Serinc, an activity-regulated protein family, incorporates serine into membrane lipid synthesis.

Cell membranes contain various transporter proteins, some of which are responsible for transferring amino acids across membrane. In this study, we report another class of carrier proteins, termed Serinc1-5, that incorporates a polar amino acid serine into membranes and facilitates the synthesis of two serine-derived lipids, phosphatidylserine and sphingolipids. Serinc is a unique protein family that shows no amino acid homology to other proteins but is highly conserved among eukaryotes. The members contain 11 transmembrane domains, and rat Serinc1 protein co-localizes with lipid biosynthetic enzymes in endoplasmic reticulum membranes. A Serinc protein forms an intracellular complex with key enzymes involved in serine and sphingolipid biosyntheses, and both functions, serine synthesis and membrane incorporation, are linked to each other. In the rat brain, expression of Serinc1 and Serinc2 mRNA was rapidly up-regulated by kainate-induced seizures in neuronal cell layers of the hippocampus. In contrast, myelin throughout the brain is enriched with Serinc5, which was down-regulated in the hippocampus by seizures. These results indicate a novel mechanism linking neural activity and lipid biosynthesis.

Rapid macromolecular synthesis is a necessary component underlying long-term neuronal plasticity (1). In an attempt to identify molecules that are involved in this process we, as well as others, have used differential cloning strategies to identify genes that are rapidly induced in neurons of the hippocampus by excitatory neuronal activity (2,3). The set of genes identified includes transcription factors such as c-fos, c-jun, and zif268 (4), as well as effector molecules that may directly modify cellular and synaptic functions. Examples of this latter class of proteins include the inducible form of regulators of G-protein signaling (RGS2) and a key regulatory enzyme of polyamine metabolism (spermidine/spermine N 1 -acetyltransferase) (3,5). These proteins are rapidly induced in neurons and interact with constitutively expressed cellular and synaptic proteins to modify neuronal properties. In the present study, a novel activity-regulated protein family has been identified that serves as an effector molecule that facilitates the biosynthesis of lipids, a major macromolecular component of the nervous system. This effector protein localizes in the endoplasmic reticulum (ER) 2 membranes, where most of the membranes including the plasma membrane, are synthesized. Most of lipid biosynthetic enzymes are also embedded in and associated with ER membranes. Because the action of the protein is to incorporate serine into newly forming membrane lipids, it is different from a transporter that transfers amino acids across membranes.
Serine is a nonessential amino acid that can be synthesized by many cells. In the cell, serine serves as a building block for the synthesis of two major classes of membrane lipids, phosphatidylserine and sphingolipids. Although it is well known that cell membranes efficiently incorporate serine into their lipid bilayer despite the hydrophilicity, the molecular mechanism remained unclear. This study has demonstrated for the first time that a protein family having 11 transmembrane domains facilitates the incorporation of serine into both phosphatidylserine and sphingolipids. This is a function novel to proteins and is henceforth referred to as Serinc (serine incorporator). Because the Serinc family showed no amino acid homology to any known proteins, nothing was known about their function, although there have been several studies on this family. First, the predicted membrane topology has 11 transmembrane segments similar to amino acid transporters. Therefore, a recent report classified TMS1, a yeast member of the Serinc family, as an "uncharacterized (orphan) transporter" (6). Secondly, two mouse homolog genes for yeast TMS1, denoted as mouse TMS-1 and TMS-2 (corresponding to Serinc3 and Serinc1, respectively), were identified by another research group, and their neuronal expression and membrane localization were shown experimentally (7). That group also performed experiments to detect amino acid transport activity using TMS1 homolog-expressing cells, but all attempts were unsuccessful (7). Thirdly, some of the family members were reported to be differentially expressed in some environments; TDE1 (corresponding to Serinc3) expression was increased in both human lung tumors and mouse testicular tumors (8,9); and TPO1 (corresponding to Serinc5) expression was developmentally induced in a terminal differentiation stage of cultured oligodendrocytes (10). These findings implied the important role of this family in multiple aspects of cell physiology, but the function of this family remained unknown.
In this study, yeast proteomic data and serine-incorporating experiments using Escherichia coli offered the key to an understanding of the function of Serinc in the central nervous system. The background is that serine is universally used for the synthesis of membrane lipids in a broad range of organisms. Phosphatidylserine is present in both prokaryotic and eukaryotic cells, but it is formed in different manners among diverse species. In E. coli and yeast, phosphatidylserine synthase catalyzes the formation of phosphatidylserine from serine and CDP-diacylglycerol (11,12). In mammalian cells, phosphatidylserine is synthesized by a calcium-dependent base exchange reaction in which the head group of a preexisting phospholipid is replaced by serine (13). This serine base exchange enzyme (recently called mammalian phosphatidylserine synthase) is the sole mechanism available for phosphatidylserine formation by mammalian cells. On the other hand, sphingolipids are not found in most prokaryotes but are distributed in all eukaryotic cells. In various eukaryotic species, the first and committed step in de novo sphingolipid synthesis is catalyzed by serine palmitoyltransferase, which condenses serine and palmitoyl-CoA to form 3-ketodihydrosphingosine in a pyridoxal 5Ј-phosphate-dependent reaction (14).
Sphingolipid molecules (particularly sphingomyelin and glycolipids) are found predominantly in the noncytoplasmic half of membranes, where they self-associate into microaggregates together with cholesterol molecules. Closely packed sphingolipid-cholesterol clusters behave as rafts that move within the fluid bilayer (accordingly termed lipid rafts or microdomains) and function as platforms for the attachment of proteins when selected membranes are transported by some routes of intracellular membrane trafficking (15). An increasing number of recent studies indicate a functional role of the lipid raft and other microdomains during neural signaling, axonal guidance, and malignant tumorigenesis (16 -18). Here we demonstrate that three Serinc proteins specifically enhance the synthesis of phosphatidylserine and sphingolipids. Because sphingolipids are major components of lipid rafts, Serinc is likely to function as a lipid raft maker in activity-dependent neural plasticity and oncogenic transformation.

MATERIALS AND METHODS
Animals and Differential Screening of cDNA Library-Adult male rats (Sprague-Dawley) were used in studies of Serinc regulation. Kainate stimulation was performed as follows. Rats were injected subcutaneously with kainate (8 mg/kg of body weight). After this treatment, rats were sacrificed after 3.5 h under anesthesia with an overdose of diethyl ether. All parts of the procedures were approved by the Niigata University Committee on Animal Research. A cDNA library was construction using 5 g of poly(A) ϩ RNA isolated from kainate-stimulated rat hippocampus as described in the ZAP-cDNA synthesis kit (Stratagene, La Jolla, CA). The library was screened with [ 32 P]cDNA prepared by reverse transcription of poly(A) ϩ RNA from the hippocampus of control or kainate-stimulated rats as described previously (3).
In Situ Hybridization-Freshly dissected brain tissue was rapidly frozen in plastic molds placed on a dry ice/ethanol slurry. Control and experimental tissues were frozen in the same tissue block to assure identical conditions during storage and in situ hybridization. Frozen 10-m thick sections were mounted on gelatin-coated glass slides desiccated at Ϫ20°C. Hybridization was performed using 35 S-labeled riboprobes of three rat Serinc subtypes as described previously (19).
Two-hybrid Experiments-Experiments were performed as described previously (20) with some modifications. Transformation of pOBD2-vector into each open reading frame transformant was performed using an ScEasyComp transformation kit (Invitrogen, Carlsbad, CA). The yeast suspensions were plated onto synthetic plates lacking tryptophan and leucine and incubated for 7 days at 30°C. After colonies had grown they were picked and plated onto synthetic plates lacking tryptophan, leucine, and histidine to detect two-hybrid activation. Control transformations were included in each assay, without insert and with only pOBD2-serinc1.
E. coli Cultures, Preparation of Lysates and Membranes, and Western Blot Analysis-The cDNAs of rat Serinc1, Serinc2, and Serinc5 were subcloned into the E. coli expression vectors of pDEST14 and pDEST15 (Invitrogen). E. coli strain BL21-AI (Invitrogen) was transformed with these plasmids, and 100 ml of the culture was induced by 0.2% arabinose to express proteins. The cells were harvested 2 h after induction by centrifugation and resuspended at 10 ml/g wet cell weight in each assay buffer or Tris-buffered saline. 3 ml of the resuspended cells was sonicated on ice using a microprobe for 20 min. Unbroken cells were removed from the lysates by centrifugation (5000 ϫ g for 10 min), and the low speed supernatant was centrifuged at 4 ϫ 10 4 ϫ g for 40 min to provide the crude membrane pellet. Western blot analysis using anti-glutathione S-transferase (GST) antibody (Amersham Biosciences) was performed at a 1:1000 dilution as described in the manufacturer's protocol.
Yeast Cultures and Preparation of Microsomes-The wild-type and tms1⌬ strain of yeast BY4743 were transformed with each plasmid of pYES/DEST52-rat serinc1 and its empty vector (Invitrogen). The transformants were selected on synthetic plates lacking uracil, and the culture was induced with 2% galactose to express proteins. The cells were harvested 12 h after induction and resuspended at 2 ml/g wet cell weight in a buffer composed of 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride. Glass beads were added to the suspension, and cells were lysed by 10 cycles (60 s each) of vortexing with cooling on ice. Unbroken cells and beads were removed by centrifugation (5000 ϫ g for 10 min), and the low speed supernatant was centrifuged at 4 ϫ 10 4 ϫ g for 40 min to provide the microsomal pellet. The pellet was resuspended at 1 ml/g wet cell weight (ϳ10 mg/ml protein) in the same buffer containing 33% glycerol and stored at Ϫ80°C.
Mammalian Cell Cultures and Preparation of Lysates-SV40-transformed African green monkey kidney (COS-7) cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Approximately 1 ϫ 10 6 cells in a 10-cm dish were transfected with the lipoplexes containing 8 g of each or combinatorial DNA of pcDNA/DEST47-rat serinc, pcDNA3.2/V5DEST-rat serinc, pAsRed2-N1-mouse sptlc1, and their empty vectors (Invitrogen and BD Biosciences). The cells were harvested 60 h following transfection and resuspended in 240 l of each assay buffer. 0.25 ml of the resuspended cells was sonicated on ice using a microprobe for 1 min. Base Exchange Enzyme Activity and E. coli Serine-incorporating Activity-The base exchange reaction mixture contained 50 mM Tris-HCl, pH 7.4, 50 mM KCl, 15 mM CaCl 2 , 0.5 mM [ 3 H]serine (60 Ci), and the indicated amount of cell lysates in a total volume of 0.35 ml. After incubation at 37°C for 20 min, the reaction was terminated by adding 1.5 ml of chloroform/methanol (1/2, v/v), and phospholipids were extracted by the method of Bligh and Dyer (21). In screening assays of serine-incorporating activity using Serinc1-expressing E. coli, 1.4 mg of E. coli total lysates was incubated for 20 min at 37°C with 30 Ci of Serine Palmitoyltransferase Activity-The reaction mixture contained 0.1 M HEPES, pH 8.3, 5 mM dithiothreitol, 2.5 mM EDTA, 50 M pyridoxal 5Ј-phosphate, 0.2 mM palmitoyl-CoA, 1 mM [ 3 H]serine (60 Ci), and the indicated amount of membrane proteins or cell lysates in a total volume of 0.3 ml. After incubation at 37°C for 10 min, the reaction was terminated by the addition of 1.5 ml of chloroform/methanol (1/2). After 25 g of sphinganine was added as carrier, the lipid products were extracted by the method described previously (22).

RESULTS
Expression and Activity-dependent Induction of Serinc1 mRNA in the Hippocampus-Adult rats were injected subcutaneously with the glutamate analogue kainate (8 mg/kg of body weight), which induce seizures and long-term synaptic enhancement in the brain (2, 5), and then sacrificed 3 h after the kainate-induced seizures. A cDNA library was prepared from the hippocampus and differentially screened using radiolabeled cDNA prepared from untreated control and kainate-stimulated rat hippocampus. Nucleotide sequence analysis of one of the differentially expressed cDNAs identified it as an unknown protein (Gen-Bank TM accession number DQ103708), and it was named Serinc1.
To further investigate the neural activity-dependent regulation of Serinc1, we examined the cellular distribution of Serinc1 mRNA in control and seizure-treated rat brains by performing in situ hybridization. Expression was examined in the half-brains of naive rats (Fig. 1, C-side) and compared with expression in rats that had received kainateinduced seizures and were then sacrificed 3 h later (S-side). Serinc1 mRNA is normally expressed in the cerebral cortex, hippocampus, and cerebellar granule cell layer (Fig. 1, left panels, C-side), which is closely related to the localization of glutamatergic excitatory neurons (23). This result agrees with a recent report detailing the neuronal localization of mouse TMS-2 mRNA using in situ hybridization (7). Within 3 h after kainate-induced seizure, Serinc1 mRNA is significantly induced in neuronal cell layers of the hippocampal CA1-3 fields (Fig. 1, left panels,  arrow). Thus, following the kainate-induced seizures, Serinc1 mRNA levels increase rapidly in discrete brain regions.
The Serinc Family of Various Eukaryotic Species Exhibit Transmembrane Domains-Serinc1 mRNA encodes a protein of 453 amino acids ( Fig. 2A) with 98% identity to mouse TMS-2 protein. This result, together with the identical neuronal localization, suggests that they are counterparts. Two other reported proteins, human TDE1 and rat TPO1, show homology to Serinc1 with amino acid identities of 58 and 38%, henceforth referred to as Serinc3 and Serinc5, respectively ( Fig.  2A). Moreover, four novel mammalian family proteins and five Arabidopsis thaliana family proteins were identified by an NCBI data base search and were termed Serinc2, Serinc3B, Serinc4, Serinc4B, and A. thaliana Serinc I-V, respectively (Fig. 2). Thus, the Serinc family is highly conserved among eukaryotes, whereas no homologs were found in prokaryotic data bases. The mammalian family members share 58 -31% amino acid identity (Fig. 2C) and have similar molecular masses ( Fig. 2A, 432-473 amino acids). Furthermore, hydropathy analysis revealed that these member proteins contain 58 -53% hydrophobic amino acids, clustered into 11 regions of up to 30 amino acids long, suggesting membrane-spanning domains ( Fig. 2A, I-XI regions). However, none of the protein functions were known.
Comparative Localization and Regulation of Serinc2 and Serinc5 mRNA in Brain-We further examined the mRNA distribution of Serinc2 and Serinc5 in control and seizure-treated rat brains (Fig. 1). Overall levels of Serinc2 hybridization are less than either Serinc1 or Serinc5, although intense staining was apparent in the dentate gyrus of the hippocampus and the cerebellar Purkinje cell layer. Seizures strongly induced Serinc2 mRNA, which is restricted to the dentate gyrus ( Fig. 1, center panels, arrow). Serinc5 is distinct from Serinc1 and Serinc2 in the dense staining pattern of the white matter such as the external capsule, corpus callosum, and arbor vitae of the cerebellum. Moreover, in contrast to the up-regulation pattern of Serinc1 and Serinc2, expression of Serinc5 is down-regulated in the hippocampal CA fields and the dentate gyrus by seizures (Fig. 1, right panels, arrow). Thus, the divergent spatial and cellular expression and their different transcriptional controls indicate that the three subtype proteins play multiple distinct roles in the activity-dependent plasticity of the central nervous system.
Serinc Protein and Serine Synthetic Enzymes Form an Intracellular Protein Complex-The clue to elucidate the Serinc function came from its interacting proteins. We searched the yeast proteome data base and identified two yeast proteins (SER3 and YGP1) that directly interact with yeast TMS1 (24) (Fig. 3A). SER3 and YGP1 function in the biosynthesis of serine (Fig. 3A). Serine is synthesized from 3-phosphoglycerate, an intermediate in glycolysis. The first and committed step in this pathway is oxidation to 3-phosphohydroxypyruvate, catalyzed by the enzyme 3-phosphoglycerate dehydrogenase (SER3). This ␣-ketoacid is transaminated to 3-phosphoserine, which is then hydrolyzed to serine by a phosphatase. YGP1 is a glycoprotein of unknown function (25) but has a highly conserved domain of asparaginase. The putative asparaginase activity accelerates the serine synthetic pathway through the subsequent transamination reaction to glutamate. These findings suggest the involvement of TMS1 in cellular synthesis of serine. To explore whether mammalian Serinc proteins bind to these serine synthetic FIGURE 1. Regulation of Serinc1, Serinc2, and Serinc5 mRNA in the rat brain by seizure. In situ hybridization of the three Serinc subtypes in rat brain coronal sections is shown. The brains are composites of half-brains from a naive control rat (C-side) and a rat that received the kainate-induced seizure stimulation 3 h before being killed (S-side). The three Serinc mRNAs individually show unique neural localization throughout the brain. Identical hybridization patterns were detected in independent experiments (n ϭ 5-6). arb, arbor vitae of the cerebellum; cc, corpus callosum; CA, hippocampal CA fields; dg, dentate gyrus; ec, external capsule; gcl, cerebellar granule cell layer; hip, hippocampus; neo, neocortex; pcl, cerebellar Purkinje cell layer.
enzymes, cDNAs of SER3, YGP1, and their associated proteins were screened by two-hybrid methods using rat Serinc1 as bait (Fig. 3B). Both SER3 and YGP1 were identified as strong interacting partners of rat Serinc1, and PHO12 was identified as a weak interactor. The result indicates that the specific binding to serine synthetic enzymes is conserved among distant members of the Serinc family.  ). Thus, the serine transport across these membranes was unaffected by Serinc1, which supports the idea that Serinc is not an amino acid transporter. However, when the E. coli lysate was incubated with [ 3 H]serine, we found that expression of Serinc1 remark-ably enhanced the incorporation of [ 3 H]serine into its chloroform-soluble fraction. Examination of the radiolabeled products by thin-layer chromatography revealed that the synthesis of phosphatidylethanolamine increased by more than 10-fold over the base level (Fig. 4B,  middle panel, left two lanes). In E. coli cells, serine is incorporated into phosphatidylserine by the catalytic action of phosphatidylserine synthase (Fig. 4A). But phosphatidylserine is found only in trace amounts in the cell because it is rapidly converted to phosphatidylethanolamine, which constitutes about 70% of the phospholipid of this organism (Fig.  4B, left panel), by the presence of phosphatidylserine decarboxylase (26) (Fig. 4A). Therefore, this finding indicates that cellular expression of Serinc1 enhances phosphatidylserine synthesis from [ 3 H]serine. Similarly, when [ 14 C]glucose was incubated with the E. coli lysate, Serinc1 enhanced the radioactivity of phosphatidylethanolamine by more than 16-fold over the base level (Fig. 4B, middle panel, right two lanes), which reflects the functional link between a Serinc protein and serine synthetic enzymes. Overexposure of a film revealed that Serinc1 enhanced the [ 3 H]phosphatidylserine synthesis to a detectable level (Fig. 4B, right   FIGURE 2. Structure and conservation of the Serinc family. A, amino acid alignment of the five mammalian Serinc proteins (Serinc1-5) and yeast TMS1. Gray boxes highlight residues that are conserved among more than three members. The predicted transmembrane domains are underlined and numbered I-XI. GenBank TM accession numbers: Serinc1, DQ103708; Serinc2, DQ103709; Serinc3, NM_006811; Serinc4, BC026459 and DQ103711; Serinc5, DQ103710; yeast TMS1, NP_010390. B, schematic structures of mammalian Serinc3, Serinc4, and their alternative splicing isoforms (Serinc3B and Serinc4B). Filled boxes, 100% identical regions; open boxes, partially homologous regions; solid lines, nonhomologous regions; broken lines, deletions. GenBank TM accession numbers: Serinc3B, U49188; Serinc4B, AK044479. C, percent identity between each amino acid sequences of the seven mammalian Serinc proteins, yeast TMS1, and Arabidopsis SerincI. Bold type labels a group of high percent identity, and parentheses indicate alternative splicing isoforms. GenBank TM accession numbers: A. thaliana SerincI, NP_187268; SerincII, NP_173069; SerincIII, NP_189089; SerincIV, NP_567403; SerincV, NP_850202.

panel, 2nd lane).
To verify further that Serinc1 enhances phosphatidylserine biosynthesis, the conversion of phosphatidylserine to phosphatidylethanolamine was blocked by utilizing the decarboxylase inhibitor hydroxylamine (NH 4 OCl) (11) (Fig. 4B, right panel, 3rd and 6th lanes). Although Serinc1-dependent enhancements of phosphatidylethanolamine completely disappeared in the presence of 10 mM hydroxylamine, [ 3 H]phosphatidylserine accumulated further (Fig. 4B, right panel, 3rd lane) through the action of Serinc1. The imbalance between levels of [ 3 H]phosphatidylserine accumulation and [ 3 H]phosphatidylethanolamine disappearance reflected a cellular feedback regulatory mechanism that reduces the rate of [ 3 H]phosphatidylserine synthesis compared with the amount of accumulation in the presence of hydroxylamine. Although other E. coli lipids were faintly radiolabeled by way of other serine metabolic pathways (Fig. 4B, left two panels, asterisk), these activities were unaffected by Serinc1 (right panel, asterisk), indicating the specific action on phosphatidylserine synthesis.
Membrane Localization of Serinc1 Protein and Function-Specific membrane localization of Serinc1 protein was demonstrated by immunoblotting the total, soluble, and membrane preparations from E. coli cells expressing GST-tagged Serinc1 (Fig. 4C). We incubated the membrane fraction of native Serinc1-expressing E. coli with [ 3 H]serine and CDP-diacylglycerol and monitored the activity of phosphatidylserine synthase (Fig. 4D). Consistent with the result of the experiment using lysates, activity enhancement of phosphatidylserine synthase to 3.1-fold of the base level (190 Ϯ 10 pmol/min/mg of protein) was detected in the membranes through the action of Serinc1. These results, together with the predicted topography of 11 transmembrane domains, indicate that a Serinc1 protein localizes in cell membranes, resulting in an acceleration of their phosphatidylserine synthesis rates.

Enhancement of the Phosphatidylserine and Sphingolipid Biosynthesis by Expression of Serinc1 in Mammalian and Yeast ER Membranes-
Because Serinc family proteins are found in various eukaryotes, the cellular function was reinvestigated with experiments using cultured mammalian COS cells. COS cells were transfected with green fluorescent protein (GFP)-tagged and native Serinc1 plasmids to examine the subcellular distribution and function in mammalian cells. Serinc1-GFP, distributed in a reticular pattern localized around the nuclear envelope, was identical to the intracellular distribution of ER membranes labeled with the dye ER-Tracker (Fig. 5A, left two panels). This result indicates the exclusive localization of Serinc1 in the mammalian ER membranes, where most of the membranes in a eukaryotic cell are synthesized. The activity of the serine base exchange enzyme was measured in lysates of Serinc1-expressing COS cells (Fig. 5B). Consistent with the results of experiments using E. coli, cellular expression of Serinc1 enhanced the synthesis of [ 3 H]phosphatidylserine to 2.8-fold of the base level (2.5 Ϯ 0.3 pmol/min/mg of protein). Because the structure and catalytic reaction of a serine base exchange enzyme are completely different from those of an E. coli phosphatidylserine synthase, these results suggest that the function of Serinc1 is to provide serine molecules not for a particular enzyme but for some distinct enzymes in the membranes. The binding strengths of Serinc1 to SER3 and YGP1 are specific and stronger than to PHO12. PHO12 is the secreted acid phosphatase that directly binds to SER3. The DNA-binding domain vector pOBD2 and the Gal4 activation domain vector pOAD (20) were used. The screening was performed by transforming a plasmid of pOBD2-rat serinc1 into a yeast reporter strain PJ69-4a expressing the following Saccharomyces cerevisiae proteins as Gal4 activation domain fusions: Control (no protein), SER3, YGP1, and other proteins. In SER3, YGP1, and PHO12, colonies were detected after 3 weeks of growth on medium lacking tryptophan, leucine, and histidine and supplemented with 3 mM 3-amino-1,2,4-triazole, thus allowing growth only of cells that express the his3 two-hybrid reporter gene. No colony was detected in the control well. To demonstrate the growth rates, the same numbers of positive colonies were picked and replated onto the selection medium; the result of their growth is shown here. ]glucose for 20 min at 37°C, and the lipid extracts were developed on a silica gel plate with chloroform/methanol/acetic acid/ formic acid (50/30/4.5/6.5). Lipid bands and their radioactive signals on the plate were visualized with iodine vapor (left panel) and fluorography (right two panels), respectively. Lipids were identified with authentic standards phosphatidylethanolamine and phosphatidylserine. The middle and right plates were exposed to x-ray film for 4 and 35 days, respectively. The levels of radioactivity in each band were quantitated using a scintillation counter. C, Western blot analysis of total, soluble, and membrane fraction of GST-Serinc1-expressing E. coli probed with anti-GST antibodies. Each lane contained 0.4 g of total cell proteins or their fractions. The GST-tagged Serinc1 (78 kDa) was detected exclusively in the membrane fraction and not in the soluble one. D, Serinc1-dependent activity enhancement of phosphatidylserine synthase in E. coli membrane fractions. 0.45 mg of native Serinc1-expressing E. coli membranes was incubated with 0.5 mM [ 3 H]serine and 0.2 mM CDP-diacylglycerol at 37°C for 20 min, and the lipid extracts were analyzed by thin-layer chromatography.
In mammals, another class of membrane lipid, the sphingolipids, is formed from serine by the action of serine palmitoyltransferase. This enzyme catalyzes the condensation of serine and palmitoyl-CoA to 3-ketodihydrosphingosine and is the most important control element in the sphingolipid biosynthetic pathway. As shown in Fig. 5C, expression of Serinc1 in COS cells enhanced the incorporation of [ 3 H]serine into 3-ketodihydrosphingosine by 2.1-fold over the base level (12 Ϯ 1.8 pmol/min/mg of protein), which indicates that Serinc1 enhances the activity of mammalian serine palmitoyltransferase. Together with Serinc1-GFP plasmid, red fluorescent protein (RFP)-tagged serine palmitoyltransferase plasmid was co-transfected into COS cells to examine subcellular localization (Fig. 5A, 2nd panel from right). Colocalization of Serinc1 and serine palmitoyltransferase in the cell was evaluated using a merged image (Fig. 5A, right panel). Subcellular distribution of RFP-tagged serine palmitoyltransferase revealed intensive co-localization with Serinc1 in ER membranes. Moreover, direct inter-action of Serinc1 with serine palmitoyltransferase was demonstrated by two-hybrid experiments (Fig. 5D). These results provide the molecular basis for the influence of Serinc1 on serine palmitoyltransferase activity. Because the E. coli lacked serine palmitoyltransferase activity, we performed an experiment using the yeast TMS1 deletion mutant strain (tms1⌬) to further establish that Serinc proteins activate serine palmitoyltransferase in ER membranes (Fig. 5E). The serine palmitoyltransferase activity of the microsome of tms1⌬ was 15 Ϯ 0.8 pmol/min/mg of protein and was found to be 52% of the base level of the wild type, which indicates that TMS1 protein increases the activity of yeast serine palmitoyltransferase. Cellular expression of Serinc1 restored the serine palmitoyltransferase activity to 96% of the base level, indicating the stimulating action of Serinc1. Together, these results suggest that Serinc proteins facilitate the cellular biosynthesis of sphingolipids.
Serine-incorporating Activities of Serinc2 and Serinc5-To investigate the cellular function of other Serinc subtypes, we expressed rat Serinc2 and Serinc5 in both E. coli and COS cells and monitored the incorporation of [ 3 H]serine into their respective phosphatidylserine and mammalian 3-ketodihydrosphingosine. In a manner similar to that of Serinc1, expression of Serinc2 and Serinc5 caused more than a 10-fold increases in E. coli phosphatidylserine synthase activity (Fig. 6A). In mammalian cells, Serinc2 and Serinc5 significantly enhanced the activities of both serine base exchange enzyme (Fig. 6B) and serine palmitoyltransferase (Fig. 6C).
Model of the Serinc Complex in Membranes-Serinc protein is likely to function by the following two mechanisms (Fig. 7). The first mechanism involves Serinc to act as a scaffold for serine synthetic enzymes on the surface of lipid-synthesizing membranes. The key enzyme of serine biosynthesis, 3-phosphoglycerate dehydrogenase, is located and diffuses freely throughout the cytosol, whereas serine palmitoyltransferase is a membrane protein that diffuses freely within lipid bilayers. Because Serinc protein interacts with both types of diffusing enzymes, it is able to provide a scaffold for 3-phosphoglycerate dehydrogenase on the cytosolic surface of membranes where lipid synthetic enzymes are located. The second mechanism involves Serinc acting as a carrier protein that carries a polar serine molecule into lipid bilayers. During synthesis of an Plasmid of pOBD2-rat serinc1 was transformed into a yeast reporter strain, PJ69-4a, expressing the following S. cerevisiae proteins as Gal4 activation domain fusions: control (no protein) and SPT (LCB1 subunit of serine palmitoyltransferase). E, serine palmitoyltransferase activity complementation of a yeast TMS1 deletion mutant by expression of the Serinc1. The wild-type strain of BY4743 (EUROSCARF accession number Y20000) and tms1⌬ strain (accession number Y34040) were obtained from EUROSCARF. One mg of yeast microsomal proteins was used in the assay, and the lipid extracts were analyzed by thin-layer chromatography. amphipathic molecule in membrane lipids, a polar head group has to bind to two hydrophobic hydrocarbon tails. However, polar materials such as serine neither dissolve well nor diffuse in the layers of hydrophobic hydrocarbon tails. Because the membrane topology of Serinc contains 11 transmembrane segments similar to amino acid transporters, Serinc may function by carrying a serine molecule into the hydrophobic milieu of membrane lipid bilayers, which is responsible for efficient reactions occurring among serine, hydrocarbon tails, and lipid synthetic enzymes.

DISCUSSION
The present study suggests that Serinc proteins play a pivotal role in the regulation of lipid biosynthesis. Eukaryotic ER membranes were enriched with Serinc protein, where it bound directly to the key enzyme of sphingolipid biosynthesis, serine palmitoyltransferase. Moreover Serinc directly interacted with an intracellular serine-synthesizing protein complex that included 3-phosphoglycerate dehydrogenase. Thus, synthetic activities of serine and serine-derived lipids were bridged by an intermediary protein, Serinc, and both activities were linked to each other. Because the 3-ketodihydrosphingosine synthesis is the first committed step in sphingolipid biosynthesis, Serinc is thought to function by increasing the cellular sphingolipid content.
One of the notable features of Serinc localization is that the highest concentration of Serinc5 is found in white matter, and the distribution patterns of Serinc5 and white matter are virtually identical throughout the brain. The predominant element of white matter is the myelin sheath, which is characterized by a high proportion of lipid (80% of the total dry weight). Myelin membranes contain strikingly large amount of glycolipids such as galactocerebrosides, and in this respect, the lipid composition of white matter (glycolipid is 27% of the total lipid) is significantly different from that of gray matter (7%). Therefore, this observation suggests that a major role of Serinc5 is to provide serine molecules for the formation of myelin glycosphingolipids in oligodendrocytes. Another notable feature is the localization of Serinc1 in glutamatergic neurons. This observation, together with the activity-dependent mRNA induction in these neurons, suggests that the action of Serinc1 may be coupled with synaptic glutamate transmission, because a transamination reaction from a glutamate molecule to 3-phosphoserine enhances the biosynthesis of serine (Fig. 3A).
Mammalian Serinc subtypes may be divided into two main groups based on structural similarity; one group consists of subtypes 1, 2, 3, and 3B, which share more than 42% amino acid identity (Fig. 2C, bold numbers), whereas subtypes 4, 4B and 5 belong to the other group. Between the two groups, neural localization and regulation of the mRNAs are different. Although Serinc5 is expressed prominently in oligodendroglial cells throughout the brain, the high concentrations of Serinc1 and Serinc2 are seen in neuronal cell populations of the hippocampus and cerebellar cortex, which is supported by a recent report indicating neuronal localization of mouse Serinc1 (TMS-2) and Serinc3 (TMS-1) (7). Moreover, in the hippocampus, the seizure up-regulated the mRNA levels of Serinc1 and Serinc2, which contrasts with the down-regulated pattern of Serinc5. These changes may be a reflection of the neuronal process extension and myelin sheath degradation, respectively, which occur at the site of neural circuit reconstruction.
The phylogenetic analyses of the Serinc family and its interacting proteins provide a deeper insight into the function of Serinc protein complexes and their role in cell-specific maturation of membranes. First, 3-phosphoglycerate dehydrogenase (SER3) is found in both prokaryotic and eukaryotic cells; the structure and catalytic reaction are highly conserved throughout evolution, which reflects the fact that phosphatidylserine and phosphatidylethanolamine are utilized as major classes of membrane lipids by a broad range of organisms. Secondly, Serinc proteins are evolutionarily conserved among eukaryotes such as animals and fungi, but no counterparts are found in prokaryotes, which is consistent with the prevalence of sphingolipids in eukaryotes. Although sphingolipids are found in the membranes of all eukaryotic cells, the concentration is highest in the cells of the central nervous system. Moreover, some glycosphingolipids included in the brain, such as galactocerebrosides and gangliosides, are not found in as high quantities elsewhere. Furthermore, localization of the highest concentration of Serinc5 mRNA in the brain was identical to the distribution of the white matter, a glycosphingolipid-rich tissue. This line of co-localizations suggests that Serinc proteins had emerged to meet the particular lipid requirements of eukaryotic organisms. Abundant evidence indicates that sphingolipids form a lipid raft and play pivotal roles in the sophisticated communication among mammalian cells, such as neuronal networks, and in their pathologic state (malignant tumors).
Thirdly, YGP1 and PHO12 are yeast-specific proteins because their counterparts are not found in other species, which suggests involvement in the formation of yeast-specific sphingolipids. It is well known that mammals and yeasts produce structurally different types of sphingolipids, although the early steps of their synthetic pathway are similar (27). Although animal cells contain sphingomyelin and a large number of complex glycosphingolipids, yeasts synthesize only three types of complex sphingolipids: inositol-phosphorylceramide, mannose-inositol-P-ceramide, and mannose-(inositol-P) 2 -ceramide. Recent studies identified YGP1 and its homolog (SPS100) as maturation factors that induce cellular adaptation into stationary phase and facilitate spore wall formation (28). Because YGP1 is a highly glycosylated secreted protein, it may function as the mannose donor for yeast-specific sphingolipids in the spore walls. Further studies of the Serinc complex will be performed to elucidate the elementary mechanism of the neural signaling and plasticity, as well as other complicated intercellular communications.