Phospholipid transport protein function at organelle contact sites

Phospholipids are synthesized at the endoplasmic reticulum (ER), the largest membrane bound organelle that forms membrane contact sites (MCS) with almost every other organelle. MCS are locations at which the membrane es of two organelles are closely positioned to provide a microenvironment where proteins in one membrane can interact with the opposite membrane. Thus, MCS provide an ideal location at which lipid transfer proteins (LTPs) can achieve the efficient transfer of individual classes of lipids from the ER to other organelles via non-vesicular transport. Here we provide an overview of emerging findings on the localization and biochemical activity of LTPs at MCS between the ER and other cellular membranes. The localization of LTPs at MCS offers an elegant cell biological solution to tune local lipid composition to ongoing cell physiology.


Introduction
The endoplasmic reticulum (ER) is the main site of phospholipid synthesis and provides lipids to other membrane compartments by vesicular and non-vesicular transport. Non-vesicular transport relies on lipid transfer proteins (LTPs) that can move lipids between membranes through aqueous cytosol. The ER is an elaborate network of membranes making contact with nearly all organelles including mitochondria, plasma membranes (PM), endosomes, lysosomes, peroxisomes, Golgi apparatus, lipid droplets and autophagosomes ( Figure 1). These areas of close contact, referred to as membrane contact sites (MCS), are formed by transient associations or can be stably present depending on cell type and context. The gap between two membranes at MCS is generally 10-30 nm spanned by tethering proteins. One of the many functions of MCS is the transfer of lipids by LTPs. LTPs are distinguished by the presence of domains such as the START (StaR related lipid-transfer), ORD (OSBPrelated domain), Acyl-CoA, SMP (synaptotagmin-like mitochondrial-lipid binding protein) and PITP ( phosphatidylinositol transfer protein) domains ( Figure 1). Most LTP domains contain hydrophobic cavities that can accommodate a single lipid and are highly selective. LTPs fall into two categories: single domain proteins constituted of solely the lipid binding domain and multi-domain proteins where an LTP domain is associated with additional domains [1,2] (see Figure 3 for examples).
The purpose of this review is to discuss emerging concepts of how lipid transfer between membrane compartments is facilitated at these MCS. In depth reviews on LTPs and MCS can be consulted for background information [2][3][4][5].

Lipid exchange at ER-PM contact sites
The PM of cells has a unique lipid composition being enriched in phosphoinositides and phosphatidylserine. Phosphoinositides are low abundance lipids generated by the phosphorylation of the precursor lipid phosphatidylinositol (PI) which is synthesized in the ER ( Figure 2). The two most abundant phosphoinositides, phosphatidylinositol (4,5)bisphosphate (PI(4,5)P 2 ) and its precursor phosphatidylinositol 4-phosphate (PI4P) are enriched in the inner leaflet of the PM where they serve many functions including regulation of the actin cytoskeleton, ion channel activity and exo-endocytosis. In addition, receptor-regulated phospholipase C (PLC) hydrolyses PI(4,5)P 2 to generate the second messengers, inositol (1,4,5)trisphosphate (IP 3 ) and diacylglycerol (DAG). During PLC signaling, PI(4,5)P 2 levels can drop rapidly at the PM requiring compensatory resynthesis to ensure stable levels of this key lipid. The biochemical pathway triggered by PI(4,5)P 2 hydrolysis and leading to its resynthesis includes five lipid intermediates that are distributed between the ER and the PM (PI(4,5)P 2 cycle); this leads to a topological constraint requiring transfer of lipid intermediates between the ER and PM [4] (Figure 2). vivo evidence of their requirement for PI transfer from the ER to the PM comes from studies in Drosophila photoreceptors. In fly photoreceptors, the microvillar PM is arranged in close contact with the ER-derived sub-microvillar cisternae (SMC); this is reminiscent of an ER-PM MCS [8]. PLCb is activated at the microvillar PM, whereas RdgBa, a multi-domain protein with an N-terminal PITP domain (Figure 3), is localized to the SMC. RdgBa mutants show depletion of PM PI(4,5)P 2 in the resting state and reduced rates of PI(4,5)P 2 resynthesis during PLC activation [9,10]. These biochemical defects affect photoreceptor function resulting in reduced responses to light and retinal degeneration. The PITP domain of RdgBa can bind and transfer PI in vitro, and in Lipid transfer at membrane contact sites Cockcroft and Raghu 53

ER
Lipid transfer at membrane contact sites. The ER is the main site of lipid synthesis and makes contact with many organelles. At these membrane contact sites, lipid transfer proteins from different families defined by the presence of specific domains such as the ORD, START, Acyl-CoA, SMP and PITP domains mediate lipid exchange. Abbreviations: ORD, OSBP-related domain; START, StAR-related lipid-transfer; SMP, synaptotagminlike mitochondrial; PITP, phosphatidylinositol transfer protein domain.

Current Opinion in Cell Biology
Transfer of PI and PA during the PI(4,5)P 2 cycle triggered by PLC activation. The enzymes are distributed between two membrane compartments, the ER and PM. Lipid transfer between these compartments is required at two points in the cycle; transfer of PI from the ER to the plasma membrane and transfer of phosphatidic acid (PA) from the plasma membrane to the ER.  (Figure 2).

Diacylglycerol transfer activity
A recent study in mammalian cells depleted of all E-Syts demonstrated sustained accumulation of PM DAG following stimulation by histamine [39,40]. These studies assumed DAG to be derived from PI(4,5)P 2 hydrolysis. However, DAG at the PM can be derived from both PLC (directly) or phospholipase D (indirectly via PA) activation and both these phospholipases are activated by histamine receptor activation. The accumulation of DAG was rescued by expression of E-Syt1, but not by mutant E-Syt1 lacking the SMP domain. As the SMP domain was found to transfer DAG, this is a potential mechanism for E-Syt1 to regulate the PI(4,5)P 2 cycle [25 ].
Lipid transfer from the ER to other organelles ER-Golgi PI synthesized at the ER ( Figure 2) is also used for non-PLC dependent processes at other organelle membranes.
In this case, PI transfer takes place at MCS using the single domain PITPb. Oxysterol binding protein (OSBP) is a modular LTP (Figure 3) localized to ER-Golgi contact sites; the FFAT motif permits OSBP to bind VAP at the ER while the PH domain binds to PI4P at the Golgi membrane (Figure 4a) [42]. Thus PITPb is ideally localized for maintaining Golgi PI4P levels that is consumed during cholesterol transfer. Evidence to support this comes from recent studies which has identified PITPb as a host factor required for positive-strand RNA viral replication [43,44]. For replication, the virus builds a membrane-associated replication complex which is Golgi-derived that is tightly associated with the ER. Interestingly OSBP, PI4KIIIb and Sac1 all of which are also localized at this MCS, are required for both PI4P homeostasis and viral replication [43,44] (Figure 4a). What recruits PITPb to these MCS is unclear. In Toxoplasma gondii, a multi-domain protein of 1912 a.a. incorporates a PITP domain with a PH domain, 4 transmembrane domains and an oxysterol binding domain (PITP-PH-4TM-OSBP) ( Figure 3). Thus the concept that a PITP domain works in concert with an OSBP domain protein appears to be amalgamated into a single protein in some organisms.

ER-peroxisomes
Peroxisomes depend on the ER for their lipid composition and the ER receives lipid precursors for plasmalogen biosynthesis (ether phospholipids) from peroxisomes. The tether that links these two organelles is the ER protein VAP-B interacting via its MSP domain with the FFAT-like motif of acyl-CoA binding domain-containing 5 (ACBD5), a peroxisomal tail-anchored membrane protein [45 ,46 ,47] (Figure 4b). An intact VAP-ACBD5 tether is required for peroxisome growth, plasmalogen synthesis and maintenance of cellular cholesterol levels [45 ]. The ACB domain of ACBD5 preferentially binds very long chain fatty acyl-CoAs and transfers them to peroxisomes. Mutations in ACBD5 show elevated levels of very long chain fatty acids and a defect in peroxisomal b-oxidation of very long chain fatty acids [48]; patients with ACBD5 deficiency manifest with retinal dystrophy [49].

ER-autophagosome
The transfer of PI from the ER is also required for autophagosome biogenesis [50]. PI is synthesized in the ER from CDP-DAG by PI synthase (PIS) (Figure 2). Overexpressed PIS localizes to a highly dynamic compartment of the ER and at leading edges of tubules [51,52]. Early autophagic structures are formed in close apposition to the ER and recent studies reveal that autophagosome formation requires a subdomain of the ER, which is highly enriched in PIS. The ULK complex first localizes to the PIS-enriched ER subdomain and then translocates to the ATG9A-positive autophagosome precursors in a PI3Pdependent manner. PI in the PIS-enriched membrane is  [50,53] remain to be identified.

ER-mitochondria
Mitochondria can synthesize PA, PG, cardiolipin and PE. However, PC, PI and PS have to be imported from the ER. PS imported into mitochondria is used by PS decarboxylase to produce PE at the inner mitochondrial membrane. Close contacts between the ER and mitochondria are important for lipid transfer; ER to mitochondria PS transfer slows down significantly in yeast cells missing both the ER-shaping reticulon proteins and the ERMES complex. This defect in PS transfer could be corrected by expression of a protein that artificially tethers the ER and mitochondria [54]; these findings have now been extended to mammalian cells [55]. The ER that associates with mitochondria is enriched in PS synthase [56,57]. Recent studies have identified ORP5/ORP8 ( Figure 3) to localize to ER-mitochondria contacts, interact with the outer mitochondrial protein, PTP1P5 and transfer PS from the ER to mitochondria [58 ]. Depletion of ORP5/ORP8 leads to altered mitochondrial morphology and function. Together, these findings indicate that PS production and transport at the ER-mitochondrial MCS is required to support mitochondrial function.
PC is the dominant lipid of mitochondria and recent studies emphasize StarD7, a member of the START family facilitates PC transfer from the ER to the outer mitochondrial membrane (OMM). StarD7-I, the longer isoform, contains a mitochondrial targeting sequence followed by a transmembrane domain anchoring the protein to the OMM [59] (Figure 3). The C-terminal START domain would then extend into the cytoplasm and shuttle PC from the ER to OMM at the ER-mitochondria contact sites [60 ] (Figure 4c). Loss of StarD7 results in embyronic lethality and compromised mitochondrial function [61 ]. Interestingly, loss of StarD7 results in only a partial loss of PC in mitochondria and suggests that other PC transfer proteins such as PCTP (StarD2) and StarD10 may have a role in PC transfer.

General conclusions
LTPs were initially identified as soluble single domain proteins that could transfer lipids between membrane compartments in vitro. However, in recent years, it has emerged that such lipid transfer domains occur in a diverse range of proteins in conjunction with other protein domains that in themselves appear to have no LTP activity. One emerging function of these additional domains is their ability to act as protein targeting signals thus ensuring the positioning of lipid transfer activity at specific and in some cases unique locations with cells. One such location that has emerged are MCS between the ER and multiple cellular organelles where LTPs seem to localize suggesting their ability to transfer lipid locally at these subcellular locations.
Numerous cell biological studies have informed on the localization and biochemical activity of LTPs in cultured cells. However, most are performed with the LTP overexpressed; while this has provided initial insights, it will be essential that going forward the localization of the endogenous LTPs in cells be established. Such studies will help to identify in vivo cell types where a given LTP is enriched leading to the development of model cell types where the function and regulation of endogenous LTPs can be studied.
In contrast to cell biological studies, there have been limited analyses of the role of LTPs in physiological processes in vivo. Where they have been done, in many cases, phenotypes have been surprisingly limited. This observation may reflect functional redundancy between multiple LTPs that can perform the same biochemical activity; this possibility is reflected in the finding that multiple genes encoding an LTP are found in mammalian genomes. Studies to address this redundancy will be important to understand the overall contribution of this class of molecules in regulating lipid homeostasis in cell physiology. Release of insulin occurs in two phases; in the second phase granules are docked and primed and then released in a series of bursts, each triggered by a spike of cytosolic Ca 2+ . TMEM24, an ER-localized protein is associated with PM by binding to PI(4,5)P 2 under resting conditions. The SMP domain can transfer PI from the ER to the PM to maintain PI4P and PI(4,5) P 2 concentrations. When cytosol Ca 2+ levels increase, TMEM24 is phosphorylated by PKC and dissociates from the PM. Only after dephosphorylation by the Ca 2+ -dependent phosphatase, PP2B, can TMEM24 re-associate with the PM and permit PI transfer to occur.