Inter- membrane contact sites: through a glass, darkly Tim Levine1 and Chris Loewen2

Inter-organelle membrane contact sites are zones where However, where studied, membrane contact sites are heterologous membranes, usually the relatively stable [2] and the portion of ER involved is plus a partner organelle, come into close apposition. These biochemically distinct from the bulk of the ER [3,4]. sites are very poorly understood because so few of their Instead of being random, membrane contact sites are components have been identified; however, it is clear that they specialised for communication, in particular the efficient are specialised for traffic of material and information between traffic of small molecules such as Ca2+ ions and , as the two membranes. There have been recent advances in the well as –substrate interactions occurring in trans study of transfer , such as ceramide transfer (Figure 1a). The permanence of membrane contact sites (CERT) and homologues of oxysterol binding protein indicates that they are structured by bridging complexes; (OSBP). Not only can these proteins carry lipids across the however, only a single example demonstrates how these cytoplasm, but they have been found to target both the bridges are constructed (Figure 1b) [5]. endoplasmic reticulum and a partnering organelle, and in some cases have been localised to membrane contact sites. Further Although no new structural components have been dis- work will be needed to test whether these lipid transfer proteins covered recently, an increasing number of proteins are act when anchored at inter-organelle contact sites. now known to function on two contacting . This Addresses review will focus on lipid traffic, since this is often 1 Division of Cell Biology, UCL Institute of Ophthalmology, Bath St, independent of NSF/Sec18-mediated vesicular traffic London EC1V 9EL, UK [6]. The hydrophobicity of lipids and the existence of 2 Department of Cellular and Physiological Science and the Brain lipid transfer proteins (LTPs), which reversibly bind Research Centre, Life Sciences Institute, 2350 Health Sciences Mall, University of British Columbia, Vancouver, BC, Canada, V6T 1Z3 selected lipids with 1:1 stochiometry in hydrophobic pockets, suggest that non-vesicular transport is mediated Corresponding author: Levine, Tim ([email protected]) by LTPs. While it is possible that LTPs act by diffusing to-and-fro across relatively large cytoplasmic gaps, an alternative hypothesis is that LTPs are firmly attached Current Opinion in Cell Biology 2006, 18:371–378 to a membrane and yet efficiently transport lipids [7], This review comes from a themed issue on implying that they function at a . Membranes and organelles Here an LTP might provide an almost static hydrophobic Edited by Pietro de Camilli and Antonella de Matteis crossing point. Data supporting this hypothesis has been

Available online 27th June 2006 accumulating over the past few years (Figure 1c), as will be discussed below. 0955-0674/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. Plasma membrane–ER contacts DOI 10.1016/j.ceb.2006.06.011 One organisational feature of the ER is its relationship in the cell periphery with the plasma membrane (PM). The interaction of the two organelles has been studied bio- chemically in only one instance, which showed the por- Introduction tion of ER adherent to the PM in is enriched in Several organelles make close contacts with each other at synthases for phosphatidylserine (PS) and phosphatidy- zones of apposition called membrane contact sites, which linositol (PI), lipids that are needed in the PM [4]. Several provide an alternative means of communication to mem- complexes bridge between the ER and the PM, although brane-bound carriers. Membrane contact sites are found all are likely to be transient, in other words not structural in all , from simple prokaryotes with two mem- [8–10], implying that the critical bridging components branes (and hence also between the two mitochondrial have yet to be found. Myocytes have enormously membranes) to eukaryotes, where one of the partnering enlarged PM and ER contact sites called triad junctions, organelles is always the endoplasmic reticulum (ER). and a family of ER membrane proteins called junctophi- Since the ER network spreads into all corners of a cell, lins may be structural components [11], but their binding including highly asymmetric cellular projections such as partners have yet to be identified. Another reported PM– neuronal dendrites, axons and synapses [1], it is not ER interaction is between SNAREs that might mediate surprising that strands of the ER should be found near fusion of ER cisternae with large phagocytic cups [12]. other organelles. For this reason, the significance of zones Although the importance of this process has been called of very close apposition (10 nm) is still open to question. into question [13], findings in divergent systems suggest www.sciencedirect.com Current Opinion in Cell Biology 2006, 18:371–378 372 Membranes and organelles

Figure 1

Components of membrane contact sites (MCSs). (a) Generic composition of a membrane contact site. In addition to a relatively stable bridging complex, unstable interactions fall into one of three categories: (1) channels connecting two organelles (for example, TOM and TIM23 for protein import into mitochondria, ANT/mtCK/porin for creatine phosphate production by mitochondria, DHPR and RyR for excitation of striated muscle, or InsP3R and TRP-C for store operated calcium entry) [46,47,64]; (2) lipid transfer proteins binding to both membranes (for example OSBP homologues, CERT and RdgBa)[38,43,49]; (3) enzyme–substrate pairs (for example PTP-1B and either EGF receptor on or Src on the plasma membrane) [45,58]). (b) Components of the nucleus vacuole junction (NVJ) in yeast. The electron micrograph shows the typical apposition of the nucleus and vacuole in S. cerevisiae, with a gap as small as 10 nm. The bridging complex consists of Vac8 bound to the limiting membrane of the vacuole and Nvj1 integral to the outer , the amount of which determines the extent of the NVJ [5]. Other proteins recruited to the NVJ by binding to Nvj1 are Tsc13 (a lipid synthase subunit) and Osh1 (an OSBP homologue that also binds yeast VAP, short for vesicle-associated [VAMP]-associated protein) [9,54,55]. Image courtesy of Anjana Roy (UCL Institute of Ophthalmology). Scale bar = 500 nm. (c) Detailed mechanism by which lipid transfer proteins might act at membrane contact sites. OSBP homologues (left) and CERT (ceramide transfer protein, right) have a similar domain structure, with targeting domains for two membranes: an N-terminal pleckstrin homology (PH) domain typically binds both a PIP lipid and a protein factor (ARF1 for OSBP PH, not yet determined for CERT PH) to specify targeting within the late secretory pathway [34,76]; a central FFAT motif binds VAP on the ER [9]. The C-terminal lipid transfer domain transfers [29,30] or ceramide [49] respectively. Osh4, the OSBP homologue studied in greatest detail, is a short form consisting of the sterol transfer domain alone, which interacts with PIPs, possibly on both donor and acceptor membranes [30]. In addition, functions of the two LTPs are linked, as OSBP recruited to the TGN acts (either as a sterol sensor [28], or as a sterol transporter, or as a modulator of the contact site) to recruit and activate CERT [38]. Double headed arrows indicate the non-directionality of the transport mechanism, which is driven by gradient of free lipid [23,24].

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temporary fusion of PM and ER as a possible explanation Importantly, this identification of OSBPs as putative for non-vesicular traffic of specific polytopic membrane sterol transfer proteins leads to testable predictions. First, proteins [14,15] and membrane lipids [16]. OSBP homologues are recruited to membranes by phos- phoinositides such as PI(4)P and PI(4,5)P2 [32–34], and The formation of cortical ER is best understood in new so these signalling lipids should be important for sterol yeast buds [17]. One of the latest proteins to be impli- traffic. This prediction has now been borne out experi- cated in this process is Ice2, a polytopic ER protein found mentally [30], although the precise role of individual only in yeast, which regulates cortical ER morphology as phosphoinositides is not yet clear (Figure 1c). Second, well as its inheritance into buds [18]. In addition, reticu- OSBP homologues contain domains that target both lons have been shown to regulate ER morphology by plasma membrane and ER, which places them at mem- tubulating membranes and switching the ER from cis- brane contact sites [9,32,34,35]. Given the profusion of ternal to reticular morphology [19], and these conserved OSBP homologues in a single cell [32], they may be able proteins bind the exocyst at the plasma membrane, to pass between different membranes of the late suggesting a possible role in PM–ER bridging [20]. secretory pathway, for example from the PM to recycling endosomes [22], via a two-step process using the ER as Sterol traffic and oxysterol binding proteins conduit [36]. Other advances in our knowledge of OSBP Sterols are the single most common PM lipid, and are homologues have included the demonstration that their critical for PM function. Sterol traffic is of great medical dissociation from membranes is mediated by AAA- importance as aberrant handling of excess sterols by ATPases [37], and that OSBP itself is a sterol sensor macrophages underlies atherosclerosis [21,22]. Recent [28,38]. studies in yeast have confirmed earlier findings in mam- malian cells that sterol traffic is non-vesicular [23,24]. Other PM–ER communication The proposed mechanism for this traffic is passive diffu- also traffic between the ER and plasma sion down a concentration gradient facilitated by LTPs. membrane; for example, during PI(4,5)P2 production, A passive mechanism that might concentrate sterols (PI) is transferred from the ER to in the PM over the ER (observed PM concentration the PM rapidly and independently of carrier vesicles 10x that of ER) is mediated by in the [7,39]. This requires proteins that can transport PI, which exofacial leaflet of the PM forming complexes with sterol fall into two families that share no : [25], although this is at odds with old data suggesting those related to mammalian PI transfer protein (PITP), that the bulk of cholesterol is in the cytofacial leaflet and those related to yeast Sec14. The striking functional [26]. The advantage of yeast studies is the relative ease convergence between these families was confirmed with which genetic experiments can be carried out, for recently by the demonstration that a mammalian homo- instance demonstrating the importance of the lipid logue of RdgBa (a PITP family member, also known as environment in regulating sterol traffic to the PM Nir2) regulates the critical lipid diacylglycerol in Golgi [23,24]. membranes by the same mechanism used by Sec14 in yeast [40]. Similarly, while mammalian PI(4,5)P2 produc- Exciting developments in this field centre on the inter- tion at the PM relies on PITP family members [7], plant actions of the likely sterol-specific LTPs. Oxysterol bind- root hairs and budding yeast use Sec14 homologues for ing protein (OSBP) was originally identified as the the same purpose [41,42]. The possibility of non-vesicular cytoplasmic receptor for 25-hydroxycholesterol, a rela- PI transport across PM-ER membrane contact sites, sug- tively water-soluble oxysterol, and OSBP was thought not gested by old morphological studies of Drosophila RdgBa to bind cholesterol itself [27]. However, a study on the [43], has been supported by biochemical studies of mam- regulation of extracellular-signal-regulated kinase (ERK) malian PITP-a [7], as well as being envisaged for the by sterols showed that OSBP in a complex with ERK yeast Sec14-homologue Sfh5 [39]. phosphatases solubilises unmodified cholesterol [28]. This has now been followed by a structural analysis of Another type of link between the PM and the ER an OSBP homologue, both empty and complexed to four involves the interaction of across membrane different (oxy-)sterols, which indicates that most OSBPs contact sites. The only clearly described case is the can act as sterol transfer proteins [29]. The same team interaction between PTP-1B, a tyrosine phosphatase has also shown that sterol traffic in vivo is in part mediated embedded in the ER, which has substrates on the PM by OSBP homologues [30], extending their earlier study including insulin receptor [44], and multiple components on retrograde traffic of sterols in yeast [23]. As a result of of focal adhesions. These have now been studied in living this work, these proteins have been tested for a role in cells using a substrate-trapping mutant form of the phos- anterograde ergosterol traffic, which was reduced five-fold phatase [45]. ER tubules grow out on microtubules to upon inactivation of OSBP homologues [31], a partial attach to a sub-population of cell–matrix adhesions, and effect that does not clarify whether their involvement is these PM structures are stabilised as a result of the direct or indirect. enzymatic action of PTP-1B on the ER. This suggests www.sciencedirect.com Current Opinion in Cell Biology 2006, 18:371–378 374 Membranes and organelles

that proximity to the ER may define sub-domains of the in particular Nvj1 on the ER. As the first membrane PM. This idea is also envisaged for zones of calcium contact site to be defined at the molecular level, the signalling, where channels on the PM and ER form NVJ forms the basis for our ideas of how other membrane complexes and activate each other, particularly in muscle contact sites may be constructed by a simple protein cells during excitation [46,47]. bridge.

Golgi–ER contacts The only function so far ascribed to the NVJ is that it acts Ultrastructural evidence shows some ER cisternae in very as a zone through which nuclear material buds to reach close contact with elements of the trans side of Golgi the vacuole for autophagic recycling, and this is affected stacks [48], but the nature of the link is unclear. Non- by the interaction of Nvj1 both with the lipid synthase vesicular lipid traffic from the ER direct to the trans-Golgi Tsc13 and with Osh1, an OSBP homologue [53–55]. network (TGN) is documented for ceramide, being Interestingly, ORP1, the closest mammalian homologue mediated by ceramide transfer protein (CERT), an of Osh1, binds to Rab7 on multivesicular late endosomes LTP with targeting domains to both TGN and ER [56] and has a domain that targets the ER [9]. Thus ORP1 [49], similar to OSBP [32]. It has now been shown that might target mammalian ER– contacts, which binding to both membranes is important for the function contrast with their yeast counterparts by being very poorly of CERT [49] and OSBP [38]. There is also a functional documented, although clearly detectable under some link, as OSBP acts as sterol sensor, and recruits CERT, circumstances [57,58]. providing a means by which sterol metabolism post- translationally modulates synthesis [38]. Mitochondrial–ER contacts The precise origin within the ER of lipids trafficked to Organelles derived from endosymbiotic prokaryotes are Golgi membranes is still open to question. It might be ER not connected to the secretory pathway by vesicular outside the Golgi region [38]. Alternatively, it may be traffic, and so mitochondria [50] and [59] the intra-Golgi ER, as both OSBP and CERT could acquire a large proportion of their lipids from the ER bridge across Golgi–ER membrane contact sites [32] by non-vesicular routes. For mitochondria, there is con- (Figure 1c). This model, in which LTPs would function siderable evidence that a specialised sub-domain of the very efficiently without needing to diffuse between donor ER, called the mitochondrial-associated membrane and acceptor, has yet to be proven. However, it has been (MAM), adheres to mitochondria. Importantly, MAM is shown that full-length OSBP homologues in yeast are enriched in synthases of lipids required by mitochondria recruited to membrane contact sites by their interaction [3,60], indicating specialisation for non-vesicular traffic. with VAP on the ER (Figure 1c) [9]. Another role for ER–mitochondrial membrane contact sites is in calcium traffic [2,46,47]. Because gross manip- Such a static model has also been postulated for the yeast ulation of these contacts does not necessarily affect cal- Sec14-homologue Sfh4, which is required for non-vesi- cium traffic from the ER to mitochondria [61], an cular PS traffic in the late secretory pathway, although alternative view is that calcium entry into mitochondria Sfh4 does not itself bind PS [50]. Interestingly, this lipid is determined by proximity to the PM. However, this traffic step can be reconstituted in vitro, and requires does not necessarily imply a direct PM–mitochondrial bridging from the acceptor to the donor by the very membrane contact site, as ER elements are often found enzyme that utilises the lipid [51], suggesting that both interposed between the PM and the [46]. the enzyme and Sfh4 act at Golgi–ER contacts [39], In addition to the traffic of small molecules, a number of although these contacts have yet to be demonstrated in proteins translocate from the ER to mitochondria, one of yeast. Another good candidate for a component of Golgi– which modulates the strength of ER–mitochondrial con- ER contacts is RdgBa/Nir2, which interacts with both the tacts [62]. Golgi and the ER [43,52]. Despite a relatively large amount being known about ER Endosome/–ER contacts specialisation in mitochondrial–ER membrane contact Membrane contact sites between the ER and organelles sites, no bridging components between ER and mito- of the endocytic pathway lie at the extremes of how chondria are known. A ubiquitin ligase integral to mam- clearly these structures have been defined. In budding malian ER membranes localises to MAM [63], and studies yeast, the nucleus–vacuole junction is a membrane con- in yeast have demonstrated the involvement of ubiqui- tact site between a portion of the ER (on the outer nuclear tination in the normal functioning of both MAM and envelope) and the vacuole (equivalent to the lysosome). mitochondria [64,65], but the mechanism is still unclear This is the sole membrane contact site whose compo- [50]. nents are known: single proteins embedded in each of the opposing membranes bind each other directly, forming Intra-mitochondrial contacts velcro-like patches [5](Figure 1b). NVJ size depends Contacts between the outer mitochondrial membrane merely on the quantity of component proteins available, (OMM) and the inner mitochondrial membrane (IMM)

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parallel similar contacts between the two membranes of mtCK, its very absence from wild-type liver indicates that many bacteria, and have the full range of activities envi- it is unlikely to form constitutive contacts. Furthermore, saged to occur at membrane contact sites between het- the large size of octameric mtCK is incompatible with the erologous membranes (Figure 1a) [46,66]. During import absence of any gap between OMM and IMM at contacts. of cytoplasmic proteins into the mitochondrial matrix, TOM complexes on the OMM pass polypeptides directly Conclusions to TIM23 complexes on the IMM. A new, conserved sub- The seeds of our current understanding of vesicular traffic unit of the TIM23 complex, Tim21 is embedded in the were sown with ground-breaking genetic and cell-free IMM and directly links the two complexes by binding reconstitution studies that identified key components in Tom22 [67,68]. Despite interest in the TOM–TIM23 the trafficking machine. For membrane contact sites, such interaction, it is unlikely to be responsible for the adhe- discoveries are still to be made, and we remain in an sion of OMM and IMM [64]. Firstly, the TOM–TIM23 earlier phase where we are still defining the very nature of interaction is dynamic, only being detected when arrested the non-vesicular traffic. This article documents the protein intermediates are present. Also, when intra-mito- increasing numbers of proteins that localise to membrane chondrion contacts are purified biochemically, they have contact sites, providing candidates that might modulate a unique protein profile that does not include either TOM membrane contact site structure and function. However, or TIM complexes. the Holy Grail still remains: to identify components that constitutively form membrane contact sites. When these So what molecular mechanism brings about OMM–IMM are known, it will be possible to perform the critical contacts 15–20 nm in diameter, occupying 5% of the experiments, both in vivo and in cell-free reconstitution mitochondrial surface? Importantly, the thickness of systems, where membrane contact sites are taken apart these adhesion sites is almost uniformly reported to be and put back together in order to properly dissect their equal to the sum of the thicknesses of the OMM and functions. IMM together [64]. This suggests that there is no hemi- fusion of OMM and IMM, and also that the adhering The problems faced in this field are exemplified by a proteins do not have large domains in the intermembrane thorough study reconstituting traffic space (IMS). One possibility is that Tim23 alone forms between MAM and mitochondria in vitro [73]. Here, contacts, as it spans both membranes [69]. However, all current data predict that a protein bridge mediates Tim23 is not enriched in contacts either biochemically efficient non-vesicular lipid traffic [74,75]; however, lipid or morphologically, and the importance of its interaction traffic was only slightly reduced by protease treatment of with the OMM is called into question by studies that both membranes. This suggests that the purification of show this is not essential for protein import [70]. the two organelles has disrupted bridging complexes. Indeed, any purification to homogeneity of an organelle OMM–IMM contacts facilitate traffic of both large poly- that normally adheres firmly to another will disrupt peptides and small metabolites between the mitochon- bridges, possibly irreversibly by proteolysis. For many drial matrix and the cytoplasm. In addition to inward years, we have had only a partial knowledge of membrane traffic of calcium ions [46], OMM–IMM contacts are the contact sites, and I suggest that if we shall know fully how site for production of the energy storage molecule phos- they function, we will first need to identify the structural phocreatine. A complex forms from the adenine nucleo- bridging components and then manipulate their activity tide translocase in the IMM (which provides ATP from to see these fundamental, conserved structures clearly. the matrix), octameric mitochondrial creatine kinase (mtCK) in the IMS, and porin in the OMM (porin is also Acknowledgements known as voltage-dependent anion channel, VDAC, We thank Anjana Roy for the use of the electron micrograph image, and we apologize to our colleagues whose work we have not been able to which shuttles [phospho]-creatine to the cytoplasm). include because of space constraints. Recent work has shown that over-expression of mtCK in a tissue where it is normally absent (mouse liver) References and recommended reading induces more OMM–IMM contacts and makes the mito- Papers of particular interest, published within the annual period of chondria more resistant to disruption by detergents [71]. review, have been highlighted as: This is similar to the effect of over-expressing a protein  of special interest that arrests during mitochondrial import, increasing  of outstanding interest TOM–TIM23 supercomplex formation [72]. Both cases 1. Collin T, Marty A, Llano I: Presynaptic calcium stores show that a single set of bridging proteins can ‘zipper up’ and synaptic transmission. Curr Opin Neurobiol 2005, OMM and IMM, and so it is tempting to speculate that 15:275-281. these proteins have structural roles in the membrane 2. Filippin L, Magalhaes PJ, Di Benedetto G, Colella M, Pozzan T: contact site [71] and might affect other functions such Stable interactions between mitochondria and endoplasmic reticulum allow rapid accumulation of calcium in a as phospholipid traffic [50]. But neither complex is likely subpopulation of mitochondria. J Biol Chem 2003, to be the glue that forms the membrane contact site. For 278:39224-39234. www.sciencedirect.com Current Opinion in Cell Biology 2006, 18:371–378 376 Membranes and organelles

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