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Identification of a family of animal sphingomyelin synthases

2003; Springer Nature; Volume: 23; Issue: 1 Linguagem: Inglês

10.1038/sj.emboj.7600034

1460-2075

Klazien Huitema, Joep van den Dikkenberg, Jos F. Brouwers, Joost C. M. Holthuis,

Lysosomal Storage Disorders Research

Article18 December 2003free access Identification of a family of animal sphingomyelin synthases Klazien Huitema Klazien Huitema Department of Membrane Enzymology, Faculty of Chemistry, Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Joep van den Dikkenberg Joep van den Dikkenberg Department of Membrane Enzymology, Faculty of Chemistry, Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Jos FHM Brouwers Jos FHM Brouwers Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Joost CM Holthuis Corresponding Author Joost CM Holthuis Department of Membrane Enzymology, Faculty of Chemistry, Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Klazien Huitema Klazien Huitema Department of Membrane Enzymology, Faculty of Chemistry, Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Joep van den Dikkenberg Joep van den Dikkenberg Department of Membrane Enzymology, Faculty of Chemistry, Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Jos FHM Brouwers Jos FHM Brouwers Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Joost CM Holthuis Corresponding Author Joost CM Holthuis Department of Membrane Enzymology, Faculty of Chemistry, Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Author Information Klazien Huitema1, Joep van den Dikkenberg1, Jos FHM Brouwers2 and Joost CM Holthuis 1 1Department of Membrane Enzymology, Faculty of Chemistry, Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands 2Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands *Corresponding author. Department of Membrane Enzymology, Faculty of Chemistry, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands. Tel.: +31 30 253 6630; Fax: +31 30 252 2478; E-mail: [email protected] The EMBO Journal (2004)23:33-44https://doi.org/10.1038/sj.emboj.7600034 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Sphingomyelin (SM) is a major component of animal plasma membranes. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, yielding diacylglycerol as a side product. This reaction is catalysed by SM synthase, an enzyme whose biological potential can be judged from the roles of diacylglycerol and ceramide as anti- and proapoptotic stimuli, respectively. SM synthesis occurs in the lumen of the Golgi as well as on the cell surface. As no gene for SM synthase has been cloned so far, it is unclear whether different enzymes are present at these locations. Using a functional cloning strategy in yeast, we identified a novel family of integral membrane proteins exhibiting all enzymatic features previously attributed to animal SM synthase. Strikingly, human, mouse and Caenorhabditis elegans genomes each contain at least two different SM synthase (SMS) genes. Whereas human SMS1 is localised to the Golgi, SMS2 resides primarily at the plasma membrane. Collectively, these findings open up important new avenues for studying sphingolipid function in animals. Introduction Sphingomyelin (SM) is an abundant constituent of cellular membranes in a wide range of organisms, from mammals (Ullman and Radin, 1974) and nematodes (Satouchi et al, 1993) to protozoa like the human malaria parasite Plasmodium falciparum (Elmendorf and Haldar, 1994). SM is preferentially concentrated in the outer leaflet of the plasma membrane. Its high packing density and affinity for sterols help provide a rigid barrier to the extracellular environment and play a role in the formation of lipid rafts, specialised areas in cellular membranes with important functions in signal transduction and membrane trafficking (Simons and Toomre, 2000; Holthuis et al, 2001). Since the discovery of the ‘SM cycle’ as a putative signalling system analogous to well-known second messenger systems like the phosphoinositide pathway, SM has emerged to the focus of interest in many research laboratories (Kolesnick and Hannun, 1999; Andrieu-Abadie and Levade, 2002). SM synthesis is mediated by a phosphatidylcholine:ceramide cholinephosphotransferase, or SM synthase, which transfers the phosphorylcholine moiety from phosphatidylcholine (PC) onto the primary hydroxyl of ceramide, thus generating SM and diacylglycerol (DAG; Ullman and Radin, 1974; Voelker and Kennedy, 1982; Marggraf and Kanfer, 1984). Since the enzyme is also able to catalyse the reverse reaction, namely the formation of PC from SM and DAG (Marggraf and Kanfer, 1984; van Helvoort et al, 1994), it may regulate, in opposite directions, the cellular levels of the bioactive lipids ceramide and DAG. The latter two are important regulators of membrane trafficking, cell proliferation and apoptosis (Kolesnick and Hannun, 1999; Bankaitis, 2002; Brose and Rosenmund, 2002; Pettus et al, 2002). Hence, one may anticipate that the physiological significance of SM synthase goes beyond the formation of SM. The subcellular localisation of the enzyme has been the subject of numerous studies. After the initial debate had focused on whether SM synthase is located in the Golgi or at the plasma membrane (Marggraf et al, 1981; Voelker and Kennedy, 1982; Lipsky and Pagano, 1985), subsequent work revealed evidence for the presence of SM synthase activity in both membranes (Futerman et al, 1990; Jeckel et al, 1990; van Helvoort et al, 1994). Whether SM synthesis detected at these locations is due to the presence of more than one isoform of the enzyme has remained an open issue. Progress in understanding the biological roles of SM synthesis and its regulation is hampered by the fact that no successful purification of the responsible enzyme has been achieved. Recent work has identified a bacterial SM synthase released from Pseudomonas aeruginosa (Luberto et al, 2003). However, this activity is a soluble protein, hence in contrast to the animal enzyme that is tightly associated with the membrane (Ullman and Radin, 1974; Voelker and Kennedy, 1982). Other efforts focused on the isolation of SM synthase mutants by screening Chinese hamster ovary cells for resistance to an SM-directed cytolysin (Hanada et al, 1998). Instead of mutants with a primary defect in SM synthase, this method yielded mutants defective in serine palmitoyltransferase activity or blocked in ceramide transport to the site of SM synthesis (Fukasawa et al, 1999). Here, we pursued a complementary approach for the identification of animal SM synthase that takes advantage of structural information available for enzymes catalysing analogous reactions. In contrast to most animal cells, plants, fungi and yeast do not produce SM. Instead, these organisms add phosphoinositol to phytoceramide to generate inositolphosphorylceramide (IPC) (Dickson, 1998). IPC production in the yeast Saccharomyces cerevisiae requires the product of the AUR1 gene (Nagiec et al, 1997). Sequence analysis of Aur1p proteins from different fungi revealed four conserved motifs (Heidler and Radding, 2000), two of which are similar to the C2 and C3 domains present in members of the lipid phosphate phosphatase (LPP) family (Waggoner et al, 1999). LPPs play a critical role in cell signalling by controlling the conversion of bioactive lipid phosphate esters such as (lyso)phosphatidic acid and sphingosine-1-phosphate to their dephosphorylated counterparts. The conserved C1, C2 and C3 domains in LPPs likely constitute the active site for cleavage of the bond between the lipid hydroxyl and phosphate groups (Neuwald, 1997). In the case of Aur1p, this reaction would represent the first step in the transfer of inositolphosphate from PI, with the resulting enzyme-phosphate intermediate being subjected to nucleophilic attack by the oxygen of ceramide rather than the oxygen of water used by LPPs. The presence of LPP-like motifs, together with the IPC synthase activity found associated with affinity-purified Aur1p (van den Dikkenberg and Holthuis, in preparation), suggests that Aur1p is directly responsible for IPC synthesis. The above considerations led us to develop a bioinformatics and functional cloning strategy to identify the enzyme responsible for SM synthesis in animals. By searching the database using a sequence motif shared by LPPs and Aur1p homologues, a set of 27 candidate SM synthase sequences from humans, mice and Caenorhabditis elegans was collected and assembled into different protein families. Several members from each family were cloned and analysed for their ability to mediate SM synthesis upon heterologous expression in S. cerevisiae, an organism lacking SM synthase activity. This approach resulted in the identification of multiple animal SM synthases. We find that animal genomes each contain at least two SM synthase genes and provide evidence that this multiplicity in enzymes is used to interconvert PC and ceramide to DAG and SM at different cellular locations. Results Identification of candidate SM synthases from the animal database Figure 1A shows an outline of the bioinformatics approach used to identify candidate sequences for SM synthases from the animal database. CSSs were identified based on the following criteria: (1) presence of a sequence motif, H-[YFWH]-X2-D-[VLI]-X2-[GA]-X3-[GSTA], shared by previously characterised LPPs and Aur1p homologues; (2) biochemical function should be unknown; (3) no structural homologues in S. cerevisiae, since this organism lacks SM; and (4) presence of multiple (>2) transmembrane domains, since the enzyme mechanism is intramembranous and because LPPs and Aur1p proteins have six predicted membrane spans. Sequences conforming to all four criteria were subsequently used as queries in a BLAST search to track down homologous sequences that were missed in the initial search due to deviations in the LPP/Aur1p motif. This approach yielded nine human, nine mouse and nine C. elegans sequences that could be grouped into three major protein families, designated CSS1, CSS2 and CSS3 (for candidate SM synthase family 1–3; Figure 1B and C). Except for the presence of a common sequence motif, CSS proteins displayed no significant sequence similarity to Aur1p proteins or to LPP family members with known biochemical functions. However, human CSS1β1 is identical to PRG1, a neuron-specific candidate phosphatidic acid phosphatase with a role in axon growth and regenerative sprouting (Brauer et al, 2003). Database accession numbers of CSS proteins are listed in the legend of Figure 1. Figure 1.Selection and phylogenetic analysis of CSSs. (A) Animal entries in SwissProt/TrEMBL were searched for the presence of a sequence motif shared by LPPs and Aur1p proteins and then further selected on the basis of three additional criteria, as indicated. (B) Phylogenetic tree of human CSSs and previously characterised members of the human LPP superfamily. (C) Phylogenetic tree of CSS proteins from human (Hs), mouse (Mm), C. elegans (Ce) and D. melanogaster (Dm), and of Aur1p proteins from S. cerevisiae (Sc), Schizosaccharomyces pombe (Sp) and Candida albicans (Ca). Asterisks denote CSS proteins expressed in S. cerevisiae and tested for SM synthase activity. SM synthases (SMS) are marked in red. SwissProt/TrEMBL accession numbers of CSS proteins are: (1) Q9TYV2; (2) Q20696; (3) Q9VS60/Q9VS61; (4) Q96LT4; (5) Q9DA37; (6) Q86VZ5; (7) Q8VCQ6; (8) Q8NHU3; (9) Q9D4B1; (10) Q9XTV2; (11) Q20735; (12) Q965Q4; (13) Q9D606; (14) Q9NXE2; (15) Q8VCY8; (16) Q96GM1; (17) Q96MP0; (18) AAP57768; (19) Q9BQF9; (20) AAP57767; (21) Q22250; (22) Q9TXU1; (23) Q9VNT9; (24) Q9VNU1; (25) Q10022; (26) Q9D4F2; (27) Q8IY26; (28) Q91WB2; (29) Q96SS7; (30) Q8T8T9; (31) Q22461. Note that Q96LT4 contains a partial protein sequence and that the complete ORF was deduced from a corresponding EST clone (Table I). Download figure Download PowerPoint A subset of CSS3 family members displays SM synthase activity To investigate whether any of the three CSS families indeed contained SM synthases, the open reading frames of human, mouse and C. elegans members for which full-length cDNAs could be obtained were cloned into a yeast multicopy, GAL1 promoter plasmid in frame with a carboxy-terminal V5 epitope. The resulting plasmids were used to transform wild-type yeast and the transformants were shifted to galactose-containing medium to induce expression of recombinant proteins. Expression of proteins was verified by Western blot analysis using anti-V5 antibodies (Figure 2A). Thus, 13 of the 27 selected CSS sequences were expressed and analysed for SM synthase activity (marked by asterisks, Figure 1C). To this end, yeast cells expressing CSS protein were lysed and incubated with fluorescent C6-NBD-ceramide (NBD-Cer), a known substrate of mammalian SM synthase. Incubations were performed in the presence of N-ethylmaleimide (NEM), a potent inhibitor of yeast-associated SMases that does not affect mammalian SM synthase (see Supplementary Figure 1). Reaction mixtures were next subjected to one-phase lipid extraction and the lipids separated by one-dimensional thin layer chromatography (TLC). Figure 2.A subset of CSS3 family members displays SM synthase activity upon expression in yeast. (A) Immunoblots of cells expressing various CSS proteins were stained with antibodies recognising the V5 epitope-tagged carboxy termini of CSS proteins. Control denotes cells transformed with empty vector. (B) TLC separation of reaction products generated when NBD-ceramide (NBD-Cer) was incubated with lysates of control or CSS-expressing cells in the presence (+) or absence (−) of the IPC synthase inhibitor aureobasidin A (Aba). (C) Metabolic labelling of cells expressing human CSS3α1/SMS1 or CSS3α2/SMS2 with [14C]-choline and NBD-ceramide. The lipids were extracted, separated by two-dimensional TLC and analysed for fluorescence and radioactivity. Note that SMS1- and SMS2-expressing cells, but not control cells, synthesised NBD-SM, and that this NBD-SM was labelled with [14C]-choline. (D) Metabolic labelling of cells expressing human CSS3α2/SMS2 with [14C]-choline. The lipids were extracted, deacylated by mild alkaline hydrolysis (+NaOH) or control incubated (−NaOH) and separated by two-dimensional TLC before autoradiography. Note that SMS2-expressing cells synthesised alkaline-resistant species of [14C]-choline-labelled lipids (phytoSM) that were absent in control cells. Download figure Download PowerPoint Figure 2B shows that in lysates of control cells, NBD-Cer was converted exclusively into NBD-IPC. The same was true for lysates of cells expressing members of the CSS1 and CSS2 protein families. However, in lysates of cells expressing human CSS3α1, human CSS3α2, C. elegans CSS3α1 or C. elegans CSS3α2, NBD-Cer was converted to a second product with an Rf value identical to that of NBD-SM. The identity of this product as NBD-SM was confirmed by electrospray ionisation tandem mass spectrometry (ESI-MS/MS; data not shown). Moreover, metabolic labelling of cells expressing human CSS3α1 or CSS3α2 with [14C]-choline in the presence of NBD-Cer resulted in the production of radiolabelled NBD-SM (Figure 2C). When NBD-Cer was omitted, we noticed that CSS3α2-expressing cells generated small amounts of [14C]-choline-labelled lipids that were absent in control cells and distinct from PC and lysoPC. In addition, these lipids were resistant to mild alkaline hydrolysis, suggesting that they represent phytoceramide-based SMs (phytoSM; Figure 2D). Importantly, ESI-MS revealed that bovine brain ceramides incubated with detergent extracts of CSS3α2-expressing cells, but not control cells, are converted to SM (Figure 3). Together, these results indicate that CSS3α proteins not only use NBD-Cer but also recognise naturally occurring ceramides as substrates for SM synthesis. Figure 3.Detergent extracts of human CSS3α2/SMS2-expressing yeast cells support SM formation from bovine brain ceramides. Lines A and B show the reconstituted ion chromatograms of m/z 731.6, corresponding to the m/z ratio of protonated 18:0 SM, during the separation of molecular species of choline-containing phospholipids after solid-phase extraction of membrane extracts that had been incubated with bovine brain ceramides and egg PC. The elution time of an authentic standard of 18:0 SM is indicated in the chromatogram by an arrow (42.3 min). Line A was derived from detergent extracts of human CSS3α2/SMS2-expressing cells and line B from detergent extracts of control cells. Mass spectra recorded at the elution time of 18:0 SM confirmed that formation of 18:0 SM occurred in CSS3α2/SMS2-containing extracts (top right panel) but not in control extracts (bottom right panel). Download figure Download PowerPoint Human CSS3α1 and CSS3α2 share 57% sequence identity and are highly conserved in mammals (human–mouse: >90%). Based on the results presented herein, we propose to rename these proteins as SMS1 and SMS2, respectively. C. elegans CSS3α1 (ceSMS1) and CSS3α2 (ceSMS2) share 22–27% sequence identity with human SMS1 and SMS2, and apparently represent their functional counterparts in the nematode (Figure 2B). C. elegans contains a third, CSS3α-related protein, termed ceCSS3α3. Although we have not tested this protein for SM synthase activity, the finding implies that C. elegans is equipped with three independent SM synthases (Figure 1C). BLAST searches for orthologous sequences identified two proteins in the human malaria parasite P. falciparum (PlasmoDB accession numbers MAL6P1.177 and MAL6P1.178; also see below). Hence, a multiplicity of SM synthase genes appears to be a general feature of organisms generating SM. Apart from CSS3α/SMS proteins, the CSS3 family contains a second cluster of CSS3β or SMS-related (SMSr) proteins with members in humans, mice, C. elegans and Drosophila (Figure 1C). Human SMSr is highly conserved in mammals (human–mouse: 95%), shares >40% sequence identity with its orthologues in C. elegans and Drosophila, and is about 34% identical to human SMS1 and SMS2. Heterologous expression of human or C. elegans SMSr did not yield any detectable SM synthase activity (Figure 2B, right panel). The C. elegans CSS3γ protein, renamed SMSdr, is only distantly related to SMS and SMSr proteins (<22% identical; see also Figure 1C) and its ability to mediate SM synthesis was not tested. Human SMS1 and SMS2 function as PC:ceramide cholinephosphotransferases with reverse activity SM synthesis in animals proceeds by the liberation of phosphorylcholine from PC and its subsequent transfer onto the primary hydroxyl of ceramide. To establish whether human SMS1 and SMS2 function as PC:ceramide cholinephosphotransferases, their enzymatic characteristics were analysed in more detail. Hence, different donors of choline-P were tested as substrates for SM synthesis. To this end, detergent extracts of cells expressing human SMS1 or SMS2 were incubated with NBD-Cer and the formation of NBD-SM was monitored by TLC. A dilution of extracts proved necessary to render SMS-mediated synthesis of NBD-SM dependent on externally added head group donors (see Supplementary Figure 2). Under these conditions, free phosphorylcholine (Ch-P) and CDP-choline (CDP-Ch) did not support SMS1- or SMS2-mediated SM synthesis (Figure 4A). PC, on the other hand, was efficiently recognised as a substrate. SM itself was also used as a donor of the phosphorylcholine group. LysoPC was a very poor substrate. These results suggest that human SMS1 and SMS2 are transferases that require two fatty chains on the choline-P donor molecule in order to be recognised efficiently as a substrate. Figure 4.Human SMS1 and SMS2 function as PC:ceramide cholinephosphotransferases. (A) Detergent extracts of yeast cells expressing human SMS1, SMS2 or transformed with empty vector (control) were incubated with NBD-ceramide (18 μM) in the presence or absence of different potential head group donors (220 μM), as indicated. Formation of NBD-SM or NBD-IPC was monitored by one-dimensional TLC and quantified as described in Materials and methods. Note that addition of PI stimulated formation of NBD-IPC in all three extracts (asterisks). PC, phosphatidylcholine; SM, sphingomyelin; Ch, choline; Ch-P, phosphorylcholine; CDP-Ch, cytidine-5′ diphosphocholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PA, phosphatidic acid; PG, phosphatidylglycerol. (B) Detergent extracts of SMS2-expressing and control cells were incubated with NBD-ceramide and [3H]-choline-labelled PC. The lipids were extracted, separated by two-dimensional TLC and analysed for fluorescence and radioactivity. Note that only SMS2-expressing cells synthesised NBD-SM, and that this NBD-SM was labelled with [3H]-choline. Download figure Download PowerPoint We next investigated the possibility that human SMS1 or SMS2 would use non-choline phospholipids as substrates. None of the phospholipids tested (PE, PI, PS, PA, PG) other than PC supported SM formation (Figure 4A). Moreover, when extracts of cells expressing SMS2 were incubated with PC containing [3H]-choline, the formation of radiolabelled NBD-SM was observed (Figure 4B). Hence, it appears that SMS1 and SMS2 directly and specifically recognise the choline head group on their substrates. Previous work suggested that mammalian SM synthase is also capable of catalysing the reverse reaction, namely the formation of PC from SM and DAG (Marggraf and Kanfer, 1984; van Helvoort et al, 1994). To investigate whether this was also the case for human SMS1 and SMS2, extracts of cells expressing these proteins were incubated with NBD-DAG and the formation of NBD-PC monitored by TLC. Addition of SM induced NBD-PC formation in extracts of SMS1- or SMS2-expressing cells, but not in control cell extracts (Figure 5). Strikingly, PC itself proved more efficient than SM in stimulating SMS1- or SMS2-dependent NBD-PC formation. In contrast, addition of non-choline phospholipids (e.g. PI) had no effect. These data suggest that human SMS1 and SMS2, rather than functioning strictly as SM synthases, are transferases capable of using PC and SM as phosphocholine donors to produce PC or SM, dependent on the relative concentrations of DAG and ceramide as phosphocholine acceptors, respectively. Figure 5.Human SMS1 and SMS2 exhibit reverse activity. Detergent extracts of yeast cells expressing human SMS1, SMS2 or transformed with empty vector (control) were incubated with NBD-diacylglycerol (NBD-DAG; 18 μM) in the presence or absence of different head group donors (220 μM), as indicated. Formation of NBD-PC was monitored by one-dimensional TLC and quantified as described in Materials and methods. Download figure Download PowerPoint Mammalian SM synthase was found to be sensitive to the bacterial PC-phospholipase C inhibitor, D609 (Luberto and Hannun, 1998). In lysates of SMS1- or SMS2-expressing yeast cells, D609 inhibited SM synthesis in a dose-dependent manner (data not shown). The extent of inhibition observed for SMS1-mediated SM synthesis (50% at 100 μg/ml D609) was comparable to that reported for mammalian SM synthase (Luberto and Hannun, 1998). SMS2 proved two-fold less sensitive to the drug. Hence, human SMS1, SMS2 and the mammalian SM synthase activities described in the literature share many enzymatic characteristics. Structure and expression of human SMS1 and SMS2 Figure 6A shows a sequence alignment of human SMS1, SMS2 and SMSr. Hydrophobicity analysis predicted six membrane-spanning alpha helices connected by hydrophilic regions that would form extramembrane loops. A comparative analysis with SMS and SMSr sequences from mice, C. elegans, P. falciparum and Drosophila revealed that the number and spacing properties of transmembrane helices are well conserved. In addition, SMS proteins contain four highly conserved sequence motifs, designated D1, D2, D3 and D4 (Figure 6A and B, residues highlighted in blue). Motifs D3 (C-G-D-X3-S-G-H-T) and D4 (H-Y-T-X-D-V-X3-Y-X6-F-X2-Y-H) are similar to the C2 and C3 motifs in LPPs (shared residues highlighted in red) and include the histidine and aspartate residues (underlined) that form a catalytic triad mediating the nucleophilic attack on the lipid phosphate ester bond (Neuwald, 1997). As in LPPs, these residues are juxtaposed to transmembrane segments 4 and 6 of the SMS proteins and consequently would be oriented towards the same side of the membrane. This would suggest that motifs D3 and D4 are part of the catalytic site responsible for liberating cholinephosphate from PC during SM synthesis. Motifs D1 (P-L-P-D) and D2 (R-R-X8-Y-X2-R-X6-T), on the other hand, appear entirely unique to SMS proteins and are located in the first extramembrane loop and third transmembrane helix, respectively (Figure 6A). The SMSr proteins found in humans, mouse, Drosophila and C. elegans each contain exact copies of the D1 and D3 motifs, yet exhibit one or more conserved amino-acid substitutions in motifs D2 and D4 (Figure 6A and B, residues highlighted in green). Figure 6.Conserved sequence motifs in SMS1, SMS2 and SMSr proteins. (A) Alignment of human SMS1, SMS2 and SMSr amino-acid sequences. Identical residues are highlighted in black and conservative amino-acid substitutions in grey. Conserved residues within four homology motifs, designated D1–D4, are highlighted in blue and conservative amino-acid substitutions in the D2 and D4 motifs of SMSr proteins in green. Note that motifs D3 and D4 display similarity to the C2 and C3 domains in LPPs, with identical residues highlighted in red. Regions predicted to form transmembrane domains, TM1–TM6, are marked by a black line. (B) Alignment of the four homology motifs in SMS and SMSr proteins from humans (Hs), C. elegans (Ce), P. falciparum (Pf) and D. melanogaster (Dm). PlasmoDB accession numbers of PfSMS1 and PfSMS2 are MAL6P1.178 and MAL6P1.177, respectively. Putative active site residues in consensus sequences are underlined. Symbols used are: n, small neutral amino acid; Φ, aromatic amino acid; b, branched amino acid; x, any amino acid. Download figure Download PowerPoint Previous work showed that some members of the mammalian LPP superfamily are expressed in only a limited set of tissues (Waggoner et al, 1999; Brauer et al, 2003). Since differences in tissue distribution would provide a possible explanation for the existence of two different SM synthase isoforms in mammals, we investigated the expression profiles of human SMS1 and SMS2. As shown in Figure 7, northern blot analysis detected the presence of a low abundant 3.8 kb transcript for SMS1 in human brain, heart, kidney, liver, muscle and stomach. A major 1.9 kb transcript for SMS2 was expressed to a similar level in all of the above human tissues. These results suggest that human SMS1 and SMS2 are encoded by ubiquitously expressed genes. Figure 7.Human SMS1 and SMS2 are encoded by ubiquitously expressed genes. Northern blot analysis of SMS1 and SMS2 transcripts (arrows) in various human tissues. Random prime-labelled human SMS1 or SMS2 cDNA was hybridised to a human poly(A)+ RNA blot (Origene, Rockville, MD). As a control for loading, the RNA blot was stripped and rehybridised with a human β-actin cDNA probe. Mobilities of RNA size markers are indicated. Download figure Download PowerPoint Subcellular localisation and membrane topology SM synthesis occurs in the Golgi complex as well as at the plasma membrane of mammalian cells. Endosomes have been put forward as another major site of SM synthesis (Kallen et al, 1994), but this has been disputed (van Helvoort et al, 1997). It is not known whether SM synthase activity detected at these locations is due to the presence of more than one isoenzyme in the cell. This led us to examine the subcellular distribution of V5-tagged versions of human SMS1 and SMS2 in transfected HeLa cells using immunofluorescence microscopy. As shown in Figure 8A, SMS1 was concentrated in the perinuclear region where it displayed extensive colocalisation with sialyltransferase, a marker of trans Golgi cisternae. This colocalisation was also observed in cells treated with nocodazole, a drug causing fragmentation of the Golgi by disrupting the microtubular network, hence confirming the association of SMS1 with the Golgi apparatus. SMS2 displayed a different localisation pattern and was primarily concentrated at the plasma membrane (Figure 8B). A portion of SMS2 was also found in the perinuclear region where it colocalised with sialyltransferase (see Supplementary Figure 3). This Golgi-associated pool of SMS2 unlikely represents newly synthesised material en route to the cell surface, since it was also observed in cells after a 4-h chase with cycloheximide. There was no substantial colocalisation of SMS2 with markers of the endosomal/lysosomal system (e.g. EEA1, CD63, internalised transferrin; data not shown). Whether SMS2 cycles between the Golgi and the plasma membrane, and thereby passes through endosomes remains to be established. Figure 8.Human SMS1 and SMS2 localise to different cellular organelles. (A) HeLa cells co-transfected with