Phospholipid Synthesis and Transport in Mammalian Cells

Membranes of mammalian subcellular organelles contain defined amounts of specific phospholipids that are required for normal functioning of proteins in the membrane. Despite the wide distribution of most phospholipid classes throughout organelle membranes, the site of synthesis of each phospholipid class is usually restricted to one organelle, commonly the endoplasmic reticulum (ER). Thus, phospholipids must be transported from their sites of synthesis to the membranes of other organelles. In this article, pathways and subcellular sites of phospholipid synthesis in mammalian cells are summarized. A single, unifying mechanism does not explain the inter‐organelle transport of all phospholipids. Thus, mechanisms of phospholipid transport between organelles of mammalian cells via spontaneous membrane diffusion, via cytosolic phospholipid transfer proteins, via vesicles and via membrane contact sites are discussed. As an example of the latter mechanism, phosphatidylserine (PS) is synthesized on a region of the ER (mitochondria‐associated membranes, MAM) and decarboxylated to phosphatidylethanolamine in mitochondria. Some evidence is presented suggesting that PS import into mitochondria occurs via membrane contact sites between MAM and mitochondria. Recent studies suggest that protein complexes can form tethers that link two types of organelles thereby promoting lipid transfer. However, many questions remain about mechanisms of inter‐organelle phospholipid transport in mammalian cells.     

Peroxisome assembly: matrix and membrane protein biogenesis

Mitochondria are dynamic organelles whose functional integrity requires a coordinated supply of proteins and phospholipids. Defined functions of specific phospholipids, like the mitochondrial signature lipid cardiolipin, are emerging in diverse processes, ranging from protein biogenesis and energy production to membrane fusion and apoptosis. The accumulation of phospholipids within mitochondria depends on interorganellar lipid transport between the endoplasmic reticulum (ER) and mitochondria as well as intramitochondrial lipid trafficking. The discovery of proteins that regulate mitochondrial membrane lipid composition and of a multiprotein complex tethering ER to mitochondrial membranes has unveiled novel mechanisms of mitochondrial membrane biogenesis.     

Intramitochondrial phospholipid trafficking

Mitochondrial functions and architecture rely on a defined lipid composition of their outer and inner membranes, which are characterized by a high content of non-bilayer phospholipids such as cardiolipin (CL) and phosphatidylethanolamine (PE). Mitochondrial membrane lipids are synthesized in the endoplasmic reticulum (ER) or within mitochondria from ER-derived precursor lipids, are asymmetrically distributed within mitochondria and can relocate in response to cellular stress. Maintenance of lipid homeostasis thus requires multiple lipid transport processes to be orchestrated within mitochondria. Recent findings identified members of the Ups/PRELI family as specific lipid transfer proteins in mitochondria that shuttle phospholipids between mitochondrial membranes. They cooperate with membrane organizing proteins that preserve the spatial organization of mitochondrial membranes and the formation of membrane contact sites, unravelling an intimate crosstalk of membrane lipid transport and homeostasis with the structural organization of mitochondria.This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.     

Phosphatidylethanolamine Biosynthesis in Mitochondria: PHOSPHATIDYLSERINE (PS) TRAFFICKING IS INDEPENDENT OF A PS DECARBOXYLASE AND INTERMEMBRANE SPACE PROTEINS UPS1P AND UPS2P*

Phosphatidylethanolamine (PE) plays important roles for the structure and function of mitochondria and other intracellular organelles. In yeast, the majority of PE is produced from phosphatidylserine (PS) by a mitochondrion-located PS decarboxylase, Psd1p. Because PS is synthesized in the endoplasmic reticulum (ER), PS is transported from the ER to mitochondria and converted to PE. After its synthesis, a portion of PE moves back to the ER. Two mitochondrial proteins located in the intermembrane space, Ups1p and Ups2p, have been shown to regulate PE metabolism by controlling the export of PE. It remains to be determined where PS is decarboxylated in mitochondria and whether decarboxylation is coupled to trafficking of PS. Here, using fluorescent PS as a substrate in an in vitro assay for Psd1p-dependent PE production in isolated mitochondria, we show that PS is transferred from the mitochondrial outer membrane to the inner membrane independently of Psd1p, Ups1p, and Ups2p and decarboxylated to PE by Psd1p in the inner membrane. Interestingly, Ups1p is required for the maintenance of Psd1p and therefore PE production. Restoration of Psd1p levels rescued PE production defects in ups1Δ mitochondria. Our data provide novel mechanistic insight into PE biogenesis in mitochondria.     

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.     

A Conserved Endoplasmic Reticulum Membrane Protein Complex (EMC) Facilitates Phospholipid Transfer from the ER to Mitochondria

Mitochondrial membrane biogenesis and lipid metabolism require phospholipid transfer from the endoplasmic reticulum (ER) to mitochondria. Transfer is thought to occur at regions of close contact of these organelles and to be nonvesicular, but the mechanism is not known. Here we used a novel genetic screen in S. cerevisiae to identify mutants with defects in lipid exchange between the ER and mitochondria. We show that a strain missing multiple components of the conserved ER membrane protein complex (EMC) has decreased phosphatidylserine (PS) transfer from the ER to mitochondria. Mitochondria from this strain have significantly reduced levels of PS and its derivative phosphatidylethanolamine (PE). Cells lacking EMC proteins and the ER–mitochondria tethering complex called ERMES (the ER–mitochondria encounter structure) are inviable, suggesting that the EMC also functions as a tether. These defects are corrected by expression of an engineered ER–mitochondrial tethering protein that artificially tethers the ER to mitochondria. EMC mutants have a significant reduction in the amount of ER tethered to mitochondria even though ERMES remained intact in these mutants, suggesting that the EMC performs an additional tethering function to ERMES. We find that all Emc proteins interact with the mitochondrial translocase of the outer membrane (TOM) complex protein Tom5 and this interaction is important for PS transfer and cell growth, suggesting that the EMC forms a tether by associating with the TOM complex. Together, our findings support that the EMC tethers ER to mitochondria, which is required for phospholipid synthesis and cell growth.     

VPS13D bridges the ER to mitochondria and peroxisomes via Miro

Mitochondria, which are excluded from the secretory pathway, depend on lipid transport proteins for their lipid supply from the ER, where most lipids are synthesized. In yeast, the outer mitochondrial membrane GTPase Gem1 is an accessory factor of ERMES, an ER–mitochondria tethering complex that contains lipid transport domains and that functions, partially redundantly with Vps13, in lipid transfer between the two organelles. In metazoa, where VPS13, but not ERMES, is present, the Gem1 orthologue Miro was linked to mitochondrial dynamics but not to lipid transport. Here we show that Miro, including its peroxisome-enriched splice variant, recruits the lipid transport protein VPS13D, which in turn binds the ER in a VAP-dependent way and thus could provide a lipid conduit between the ER and mitochondria. These findings reveal a so far missing link between function(s) of Gem1/Miro in yeast and higher eukaryotes, where Miro is a Parkin substrate, with potential implications for Parkinson’s disease pathogenesis.     

Continuous equilibration of phosphatidylcholine and its precursors between endoplasmic reticulum and mitochondria in yeast

In Saccharomyces cerevisiae phosphatidylcholine (PC) is synthesized in the ER and transported to mitochondria via an unknown mechanism. The transport of PC synthesized by the triple methylation of phosphatidylethanolamine was investigated by pulsing yeast spheroplasts with l-[methyl-3H]methionine, followed by a chase with unlabeled methionine and subcellular fractionation. During the pulse, increasing amounts of PC and its mono- and dimethylated precursors (PMME and PDME, respectively) appear in similar proportions in both microsomes and mitochondria, with the extent of incorporation in microsomes being twice that in mitochondria. During the chase, the [3H]-methyl label from the precursors accumulates into PC with similar kinetics in both organelles. The results demonstrate that transport of methylated phospholipids from ER to mitochondria is 1) coupled to synthesis, 2) not selective for PC, 3) at least as fast as the fastest step in the methylation of PE, and 4) bidirectional for PMME and PDME. The interorganellar equilibration of methylated phospholipids was reconstituted in vitro and did not depend on ongoing methylation, cytosolic factors, ATP, and energization of the mitochondria, although energization could accelerate the reaction. The exchange of methylated phospholipids was reduced after pretreating both microsomes and mitochondria with trypsin, indicating the involvement of membrane proteins from both organelles.     

Cnm1: A bridge between mitochondria and nuclear ER

Few membrane contact sites have been defined at the molecular level. By using a high-throughput, microscopy-based screen, Eisenberg-Bord, Zung et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202104100) identify Cnm1 as a novel tethering protein that mediates contact between mitochondria and the nuclear ER in response to phospholipid levels.

Organelles communicate through the exchange of biological materials by vesicular trafficking or at sites of close membrane apposition known as membrane contact sites (MCSs). While the molecular machinery mediating vesicular trafficking has been well characterized, our knowledge of the molecules involved in forming and regulating MCSs is limited. MCSs physically tether two or more organelles via protein–protein or protein–lipid interactions, contain defined proteomes, and perform specific biological functions (1). While MCSs have been appreciated microscopically since the 1950s, only recently have advances in technology permitted the discovery of the molecular composition of some MCSs (2). A major breakthrough occurred when a synthetic biology screen identified the ER–mitochondria encounter structure (ERMES), which forms an MCS between the ER and mitochondria (3). ERMES has since been shown to be involved in phospholipid transport between mitochondria and the ER (4). While ERMES is one of the best characterized MCSs, there are still many questions as to the precise molecules being transported at ER–mitochondria contacts and how directionality of transport is achieved. Subsequent studies using split fluorescent proteins revealed that nearly all organelles appear to form MCSs of some kind (5). Thus, despite progress in defining the components and functions of a few MCSs, there are still many MCSs whose molecular identities are completely unknown.Recently, a study in mammalian cells identified an MCS between the nucleus and mitochondria that plays a role in adapting cells to stress via the mitochondrial retrograde signaling response (6). The proteins that form this MCS are not conserved in yeast, however, suggesting that alternative mechanisms for nucleus–mitochondria contacts exist in other organisms. In this issue, Eisenberg-Bord, Zung et al., set out to identify proteins involved in forming an MCS between mitochondria and the nuclear ER that is distinct from ERMES-mediated ER–mitochondria contacts (7). First, high-resolution cryo-electron tomographs revealed that mitochondria form contacts with the nucleus that have an average separation of ∼20 nm, which is within the expected range for a bona fide MCS (1). To identify the molecular composition of this contact site, the authors generated a synthetic reporter that is specific to nucleus-mitochondria contacts by fusing one part of a split fluorescent protein to an outer mitochondrial membrane protein and the other to a peripheral nuclear protein. A high-throughput, microscopy-based genetic screen was then used to compare the localization of the synthetic reporter to fluorescently tagged versions of all yeast proteins. Candidates were refined by determining which proteins caused an expansion of the nucleus–mitochondria contact site upon overexpression, a phenotype that has been observed with other MCS proteins (8). Based on these results, the best candidate for a molecular tether between mitochondria and the nucleus was Ybr063c.Ybr063c is a 46-kD nonessential protein of uncharacterized function that contains predicted transmembrane domains. The authors first demonstrated that Ybr063c is an integral membrane protein residing on the nuclear membrane. In support of Ybr063c forming a nucleus–mitochondria contact site that is distinct from ERMES, Ybr063c did not colocalize with ERMES subunits nor did overexpression of Ybr063c alter the size of ERMES patches. Remarkably, overexpression of Ybr063c resulted in the mitochondrial network becoming tightly associated with the nuclear membrane. Based on these results, the authors concluded that Ybr063c functions as a molecular tether between mitochondria and the nucleus and the protein was renamed Cnm1 for contact nucleus mitochondria 1.Through further genetic screens, Eisenberg-Bord, Zung et al., identified several genes that are required to cluster mitochondria around the nucleus when Cnm1 is overexpressed. Interestingly, several of these genes are known to function in phosphatidylcholine (PC) metabolism. Deletion of these components resulted in a decrease in Cnm1 expression, which alters the extent of nucleus-mitochondria contacts. Overexpression of Cnm1 in genetic conditions that reduce PC levels resulted in exaggerated growth defects. These results raise the possibility that Cnm1-mediated nuclear–mitochondria contacts may be involved in the transport of PC from the ER to mitochondria. Thus, while the functional importance is unknown, Cnm1-mediated nuclear–mitochondria contacts respond to PC levels.The genetic screens also identified a single resident mitochondrial protein, Tom70, as affecting the ability of overexpressed Cnm1 to cluster mitochondria around the nucleus. Subsequent experiments demonstrated that localization of Cnm1 to the nuclear membrane and Tom70 to the mitochondrial membrane is required to tether mitochondria to the nucleus upon overexpression of Cnm1. Thus, Cnm1 and Tom70 mediate an MCS between mitochondria and the nucleus.The identification of Cnm1-mediated nucleus–mitochondria contacts opens many questions about the function and composition of the contact site and how it operates in the broader context of mitochondrial–nuclear communication. While identifying the functions of MCSs has proven challenging, the genetic screens conducted in this study provide an excellent starting point by elucidating a link between Cnm1 and PC metabolism. The authors propose that Cnm1-mediated contacts could function in the direct transport of PC from the ER to mitochondria (Fig. 1). In this model, ERMES, which likely functions in earlier steps of PC synthesis by transporting phosphatidylethanolamine (PE) or phosphatidylserine (PS), would have a distinct but related function in organizing and maintaining a pipeline for the transport of lipids between the ER and mitochondria (Fig. 1). This model is speculative, however, and future experiments will be necessary to define the role of Cnm1 in PC metabolism.Open in a separate windowFigure 1.The ER and vacuole form multiple MCSs with mitochondria in budding yeast. The ER is depicted in green, and the mitochondrial network is depicted in gray. ERMES mediates an MCS between tubular ER and mitochondria. In addition to functions that are distinct from lipid trafficking, ERMES-mediated MCSs likely function to transport PS or PE between the organelles. Cnm1 mediates an MCS specifically between the nuclear ER and mitochondria and potentially functions in PC transport. The Vps13-Mcp1 vCLAMP mediates an MCS between mitochondria and the vacuole that likely functions in lipid transport and may have redundant functions with ERMES. The Vps39-Tom40 vCLAMP is a separate MCS between mitochondria and the vacuole that responds to different stress conditions, though its function is unknown.There is a growing body of evidence that two organelles can form multiple MCSs that are spatially and functionally distinct. In addition to ERMES and Cnm1-mediated mitochondria–ER contacts, in yeast, two distinct MCSs have been described between mitochondria and the vacuole that are referred to as vacuolar and mitochondrial patches, or vCLAMPs. One, mediated by Vam6 and Tom40, has been implicated in responding to cellular stress while the other, mediated by Mcp1 and Vps13, may have overlapping functions with the ERMES complex (8, 9; Fig. 1). Interestingly, many of the proteins present at MCSs have been shown to be multifunctional (2). For example, the vCLAMP component Vam6 is also a subunit of the homotypic fusion and protein-sorting (HOPS) complex while its binding partner Tom40 is the central subunit of the translocase of outer membrane (TOM) complex (8). Thus, while these complexes have distinct biological functions in vacuolar protein sorting and mitochondrial protein import respectively, individual subunits have moonlighting functions in the formation, and perhaps function, of MCSs. Eisenberg-Bord, Zung et al., now reveal that Tom70, another component of the TOM complex, also plays a role in the formation of nucleus–mitochondria contacts. This raises the exciting possibility that cells use these multifunctional proteins to coordinate functions such as mitochondrial protein import with lipid trafficking. A crucial next step will be to determine how the multiple functions of these proteins are coordinated to maintain organelle homeostasis.Nuclear–mitochondrial communication is a critical aspect of eukaryotic cellular life that allows cells to adapt to different environmental conditions and energy needs. A breakdown in communication between mitochondria and the nucleus has been implicated in several diseases, including cancers (10). The formation of a nucleus–mitochondria MCS likely facilitates the exchange of lipids or small molecules that stimulate signaling pathways to help cells respond to environmental changes or mitochondrial damage (6, 7). Identifying the molecules that regulate these contacts and clarifying the physiological contexts under which these contacts function is crucial to our understanding of human disease. Thus, the identification of a nucleus-mitochondria MCS represents a significant breakthrough in our understanding of nucleus–mitochondria communication.     

酵母线粒体的磷脂转运

线粒体是一种由两层膜包被的细胞器,其功能和结构的稳定性取决于线粒体膜上精确的磷脂组成及分布。线粒体膜上的大部分脂类物质由内质网合成,既而转运到线粒体。而部分脂类利用内质网上产生的前体,在线粒体内膜上合成。由此可见,线粒体膜脂的生物合成需要线粒体与内质网以及线粒体外膜(outer mitochondrial membrane, OMM)与内膜(inner mitochondrial membrane, IMM)之间进行大量的脂质转运。目前认为,这种运输过程既可在拴系因子(tether factors)形成的膜结合部位(membrane contact sites, MCSs)内发生,也可借助脂质转运蛋白(lipid transfer proteins, LTPs)完成。近年来,研究者以酵母为对象,建立了多种线粒体磷脂转运(phospholipid trafficking)的模型,这使人们初步理解了线粒体磷脂转运的机制。本综述总结了酵母线粒体磷脂转运的最新发现,并对这些磷脂转运的模型进行了讨论,以期为今后深入了解线粒体脂类代谢提供参考。     

Mitofusin‐2 knockdown increases ER–mitochondria contact and decreases amyloid β‐peptide production

Mitochondria are physically and biochemically in contact with other organelles including the endoplasmic reticulum (ER). Such contacts are formed between mitochondria‐associated ER membranes (MAM), specialized subregions of ER, and the outer mitochondrial membrane (OMM). We have previously shown increased expression of MAM‐associated proteins and enhanced ER to mitochondria Ca²⁺ transfer from ER to mitochondria in Alzheimer's disease (AD) and amyloid β‐peptide (Aβ)‐related neuronal models. Here, we report that siRNA knockdown of mitofusin‐2 (Mfn2), a protein that is involved in the tethering of ER and mitochondria, leads to increased contact between the two organelles. Cells depleted in Mfn2 showed increased Ca²⁺ transfer from ER to mitchondria and longer stretches of ER forming contacts with OMM. Interestingly, increased contact resulted in decreased concentrations of intra‐ and extracellular Aβ₄₀ and Aβ₄₂. Analysis of γ‐secretase protein expression, maturation and activity revealed that the low Aβ concentrations were a result of impaired γ‐secretase complex function. Amyloid‐β precursor protein (APP), β‐site APP‐cleaving enzyme 1 and neprilysin expression as well as neprilysin activity were not affected by Mfn2 siRNA treatment. In summary, our data shows that modulation of ER–mitochondria contact affects γ‐secretase activity and Aβ generation. Increased ER–mitochondria contact results in lower γ‐secretase activity suggesting a new mechanism by which Aβ generation can be controlled.     

Phospholipid Transport via Mitochondria

In eukaryotic cells, complex membrane structures called organelles are highly developed to exert specialized functions. Mitochondria are one of such organelles consisting of the outer and inner membranes (OM and IM) with characteristic protein and phospholipid compositions. Maintaining proper phospholipid compositions of the membranes is crucial for mitochondrial integrity, thereby contributing to normal cell activities. As cellular locations for phospholipid synthesis are restricted to specific compartments such as the endoplasmic reticulum (ER) membrane and the mitochondrial inner membrane, newly synthesized phospholipids have to be transported and distributed properly from the ER or mitochondria to other cellular membranes. Although understanding of molecular mechanisms of phospholipid transport are much behind those of protein transport, recent studies using yeast as a model system began to provide intriguing insights into phospholipid exchange between the ER and mitochondria as well as between the mitochondrial OM and IM. In this review, we summarize the latest findings of phospholipid transport via mitochondria and discuss the implicated molecular mechanisms.      

Phosphatidylethanolamine and Cardiolipin Differentially Affect the Stability of Mitochondrial Respiratory Chain Supercomplexes

The mitochondrial inner membrane contains two non-bilayer‐forming phospholipids, phosphatidylethanolamine (PE) and cardiolipin (CL). Lack of CL leads to destabilization of respiratory chain supercomplexes, a reduced activity of cytochrome c oxidase, and a reduced inner membrane potential Δψ. Although PE is more abundant than CL in the mitochondrial inner membrane, its role in biogenesis and assembly of inner membrane complexes is unknown. We report that similar to the lack of CL, PE depletion resulted in a decrease of Δψ and thus in an impaired import of preproteins into and across the inner membrane. The respiratory capacity and in particular the activity of cytochrome c oxidase were impaired in PE-depleted mitochondria, leading to the decrease of Δψ. In contrast to depletion of CL, depletion of PE did not destabilize respiratory chain supercomplexes but favored the formation of larger supercomplexes (megacomplexes) between the cytochrome bc₁ complex and the cytochrome c oxidase. We conclude that both PE and CL are required for a full activity of the mitochondrial respiratory chain and the efficient generation of the inner membrane potential. The mechanisms, however, are different since these non-bilayer‐forming phospholipids exert opposite effects on the stability of respiratory chain supercomplexes.     

Cell death regulation by MAMs: from molecular mechanisms to therapeutic implications in cardiovascular diseases

The endoplasmic reticulum (ER) and mitochondria are interconnected intracellular organelles with vital roles in the regulation of cell signaling and function. While the ER participates in a number of biological processes including lipid biosynthesis, Ca²⁺ storage and protein folding and processing, mitochondria are highly dynamic organelles governing ATP synthesis, free radical production, innate immunity and apoptosis. Interplay between the ER and mitochondria plays a crucial role in regulating energy metabolism and cell fate control under stress. The mitochondria-associated membranes (MAMs) denote physical contact sites between ER and mitochondria that mediate bidirectional communications between the two organelles. Although Ca²⁺ transport from ER to mitochondria is vital for mitochondrial homeostasis and energy metabolism, unrestrained Ca²⁺ transfer may result in mitochondrial Ca²⁺ overload, mitochondrial damage and cell death. Here we summarize the roles of MAMs in cell physiology and its impact in pathological conditions with a focus on cardiovascular disease. The possibility of manipulating ER-mitochondria contacts as potential therapeutic approaches is also discussed.Subject terms: Cardiovascular diseases, Cardiomyopathies     

Phosphatidylethanolamine synthesized by three different pathways is supplied to peroxisomes of the yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae three pathways lead to the formation of phosphatidylethanolamine (PE), namely decarboxylation of phosphatidylserine (PS) (i) by Psd1p in mitochondria, and (ii) by Psd2p in a Golgi/vacuolar compartment; and (iii) synthesis via CDP–ethanolamine pathway in the endoplasmic reticulum. To determine the contribution of these pathways to the supply of PE to peroxisomes, we subjected mutants bearing defects in the respective metabolic routes to biochemical and cell biological analysis. Despite these defects in PE formation mutants were able to grow on oleic acid indicating induction of peroxisome proliferation. Biochemical analysis revealed that PE formed through all three pathways was supplied to peroxisomes. These analyses also demonstrated that selective as well as equilibrium interorganelle flux of PE appear to be equally important for cellular homeostasis of this phospholipid. Electron microscopic inspection confirmed that defects in PE synthesis still allowed formation of peroxisomes, although these organelles from strains lacking PSD1 were significantly smaller than wild type. The fact that peroxisomes were always found in close vicinity to mitochondria, ER and lipid particles supported the view that membrane contact may play a role in lipid traffic between these organelles.     

The small GTPase Arf1 modulates mitochondrial morphology and function

The small GTPase Arf1 plays critical roles in membrane traffic by initiating the recruitment of coat proteins and by modulating the activity of lipid-modifying enzymes. Here, we report an unexpected but evolutionarily conserved role for Arf1 and the ArfGEF GBF1 at mitochondria. Loss of function of ARF-1 or GBF-1 impaired mitochondrial morphology and activity in Caenorhabditis elegans. Similarly, mitochondrial defects were observed in mammalian and yeast cells. In Saccharomyces cerevisiae, aberrant clusters of the mitofusin Fzo1 accumulated in arf1-11 mutants and were resolved by overexpression of Cdc48, an AAA-ATPase involved in ER and mitochondria-associated degradation processes. Yeast Arf1 co-fractionated with ER and mitochondrial membranes and interacted genetically with the contact site component Gem1. Furthermore, similar mitochondrial abnormalities resulted from knockdown of either GBF-1 or contact site components in worms, suggesting that the role of Arf1 in mitochondrial functioning is linked to ER–mitochondrial contacts. Thus, Arf1 is involved in mitochondrial homeostasis and dynamics, independent of its role in vesicular traffic.     

Plasma membrane localization of Ras requires class C Vps proteins and functional mitochondria in Saccharomyces cerevisiae

Ras proteins are synthesized as cytosolic precursors, but then undergo posttranslational lipid addition, membrane association, and subcellular targeting to the plasma membrane. Although the enzymes responsible for farnesyl and palmitoyl lipid addition have been described, the mechanism by which these modifications contribute to the subcellular localization of Ras is not known. Following addition of the farnesyl group, Ras associates with the endoplasmic reticulum (ER), where palmitoylation occurs in Saccharomyces cerevisiae. The subsequent translocation of Ras from the ER to the plasma membrane does not require the classical secretory pathway or a functional Golgi apparatus. Vesicular and nonvesicular transport pathways for Ras proteins have been proposed, but the pathway is not known. Here we describe a genetic screen designed to identify mutants defective in Ras trafficking in S. cerevisiae. The screen implicates, for the first time, the class C VPS complex in Ras trafficking. Vps proteins are best characterized for their role in endosome and vacuole membrane fusion. However, the role of the class C Vps complex in Ras trafficking is distinct from its role in endosome and vacuole vesicle fusion, as a mitochondrial involvement was uncovered. Disruption of class C VPS genes results in mitochondrial defects and an accumulation of Ras proteins on mitochondrial membranes. Ras also fractionates with mitochondria in wild-type cells, where it is detected on the outer mitochondrial membrane by virtue of its sensitivity to protease treatment. These results point to a previously uncharacterized role of mitochondria in the subcellular trafficking of Ras proteins.     

Structural and functional link between the mitochondrial network and the endoplasmic reticulum

Mitochondrial and endoplasmic reticulum (ER) networks are fundamental for the maintenance of cellular homeostasis and for determination of cell fate under stress conditions. Recent structural and functional studies revealed the interaction of these networks. These zones of close contact between ER and mitochondria called MAM (mitochondria associated membranes) support communication between the two organelles including bioenergetics and cell survival. The existence of macromolecular complexes in these contact sites has also been revealed. In this contribution, we will review: (i) the ER and mitochondria structure and their dynamics, (ii) the basic principles of ER mitochondrial Ca²⁺ transport, (iii) the physiological/pathological role of this cross-talk.     

GRP75 mediates endoplasmic reticulum–mitochondria coupling during palmitate-induced pancreatic β-cell apoptosis

The endoplasmic reticulum (ER) and mitochondria are structurally connected with each other at specific sites termed mitochondria-associated membranes (MAMs). These physical links are composed of several tethering proteins and are important during varied cellular processes, such as calcium homeostasis, lipid metabolism and transport, membrane biogenesis, and organelle remodeling. However, the attributes of specific tethering proteins in these cellular functions remain debatable. Here, we present data to show that one such tether protein, glucose regulated protein 75 (GRP75), is essential in increasing ER–mitochondria contact during palmitate-induced apoptosis in pancreatic insulinoma cells. We demonstrate that palmitate increased GRP75 levels in mouse and rat pancreatic insulinoma cells as well as in mouse primary islet cells. This was associated with increased mitochondrial Ca²⁺ transfer, impaired mitochondrial membrane potential, increased ROS production, and enhanced physical coupling between the ER and mitochondria. Interestingly, GRP75 inhibition prevented these palmitate-induced cellular aberrations. Additionally, GRP75 overexpression alone was sufficient to impair mitochondrial membrane potential, increase mitochondrial Ca²⁺ levels and ROS generation, augment ER–mitochondria contact, and induce apoptosis in these cells. In vivo injection of palmitate induced hyperglycemia and hypertriglyceridemia, as well as impaired glucose and insulin tolerance in mice. These animals also exhibited elevated GRP75 levels accompanied by enhanced apoptosis within the pancreatic islets. Our findings suggest that GRP75 is critical in mediating palmitate-induced ER–mitochondrial interaction leading to apoptosis in pancreatic islet cells.     

Mitochondrially-targeted bacterial phosphatidylethanolamine methyltransferase sustained phosphatidylcholine synthesis of a Saccharomyces cerevisiae Δpem1 Δpem2 double mutant without exogenous choline supply

In eukaryotic cells, phospholipids are synthesized exclusively in the defined organelles specific for each phospholipid species. To explain the reason for this compartmental specificity in the case of phosphatidylcholine (PC) synthesis, we constructed and characterized a Saccharomyces cerevisiae strain that lacked endogenous phosphatidylethanolamine (PE) methyltransferases but had a recombinant PE methyltransferase from Acetobacter aceti, which was fused with a mitochondrial targeting signal from yeast Pet100p and a 3 × HA epitope tag. This fusion protein, which we named as mitopmt, was determined to be localized to the mitochondria by fluorescence microscopy and subcellular fractionation. The expression of mitopmt suppressed the choline auxotrophy of a double deletion mutant of PEM1 and PEM2 (pem1Δpem2Δ) and enabled it to synthesize PC in the absence of choline. This growth suppression was observed even if the Kennedy pathway was inactivated by the repression of PCT1 encoding CTP:phosphocholine cytidylyltransferase, suggesting that PC synthesized in the mitochondria is distributed to other organelles without going through the salvage pathway. The pem1Δpem2Δ strain deleted for PSD1 encoding the mitochondrial phosphatidylserine decarboxylase was able to grow because of the expression of mitopmt in the presence of ethanolamine, implying that PE from other organelles, probably from the ER, was converted to PC by mitopmt. These results suggest that PC could move out of the mitochondria, and raise the possibility that its movement is not under strict directional limitations.