artificial tether consisting of an ER-mitochondrial contact site reporter, to evaluate the effects of artificial membrane tethers on CoQ biosynthesis in both wild-type and ERMES mutant yeast strains. Overall, this work identifies the deletion of COQ11 as a novel suppressor of phenotypes associated with ERMES deletion mutants and indicates that ER-mitochondria tethers influence CoQ content and turnover, highlighting the role of membrane contact sites in regulating mitochondrial respiratory homeostasis.
Membrane contact sites (MCS) provide specialized conduits for inter-organelle communications to maintain cellular homeostasis. Most organelles are interconnected, which supports their coordination and function. M-phase (mitosis or meiosis) is associated with dramatic cellular remodeling to support cell division, including the equal distribution of organelles to the two daughter cells. However, the fate of MCS in M-phase is poorly understood. Here we review recent advances arguing for differential remodeling of endoplasmic reticulum (ER) MCS with the plasma membrane (PM, ERPMCS) and the mitochondria (MERCS) during cell division.
Microglia, the resident immune cells of the central nervous system (CNS), play a crucial role in maintaining tissue homeostasis by monitoring and responding to environmental changes through processes such as phagocytosis, cytokine production or synapse remodeling. Their dynamic nature and diverse functions are supported by the regulation of multiple metabolic pathways, enabling microglia to efficiently adapt to fluctuating signals. A key aspect of this regulation occurs at mitochondria-associated ER membranes (MAM), specialized contact sites between the ER and mitochondria. These structures facilitate the exchange of calcium, lipids, and metabolites and serve as metabolic and signaling hubs. This review synthesizes current research on how MAM influence microglial physiology, with an emphasis on their role in immunometabolism, offering new insights into the integration of metabolic and immune functions in the CNS and its impact in the context of neurodegeneration.
