STIM1 Is a Novel Component of ER-Chlamydia trachomatis Inclusion Membrane Contact Sites

Productive developmental cycle of the obligate intracellular bacterial pathogen Chlamydia trachomatis depends on the interaction of the replicative vacuole, named the inclusion, with cellular organelles. We have recently reported the formation of ER-Inclusion membrane contact sites (MCSs), where the endoplasmic reticulum (ER) is in apposition to the inclusion membrane. These platforms contain the C. trachomatis inclusion membrane protein IncD, the mammalian ceramide transfer protein CERT and the ER resident proteins VAPA/B and were proposed to play a role in the non-vesicular trafficking of lipids to the inclusion. Here, we identify STIM1 as a novel component of ER-Inclusion MCSs. STIM1, an ER calcium (Ca²⁺) sensor that relocate to ER-Plasma Membrane (PM) MCSs upon Ca²⁺ store depletion, associated with C. trachomatis inclusion. STIM1, but not the general ER markers Rtn3C and Sec61ß, was enriched at the inclusion membrane. Ultra-structural studies demonstrated that STIM1 localized to ER-Inclusion MCSs. Time-course experiments showed that STIM1, CERT and VAPB co-localized throughout the developmental cycle. By contrast, Orai1, the PM Ca²⁺ channel that interacts with STIM1 at ER-PM MCSs, did not associate with C. trachomatis inclusion. Upon ER Ca²⁺ store depletion, a pool of STIM1 relocated to ER-PM MCSs, while the existing ER-Inclusion MCSs remained enriched in STIM1. Finally, we have identified the CAD domain, which mediates STIM1-Orai1 interaction, as the minimal domain required for STIM1 enrichment at ER-Inclusion MCSs. Altogether this study identifies STIM1 as a novel component of ER-C. trachomatis inclusion MCSs. We discuss the potential role(s) of STIM1 during the infection process.     

The yeast VAP homolog Scs2p has a phosphoinositide-binding ability that is correlated with its activity

The yeast VAMP-associated protein (VAP) homolog Scs2p is an endoplasmic reticulum (ER)/nuclear membrane protein that binds to an FFAT (diphenylalanine in an acidic tract) motif found in various lipid-metabolic proteins, including Opi1p, a negative regulator of phospholipid biosynthesis. Here, we show that Scs2p is a novel phosphoinositide-binding protein that can bind to phosphatidylinositol monophosphates and bisphosphates in vitro. The phosphoinositide-binding domain was assigned to the N-terminal major sperm protein (MSP) domain which also contains the FFAT-binding domain. When several lysine residues in the MSP domain were substituted for alanine, the resulting mutant Scs2 proteins lost the phosphoinositide-binding ability and failed to complement the inositol auxotrophy of an scs2 deletion strain. However, the mutant proteins still localized in the ER/nuclear membrane, in a similar manner to wild-type Scs2p. These results suggest the possibility that Scs2p activity is regulated by phosphoinositides to coordinate phospholipid biosynthesis in response to changes in phospholipid composition.     

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.     

Co-opted Oxysterol-Binding ORP and VAP Proteins Channel Sterols to RNA Virus Replication Sites via Membrane Contact Sites

Viruses recruit cellular membranes and subvert cellular proteins involved in lipid biosynthesis to build viral replicase complexes and replication organelles. Among the lipids, sterols are important components of membranes, affecting the shape and curvature of membranes. In this paper, the tombusvirus replication protein is shown to co-opt cellular Oxysterol-binding protein related proteins (ORPs), whose deletion in yeast model host leads to decreased tombusvirus replication. In addition, tombusviruses also subvert Scs2p VAP protein to facilitate the formation of membrane contact sites (MCSs), where membranes are juxtaposed, likely channeling lipids to the replication sites. In all, these events result in redistribution and enrichment of sterols at the sites of viral replication in yeast and plant cells. Using in vitro viral replication assay with artificial vesicles, we show stimulation of tombusvirus replication by sterols. Thus, co-opting cellular ORP and VAP proteins to form MCSs serves the virus need to generate abundant sterol-rich membrane surfaces for tombusvirus replication.

Authors Summary

Cellular proteins and cellular membranes are usurped by positive-stranded RNA viruses to assemble viral replicase complexes required for their replication. Tombusviruses, which are small RNA viruses of plants, depend on sterol-rich membranes for replication. The authors show that the tombusviral replication protein binds to cellular oxysterol-binding ORP proteins. Moreover, the endoplasmic reticulum resident cellular VAP proteins also co-localize with viral replication proteins. These protein interactions likely facilitate the formation of membrane contact sites that are visible in cells replicating tombusvirus RNA. The authors also show that sterols are recruited and enriched to the sites of viral replication. In vitro replication assay was used to show that sterols indeed stimulate tombusvirus replication. In summary, tombusviruses use subverted cellular proteins to build sterol-rich membrane microdomain to promote the assembly of the viral replicase complex. The paper connects efficient virus replication with cellular lipid transport and membrane structures.     

Bridging the gap: Membrane contact sites in signaling,metabolism, and organelle dynamics

Regions of close apposition between two organelles, often referred to as membrane contact sites (MCSs), mostly form between the endoplasmic reticulum and a second organelle, although contacts between mitochondria and other organelles have also begun to be characterized. Although these contact sites have been noted since cells first began to be visualized with electron microscopy, the functions of most of these domains long remained unclear. The last few years have witnessed a dramatic increase in our understanding of MCSs, revealing the critical roles they play in intracellular signaling, metabolism, the trafficking of metabolites, and organelle inheritance, division, and transport.

Introduction

The compartmentalization of cells allows the segregation and regulation of the myriad reactions that occur within them. The tremendous benefits of intracellular compartmentalization also come at a price; to function optimally, cells must transmit signals and exchange material between compartments. Numerous mechanisms have evolved to facilitate these exchanges. One that has not been well appreciated until the last few years is the transmission of signals and molecules between organelles that occurs at regions where the organelles are closely apposed, often called membrane contact sites (MCSs). These sites were first characterized because of their critical roles in the intracellular exchange of lipids and calcium, which can be directly channeled between organelles via MCSs. More recently, it has also become apparent that MCSs are important sites for intracellular signaling, organelle trafficking, and inheritance, and that MCSs are specialized regions where regulatory complexes are assembled (English and Voeltz, 2013; Helle et al., 2013).A hallmark of MCSs is that membranes from two organelles (or compartments of the same organelle) are tethered to one another, but not all instances in which membranes interact with or are tethered to one another are considered MCSs. True MCSs have four properties: (1) membranes from two intracellular compartments are tethered in close apposition, typically within 30 nm, (2) the membranes do not fuse (though they may transiently hemi-fuse), (3) specific proteins and/or lipids are enriched at the MCS, and (4) MCS formation affects the function or composition of at least one of the two organelles in the MCS.This review will discuss what we know about proteins that tether organelles, the exchange of small molecules at MCSs, and other emerging functions of MCSs.

MCS tethers

An MCS tether is a protein or complex of proteins (Fig. 1) that simultaneously binds the two apposing membranes at an organelle contact site and plays a role in maintaining the site (English and Voeltz, 2013; Helle et al., 2013). In many cases it is not yet clear if these proteins and complexes are genuine tethers, which are necessary to maintain MCSs, or function at MCSs but are not necessary to sustain contacts. Distinguishing between these possibilities is an important challenge for the field, especially when more than one protein or complex of proteins independently hold together the membranes at an MCS.Open in a separate windowFigure 1.Proteins proposed to mediate tethering at MCSs. Mammalian proteins are shown on a yellow background, yeast proteins on a blue background, and proteins found in both mammals and yeast are on a green background. Tethering complexes not described in the text are indicated with red numbers: (1) StARD3-VAPs (Alpy et al., 2013), (2) NPC1-ORP5 (Du et al., 2011), (3) Psd2-Pdr17 (Riekhof et al., 2014), (4) Vac8-Nvj1 (Pan et al., 2000), (5) Nvj2 (Toulmay and Prinz, 2012), (6) PTPIP51-VAPs (De Vos et al., 2012), (7) Orai1-STIM1 (Nunes et al., 2012), (8) DGAT2-FATP1 (Xu et al., 2012), and (9) IncD-CERT-VAPs (Derré et al., 2011; Elwell et al., 2011).As a growing number of potential tethers are identified, three trends are emerging. First, most MCSs are maintained by several tethers. One of the best-characterized examples of this is the junction of the ER and plasma membrane (PM) in Saccharomyces cerevisiae. Recent work showed that it was necessary to eliminate six ER resident proteins to dramatically reduce the normally extensive interactions between the ER and PM (Manford et al., 2012; Stefan et al., 2013). This suggests that these six proteins mediate tethering independently of each other. Four of the six proteins (three calcium and lipid-binding domain proteins 1–3, also called Tcb1–3, and Ist2) are integral ER membrane proteins that have cytosolic domains that bind the plasma membranes (Fischer et al., 2009; Toulmay and Prinz, 2012). The other two proteins, Scs2 and Scs22 (Scs, suppressor of Ca²⁺ sensitivity), are homologues of mammalian VAPs (vesicle-associated membrane protein–associated proteins). VAPs are integral membrane tail-anchored proteins in the ER that bind proteins containing FFAT (phenylalanines in an acid tract) motifs (Loewen et al., 2003). A number of proteins that contain these motifs also have domains that bind lipids and proteins in the PM, allowing them to simultaneously bind and tether the ER and PM. For example, some oxysterol-binding protein (OSBP)–related proteins (ORPs) have FFAT motifs and pleckstrin homology (PH) domains that bind phosphoinositides (PIPs) in the plasma membrane (Levine and Munro, 1998; Weber-Boyvat et al., 2013). Thus, ORPs and other FFAT motif-containing proteins can mediate ER–PM tethering via VAPs. It should be noted that VAPs and proteins bound by VAPs also mediate tethering between the ER and organelles in addition to the PM. These are shown in Fig. 1.A second emerging trend is that tethering seems to be a dynamic, regulated process, and we are beginning to understand the mechanisms of dynamic apposition of membranes at MCSs by tethers. One example is ER–PM tethering mediated by proteins called extended synaptotagmins (E-Syts), which are homologues of the yeast Tcb tethers. The tethering of the ER and PM by E-Syts is regulated by Ca²⁺ and the PM-enriched lipid PI(4,5)P₂ (Chang et al., 2013; Giordano et al., 2013). Binding of these molecules by E-Syts may control both the extent of ER–PM contact and the distance between these organelles at MCSs. A second example of regulated MCS formation is provided by a recent study on OSBP. This protein and other FFAT motif-containing proteins have been thought to mediate ER–Golgi tethering by simultaneously binding VAPs in the ER and PIPs in the Golgi complex (Kawano et al., 2006; Peretti et al., 2008). In an elegant set of experiments, Mesmin et al. (2013) showed that OSBP regulates its own ability to mediate ER–Golgi tethering by modulating PI4P levels in the Golgi complex. When PI4P levels in the Golgi complex are high, OSBP tethers the ER and Golgi complex and also transports PI4P from the Golgi to the ER. When the PI4P reaches the ER, it is hydrolyzed by the phosphatase Sac1, preventing it from being transferred back to the Golgi. The reduction in Golgi complex PI4P levels by OSBP causes OSBP to dissociate from the Golgi, decreasing ER–Golgi tethering. Thus, OSBP negatively regulates its own tethering of the ER and Golgi membranes. Lipid transport by OSBP and similar proteins will be discussed in more detail in the section on lipid transport at MCSs.The third important feature of many MCS tethering complexes is that most have functions in addition to tethering. This is well illustrated by complexes proposed to mediate ER–mitochondria tethering in mammalian cells, where four such complexes have been described (Fig. 1). For example, Mfn2 (mitofusin-2) acts as a tether (de Brito and Scorrano, 2008), but the primary function of this dynamin-like protein is to mediate mitochondrial fusion. Although Mfn2 is largely in the outer mitochondrial membrane (OMM), a small fraction also resides the ER, and it has been proposed that the interaction of Mfn2 in the ER with Mfn2 in the OMM tethers the ER and mitochondria (de Brito and Scorrano, 2008). The other ER–mitochondria tethering complexes proposed in mammals (Fig. 1) also have additional functions—either Ca²⁺ signaling or apoptotic signaling between these organelles.

Tethers within organelles

MCSs may form not only between organelles but also between compartments of the same organelle. In two cases, proteins necessary for these intra-organelle contacts are known. The Golgi complex is divided into a number of cisternae that remain closely apposed in some cell types, forming stacked compartments. Two tethering proteins maintain connections between Golgi cisternae. Golgi reassembly stacking protein 65 (GRASP65) forms contacts between cis- and medial-Golgi cisternae and GRASP55 mediates medial- to trans-cisternal interactions (Fig. 1; Barr et al., 1997; Shorter et al., 1999). The Golgi stack disassembles when both GRASPs are depleted, indicating that they are the primary or sole tethers (Xiang and Wang, 2010). Tethering by these proteins is regulated by kinases to allow Golgi cisternal disassembly during the cell cycle. Whether the inter-Golgi contacts formed by GRASPs mediate signaling or lipid exchange between cisternae is not yet known (Tang and Wang, 2013).MCSs also form inside organelles with internal membranes: mitochondria, chloroplasts, and multivesicular bodies. These MCSs may form between membranes within these organelles or between internal membranes and the outer membrane of the organelle. Recently, three groups discovered a tethering complex involved in forming contacts between mitochondrial cisternae and between cisternae and the mitochondrial outer membrane (Harner et al., 2011; Hoppins et al., 2011; von der Malsburg et al., 2011). This complex, called the mitochondrial contact site and cristae organizing system (MICOS), is conserved from yeast to humans and contains at least six proteins (Fig. 1). It is necessary to maintain inner membrane organization and also interacts with protein complexes in the outer membrane, including the translocase of the outer membrane (TOM) complex and the sorting and assembly machinery (SAM) complex (van der Laan et al., 2012; Zerbes et al., 2012).

Lipid exchange at MCSs

Lipid exchange between organelles at MCSs may serve a number of important functions. One is that it allows cells to rapidly modulate the lipid composition of an organelle independently of vesicular trafficking. In addition, some organelles, such as mitochondria and chloroplasts, must obtain most of the lipids they require for membrane biogenesis by nonvesicular lipid trafficking that almost certainly occurs at MCSs (Osman et al., 2011; Wang and Benning, 2012; Horvath and Daum, 2013). Finally, and perhaps most importantly, lipid transfer at MCSs may play an important role in lipid metabolism by channeling lipids to or away from enzymes in different compartments.Some lipid exchange at MCSs is facilitated by soluble lipid transport proteins (LTPs), which can shuttle lipid monomers between membranes (Fig. 2 A). In other cases, known LTPs do not seem to be required and lipids may be exchanged at MCSs by other mechanisms (Fig. 2, B and C), which will be discussed next.Open in a separate windowFigure 2.Possible mechanisms of lipid exchange at MCSs. (A) Transfer by LTPs using CERT as an example. The targeting PH domain (pink) and FFAT motif (blue) are shown. CERT could shuttle between membranes (left) or transfer while binding both membranes (right). (B) Some transfer could occur through hydrophobic channels or tunnels (in green) bridging the two membranes at a MCS. (C) Lipid exchange between hemifused membranes. Hemifusion could be promoted and regulated by proteins (red).Most LTPs fall into at least five superfamilies that differ structurally but that all have a hydrophobic pocket or groove that can bind a lipid monomer, and often have a lid domain that shields the bound lipid from the aqueous phase (D’Angelo et al., 2008; Lev, 2010). This allows LTPs to shuttle lipid monomers between membranes. LTPs probably transfer lipids between organelles in cells most efficiently at MCSs, where they have only a short distance to diffuse between membranes. LTPs that may transfer lipids at contact sites are: OSBP, ceramide transport protein (CERT), the yeast OSBP homologues Osh6 and Osh7, protein tyrosine kinase 2 N-terminal domain–interacting receptor 2 (Nir2), and Ups1 (Hanada, 2010; Connerth et al., 2012; Chang et al., 2013; Maeda et al., 2013; Mesmin et al., 2013).LTPs could function by shuttling between membranes at MCSs or while simultaneously bound to both membranes (Fig. 2 A). Many LTPs have domains that target them to the two membranes at an MCS. For example, OSBP and CERT have FFAT motifs, which bind ER resident VAPs, and PH domains that bind PIPs in the Golgi complex or PM.Another important emerging aspect of lipid exchange by some LTPs is that it may be driven by their ability to exchange one lipid for another. For example, OSBP can transfer both cholesterol and PI4P. At ER–Golgi MCSs, OSBP may facilitate the net movement of cholesterol from the ER to the Golgi and PI4P in the opposite direction (Mesmin et al., 2013). The difference in the PI4P concentrations in the ER and Golgi (lower in the ER than in the Golgi) may drive the net transfer of cholesterol to the Golgi. The ability to exchange one lipid for another has been found for other LTPs (Schaaf et al., 2008; de Saint-Jean et al., 2011; Kono et al., 2013) and may be critical for driving directional lipid exchange at MCSs.Some lipid exchange at MCSs does not seem to be facilitated by LTPs. The best evidence for this comes from studies on lipid transfer between the ER and mitochondria. It has long been known that lipids are exchanged between these two organelles; mitochondria must acquire most of the lipid it requires for membrane biogenesis from the rest of the cell. Lipid exchange at ER–mitochondria MCSs occurs by a mechanism that does not require energy, at least in vitro, and does not require any cytosolic factors (Osman et al., 2011; Vance, 2014).How this lipid transfer occurs is not known, and two possible types of mechanism are shown in Fig. 2, B and C. One is that some MCS proteins form a hydrophobic channel that allows lipids to move between membranes. Such a channel would be similar to an LTP, but whereas lipids enter and exit LTPs by the same opening, they enter and exit channels by different openings. This difference could allow lipid exchange by a channel to be regulated and, if the channel could bind two different lipids simultaneously, it might couple the transfer of the lipids. A domain that may form channels at MCSs has been identified. Called the synaptotagmin-like mitochondrial lipid-binding protein (SMP) domain, it has been predicted to be part of a superfamily of proteins that includes cholesterol ester transfer protein (CETP; Kopec et al., 2010). CETP has a tubular lipid-binding domain that transfers lipids between high-density and low-density lipoproteins, probably while simultaneously bound to both (Qiu et al., 2007; Zhang et al., 2012). SMP domains could transfer lipids between membranes by a similar mechanism. Consistent with this possibility, all SMP-containing proteins in budding yeast localize to MCSs and many mammalian SMP-containing proteins do as well (Toulmay and Prinz, 2012). Interestingly, SMP domains are present in three of the five proteins in a yeast ER–mitochondria tethering complex called ERMES (Kornmann et al., 2009). Whether ERMES facilitates lipid exchange between the ER and mitochondria is not yet clear. Mitochondria derived from cells missing ERMES have altered lipid composition (Osman et al., 2009; Tamura et al., 2012; Tan et al., 2013), indicating that lipid exchange between the ER and mitochondria could be altered in these strains. On the other hand, little or no defect in the rates of phospholipid exchange between ER and mitochondria were found in ERMES mutants (Kornmann et al., 2009; Nguyen et al., 2012; Voss et al., 2012). Thus, whether proteins that contain SMP domains actually facilitate lipid exchange remains to be determined.As second possible mechanism of lipid transfer at MCSs that does not require LTPs is membrane hemifusion (Fig. 2 C), which could allow rapid exchange of large amounts of lipids between compartments. Recent indirect evidence suggests that hemifusion may occur between the ER and chloroplasts (Mehrshahi et al., 2013). This is consistent with an earlier study using optical tweezers that found the ER and chloroplasts remained attached to one another even when a stretching force of 400 pN was applied (Andersson et al., 2007). Whether hemifusion occurs at MCSs in animal cells remains to be determined.

Calcium signaling at MCSs

MCSs between the ER and PM and the ER and mitochondria play central roles in intracellular Ca²⁺ storage, homeostasis, and signaling in mammalian cells. MCSs between the ER and lysosomes may also be important, though they are less well understood (Helle et al., 2013; Lam and Galione, 2013).One of the best-characterized MCSs is the one formed between the PM and ER in muscle cells. In both cardiac and skeletal muscle cells, deep invaginations of the PM, called T (transverse)-tubules, allow it to form extensive contacts with the ER, called the sarcoplasmic reticulum (SR) in muscle cells. These contacts are essential for coupling excitation and contraction. Before excitation, Ca²⁺ levels in the cytoplasm of muscle cells are low, whereas the Ca²⁺ concentrations in the SR and outside muscle cells are high. During muscle excitation, Ca²⁺ rapidly flows into the cytosol through channels in the PM and the SR (Fig. 3 A). The channels in the PM, called dihydropyridine receptors (DHPRs), and those in the SR, known ryanodine receptors RyRs, directly interact with each other where the SR and PM are closely apposed, allowing the opening of both types of channels to be coordinated (Fabiato, 1983; Bannister, 2007; Beam and Bannister, 2010; Rebbeck et al., 2011).Open in a separate windowFigure 3.Ca²⁺ trafficking at ER–PM MCSs. (A) In muscle cells, the interaction of the RyR in the SR and with DHPR in the PM allows the coordinated release of Ca²⁺ during muscle excitation and contraction. See text for details. (B) When STIM1 senses low Ca²⁺ concentration in the ER, it undergoes a conformational change that allows it to oligomerize and bind to the PM, to the protein Orai1, and to accumulate at ER–PM MCSs. Ca²⁺ influx at these sites facilitates Ca²⁺ import into the ER by sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA). (C) Calcium channeling from the ER lumen to the mitochondrial matrix. Calcium exits the ER through the inositol trisphosphate receptor (IP3R) channel, enters mitochondria via VDAC, and then uses the mitochondrial Ca²⁺ uniporter (MCU) to move into the mitochondrial matrix.The extensive contacts between the SR and PM in muscle cells are largely maintained by tethering proteins called junctophilins, which have a single transmembrane domain in the SR and a large cytosolic domain that interacts with the PM. Expression of junctophilins in cells lacking them induces ER–PM contacts (Takeshima et al., 2000) and cells lacking junctophilins have abnormal SR–PM MCSs and defects in Ca²⁺ signaling (Ito et al., 2001; Komazaki et al., 2002; Hirata et al., 2006). Thus, junctophilins are both necessary and sufficient for generating functional SR–PM contacts. However, cells lacking junctophilins still maintain some SR–PM contacts, indicating that other proteins also tether the SR and the PM. Some of this residual tethering probably comes from the interaction of DHPRs and RyRs.ER–PM contacts also play a role in regulating intracellular Ca²⁺ levels in non-excitable cells. When the Ca²⁺ concentration in the ER lumen is low it triggers Ca²⁺ entry into the cytosol and ER from outside cells (Fig. 3 B), a process known as store-operated Ca²⁺ entry (SOCE). The PM channel responsible for Ca²⁺ entry is Orai1, and the sensor of Ca²⁺ concentration in the ER lumen is the integral membrane protein stromal interaction molecule-1 (STIM1). When STIM1 senses that the Ca²⁺ concentration in the ER is low, it oligomerizes and undergoes a conformational change that exposes a basic cluster of amino acids in its C terminus that binds PIPs in the PM (Stathopulos et al., 2006, 2008; Liou et al., 2007; Muik et al., 2011). STIM1 also binds to Orai1 in the PM and activates it (Kawasaki et al., 2009; Muik et al., 2009; Park et al., 2009; Wang et al., 2009). Activation of STIM1 causes it to shift from being relatively evenly distributed on the ER to forming a number of puncta, which are regions were the ER and PM are closely apposed. It seems likely that STIM1 accumulates at and expands preexisting ER–PM MCSs and may also drive the formation of new MCSs (Wu et al., 2006; Lur et al., 2009; Orci et al., 2009).The interaction of STIM1 and Orai1 at ER–PM contacts during SOCE is an elegant mechanism for channeling both signals and small molecules at an MCS. The signal that ER luminal Ca²⁺ concentration is low is transmitted directly from STIM1 in the ER to Orai1 in the PM. The close contact of PM and ER also allows Ca²⁺ to move from outside the cell into the lumen of the ER without significantly increasing cytosolic Ca²⁺ levels (Jousset et al., 2007). During SOCE, ER Ca²⁺ levels are restored by the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) pump (Sampieri et al., 2009; Manjarrés et al., 2011). This pump is enriched in ER–PM contacts with STIM1 and may interact directly with it, suggesting how Ca²⁺ can be effectively channeled from outside cells directly into the ER lumen at ER–PM MCSs (Fig. 3 B).Interestingly, it has become clear that proteins that are not part of the SOCE pathway also facilitate ER–PM connections during Ca²⁺ signaling. The E-Syts have multiple domains that probably bind Ca²⁺. They have been shown to regulate both the number of the ER–PM contacts and the distance between the ER and PM at MCSs during Ca²⁺ signaling (Chang et al., 2013; Giordano et al., 2013).MCSs between the ER and mitochondria similarly facilitate Ca²⁺ movement from the ER lumen to mitochondria (Rizzuto et al., 1998; Csordás et al., 2006). Ca²⁺ channels in the ER and OMM interact with each other at MCSs (Fig. 3 C). The channel in the ER is called the inositol trisphosphate receptor (IP3R), while the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane is a nonspecific pore that allows Ca²⁺ entry into mitochondria. These proteins, together with the cytosolic chaperone Grp75, form a complex that links the ER and mitochondria and facilitates Ca²⁺ exchange (Szabadkai et al., 2006).More evidence that Ca²⁺ transfer from the ER to mitochondria occurs at MCSs came from studies on the channel that allows Ca²⁺ to move across the inner mitochondrial membrane, called the mitochondrial Ca²⁺ uniporter (MCU). Surprisingly, this channel has an affinity for Ca²⁺ that is lower than the typical Ca²⁺ concentration in the cytosol (Kirichok et al., 2004). However, Ca²⁺ release by the ER at ER–mitochondrial MCSs suggests a solution to this puzzle; the local Ca²⁺ concentration at these MCSs is probably high enough for MCU to function (Csordás et al., 2010). Close contacts between the ER and mitochondria are therefore essential for channeling Ca²⁺ from the ER lumen to the mitochondrial matrix.It is thought that MCSs between the ER (or SR) and lysosomes regulate Ca²⁺ release by lysosomes, but the mechanism is not yet understood (Kinnear et al., 2004, 2008; Galione et al., 2011; Morgan et al., 2011).

Enzymes working in trans and signaling at MCSs

MCSs allow rapid and efficient signaling between intracellular compartments. We are still just beginning to understand the mechanisms and functions of this signaling. One way that signals are transmitted between the two compartments at an MCS is for an enzyme in one compartment to modify substrates in the second; that is, for the enzyme to work in trans. Although there are currently only a few examples of this, which are discussed here, it seems likely that many more will be uncovered.The protein tyrosine phosphatase PTP1B regulates a number of receptor tyrosine kinases. PTP1B resides on the surface of the ER with its active site in the cytosol, and yet the receptor tyrosine kinases it modifies are in the PM. Although this was initially puzzling, it was found that PTP1B probably encounters its substrates at MCSs, either at ER–PM junctions or at contacts between the ER and endocytic recycling compartments (Haj et al., 2002; Boute et al., 2003; Anderie et al., 2007; Eden et al., 2010; Nievergall et al., 2010). Interestingly, in some cases the interaction of PTP1B with substrates in the PM occurs on portions of the PM that are part of cell–cell contacts (Haj et al., 2012), suggesting that ER–PM contacts could play a role in signaling, not only between the ER and PM but between cells as well. Dephosphorylation of receptor tyrosine kinases by PTP1B at contact sites probably allows their kinase activity to be regulated in response to changes in the ER or changes in cellular architecture that alter MCSs. For example, the dephosphorylation of epidermal growth factor receptor (EGFR) by PTP1B occurs at regions of close contact between the ER and multivesicular bodies, causing EGFR to become sequestered with multivesicular bodies (Eden et al., 2010). This may provide a mechanism for cells to regulate EGFR levels on the PM in response to signals in the ER.Lipid metabolism enzymes can also work in trans at MCSs. In two cases, both in yeast, enzymes that reside in the ER have been found to modify lipids in the PM at MCSs. In one instance, the phosphatase Sac1, which is on the surface of the ER, can dephosphorylate PIPs in the PM (Stefan et al., 2011). In the second, the ER enzyme Opi3 methylates phosphatidylethanolamine in the PM, a reaction that is required for the conversion of phosphatidylethanolamine to phosphatidylcholine (Tavassoli et al., 2013). Remarkably, the PIP-binding protein Osh3 (Tong et al., 2013) regulates both reactions, suggesting that lipid metabolism at ER–PM junctions is regulated by PIPs. It seems likely that ER–PM junctions play important roles in integrating lipid metabolism in both organelles.

MCSs and organelle trafficking and inheritance

In addition to being sites at which signals and small molecules are exchanged between cellular compartments, there is growing evidence that MCS formation also regulates organelle trafficking and inheritance.In budding yeast, organelle transport is polarized from the mother cell to the growing bud and is required for proper organelle inheritance. The transport of peroxisomes and mitochondria to the bud is regulated by their association with the ER or PM.Knoblach et al. (2013) found that tethering of the ER to peroxisomes requires Pex3, an integral membrane protein that resides in both compartments, and Inp1, a cytosolic protein that binds to Pex3. This tether keeps peroxisomes in mother cells. When peroxisomes divide they are transferred to the bud by the myosin V motor Myo2 and become attached to the ER in the bud. In cells lacking the ER–peroxisome tether, peroxisomes accumulate in daughter cells. Thus, tethering plays a critical role in ensuring that some peroxisomes are retained in mother cells and that both cells inherit peroxisomes.Mitochondrial inheritance in yeast is regulated by close contacts with both the ER and PM. Mitochondria–PM contacts mediated by a complex containing Num1 and Mdm36 ensure that mitochondria are properly distributed between mother and daughter cells and seem to be particularly important for retaining mitochondria in the mother cells (Klecker et al., 2013; Lackner et al., 2013). Interestingly, Num1–Mdm36-mediated contacts also associate with the ER (Lackner et al., 2013), suggesting that three membranes may somehow associate at these MCSs. An ER–mitochondria tether containing the protein Mmr1, which anchors mitochondria to bud tips, also plays a role in mitochondrial inheritance (Swayne et al., 2011). Thus, the Num1-tethering complex and Mmr1-tethering complex seem to play antagonistic roles in mitochondrial distribution; the Num1 complex promotes mitochondrial retention in the mother, whereas the Mmr1 complex favors retention in the bud.MCSs also play a role in endosomal trafficking in mammalian cells. One of the complexes that tethers the ER to endosomes contains VAPs and ORP1L, which is an OSBP homologue that can bind cholesterol (Fig. 1). ORPlL can also binds the p150^Glued subunit of the dynein–dynactin motor that participates in endosome transport along microtubules (Johansson et al., 2007). When cellular cholesterol levels are high, ORP1L associates with p150^Glued but not VAPs and endosomes are transported on microtubules. However, when cholesterol levels decrease, ORP1L undergoes a conformation change that dissociates it from p150^Glued and allows it to bind to VAPs on the surface of the ER, thus forming a tether between endosomes and the ER (Rocha et al., 2009). Under these conditions, endosome transport on microtubules is blocked. ORPlL is therefore a cholesterol sensor that regulates a switch between the association of endosomes with either motors or the ER.

MCSs and organelle division

A groundbreaking study revealed a new and unexpected role for MCSs between the ER and mitochondria: the ER regulates mitochondrial fission (Friedman et al., 2011). Although a mechanistic understanding of how ER participates in mitochondrial fission is not yet available, the sequence of events is beginning to come into focus (Fig. 4). The ER encircles mitochondria at sites where scission will occur. The ERMES complex is present at these sites (Murley et al., 2013). Because mammalian cells lack ERMES, another tethering complex must perform the same function in higher eukaryotes. Mitochondrial division requires membrane scission by the dynamin-like protein Dnm1/Drp1, which multimerizes on the outer mitochondria membrane. Close contacts between the ER and mitochondria occur before Dnm1/Drp1 assembly, suggesting that these contacts promote or regulate the association of Dnm1/Drp1 with mitochondria and hence mitochondrial division. It is possible that when the ER encircles mitochondria it causes mitochondria to constrict to a diameter that allows Dnm1/Drp1 to assemble. The force necessary to drive constriction may come from actin polymerization. A recent study found that the ER protein, inverted formin-2, probably drives actin polymerization at these sites and is necessary for mitochondria fusion (Korobova et al., 2013).Open in a separate windowFigure 4.Model of ER-mediated regulation of mitochondrial fission at sites of contact. (A) The ER and mitochondria are tethered by ERMES in yeast (other tethers are used in higher eukaryotes). (B) The ER encircles mitochondria at sites where division will occur. (C) Actin polymerization facilitated by formin 2 may cause mitochondrial constriction. (D) The dynamin-like protein Drp1 is recruited to the mitochondrial surface, where it multimerizes and causes mitochondrial scission. (E) After fission, the ER remains associated with the mitochondrion that retains the ERMES complex.Understanding the assembly and regulation of the mitochondrial division machinery at ER–mitochondria MCSs and how this is linked to mitochondrial and perhaps ER function remain fascinating questions for the future. Another interesting question is whether other MCSs play roles in the fission of other organelles.

Proposed functions of ER–mitochondrial MCSs

A growing number of studies have suggested that ER–mitochondria MCSs play critical roles in autophagy, apoptosis, inflammation, reactive oxygen species signaling, and metabolic signaling. ER–mitochondria MCSs have also been implicated in Alzheimer’s disease, Parkinson’s disease, and some viral infections. These topics have been recently reviewed (Eisner et al., 2013; Raturi and Simmen, 2013; Marchi et al., 2014; Vance, 2014) and will not be discussed in detail here.One issue with most of the studies on the functions of ER–mitochondria junctions is that they rely, at least in part, on density gradient purification of the ER that associates with mitochondria. These operationally defined membranes, often called mitochondrial-associated membranes (MAMs), remain poorly defined. In fact, a significant number of proteins that are enriched in MAMs do not seem to be enriched at ER–mitochondria junctions when their localization is determined by other methods (Helle et al., 2013; Vance, 2014). Therefore, it remains unclear why some proteins and lipids are enriched in MAMs.Here, two interesting findings will be discussed that suggest the importance of ER–mitochondrial junctions in signaling in addition to their well-known role in Ca²⁺ signalling.The induction of apoptosis requires signal transmission between the ER and mitochondria. Part of this signaling process occurs through an interaction between the ER protein Bap31 and the mitochondrial fission protein Fission 1 homologue (Fis1; Iwasawa et al., 2011). This interaction occurs at ER–mitochondria MCSs and results in the cleavage of Bap31 by caspase-8 to form p20Bap31, which is pro-apoptotic. Both Bap31 and Fis1 are parts of larger complexes that are still being characterized. Interestingly, it has recently been found that a protein called cell death–involved p53 target-1 (CDIP1) binds to Bap31 during ER stress and promotes apoptotic signaling from the ER to mitochondria (Namba et al., 2013), suggesting how ER stress signals are transmitted from the ER to mitochondria through MCSs.Another important connection between ER–mitochondrial MCSs and signaling has to do with the target of rapamycin (TOR) kinase complexes, which are critical regulators of growth and metabolism. The mammalian TOR complex 2 (mTORC2) was found to interact with the IP3R–Grp75–VDAC complex that tethers the ER and mitochondria (Betz et al., 2013). Remarkably, this study presents evidence that mTORC regulates both the formation of ER–mitochondrial MCSs and mitochondrial function, suggesting an interesting new mechanism for how metabolic signaling can impact mitochondrial function via MCSs.

Conclusions and perspectives

The potential of MCSs to facilitate Ca²⁺ signaling and channel lipids between organelles was recognized some time ago (Levine and Loewen, 2006), but it has only been in the last few years that we have finally begun to have some mechanistic insight into how these processes occur and how MCSs are formed. Many fundamental questions remain to be addressed. How lipid exchange at MCSs that does not require soluble LTPs occurs or whether transient hemifusion of membranes at MCS ever occurs remain open questions. Another is the mechanisms by which Ca²⁺ regulates MCS formation between the ER and other organelles. One major challenge for the field will be devising better methods to visualize MCSs and identify proteins and lipids enriched at these sites. It is particularly important to better understand what the MAM fraction is and what it means for proteins and lipids to be enriched in this fraction.One of the most exciting developments in the study of MCSs in the last few years has been the discovery of the role of MCSs in organelle trafficking, inheritance, and dynamics. These studies have revealed that MCSs not only play critical roles in signaling and metabolism, but also modulate the intracellular distribution of organelles and organelle architecture. Understanding how MCSs perform these functions will probably shed light on the connection between the still murky relationship between organelle structure and function as well as the role of the ER as a regulator of other organelles. Given the current pace of discovery, it seems likely that in the next few years our knowledge of the functions of MCSs will grow dramatically.     

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.     

Osh proteins regulate phosphoinositide metabolism at ER-plasma membrane contact sites

Sac1 phosphoinositide (PI) phosphatases are essential regulators of PI-signaling networks. Yeast Sac1, an integral endoplasmic reticulum (ER) membrane protein, controls PI4P levels at the ER, Golgi, and plasma membrane (PM). Whether Sac1 can act in trans and turn over PI4P at the Golgi and PM from the ER remains a paradox. We find that Sac1-mediated PI4P metabolism requires the oxysterol-binding homology (Osh) proteins. The PH domain-containing family member, Osh3, localizes to PM/ER membrane contact sites dependent upon PM PI4P levels. We reconstitute Osh protein-stimulated Sac1 PI phosphatase activity in vitro. We also show that the ER membrane VAP proteins, Scs2/Scs22, control PM PI4P levels and Sac1 activity in vitro. We propose that Osh3 functions at ER/PM contact sites as both a sensor of PM PI4P and an activator of the ER Sac1 phosphatase. Our findings further suggest that the conserved Osh proteins control PI metabolism at additional membrane contact sites.     

Chlamydiae interaction with the endoplasmic reticulum: contact,function and consequences

Chlamydiae and chlamydiae‐related organisms are obligate intracellular bacterial pathogens. They reside in a membrane‐bound compartment termed the inclusion and have evolved sophisticated mechanisms to interact with cellular organelles. This review focuses on the nature, the function(s) and the consequences of chlamydiae–inclusion interaction with the endoplasmic reticulum (ER). The inclusion membrane establishes very close contact with the ER at specific sites termed ER–inclusion membrane contact sites (MCSs). These MCSs are constituted of a specific set of factors, including the C. trachomatis effector protein IncD and the host cell proteins CERT and VAPA/B. Because CERT and VAPA/B have a demonstrated role in the non‐vesicular trafficking of lipids between the ER and the Golgi, it was proposed that Chlamydia establish MCSs with the ER to acquire host lipids. However, the recruitment of additional factors to ER–inclusion MCSs, such as the ER calcium sensor STIM1, may suggest additional functions unrelated to lipid acquisition. Finally, chlamydiae interaction with the ER appears to induce the ER stress response, but this response is quickly dampened by chlamydiae to promote host cell survival.     

VPS13D interacts with VCP/p97 and negatively regulates endoplasmic reticulum–mitochondria interactions

Membrane contact sites (MCSs) between the endoplasmic reticulum (ER) and mitochondria are emerging as critical hubs for diverse cellular events, and alterations in the extent of these contacts are linked to neurodegenerative diseases. However, the mechanisms that control ER–mitochondria interactions are so far elusive. Here, we demonstrate a key role of vacuolar protein sorting–associated protein 13D (VPS13D) in the negative regulation of ER–mitochondria MCSs. VPS13D suppression results in extensive ER–mitochondria tethering, a phenotype that can be substantially rescued by suppression of the tethering proteins VAPB and PTPIP51. VPS13D interacts with valosin-containing protein (VCP/p97) to control the level of ER-resident VAPB at contacts. VPS13D is required for the stability of p97. Functionally, VPS13D suppression leads to severe defects in mitochondrial morphology, mitochondrial cellular distribution, and mitochondrial DNA synthesis. Together, our results suggest that VPS13D negatively regulates the ER–mitochondria MCSs, partially through its interactions with VCP/p97.     

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.Open in a separate windowClick here to view.^{(28M, flv)}     

PI4P/PS countertransport by ORP10 at ER–endosome membrane contact sites regulates endosome fission

Membrane contact sites (MCSs) serve as a zone for nonvesicular lipid transport by oxysterol-binding protein (OSBP)-related proteins (ORPs). ORPs mediate lipid countertransport, in which two distinct lipids are transported counterdirectionally. How such lipid countertransport controls specific biological functions, however, remains elusive. We report that lipid countertransport by ORP10 at ER–endosome MCSs regulates retrograde membrane trafficking. ORP10, together with ORP9 and VAP, formed ER–endosome MCSs in a phosphatidylinositol 4-phosphate (PI4P)-dependent manner. ORP10 exhibited a lipid exchange activity toward its ligands, PI4P and phosphatidylserine (PS), between liposomes in vitro, and between the ER and endosomes in situ. Cell biological analysis demonstrated that ORP10 supplies a pool of PS from the ER, in exchange for PI4P, to endosomes where the PS-binding protein EHD1 is recruited to facilitate endosome fission. Our study highlights a novel lipid exchange at ER–endosome MCSs as a nonenzymatic PI4P-to-PS conversion mechanism that organizes membrane remodeling during retrograde membrane trafficking.     

Thank ORP9 for FFAT: With endosomal ORP10, it’s fission accomplished!

Heterogeneity in endosomal membrane phospholipid content is emerging as a regulator of endocytic trafficking pathways. Kawasaki et al. (2021. J. Cell. Biol. https://doi.org/10.1083/jcb.202103141) demonstrate exchange of endosomal PI4P for PS by ORP10 at ER–endosome contact sites, with the consequent recruitment of endosomal fission factors.

Most cellular lipids are synthesized in the ER, often undergoing rapid redistribution to other cellular membranes, thereby maintaining low concentrations at the ER. Consequently, lipids exiting the ER may need to be transported against their concentration gradient. Lipid flow along a gradient to the ER can drive countertransport of ER-derived lipid to membranes with a higher lipid concentration. This nonvesicular lipid exchange occurs at membrane contact sites (MCS), where different organelles are closely apposed, providing a platform for lipid transport proteins including oxysterol-binding protein (OSBP)-related proteins (ORPs). Lipid specificity, which varies between ORPs, is defined by the OSBP-related domain (ORD). The ORD of ORP10 shares phosphatidylinositol-4-phosphate (PI4P) and phosphatidylserine (PS) binding residues with ORP5/8 and can bind and extract PS from liposomes (1), suggesting a potential role in PI4P-PS counter transport, analogous to that of ORP5/8 at ER–plasma membrane MCS (2). ORPs are targeted to specific organelles by interaction between their PH domain and membrane phospholipids. Most ORPs also possess a FFAT motif (two phenylalanines in an acidic tract), which simultaneously targets the ORP to ER-localized VAMP-associated proteins (VAPs) at MCS between the ER and other organelles. ORP10, however, lacks a FFAT motif, yet was found to stabilize ER–Golgi MCSs (Fig. 1 A) for PI4P transport to the ER (2). Kawasaki et al. have now uncovered a novel function for ORP10 in PI4P–PS lipid exchange at the ER–endosome interface (Fig. 1 B), with downstream effects on endosomal fission and retrograde transport (3).Open in a separate windowFigure 1.Regulation of retrograde and secretory traffic by ORP10-mediated lipid exchange. (A) ORP10 interacts with VAP-bound ORP9 at ER–endosome and ER–Golgi MCSs, with downstream effects on retrograde transport of mannose 6-phosphate receptor (M6PR). Boxed region (detailed in B) depicts ORP10 at the ER–endosome interface. (B) ORP10 functions in lipid exchange between the ER and endosomes, transporting endosomal PI4P to the ER in exchange for ER-derived PS. Production of PI4P in endosomes by PI4KIIα-dependent phosphorylation of phosphatidylinositol (PI), coupled with its consumption in the ER by ER-localized Sac1, generates a PI4P concentration gradient from the endosome to the ER. Low membrane PS concentrations in the ER are maintained by PS inhibition of PS synthesis from phosphatidylcholine (PC) by Pss1 or from PE by Pss2, with PS synthesis at ER–endosome contact sites promoting rapid PS export from the ER in yeast (not yet known if a similar mechanism operates in mammalian cells). ORP10 mediates PI4P transport along its gradient to the ER, driving countertransport of PS by ORP10 against its concentration gradient to the endosome. PS enrichment at the endosome leads to recruitment of the ATPase EHD1 to facilitate endosome fission for retrograde transport. (C) Depletion of ORP10 prevents lipid exchange at ER–endosome contact sites, resulting in a loss of retrograde transport of M6PR. Additionally, ER–Golgi MCSs are diminished, and secretion of ApoB-100 is increased.The PH domain of ORP10 selectively binds PI4P and is required for ORP10 recruitment to the TGN (2) and endosomes (3), both home to PI4KIIα, a PI4P-producing kinase. Rapid PI4P degradation at the ER by the phosphatase Sac1 generates a PI4P gradient at the ER–endosome or TGN interface, with PI4P flow to the ER driving countertransport of PS to the endosome (as also predicted for the Golgi). Activity of endosomal PI4P phosphatase Sac2 (4) may hamper formation of an endosome–ER PI4P gradient, but since ORP10 did not colocalise well with Sac2 (3), they likely function at different endosome populations.PS synthesis at MCSs may also contribute to ORP10-mediated lipid exchange. Targeting PS synthase to ER:mitochondria contacts in yeast was found topromote PS transport out of the ER to mitochondria (5). Similarly, ER to endosome PS transport was increased when PS synthase was targeted to ER:endosome MCS. Localized PS gradients from PS synthesis in the ER at MCSs, coupled with rapid decarboxylation of PS to phosphatidylethanolamine (PE) in mitochondria/endosomes by yeast PS decarboxylases Psd1/Psd2, could contribute to lipid exchange. In mammalian cells, though, since no endosomal decarboxylase has been identified, ORP10-mediated lipid exchange is likely to be primarily driven by the PI4P gradient. Whether this process is facilitated by localized activation of PS synthase at MCS has not yet been demonstrated. Since PS synthase activity is negatively regulated by PS, exit from the ER is a key factor in its biogenesis. Recruitment of specific ORPs to endosomes/TGN by PI4P for ER tethering and consequent lipid exchange provides an elegant regulatory pathway for PI4P–PS homeostasis in cellular membranes.ORP10 shares functional similarities with ORP11: both proteins comprise an N-terminal PH domain and a C-terminal ORD, with a linker region in between harboring a coiled-coiled domain. Unlike other ORPs, ORP10 and ORP11 possess neither a FFAT motif nor a membrane spanning domain to enable ER interaction, but heterotypic interaction with ORP9, which does contain a FFAT motif, has been demonstrated for both proteins. Kawasaki et al. identified an ORP9-ORP10 interaction at ER–endosome MCSs that is dependent on the ORP10 linker region. ORP9 was also implicated in ORP10-mediated lipid exchange at the TGN, where it may play a redundant role with OSBP in maintaining ER contact. Similarly, ORP11 is also recruited to the TGN and, to a lesser extent, the endosome, by ORP9, with the interaction depending on the linker region of both proteins (6).The finely tuned regulation of PI4P/PS is emerging as an important determinant of endocytic traffic. Previous studies have shown that endosomal PI4P accumulation inhibits retrograde transport from endosomes to the TGN (7), while endosomal PS regulates endosome to Golgi retrograde traffic. As depicted in Fig. 1 B, Kawasaki et al. have built on this to show that through interaction with VAP-bound ORP9, ORP10 mediates lipid countertransport at ER–endosome MCSs, removing PI4P from, and supplying PS to, the endosome, with consequent recruitment of the membrane scission protein EHD1 to control endosomal fission and retrograde transport (3). Spatial and temporal regulation of endosome fission by ER–endosome MCSs involves recruitment of the ER membrane protein TMCC1 to the budding endosome by the actin regulator Coronin 1C, stabilizing the MCS (7), but the mechanism by which MCS might effect scission has remained elusive. The findings of Kawaski et al. present an explanation: by providing a platform for lipid exchange, MCS promote the recruitment of EHD1, which belongs to a conserved class of ATPases that can oligomerise in ring-like structures around tubules to mediate fission (8). VAP interaction with OSBP at ER–endosome MCSs is also required for retrograde transport (7), but potential redundancy between ORP9/OSBP in ORP10-mediated lipid exchange, or if ORP10 functions at Coronin 1C/TMCC1-regulated MCS is not yet established.Interestingly, ORP10 function at the TGN has been implicated in regulating ApoB-100 secretion (Fig. 1 C), with hypersecretion reported in ORP10-depleted cells (9). FFAP1, which promotes PI4P consumption by Sac1 at ER:TGN contacts, also negatively regulates ApoB-100 exit from the TGN in a PI4KIIIβ-dependent manner, suggesting direct regulation of ApoB-100 secretion by PI4P at the TGN (9). Could PI4P coordinate nutrient sensing with cargo sorting and secretion at the TGN? PI4P has been described as lipid biosensor of cytosolic pH, with protonation of its head group regulating protein interactions (10). The influence of cytosolic pH on ORP10-PI4P interaction may provide an additional layer of regulation of lipoprotein secretion in response to changes in cellular energy/pH.How ORP10 function is coordinated at Golgi and endosomal membranes and the significance of potential redundancy with ORP11 remains unclear. The regulation of Sac2 activity and how it relates to ER-endosome lipid exchange is also intriguing. While questions still remain, an important role for ORP10 is emerging in maintaining homoeostasis between endosome maturation, retrograde traffic and secretory transport.     

IP-FCM: Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.Download video file.^{(71M, mov)}     

Assembly-dependent trafficking assays in the detection of receptor-receptor interactions

Assembly-dependent trafficking is a property of many multimeric membrane protein complexes; this coupling of assembly and trafficking processes provides an important cellular quality control mechanism, ensuring that only properly folded and assembled complexes are expressed on the cell surface. In all membrane protein complexes whose trafficking is known to be assembly-dependent, at least one of the subunits contains an endoplasmic reticulum (ER) retention/retrieval signal that is shielded on subunit assembly, allowing the assembled protein complex to traffic to the plasma membrane. Under these conditions, presence of the normally retained subunit on the cell surface can be used as an indirect index of protein assembly in the ER. In this article, I describe the design of two complementary approaches (trafficking enhancement and trap assays) that can be used separately or in combination to determine whether two (or more) proteins assemble in the ER, i.e., whether they constitutively oligomerize. Both of the approaches are based on the measurement of plasma membrane-expressed proteins using antibody-mediated detection of extracellularly expressed epitopes and subsequent luminometric quantification. These methods provide a straightforward and relatively inexpensive way to assess protein-protein interactions early in the synthetic pathway.     

Three-dimensional spatio-temporal modelling of store operated Ca2+ entry: Insights into ER refilling and the spatial signature of Ca2+ signals

The spatial organisation of Orai channels and SERCA pumps within ER-PM junctions is important for enhancing the versatility and specificity of sub-cellular Ca²⁺ signals generated during store operated Ca²⁺ entry (SOCE). In this paper, we present a novel three dimensional spatio-temporal model describing Ca²⁺ dynamics in the ER-PM junction and sub-PM ER during SOCE. We investigate the role of Orai channel and SERCA pump location to provide insights into how these components shape the Ca²⁺ signals generated and affect ER refilling. We find that the organisation of Orai channels within the ER-PM junction controls the amplitude and shape of the Ca²⁺ profile but does not enhance ER refilling. The model shows that ER refilling is only weakly affected by the location of SERCA2b pumps within the ER-PM junction and that the placement of SERCA2a pumps within the ER-PM junction has much greater control over ER refilling.