Molecular mechanisms and regulation of ceramide transport

Biochimica et Biophysica Acta 1734 (2005) 220 – 234 http://www.elsevier.com/locate/bba

Review

Molecular mechanisms and regulation of ceramide transport Ryan J. Perry, Neale D. Ridgway* Atlantic Research Centre, Dalhousie University, 5849 University Avenue, Halifax, N.S., Canada B3H 4H7 Department of Pediatrics, Dalhousie University, 5849 University Avenue, Halifax, N.S., Canada B3H 4H7 Department of Biochemistry and Molecular Biology, Dalhousie University, 5849 University Avenue, Halifax, N.S., Canada B3H 4H7 Received 14 March 2005; received in revised form 6 April 2005; accepted 7 April 2005 Available online 2 May 2005

Abstract De novo biosynthesis of sphingolipids begins in the endoplasmic reticulum (ER) and continues in the Golgi apparatus and plasma membrane. A crucial step in sphingolipid biosynthesis is the transport of ceramide by vesicular and non-vesicular mechanisms from its site of synthesis in the ER to the Golgi apparatus. The recent discovery of the ceramide transport protein CERT has revealed a novel pathway for the delivery of ceramide to the Golgi apparatus for sphingomyelin (SM) synthesis. In addition to a ceramide-binding START domain, CERT has FFAT (referring to two phenylalanines [FF] in an acidic tract) and pleckstrin homology (PH) domains that recognize the ER integral membrane protein VAMP-associated protein (VAP) and Golgi-associated PtdIns 4-phosphate, respectively. Mechanisms for vectorial transport involving dual-organellar targeting and sites of deposition of ceramide in the Golgi apparatus are proposed. Similar Golgi – ER targeting motifs are also present in the oxysterol-binding protein (OSBP), which regulates ceramide transport and SM synthesis in an oxysterol-dependent manner. Consequently, this emerges as a potential mechanism for integration of sphingolipid and cholesterol metabolism. The identification of organellar targeting motifs in other related lipid-binding/transport proteins indicate that concepts learned from the study of ceramide transport can be applied to other lipid transport processes. D 2005 Elsevier B.V. All rights reserved. Keywords: Ceramide; Hydroxycholesterol; Lipid transport; CERT; Endoplasmic reticulum; Golgi apparatus

1. Introduction In general, lipid biosynthetic pathways involve different organelles, thereby necessitating the transport of metabolic intermediates and end products for the completion of synthesis and production of cellular membranes, respectively. The properties of lipids that are essential for membrane structure also render free movement across the

cytoplasmic space thermodynamically unfavorable. This restriction requires that lipid transport occur by bulk membrane fusion events and/or lipid-binding proteins. Clearly, this transport machinery is efficient and highly selective since lipid biosynthetic pathways are tightly coordinated and organelles exhibit distinct lipid compositions. Thus, compartmentalization of lipid synthesis, restricted lipid movement and the preservation of membrane

Abbreviations: ARF, ADP-ribosylation factor; CERT, ceramide transport protein; CGlcT, ceramide glucosyltransferase; CHO, Chinese hamster ovary; DAG, diacylglycerol; D609, tricyclodecan-9-yl xanthate; HPA-12, N-(3-hydroxyl-1-hydroxylmethyl-3-phenylpropyl)dodecanamide; ER, endoplasmic reticulum; ERK, extracellular signal-regulated kinase; FFAT, two phenyalanines in an acidic tract; FAPP, phosphatidylinositol 4-phosphate adapter protein; GFP, green fluorescent protein; GlcCer, glucosylceramide; GPBP, Good-pasture binding protein; GPI, glycosylphosphatidylinositol; IPC, inositol phosphorylceramide; KDS, 3-ketosphinganine; LAG, longevity assurance gene; MCS, membrane contact sites; OSBP, oxysterol-binding protein; ORP, OSBP-related protein; PA, phosphatidic acid; PCTP, phosphatidylcholine transfer protein; PH, pleckstrin homology; PITP, phosphatidylinositol transfer protein; PM, plasma membrane; PtdCho, phosphatidylcholine; PtdIns, phosphatidylinositol; SM, sphingomyelin; SMS, sphingomyelin synthase; StAR, steroidogenic acute regulatory protein; StarD4, START domain protein 4; START, StAR-related lipid transfer; SPT, serine palmitoyltransferase; SRD6, sterol regulatory defective 6; SREBP, sterol-regulation element binding protein; TGN, trans-Golgi network; VAP, vesicle-associated membrane protein-associated protein; 25-OH, 25-hydroxycholesterol * Corresponding author. Atlantic Research Centre, Dalhousie University, 5849 University Avenue, Halifax, N.S., Canada B3H 4H7. Fax: +1 902 494 1394. E-mail address: [email protected] (N.D. Ridgway). 1388-1981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2005.04.001

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compositions implies the existence of multiple discrete intracellular lipid transport pathways. A number of technical advances have overcome previous limitations for studying intracellular lipid trafficking. Improved methodologies for accurate quantitative analysis of membrane lipid composition have provided a framework for elucidating which lipids are targeted to different membranes [1 –7]. Genomics has facilitated the identification of lipid biosynthetic enzymes and entire pathways leading to a better understanding of intracellular sites of lipid synthesis. Finally, modifications to lipids to improve water solubility and fluorophore-conjugation has improved delivery and visualization in cells [8 – 10], although these modifications alter physical properties, making it difficult in some instances to extrapolate results to the natural compounds. These advances had, by far, the greatest impact on the study of sphingolipid metabolism. Entire sphingolipid biosynthetic pathways have been identified in numerous organisms, leading to the elucidation of intracellular sites of synthesis and prediction of transport pathways for metabolic intermediates and products [11 –16]. The visualization of these intracellular sites and monitoring of intermembrane movement was facilitated by the generation of fluorescent-tagged sphingolipids [17,18] and short N-acyl-chain analogs with improved solubility [19,20] (reviewed in [21]). Collectively, these studies showed that sphingolipid synthesis begins in the ER and continues in the Golgi apparatus and plasma membrane (PM) [22] by continuous and uninterrupted pathways dependent on specific intracellular lipid transport mechanisms. An early and critical step in sphingolipid synthesis involves the transport of ceramide, the precursor for glycosphingolipids and sphingomyelin (SM), from its site of synthesis in the ER to the Golgi apparatus. This review will focus on recent advances in our understanding of the molecular mechanisms and regulation of ER-toGolgi ceramide transport. The reader is referred to other reviews for a broader description of sphingolipid transport in yeast [23] and mammalian cells [24].

2. Synthesis of ceramide, sphingomyelin and glucosylceramide Johann L. W. Thudichum originally named sphingolipids after the Greek Sphinx, owing to the enigmatic chemical properties of the sphingosine backbone [25]. On a functional level, this enigma has been lifted to reveal an everexpanding role for sphingolipids in signal transduction, cell survival and death pathways, and in the formation of membrane microdomains, termed rafts. In order for these functions to be exerted in different intracellular compartments, sphingolipids must be generated locally or transported to the site of action [26,27]. De novo synthesis of sphingolipids begins in the ER with the serine palmitoyltransferase (SPT)-catalyzed condensation of serine and

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palmitoyl-CoA to generate 3-ketosphinganine (KDS) [12,28]. SPT is the rate-limiting enzyme in this biosynthetic pathway and is regulated by transcriptional [29 – 33] and post-transcriptional mechanisms [34 –37]. Next, a series of three reactions, reduction of KDS to sphinganine, N-acylation to produce dihydroceramide and desaturation to generate a 4-trans double bond, produce ceramide on the cytosolic surface of the ER [12 – 14]. Until recently, the intracellular localization and topology of these latter enzymes has been based solely on kinetic evidence. Mammalian longevity assurance gene (LAG) family members have now been shown to regulate acyl-CoA-dependent dihydroceramide synthesis in the ER [38 – 40]. However, it is not known if these proteins represent bona fide dihydroceramide synthase enzymes or specify acyl chain selectivity for another dihydroceramide synthase. Mammalian D4-desaturases have also been identified and shown to prefer dihydroceramide as a substrate compared to sphinganine [41 – 43]. Interestingly, the D4-desaturases also exhibit C-4 hydroxylase activity to generate phytoceramide. While the yeast KDS reductase has been identified [44], the mammalian homologue remains elusive. The conversion of ceramide to glucosylceramide (GlcCer) or SM requires efficient transport to the Golgi apparatus where two enzymes catalyze the addition of polar head groups to the primary hydroxyl group of ceramide. The glucosylation of ceramide is catalyzed by ceramide glucosyltransferase (CGlcT) on the cytosolic surface of the early Golgi apparatus [45,46]. CGlcT has also been identified in pre-Golgi compartments [45], including the ER [47], and thus ER-to-Golgi ceramide transport may not be necessary for GlcCer synthesis. Following synthesis on the cytosolic surface, GlcCer is translocated to the luminal surface of the Golgi apparatus where it is converted to lactosylceramide and complex glycosphingolipids (e.g. gangliosides) [48 –51]. In contrast, SM synthesis is dependent on the translocation of ceramide from the cytoplasmic to the luminal surface of Golgi membranes [52,53]. There, SM is synthesized by phosphatidylcholine:ceramide cholinephosphotransferase, also referred to as SM synthase (SMS), by the transfer of the phosphorylcholine moiety from phosphatidylcholine (PtdCho) onto the primary hydroxyl of ceramide, generating SM and diacylglycerol (DAG). Since SMS also catalyzes the reverse reaction [15,54,55], it has been suggested to function as a molecular switch between ceramide-dependent cell death pathways and pro-survival DAG-dependent pathways. Two SMS genes in mammals encode integral membrane enzymes expressed in the Golgi apparatus (SMS1) and PM (SMS2) [15]. Since Golgilocalized SMS1 is proximal to ER-derived ceramide relative to SMS2, SMS1 appears to be the principal enzyme involved in de novo SM synthesis. SMS1 was shown to co-localize with the trans-Golgi cisternae marker sialyltransferase by immunofluorescence microscopy [15]. However, previous cell fractionation studies demonstrated

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enrichment of SMS activity in cis/medial elements of the Golgi apparatus [53,56 – 59]. These differences in SMS1 activity among Golgi cisternae may be cell-type specific [56,60,61] or indicate that SMS1 is distributed throughout the Golgi apparatus. PM localization of SMS2 suggests a role in maintaining SM levels for signaling pathways involving sphingomyelinases. Sphingomyelinase-catalyzed hydrolysis of SM in response to external stimuli [62 – 67] generates ceramide that is a potent signaling molecule for intracellular signaling pathways for cell proliferation, differentiation and cell death (apoptosis) (reviewed in [68]). Thus, potential roles for SMS2 involve the regeneration of SM and/or attenuation of ceramide signaling at the PM. The pathway for ceramide synthesis in the ER of S. cerevisiae is similar to mammals, except that sphinganine is hydroxylated by Sur2p to produce phytosphingosine, which in turn is N-acylated by ceramide synthase producing phytoceramide, the precursor for complex sphingolipids [44,69 –75]. The synthesis of complex sphingolipids also occurs in the Golgi apparatus, but in this case, phosphatidylinositol (PtdIns) donates a phosphorylinositol head group to phytoceramide generating inositol phosphorylceramide (IPC), the yeast equivalent of SM. This reaction is catalyzed by Aur1p (IPC synthase), the active site of which is localized to the luminal surface of the medial cisternae of the Golgi apparatus [76]. IPC is subsequently modified by mannosylation and the addition of another inositol phosphate group [77 –81].

3. ER-to-Golgi ceramide transport pathways In order for ER-derived ceramide to reach the Golgi apparatus for conversion to complex sphingolipids, it must first traverse the cytosolic compartment. The t 1/2 for spontaneous interbilayer movement of ceramide is on the order of days [82]. Therefore, efficient, rapid and regulated movement of ceramide between the ER and Golgi apparatus must be facilitated by specific interbilayer transport mechanisms. In mammalian cells and yeast, both non-vesicular and vesicular transport pathways have been described. In yeast, the synthesis of IPC decreased by 40– 60% when ER –Golgi vesicular transport was blocked in various temperature-sensitive secretion (sec) mutants [83 – 85]. Residual IPC synthesis was not a consequence of trapping newly synthesized Aur1p in the ER but was due to an ER-to-Golgi non-vesicular ceramide transport pathway that was ATP-independent and required a heat-labile, trypsin-sensitive cytosolic factor [85]. This pathway also involved ER –Golgi membrane contact sites (MCS) since in vitro assays of ceramide transport were independent of membrane concentration. Non-vesicular ceramide transport was first described in mammalian cells undergoing mitosis [86]. In mitotic cells, a number of vesicular transport pathways are inhibited [87] (reviewed in [88,89]) due to the fragmentation of the ER

[90], nuclear envelope [91] and Golgi apparatus [92], yet synthesis of SM only decreased by 40%. It has been argued, however, that Golgi-localized enzymes (i.e. SMS1) may relocate to the ER during mitosis thereby maintaining SM synthesis. Subsequent reports supported the existence of non-vesicular ceramide transport, but yielded conflicting results regarding required factors (e.g. ATP) [93,94]. A significant breakthrough came when Hanada and colleagues isolated mutant Chinese hamster ovary (CHO) cells (LY-A) resistant to lysenin, an SM-directed cytolysin isolated from the coelomic fluid of the earthworm Eisenia foetida [95]. Resistance to lysenin was due to reduced SM levels in the PM, the result of defective ER-to-Golgi ceramide transport for SM synthesis [96]. Interestingly, neither GlcCer synthesis nor protein export from the ER was affected in LY-A cells. Similar to findings from yeast [85], the missing factor in LY-A cells was also cytosolic and heat-labile, but catalyzed transport in an ATP-dependent manner [96,97]. Recently, the cytosolic ceramide transport factor was identified by the isolation of cyclodextran-sensitive revertants of LY-A cells following retroviral transfection of a human cDNA library. A cDNA isolated by this method encoded a previously identified protein, Good-pasture antigen binding protein (GPBP), and was renamed as ceramide transport (CERT) protein to reflect this function [98]. The isolation of the CERT cDNA from LY-A cells revealed a glycine to glutamic acid (G67E) mutation within the N-terminal pleckstrin homology (PH) domain. This mutation prevented the targeting of CERT to the Golgi apparatus, due to the loss of PtdIns 4-phosphate binding (see Section 4.2), resulting in the decreased delivery of ceramide to the Golgi apparatus for SM synthesis. However, ceramide binding to CERT(G67E) was similar to wild-type, suggesting that CERT(G67E) could have a dominant effect on ceramide transport by sequestering ceramide or forming non-productive complexes with partner proteins (see Section 4.3). CERT-dependent transport between the ER and Golgi apparatus in CHO cells did not depend on Sar1, a G-protein essential for COPII-mediated vesicular transport, confirming that this process is non-vesicular [98]. However, COPIIdependent transport accounts for about 50% of the ceramide transfer between the ER and Golgi apparatus in yeast [85]. Vesicular transport of ceramide in yeast is coupled to the transport of glycosylphosphatidylinositol (GPI)-anchored proteins to the Golgi apparatus [83 – 85,99,100], and consequently may not be a regulated pathway for the biosynthesis of complex sphingolipids. It should be noted that the synthesis of IPC has not been measured in the absence of non-vesicular transport. In mammalian cells, there is conflicting evidence for ceramide inclusion in secretory vesicles. ER-derived transport vesicles containing ceramide were reported to fuse with Golgi apparatus membranes in an N-ethylmaleimide-sensitive manner [101]. In contrast, others reported that ceramide was excluded from ER-derived transport vesicles [94]. De novo

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synthesis of SM takes place in LY-A cells, albeit at a significantly reduced rate (10 – 30%), indicating that ceramide transport between the ER and Golgi apparatus occurs by other vesicular or non-vesicular mechanism(s) [98]. CERT- and ATP-independence of GlcCer synthesis indicates separate ceramide transport pathways for GlcCer and SM synthesis. GlcCer synthase has been detected in both the ER [47] and Golgi compartments [45,46] and, as a result, the synthesis of GlcCer may not depend exclusively on the ER-to-Golgi transport of ceramide. In addition, the active site of CGlcT faces the cytoplasm, whereas the SMS1 active site is luminal. Thus, the lack of dependence on CERT and ATP for GlcCer synthesis could also reflect the differences in subcellular localization and active site topology of CGlcT. The contribution to GlcCer synthesis by COPII-mediated ceramide transport to the Golgi apparatus is not known. The localization of GlcCer in both the ER and Golgi apparatus may indicate a need by cells to independently regulate ceramide consumption by the two pathways. Unlike SM synthesis, GlcCer synthesis more closely reflects rates of ER ceramide synthesis and mass [37,98,102 – 107]. This may represent a mechanism to prevent the accumulation of ceramide and attenuate its activity in a similar manner to neutralization of ER cholesterol by conversion to cholesteryl esters by acylCoA:cholesterol acyltransferase. The presence of both vesicular and non-vesicular pathways in yeast and mammalian cells raises the question as to the relative contribution and role of each pathway. In yeast, both pathways appear to contribute equally to ceramide transport between the ER and Golgi apparatus for IPC

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synthesis, while vesicular ceramide transport is also important for GPI-anchored protein transport. In mammalian cells, vesicular and non-vesicular (CERT-dependent) transports represent minor and major pathways for ER-toGolgi ceramide transport for SM synthesis, respectively. Based on this, non-vesicular transport has a primary role in ceramide delivery to the Golgi apparatus for de novo sphingolipid synthesis, whereas vesicular transport appears to be associated with ceramide functionality [85].

4. CERT 4.1. Intracellular localization and protein domain organization The identification of GPBP as the ceramide transporter CERT was unexpected since GPBP was originally described as a protein that bound and phosphorylated serine residues in the non-collagenous C-terminal region of the a3 chain of collagen IV (Good-pasture antigen) [108]. The lack of conventional protein kinase motifs in GPBP suggests that an associated kinase maybe responsible for this activity. Moreover, GPBP is an intracellular protein, whereas the Good-pasture antigen is extracellular, casting further doubt on the role of GPBP as the Good-Pasture antigen kinase. GPBP is expressed as a full-length and alternatively spliced form (GPBPD26) missing a 78 bp exon encoding a 26-amino-acid, serine-rich region [109] (Fig. 1A). CERT is

Fig. 1. Domain structure and PH domain alignment of CERT and related proteins. (A) CERT and OSBP have similar protein domain organization consisting of an N-terminal PH domain (yellow), a centrally located FFAT domain (orange), and a C-terminal lipid-binding START (red) or OHD (blue) domain that binds ceramide and 25-OH, respectively. CERT is a splice variant of CERTL resulting from the removal of a 78-bp exon that encodes a serine-rich 25 amino acid region (green). (B) Alignment of the PH domains of CERT, OSBP, ORP4, FAPP1 and FAPP2. Identical and similar amino acids are shaded black and grey, respectively. Below the alignment is a PH domain consensus sequence. The dots indicate amino acids predicted to be important for PtdIns 4,5-phosphate binding, the underlined residues are predicted to interfere with PtdIns 3-phosphate binding and the arrow indicates the mutation in CERT(G67E). The predicted PH domain secondary structure is shown above the alignment (numbering corresponds to CERT).

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identical to GPBPD26, but GPBP (designated CERTL) also has ceramide transport activity in vitro and in vivo [98]. CERT and CERTL are 598- and 624-amino-acid polypeptides, respectively, that have variable mobility on SDSPAGE due to phosphorylation and self-associate in yeasttwo hybrid assays [108,109]. Both isoforms are expressed preferentially in striated muscle and the brain, and are poorly expressed in the placenta, lung and liver [108,109]. As well, CERT is more highly expressed in the kidney and pancreas, as well as in a number of cancer cell lines, compared to CERTL. Interestingly, yeast does not possess a CERT homolog [98,108]; however, a yeast ortholog likely exists since ceramide is transported to the Golgi apparatus for IPC synthesis by a related non-vesicular mechanism [85]. When overexpressed in CHO cells, green fluorescent protein (GFP)-tagged CERT was distributed throughout the cytosol, as well as enriched in a perinuclear region, where it co-localized with the cis/medial-Golgi apparatus marker, GS28 [98]. An analysis of the amino acid sequence revealed PH and FFAT (referring to two phenylalanines [FF] in an acidic tract) domains, which are responsible for targeting CERT to the Golgi apparatus and ER, respectively (Fig. 1A). In addition, CERT contains a C-terminal START domain necessary for ceramide binding. The following three sections provide a detailed analysis of the roles of these domains in organelle targeting and lipid binding. 4.2. PH domain PH domains are ¨100 –120 amino acid protein modules that recognize phosphorylated PtdIns and recruit cytosolic proteins to membranes to participate in signal transduction, cytoskeletal re-organization and membrane trafficking (reviewed in [110,111]). In general, PH domains exhibit limited similarity in their primary amino acid sequence, but have a common secondary and tertiary structure. This shared core structure consists of a 7-stranded h-sandwich composed of two nearly orthogonal h-sheets that contain 4and 3-strands, respectively. A C-terminal a-helix caps one open end of the h-sandwich, whereas 3 interstrand variable loops cap the other open end. Amino acid residues in the PH domain h-sheets that determine specificity and affinity for the head groups of phosphorylated-derivatives of PtdIns have been identified [112– 114]. Interestingly, the PH domains of CERT, oxysterol-binding protein (OSBP), OSBP-related protein 4 (ORP4) and PtdIns 4-phosphate adapter proteins (FAPP1 and FAPP2) form a subgroup with a high degree of primary sequence similarity (Fig. 1B) and specificity for PtdIns 4phosphate and PtdIns 4,5-phosphate [115 – 117]. The K D for PtdIns 4-phosphate and PtdIns 4,5-phosphate binding to CERT and OSBP were determined to be ¨2.0 AM and ¨4.0 AM, respectively, whereas the K D for FAPP1 was ¨10-fold higher [117]. A comparison to PH domains with known ligand-bound structures [112,113] and similar phosphory-

lated PtdIns-binding affinities and specificities [116,118– 120] reveals key residues in the CERT PH domain that are important for binding 4- and 5-phosphorylated PtdIns. These residues include K32 of the h1-strand, R43 of the h2-strand, Y54 of the h3-strand and R66 at the C-terminal end of the h3/h4 loop that could interact with the 4- and 5phosphates of PtdIns 4-phosphate and PtdIns 4,5-phosphate (Fig. 1B, dots) [112,113,120]. The specificity of CERT for 4- and 5-phosphorylated rather than 3-phosphorylated PtdIns may be due to the presence of amino acids with bulky side chains at the end of the h1 loop (Fig. 1B, underlined residues). PH domains that bind 3-phosphorylated PtdIns with relatively high affinity generally have G, A, P or S at this position [112,113]. The CERT(G67E) mutation in LY-A cells introduces a negative change next to the critical R66 residue, thus abolishing PtdIns 4-phosphate specific binding and Golgi apparatus recognition (Fig. 1B, arrow). The GFP-tagged PH domains of OSBP, CERT and FAPP1 localized exclusively to the Golgi apparatus in yeast, or with the trans-Golgi network (TGN) marker TGN46 in mammalian cells [117,121]. However, full-length CERT and OSBP have been shown to co-localize with cis/ medial-Golgi apparatus markers in mammalian cells, suggesting that the overexpression of PH domains in isolation may not reflect the location of the intact protein [98,122] (Wyles and Ridgway, unpublished observations). Recent studies also localized OSBP and FAPP1 PH domains with cis/medial-Golgi markers [123,124]. With increased overexpression, the OSBP and FAPP1 PH domains redistributed from cis/medial-Golgi elements to the TGN [123]. The localization of this subfamily of PH domains to the Golgi apparatus appears to be unique, since the majority of PH domains specifically target the PM [112 –114,125 – 135] (reviewed in [136,137]). The localization of the CERT PH domain to the Golgi apparatus in yeast was dependent on PtdIns 4-phosphate synthesis, but not PtdIns 4,5-phosphate or any of the 3phosphorylated PtdIns [117], which may explain the ATPdependence of CERT-mediated ceramide transport. A similar requirement for PtdIns 4-phosphate synthesis was shown in mammalian cells for the OSBP and FAPP1 PH domains [121,123,124]. Since PtdIns 4-phosphate is also present at the PM, additional factors are involved in the Golgi-specific targeting of CERT. A second Golgi determinant for CERT has not been identified; however, Levine and Munro observed residual localization of a PtdIns 4phosphate-binding defective form of the OSBP PH-domain to the Golgi apparatus [117]. This residual localization was diminished in yeast cells lacking the small GTPase, Arf1p. Similarly, Godi et al. reported that the FAPP1 PH domain required ADP-ribosylation factor 1 (ARF1, the mammalian homolog of Arf1p) for Golgi-specific binding in COS7 cells, and that both the FAPP1 and OSBP PH domains physically interacted with ARF1 [121]. Contact sites between these PH domains and ARF1 are not known,

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but may involve conserved residues within the h7-strand [119]. Sequence similarities in the CERT PH domain h7strand (Fig. 1B) suggest that it may also interact with ARF1. Since ARF1 [138 –141] and PtdIns 4-phosphate [123,124] are also associated with the TGN, additional Golgi determinants could be necessary for specific recruitment of these PH domains to early and late Golgi cisternae. 4.3. FFAT domain In addition to a Golgi targeting domain, CERT also contains an ER localization domain termed FFAT. The FFAT domain targets soluble proteins to the ER via binding to the ER resident type II transmembrane protein vesicle-associated membrane protein (VAMP)-associated protein (VAP) in mammals [142,143] or Scs2p in yeast [144]. VAP was originally identified based on yeast 2-hybrid and in vitro interactions with proteins involved in vesicle fusion, such as syntaxin 1A, rbet1, rsec22, SNAP-25, NSF and a wide range of SNARE proteins [145,146]. The relevance of these relationships is unclear, since evidence is accruing that VAP is a key ER-binding site for various lipid-binding proteins including members of the oxysterol-binding protein (OSBP)-related protein family [142,144], human homologs of Nir/rdgB [147] and Opi1p [144]. While CERT is a lipid transport protein with a FFAT domain (Fig. 1A), evidence for an interaction with VAP has yet to be reported. However, we recently co-immunoprecipitated CERT and VAP in detergent lysates prepared from CHO cells exposed to a protein crosslinker (Perry and Ridgway, unpublished results). The ATP-requirement for CERT function could be for disruption of the CERT-VAP complex. In this regard, an AAA-ATPase (Agf2p) potentially involved in protein complex dissociation has been shown to be part of a large protein complex with Scs2p and Osh1p [148]. Recently, the FFAT motif binding site in Scs2p was localized to a highly conserved region of basic residues at the N-terminus [149]. Protein modeling predicted the clustering of these positively charged residues on one face of the protein and the presence of two shallow pits on either side of a critical lysine, which are partially lined by hydrophobic residues. Therefore, the binding of FFAT motifs most likely occurs through electrostatic interactions and the shallow pits accommodate the two aromatic residues. 4.4. START domain The steroidogenic acute regulatory (StAR)-related lipid transfer (START) domain was originally described in the StAR protein as a cholesterol-binding motif [150]. Similarly, the START domain of MLN64 also binds cholesterol [150], but these domains in other proteins have different lipid ligands. For example, the START domain of PtdChotransfer protein (PCTP) exclusively binds PtdCho [151 –

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153], and the C-terminal START domain in CERT binds ceramide but not cholesterol or phospholipids [154]. An analysis of ceramide-binding activity in a cell-free assay system for intermembrane transfer demonstrated that the CERT START domain binds and transports d-erythroceramides with C14, C16, C18, and C20, but not C22 and C24, N-acyl chains [154]. The efficient transfer of C16-dihydroceramide and C16-phytoceramide was also mediated by CERT. Diacylglycerol transport was 5 –10% of the activity for C16-ceramide, and neither sphingosine nor SM was transferred by CERT. In addition, CERT bound the shortchain fluorescent ceramide analogs C5-DMB-ceramide and C6-NBD-ceramide with a 1:1 stoichiometry, consistent with a monomeric lipid transport mechanism. StAR and MLN64 START domains bind cholesterol with a 1:1 stoichiometry in a site composed of a hydrophobic tunnel created by an a/h fold built around a U-shaped incomplete h-barrel [150]. Crystallographic analysis of mouse START domain protein 4 (StarD4) revealed a similar binding fold [155]. This suggests that the hydrophobic tunnel of START domains accommodate single lipid molecules, and the diversity of lipid-binding reflects subtle alterations in residues within the tunnel interior. Compared to StAR and MLN64, PCTP exhibits polar residues at key positions within the hydrophobic tunnel that could facilitate binding to the zwitterionic head group of PtdCho rather than the more hydrophobic cholesterol molecule [150]. The START domain of CERT possesses similar polar residues at comparable positions, consistent with specificity for ceramide rather than cholesterol. The mouse StAR4 START domain has an estimated tunnel volume that predicts the binding of a single cholesterol molecule, but displays polar amino acid residues at similar positions to PCTP and CERT [155]. Thus, this questions the importance of these particular amino acid residues in lipid-binding specificity, necessitating further studies to define the molecular determinants for binding specificity.

5. Molecular mechanisms of ceramide transport 5.1. Non-vesicular Two mechanisms can be envisioned for CERT-dependent, non-vesicular ceramide transport between the ER and Golgi apparatus. First, CERT could bridge the ER and transGolgi cisternae through MCS by simultaneously binding VAP and PtdIns 4-phosphate (and possibly ARF1) via the FFAT and PH domains, respectively (Fig. 2, reviewed in [156,157]). With CERT attached to both membranes, 1 mol of ceramide is transported to the trans-Golgi from the ER via the START domain during each cycle. In support of this model, the trans-ER was found in close proximity to the trans-cisternae of the Golgi apparatus [16]. Based on EM analysis, the ER also has contacts with the plasma membrane [158,159], mitochondria [160] and endosomes

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Fig. 2. Models for ER-to-Golgi ceramide transport and regulation by OSBP. Ceramide is transported between the ER and Golgi apparatus by non-vesicular (Panels and ) and vesicular mechanisms (Panel ). (Panel ) At a MCS, CERT simultaneously binds VAP ( ) in the ER and PtdIns 4-phosphate ( , and possibly ARF1) at the Golgi apparatus via the FFAT ( ) and PH ( ) domains, respectively. While CERT is attached to both membranes, ceramide ( ) is bound and transferred from the ER to the trans-Golgi via the START domain ( ). (Panel ) Upon 25-OH ( ) binding to the OSBP oxysterol homology ), OSBP translocates from the ER to the Golgi apparatus, where it positively regulates CERT-dependent ceramide transport through the domain ( stabilization of the MCS (Panel ) or increasing the recruitment of ceramide-bound CERT to the Golgi apparatus (Panel ). (Panel ) Recruitment of CERT to the ER occurs through interaction with VAP via the FFAT domain. At the ER, the CERT START domain extracts ceramide from the membrane bilayer, is released from the ER and transports ceramide across the cytoplasmic compartment to the cis/medial/trans-elements of the Golgi apparatus through interaction with PtdIns 4-phosphate via the PH domain. At the Golgi apparatus, ceramide is transferred into the membrane bilayer and CERT is released and recruited back to the ER to continue the cycle. In Panels and , ceramide transport to the Golgi apparatus is coupled to the SM ( ) production by SMS1 on the luminal surface of the Golgi apparatus. (Panel ) Ceramide is also transported between the ER and Golgi apparatus by COPII-dependent vesicular trafficking, and utilized by CGlcT and SMS1 for production of GlcCer ( ) and SM, respectively.

[161,162], suggesting that MCS may be specialized regions of the ER involved in inter-organelle communication and/or transport [156]. In yeast, the OSBP homolog Osh1p, which interacts with Scs2p through a FFAT domain, localized to nuclear-vacuolar MCS or junctions [163]. Although VAP or Scs2p has not been localized to the contacts sites described above, VAP co-localized with occludin at tight junctions between cells [164] and was implicated in intramembrane ER contacts [142,147]. Secondly, CERT could shuttle ceramide through the cytosol between the ER and Golgi cisternae by a mechanism involving cytoplasmic pools of ceramide-bound and -free CERT (Fig. 2), in a manner similar to that predicted for oxysterol-bound and -free OSBP [165]. When not bound to ceramide, the START domain would interact with and conceal the PH domain and/or expose the FFAT domain, resulting in the recruitment of CERT to the ER through VAP binding and extraction of 1 mol of ceramide from the membrane bilayer. The release of CERT from the ER would occur upon ceramide binding due to the induction of a

conformational change that exposes the PH domain and/or disrupts the FFAT domain– VAP interaction. However, it should be recognized that VAP could sequester both ceramide-bound and -unbound CERT, releasing CERT only when ceramide levels are above a certain threshold in a manner analogous (yet opposite) to Opi1p regulation by SCS2 and phosphatidic acid (PA) [166]. Upon release from the ER, CERT would then transport ceramide across the cytosolic compartment, where it is recruited via the PH domain to cis/medial/trans-elements of the Golgi apparatus through the recognition of PtdIns 4-phosphate and possibly ARF1. The movement of CERT through the cytoplasmic compartment could involve the actin cytoskeleton since the related OSBP PH domain has been shown to bind and induce actin bundle formation [167]. The release of ceramide from the START domain into the membrane bilayer of the Golgi apparatus would evoke a final conformational change that conceals the PH domain and/ or exposes the FFAT domain for recruitment back to the ER to continue the cycle. The delivery of ceramide to cis/

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medial/trans-Golgi cisternae is predicted in both models for the following reasons. First, PH domains similar to CERT extensively localize to these Golgi cisternae, and only with the high expression of these PH domains is TGN localization observed (see Section 4.1). Second, SMS1 resides throughout the early and late Golgi cisternae (see Section 2), but not the TGN, thereby effectively coupling CERTmediated ceramide delivery with SM synthesis in those compartments. CERT also binds DAG, albeit to a significantly lesser extent then ceramide [98,154], and could also play a role in removal of DAG from the Golgi apparatus. The binding of DAG by CERT may be physiologically relevant since the reversibility of the SMS is dependent on the relative concentrations of phosphorylcholine donors (SM and PtdCho) to acceptors (DAG and ceramide) [15]. Thus, the accumulation of DAG at the Golgi apparatus could potentially drive the reverse reaction, thereby consuming SM and producing ceramide. CERT-dependent DAG transport back to the ER for conversion to PtdCho or other glycerolipids would maintain the forward reaction of SMS1 as well as increase the production of substrate PtdCho in the ER. In contrast to the lack of experimental evidence for CERT interaction with VAP at the ER, mechanisms regulating CERT recruitment to the Golgi apparatus are becoming apparent based on studies using the related PH domains of FAPP1 and OSBP. As discussed above, the PH domains of CERT, OSBP, FAPP1 and FAPP2 are highly conserved and recruited to the Golgi apparatus via binding to PtdIns 4-phosphate (Section 4.1). PtdIns-4 kinase typeIIa (PtdIns-4KIIa) and -IIIh (PtdIns-4KIIIh) produce the majority of PtdIns 4-phosphate at the Golgi apparatus [168,169], and are localized to the TGN and cis/medialGolgi compartments, respectively [124]. A recent study demonstrated that the OSBP and FAPP1 PH domains predominantly recognize PtdIns 4-phosphate produced by PtdIns-4KIIIh and co-localize with PtdIns-4KIIIh in cis/ medial-Golgi compartments, but co-localize at the TGN with PtdIns-4KIIa when highly overexpressed [123]. The inhibition of PtdIns-4KIIIh activity by high concentrations of wortmannin (which does not inhibit PtdIns-4KIIa) or a dominant-negative, catalytically-inactive mutant of PtdIns4KIIIh (PtdIns-4KIIIhD656A) significantly decreased the localization of OSBP and FAPP1 PH domains to the cis/ medial elements of the Golgi apparatus. In the case of FAPP1, decreased PtdIns-4KIIIh activity also caused a shift in localization to the TGN, presumably due to sustained PtdIns 4-phosphate generation at the TGN by PtdIns-4KIIa [124]. In yeast, OSBP PH domain recruitment to the Golgi apparatus was dependent on Pik1p, the homolog of PtdIns4KIIIh [117]. Finally, ARF1 has been shown to regulate Golgi localization of PtdIns-4KIIIh, which may partially reflect ARF1-dependent Golgi localization of OSBP, FAPP1 and FAPP2 [121,170]. These observations strongly support a dominant role for PtdIns-4KIIIh-generated PtdIns 4-

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phosphate in the recruitment of the OSBP, FAPP and CERT to cis/medial/trans-cisternae of the Golgi apparatus. PtdIns transfer protein h (PITPh) is also implicated in the regulation of CERT-dependent ceramide transport and SM synthesis. A number of studies have shown that PITPh transfers SM, as well as PtdIns and PtdCho, between membranes [171 – 174], and the overexpression of PITPh in NIH-3T3 cells stimulated SM synthesis following exogenous sphingomyelinase treatment [175]. The PtdIns transfer activity of PITPh could indirectly effect CERT recruitment and the delivery of ceramide to SMS1, leading to increased SM synthesis through increasing PtdIns 4-phosphate generation at the Golgi apparatus. 5.2. Vesicular A portion of ceramide is transported to the Golgi apparatus from ER-derived vesicles (Fig. 2; Section 3), yet the molecular mechanism for the recruitment of ceramide into COPII-coated vesicles is unknown. Studies from yeast suggest that ceramide recruitment into vesicles is necessary to remodel the DAG-GPI-anchor to include ceramide and sustain GPI – protein transport [83]. Ceramide not used for this specific purpose could potentially reach the Golgi apparatus for production of GlcCer or SM on the cytosolic or luminal surfaces, respectively. 5.3. Vectorial transport It is presently unclear how monomeric lipid transport proteins achieve vectorial transport. In vitro assays to study lipid transport proteins usually assess lipid equilibration between donor and acceptor vesicles and omit specific targeting components and enzymes that contribute to vectorial transport in vivo. For example, VAP, PtdIns 4phosphate, SMS1 and ARF1 potentially affect CERTdependent ceramide transport between the ER and Golgi apparatus. Vectorial transport of ceramide by CERT (Fig. 2) could be achieved in the following manner. Since SM synthesis occurs in the Golgi lumen, SMS1 conversion of ceramide to SM would maintain a low ceramide concentration on the cytosolic surface of the Golgi apparatus compared to the ER, thus transport by CERT would proceed down a concentration gradient. As described in Section 5.1, ceramide binding to CERT may result in conformational changes leading to alternating exposure and concealment of PH and FFAT domains, resulting in unidirectional ceramide transport between the ER and Golgi apparatus. Finally, vectorial transport of sphingolipids to the PM would also be facilitated by their exclusion from Golgi-derived COPIcontaining retrograde transport vesicles [176]. 5.4. Regulation by sterols It is apparent that cholesterol and SM levels are coordinately regulated both in terms of metabolism and

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physical association in lipid rafts (reviewed in [177]). However, we have a limited understanding of the mechanisms that control the coordinate biosynthesis of cholesterol and SM. Oxysterols are hydroxy, keto or epoxy derivatives of cholesterol believed to be endogenous regulators of cholesterol homeostasis. Oxysterols cause a potent downregulation of de novo cholesterol synthesis and low density lipoprotein (LDL) uptake, increase the expression of proteins necessary for cholesterol removal and increase the cellular storage of cholesterol as cholesteryl esters [178 – 185]. One of the more efficacious of these oxysterols, 25hydroxycholesterol (25-OH), also triggers an increase in SM synthesis and mass in CHO cells [186]. Interestingly, other oxysterols known to suppress cholesterol synthesis did not stimulate SM production. 25-OH did not affect the activities of SPT or SMS, but stimulated conversion of ceramide to SM, indicating that ER-to-Golgi ceramide transport was enhanced [186]. Since CERT has now been identified as the ER-to-Golgi ceramide transport protein, 25-OH emerges as a novel regulator of this transport pathway. Several lines of evidence suggest that OSBP, a high affinity (K D = 10 nM) cytosolic receptor for 25-OH, mediates 25-OH-regulated, CERT-dependent transport of ceramide. Treatment of CHO cells with 25-OH resulted in the translocation of OSBP to the Golgi apparatus [122], the site of SM synthesis. When OSBP was over-expressed in CHO cells, 25-OH-mediated SM synthesis was augmented compared to mock controls [187]. Subsequently, it was shown that the overexpression of a PH domain mutant of OSBP (W174A) located constitutively to the ER and significantly reduced SM synthesis [142]. ER-to-Golgi transport of ceramide was inhibited in these cells, as indicated by the accumulation of C5-DMB-ceramide in unusual ER-associated inclusions induced by the expression of OSBP W174A and decreased [3H]serine incorporation into SM. While ceramide transport was compromised, vesicular trafficking was unaffected as determined by vesicular stomatitis virus G protein to the transport to the Golgi apparatus. Additional evidence supporting a role for OSBP in CERT-dependent ceramide transport comes from studies using sterol regulatory defective-6 (SRD6) cells, which have a mutation in the sterol-regulation element binding protein (SREBP) site 2 protease leading to chronic suppression of de novo cholesterol synthesis and uptake. The treatment of SRD6 cells with 25-OH did not cause increased SM synthesis, unless cells were supplemented with cholesterol [188]. OSBP was dephosphorylated and localized to the Golgi apparatus in sterol-depleted SRD6 cells, but was phosphorylated and localized to the ER/cytosol in SRD6 cells supplemented with sterols. These results were reproduced in CHO cells treated with the de novo cholesterol synthesis inhibitor lovastatin. Thus, cholesterol-dependent phosphorylation of OSBP may act as a switch to regulate the control of CERT-dependent ceramide transport to the Golgi apparatus by 25-OH. Finally, 25-OH did not elevate SM synthesis in CHO cells where OSBP expression was

suppressed using RNA interference (RNAi) (Perry and Ridgway, unpublished results). Based on these results, OSBP acts to integrate cholesterol and SM metabolism by sensing increased cholesterol levels through oxysterol binding, resulting in increased CERT-dependent ceramide transport to the Golgi apparatus and increased SM synthesis. The physiological relevance for increasing SM may be to buffer against increased cholesterol and/or to maintain SM:cholesterol ratios to preserve lipid raft domain function. Consistent with a role as an cholesterol/oxysterol sensor, OSBP may also function to integrate cholesterol metabolism with cell growth through the regulation of extracellular signal-regulated kinase (ERK) phosphorylation [189]. A clue to the mechanism of OSBP regulation of CERT lies in their shared domain structure (Fig. 1A) and intracellular localization (Fig. 2). Common ER and Golgi binding sites in these two proteins suggest that OSBP may increase the recruitment of CERT at either organelle to enhance ceramide transport. For example, in the scenario where CERT forms MCS between the ER and Golgi apparatus (Fig. 2), OSBP may serve to increase the surface area of the MCS by forming simultaneous contacts between VAP and PtdIns 4-phosphate. Furthermore, the avidity of OSBP and CERT for each organelle and enhancement or stabilization of the MCS could be strengthened by homodimer formation [109,122] and the potential interaction between OSBP and CERT homodimers. In the case where CERT transports ceramide through the cytosolic compartment, OSBP may augment the recruitment of ceramide-bound CERT to the Golgi apparatus by increasing PtdIns 4-phosphate synthesis through ARF1 interaction or clustering at the Golgi apparatus. 5.5. Pharmacological regulators The only known inhibitor that specifically targets ceramide trafficking is the ceramide analogue N-(3-hydroxy-1hydroxymethyl-3-phenylpropyl)dodecanamide (HPA-12), which inhibits CERT-dependent ceramide transport between membranes in vivo and in vitro [154,190]. Another potential inhibitor of CERT-dependent ceramide transport is the tricyclic xanthate derivative, tricyclodecan-9-yl xanthate (D609). D609 was reported to inhibit SMS activity in vitro and reduce SM synthesis in vivo [191]. However, the apparent inhibition of SM synthesis in CHO cells by D609 was due to the accumulation of ceramide in the ER [37]. The mechanism for ceramide accumulation was not unambiguously determined, but could have resulted from the inhibition of CERT-dependent transport, transbilayer movement, and/or saturation of CERT-dependent transport pathway.

6. Perspectives Although this review focused primarily on the molecular mechanisms and regulation of CERT-dependent ceramide

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transport, similar processes may be involved in interorganelle transport of other lipids. Lipids are small molecules that do not contain the appropriate sorting signals for specific membrane targeting, necessitating lipid transport proteins to function as surrogates for their distribution to appropriate target sites. The regulation of these independent transport pathways involves auxiliary protein partners, such as OSBP, that coordinate the delivery of lipids to target sites with the metabolic requirements of the cell. In the ER – Golgi compartment, VAP and PtdIns 4-P could act as nucleation sites for the recruitment of numerous lipidbinding/transport proteins. Interactions at these sites between lipid-binding proteins would then serve to integrate metabolisms at the level of lipid transfer. Similar interactions could occur in other membrane compartments. For instance, ORP4, which belongs to the same ORP subfamily as OSBP, binds 25-OH and has similar intracellular targeting domains [192]. But instead of interacting with the Golgi apparatus, ORP4 interacts with vimentin intermediate filaments, which are implicated in glycosphingolipid [193,194] and cholesterol [195] transport in post-Golgi compartments. Thus, in contrast to the discrete lipid transport function of CERT, the numerous members of the ORP family may function to integrate lipid metabolism at distinct intracellular compartments through the regulation of specific lipid-transport proteins. Acknowledgements We thank Monique Guilderson for the design and production of Fig. 2. Work described herein was supported by operating grants from the Canadian Institutes for Health Research (CIHR) and Heart and Stroke Foundation of New Brunswick (to NDR). RJP received a doctoral award from the Heart and Stroke Foundation of Canada (HSFC). References [1] M. Dobiasova, A simple method for the separation of minute amounts of tissue lipids by thin-layer chromatography and gas – liquid chromatography, J. Lipid Res. 58 (1963) 481 – 482. [2] N.H. Andersen, E.M. Leovey, Identification and quantitative determination of prostaglandins by high pressure liquid chromatography, Prostaglandins 6 (1974) 361 – 374. [3] M.J. Cooper, M.W. Anders, Determination of long chain fatty acids as 2-naphthacyl esters by high pressure liquid chromatography and mass spectrometry, Anal. Chem. 46 (1974) 1849 – 1852. [4] R.H. McCluer, J.E. Evans, Preparation and analysis of benzoylated cerebrosides, J. Lipid Res. 14 (1973) 611 – 617. [5] J. Gottfries, J.E. Mansson, P. Fredman, C.J. Wikstrand, H.S. Friedman, D.D. Bigner, L. Svennerholm, Ganglioside mapping of a human medulloblastoma xenograft, Acta Neuropathol. (Berl.) 77 (1989) 283 – 288. [6] M. Pollard, J. Ohlrogge, Testing models of fatty acid transfer and lipid synthesis in spinach leaf using in vivo oxygen-18 labeling, Plant Physiol. 121 (1999) 1217 – 1226. [7] G.A.J. Thompson, The Regulation of Membrane Lipid Metabolism, CRC Press, Inc., Boca Ranton, 1992, pp. 1 – 20.

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