Changes in the amount of nucleotide sugars in aged mouse tissues.

Aging affects tissue glycan profiles, which may alter cellular functions and increase the risk of age-related diseases. Glycans are biosynthesized by glycosyltransferases using the corresponding nucleotide sugar, and the availability of nucleotide sugars affects glycosylation efficiency. However, the effects of aging on nucleotide sugar profiles and contents are yet to be elucidated. Therefore, this study aimed to investigate the effects of aging on nucleotide sugars using a new LC-MS/MS method. Specifically, the new method was used to determine the nucleotide sugar contents of various tissues (brain, liver, heart, skeletal muscle, kidney, lung, and colon) of male C57BL/6NCr mice (7- or 26-month-old). Characteristic age-associated nucleotide sugar changes were observed in each tissue sample. Particularly, there was a significant decrease in UDP-glucuronic acid content in the kidney of aged mice and a decrease in the contents of several nucleotide sugars, including UDP-N-acetylgalactosamine, in the brain of aged mice. Additionally, there were variations in nucleotide sugar profiles among the tissues examined regardless of the age. The kidneys had the highest concentration of UDP-glucuronic acid among the seven tissues. In contrast, the skeletal muscle had the lowest concentration of total nucleotide sugars among the tissues; however, CMP-N-acetylneuraminic acid and CDP-ribitol were relatively enriched. Conclusively, these findings may contribute to the understanding of the roles of glycans in tissue aging.

Endogenous reductase activities for the generation of ribitol-phosphate, a CDP-ribitol precursor, in mammals.

The core M3 O-mannosyl glycan on α-dystroglycan serves as the binding epitope for extracellular matrix molecules. Defects in core M3 glycans cause congenital muscular dystrophies that are collectively known as dystroglycanopathies. The core M3 glycan contains a tandem D-ribitol-5-phosphate (Rbo5P) structure, which is synthesized by the Rbo5P-transferases fukutin and fukutin-related protein using CDP-ribitol (CDP-Rbo) as a donor substrate. CDP-Rbo is synthesized from CTP and Rbo5P by CDP-Rbo pyrophosphorylase A. However, the Rbo5P biosynthesis pathway has yet to be elucidated in mammals. Here, we investigated the reductase activities toward four substrates, including ribose, ribulose, ribose-phosphate and ribulose-phosphate, to identify the intracellular Rbo5P production pathway and elucidated the role of the aldo-keto reductases AKR1A1, AKR1B1 and AKR1C1 in those pathways. It was shown that the ribose reduction pathway is the endogenous pathway that contributes most to Rbo5P production in HEK293T cells and that AKR1B1 is the major reductase in this pathway.

An endomembrane zinc transporter negatively regulates systemic RNAi in Caenorhabditis elegans.

Double-stranded RNA (dsRNA) regulates gene expression in a sequence-dependent manner. In Caenorhabditis elegans, dsRNA spreads through the body and leads to systemic RNA silencing. Although several genes involved in systemic RNAi have been genetically identified, molecules that mediate systemic RNAi remain largely unknown. Here, we identified ZIPT-9, a C. elegans homolog of ZIP9/SLC39A9, as a broad-spectrum negative regulator of systemic RNAi. We showed that RSD-3, SID-3, and SID-5 genetically act in parallel for efficient RNAi, and that zipt-9 mutants suppress the RNAi defects of all the mutants. Analysis of a complete set of deletion mutants for SLC30 and SLC39 family genes revealed that only zipt-9 mutants showed altered RNAi activity. Based on these results and our analysis using transgenic Zn2+ reporters, we propose that ZIPT-9-dependent Zn2+ homeostasis, rather than overall cytosolic Zn2+, modulates systemic RNAi activity. Our findings reveal a previously unknown function of zinc transporters in negative RNAi regulation.

Chemical and Chemo-Enzymatic Syntheses of Glycans Containing Ribitol Phosphate Scaffolding of Matriglycan.

Ribitol phosphate modifications to the core M3 O-mannosyl glycan are important for the functional maturation of α-dystroglycan. Three sequentially extended partial structures of the core M3 O-mannosyl glycan including a tandem ribitol phosphate were regio- and stereo-selectively synthesized: Rbo5P-3GalNAcß, Rbo5P-1Rbo5P-3GalNAcß, and Xylß1-4Rbo5P-1Rbo5P-3GalNAcß (Rbo5P, d-ribitol-5-phosphate; GalNAc, N-acetyl-d-galactosamine; Xyl, d-xylose). Rbo5P-3GalNAcß with p-nitrophenyl at the aglycon part served as a substrate for ribitol phosphate transferase (FKRP, fukutin-related protein), and its product was glycosylated by the actions of a series of glycosyltransferases, namely, ribitol xylosyltransferase 1 (RXYLT1), ß1,4-glucuronyltransferase 1 (B4GAT1), and like-acetyl-glucosaminyltransferase (LARGE). Rbo5P-3GalNAcß equipped with an alkyne-type aglycon was also active for FKRP. The molecular information obtained on FKRP suggests that Rbo5P-3GalNAcß derivatives are the minimal units required as the acceptor glycan for Rbo5P transfer and may serve as a precursor for the elongation of the core M3 O-mannosyl glycan.

CDP-ribitol prodrug treatment ameliorates ISPD-deficient muscular dystrophy mouse model.

Ribitol-phosphate modification is crucial for the functional maturation of α-dystroglycan. Its dysfunction is associated with muscular dystrophy, cardiomyopathy, and central nervous system abnormalities; however, no effective treatments are currently available for diseases caused by ribitol-phosphate defects. In this study, we demonstrate that prodrug treatments can ameliorate muscular dystrophy caused by defects in isoprenoid synthase domain containing (ISPD), which encodes an enzyme that synthesizes CDP-ribitol, a donor substrate for ribitol-phosphate modification. We generated skeletal muscle-selective Ispd conditional knockout mice, leading to a pathogenic reduction in CDP-ribitol levels, abnormal glycosylation of α-dystroglycan, and severe muscular dystrophy. Adeno-associated virus-mediated gene replacement experiments suggested that the recovery of CDP-ribitol levels rescues the ISPD-deficient pathology. As a prodrug treatment strategy, we developed a series of membrane-permeable CDP-ribitol derivatives, among which tetraacetylated CDP-ribitol ameliorated the dystrophic pathology. In addition, the prodrug successfully rescued abnormal α-dystroglycan glycosylation in patient fibroblasts. Consequently, our findings provide proof-of-concept for supplementation therapy with CDP-ribitol and could accelerate the development of therapeutic agents for muscular dystrophy and other diseases caused by glycosylation defects.

Biosynthetic Mechanisms and Biological Significance of Glycerol Phosphate-Containing Glycan in Mammals.

Bacteria contain glycerol phosphate (GroP)-containing glycans, which are important constituents of cell-surface glycopolymers such as the teichoic acids of Gram-positive bacterial cell walls. These glycopolymers comprising GroP play crucial roles in bacterial physiology and virulence. Recently, the first identification of a GroP-containing glycan in mammals was reported as a variant form of O-mannosyl glycan on α-dystroglycan (α-DG). However, the biological significance of such GroP modification remains largely unknown. In this review, we provide an overview of this new discovery of GroP-containing glycan in mammals and then outline the recent progress in elucidating the biosynthetic mechanisms of GroP-containing glycans on α-DG. In addition, we discuss the potential biological role of GroP modification along with the challenges and prospects for further research. The progress in this newly identified glycan modification will provide insights into the phylogenetic implications of glycan.

PCYT2 synthesizes CDP-glycerol in mammals and reduced PCYT2 enhances the expression of functionally glycosylated α-dystroglycan.

α-Dystroglycan (α-DG) is a highly glycosylated cell-surface protein. Defective O-mannosyl glycan on α-DG is associated with muscular dystrophies and cancer. In the biosynthetic pathway of the O-mannosyl glycan, fukutin (FKTN) and fukutin-related protein (FKRP) transfer ribitol phosphate (RboP). Previously, we reported that FKTN and FKRP can also transfer glycerol phosphate (GroP) from CDP-glycerol (CDP-Gro) and showed the inhibitory effects of CDP-Gro on functional glycan synthesis by preventing glycan elongation in vitro. However, whether mammalian cells have CDP-Gro or associated synthetic machinery has not been elucidated. Therefore, the function of CDP-Gro in mammals is largely unknown. Here, we reveal that cultured human cells and mouse tissues contain CDP-Gro using liquid chromatography tandem-mass spectrometry (LC-MS/MS). By performing the enzyme activity assay of candidate recombinant proteins, we found that ethanolamine-phosphate cytidylyltransferase (PCYT2), the key enzyme in de novo phosphatidylethanolamine biosynthesis, has CDP-Gro synthetic activity from glycerol-3-phosphate (Gro3P) and CTP. In addition, knockdown of PCYT2 dramatically reduced cellular CDP-Gro. These results indicate that PCYT2 is a CDP-Gro synthase in mammals. Furthermore, we found that the expression of functionally glycosylated α-DG is increased by reducing PCYT2 expression. Our results suggest an important role for CDP-Gro in the regulation of α-DG function in mammals.

The structure of POMGNT2 provides new insights into the mechanism to determine the functional O-mannosylation site on α-dystroglycan.

Defects in the O-mannosyl glycan of α-dystroglycan (α-DG) are associated with α-dystroglycanopathy, a group of congenital muscular dystrophies. While α-DG has many O-mannosylation sites, only the specific positions can be modified with the functional O-mannosyl glycan, namely, core M3-type glycan. POMGNT2 is a glycosyltransferase which adds ß1,4-linked GlcNAc to the O-mannose (Man) residue to acquire core M3-type glycan. Although it is assumed that POMGNT2 extends the specific O-Man residues around particular amino acid sequences, the details are not well understood. Here, we determined a series of crystal structures of POMGNT2 with and without the acceptor O-mannosyl peptides and identified the critical interactions between POMGNT2 and the acceptor peptide. POMGNT2 has an N-terminal catalytic domain and a C-terminal fibronectin type III (FnIII) domain and forms a dimer. The acceptor peptide is sandwiched between the two protomers. The catalytic domain of one protomer recognizes the O-mannosylation site (TPT motif), and the FnIII domain of the other protomer recognizes the C-terminal region of the peptide. Structure-based mutational studies confirmed that amino acid residues of the catalytic domain interacting with mannose or the TPT motif are essential for POMGNT2 enzymatic activity. In addition, the FnIII domain is also essential for the activity and it interacts with the peptide mainly by hydrophobic interaction. Our study provides the first atomic-resolution insights into specific acceptor recognition by the FnIII domain of POMGNT2. The catalytic mechanism of POMGNT2 is proposed based on the structure.

Crystal structures of fukutin-related protein (FKRP), a ribitol-phosphate transferase related to muscular dystrophy.

α-Dystroglycan (α-DG) is a highly-glycosylated surface membrane protein. Defects in the O-mannosyl glycan of α-DG cause dystroglycanopathy, a group of congenital muscular dystrophies. The core M3 O-mannosyl glycan contains tandem ribitol-phosphate (RboP), a characteristic feature first found in mammals. Fukutin and fukutin-related protein (FKRP), whose mutated genes underlie dystroglycanopathy, sequentially transfer RboP from cytidine diphosphate-ribitol (CDP-Rbo) to form a tandem RboP unit in the core M3 glycan. Here, we report a series of crystal structures of FKRP with and without donor (CDP-Rbo) and/or acceptor [RboP-(phospho-)core M3 peptide] substrates. FKRP has N-terminal stem and C-terminal catalytic domains, and forms a tetramer both in crystal and in solution. In the acceptor complex, the phosphate group of RboP is recognized by the catalytic domain of one subunit, and a phosphate group on O-mannose is recognized by the stem domain of another subunit. Structure-based functional studies confirmed that the dimeric structure is essential for FKRP enzymatic activity.

CDP-glycerol inhibits the synthesis of the functional O-mannosyl glycan of α-dystroglycan.

α-Dystroglycan (α-DG) is a highly glycosylated cell-surface laminin receptor. Defects in the O-mannosyl glycan of an α-DG with laminin-binding activity can cause α-dystroglycanopathy, a group of congenital muscular dystrophies. In the biosynthetic pathway of functional O-mannosyl glycan, fukutin (FKTN) and fukutin-related protein (FKRP), whose mutated genes underlie α-dystroglycanopathy, sequentially transfer ribitol phosphate (RboP) from CDP-Rbo to form a tandem RboP unit (RboP-RboP) required for the synthesis of the laminin-binding epitope on O-mannosyl glycan. Both RboP- and glycerol phosphate (GroP)-substituted glycoforms have recently been detected in recombinant α-DG. However, it is unclear how GroP is transferred to the O-mannosyl glycan or whether GroP substitution affects the synthesis of the O-mannosyl glycan. Here, we report that, in addition to having RboP transfer activity, FKTN and FKRP can transfer GroP to O-mannosyl glycans by using CDP-glycerol (CDP-Gro) as a donor substrate. Kinetic experiments indicated that CDP-Gro is a less efficient donor substrate for FKTN than is CDP-Rbo. We also show that the GroP-substituted glycoform synthesized by FKTN does not serve as an acceptor substrate for FKRP and that therefore further elongation of the outer glycan chain cannot occur with this glycoform. Finally, CDP-Gro inhibited the RboP transfer activities of both FKTN and FKRP. These results suggest that CDP-Gro inhibits the synthesis of the functional O-mannosyl glycan of α-DG by preventing further elongation of the glycan chain. This is the first report of GroP transferases in mammals.

Cell endogenous activities of fukutin and FKRP coexist with the ribitol xylosyltransferase, TMEM5.

Dystroglycanopathies are a group of muscular dystrophies that are caused by abnormal glycosylation of dystroglycan; currently 18 causative genes are known. Functions of the dystroglycanopathy genes fukutin, fukutin-related protein (FKRP), and transmembrane protein 5 (TMEM5) were most recently identified; fukutin and FKRP are ribitol-phosphate transferases and TMEM5 is a ribitol xylosyltransferase. In this study, we show that fukutin, FKRP, and TMEM5 form a complex while maintaining each of their enzyme activities. Immunoprecipitation and immunofluorescence experiments demonstrated protein interactions between these 3 proteins. A protein complex consisting of endogenous fukutin and FKRP, and exogenously expressed TMEM5 exerts activities of each enzyme. Our data showed for the first time that endogenous fukutin and FKRP enzyme activities coexist with TMEM5 enzyme activity, and suggest the possibility that formation of this enzyme complex may contribute to specific and prompt biosynthesis of glycans that are required for dystroglycan function.

Endomembrane-associated RSD-3 is important for RNAi induced by extracellular silencing RNA in both somatic and germ cells of Caenorhabditis elegans.

RNA silencing signals in C. elegans spread among cells, leading to RNAi throughout the body. During systemic spread of RNAi, membrane trafficking is thought to play important roles. Here, we show that RNAi Spreading Defective-3 (rsd-3), which encodes a homolog of epsinR, a conserved ENTH (epsin N-terminal homology) domain protein, generally participates in cellular uptake of silencing RNA. RSD-3 is previously thought to be involved in systemic RNAi only in germ cells, but we isolated several deletion alleles of rsd-3, and found that these mutants are defective in the spread of silencing RNA not only into germ cells but also into somatic cells. RSD-3 is ubiquitously expressed, and intracellularly localized to the trans-Golgi network (TGN) and endosomes. Tissue-specific rescue experiments indicate that RSD-3 is required for importing silencing RNA into cells rather than exporting from cells. Structure/function analysis showed that the ENTH domain alone is sufficient, and membrane association of the ENTH domain is required, for RSD-3 function in systemic RNAi. Our results suggest that endomembrane trafficking through the TGN and endosomes generally plays an important role in cellular uptake of silencing RNA.

The Tumor Suppressor BCL7B Functions in the Wnt Signaling Pathway.

Human BCL7 gene family consists of BCL7A, BCL7B, and BCL7C. A number of clinical studies have reported that BCL7 family is involved in cancer incidence, progression, and development. Among them, BCL7B, located on chromosome 7q11.23, is one of the deleted genes in patients with Williams-Beuren syndrome. Although several studies have suggested that malignant diseases occurring in patients with Williams-Beuren syndrome are associated with aberrations in BCL7B, little is known regarding the function of this gene at the cellular level. In this study, we focused on bcl-7, which is the only homolog of BCL7 gene family in Caenorhabditis elegans, and analyzed bcl-7 deletion mutants. As a result, we found that bcl-7 is required for the asymmetric differentiation of epithelial seam cells, which have self-renewal properties as stem cells and divide asymmetrically through the WNT pathway. Distal tip cell development, which is regulated by the WNT pathway in Caenorhabditis elegans, was also affected in bcl-7-knockout mutants. Interestingly, bcl-7 mutants exhibited nuclear enlargement, reminiscent of the anaplastic features of malignant cells. Furthermore, in KATOIII human gastric cancer cells, BCL7B knockdown induced nuclear enlargement, promoted the multinuclei phenotype and suppressed cell death. In addition, this study showed that BCL7B negatively regulates the Wnt-signaling pathway and positively regulates the apoptotic pathway. Taken together, our data indicate that BCL7B/BCL-7 has some roles in maintaining the structure of nuclei and is involved in the modulation of multiple pathways, including Wnt and apoptosis. This study may implicate a risk of malignancies with BCL7B-deficiency, such as Williams-Beuren syndrome.

A conditional knockout toolkit for Caenorhabditis elegans based on the Cre/loxP recombination.

Conditional knockout (cKO) based on site-specific recombination (SSR) technology is a powerful approach for estimating gene functions in a spatially and temporally specific manner in many model animals. In Caenorhabditis elegans (C. elegans), spatial- and temporal-specific gene functions have been largely determined by mosaic analyses, rescue experiments and feeding RNAi methods. To develop a systematic and stable cKO system in C. elegans, we generated Cre recombinase expression vectors that are driven by various tissue-specific or heat-shock promoters. Validation using Cre-mediated fluorescence protein inactivation or activation systems demonstrated successful Cre-dependent loxP excision. We established a collection of multi-copy Cre transgenic strains for each evaluated vector. To evaluate our Cre/loxP-based cKO system, we generated sid-1 deletion mutants harboring floxed sid-1 single-copy integration (SCI) using ultraviolet trimethylpsoralen (UV/TMP) methods. sid-1 mutants that were rescued by the floxed sid-1 SCI were then crossed with the Pdpy-7::Cre strain for cKO in the hypodermis. The sid-1 cKO animals were resistant to bli-3 RNAi, which causes the Bli-phenotyple in the hypodermis, but they were sensitive to unc-22 RNAi, which leads to twitching of the body wall muscle. Our system, which is based on the combination of a transgenic Cre collection, pre-existing deletion mutants, and UV/TMP SCI methods, provided a systematic approach for cKO in C. elegans.

Methods for single/low-copy integration by ultraviolet and trimethylpsoralen treatment in Caenorhabditis elegans.

Single/low-copy transgene integration is essential for avoiding overexpression, ectopic expression and gene silencing in the germline. Here, we present an overview of a method that uses ultraviolet and trimethylpsoralen (UV/TMP) to generate single/low-copy gene integrations in Caenorhabditis elegans. Single/low-copy transgenes from extrachromosomal arrays are integrated into the genome using positive selection based on temperature sensitivity with a vps-45 rescue fragment and negative selection based on benzimidazole sensitivity with a ben-1 rescue fragment. The copy number of the integrated transgenes is determined using quantitative PCR. Our UV/TMP integration method, which is based on familiar extrachromosomal transgenics, provides a simple approach for generating single/low-copy gene integrations.

Mitochondria-type GPAT is required for mitochondrial fusion.

Glycerol-3-phosphate acyltransferase (GPAT) is involved in the first step in glycerolipid synthesis and is localized in both the endoplasmic reticulum (ER) and mitochondria. To clarify the functional differences between ER-GPAT and mitochondrial (Mt)-GPAT, we generated both GPAT mutants in C. elegans and demonstrated that Mt-GPAT is essential for mitochondrial fusion. Mutation of Mt-GPAT caused excessive mitochondrial fragmentation. The defect was rescued by injection of lysophosphatidic acid (LPA), a direct product of GPAT, and by inhibition of LPA acyltransferase, both of which lead to accumulation of LPA in the cells. Mitochondrial fragmentation in Mt-GPAT mutants was also rescued by inhibition of mitochondrial fission protein DRP-1 and by overexpression of mitochondrial fusion protein FZO-1/mitofusin, suggesting that the fusion/fission balance is affected by Mt-GPAT depletion. Mitochondrial fragmentation was also observed in Mt-GPAT-depleted HeLa cells. A mitochondrial fusion assay using HeLa cells revealed that Mt-GPAT depletion impaired mitochondrial fusion process. We postulate from these results that LPA produced by Mt-GPAT functions not only as a precursor for glycerolipid synthesis but also as an essential factor of mitochondrial fusion.

Ablation of ALCAT1 mitigates hypertrophic cardiomyopathy through effects on oxidative stress and mitophagy.

Oxidative stress causes mitochondrial dysfunction and heart failure through unknown mechanisms. Cardiolipin (CL), a mitochondrial membrane phospholipid required for oxidative phosphorylation, plays a pivotal role in cardiac function. The onset of age-related heart diseases is characterized by aberrant CL acyl composition that is highly sensitive to oxidative damage, leading to CL peroxidation and mitochondrial dysfunction. Here we report a key role of ALCAT1, a lysocardiolipin acyltransferase that catalyzes the synthesis of CL with a high peroxidation index, in mitochondrial dysfunction associated with hypertrophic cardiomyopathy. We show that ALCAT1 expression was potently upregulated by the onset of hyperthyroid cardiomyopathy, leading to oxidative stress and mitochondrial dysfunction. Accordingly, overexpression of ALCAT1 in H9c2 cardiac cells caused severe oxidative stress, lipid peroxidation, and mitochondrial DNA (mtDNA) depletion. Conversely, ablation of ALCAT1 prevented the onset of T4-induced cardiomyopathy and cardiac dysfunction. ALCAT1 deficiency also mitigated oxidative stress, insulin resistance, and mitochondrial dysfunction by improving mitochondrial quality control through upregulation of PINK1, a mitochondrial GTPase required for mitochondrial autophagy. Together, these findings implicate a key role of ALCAT1 as the missing link between oxidative stress and mitochondrial dysfunction in the etiology of age-related heart diseases.

LYCAT, a homologue of C. elegans acl-8, acl-9, and acl-10, determines the fatty acid composition of phosphatidylinositol in mice.

Mammalian phosphatidylinositol (PI) has a unique fatty acid composition in that 1-stearoyl-2-arachidonoyl species is predominant. This fatty acid composition is formed through fatty acid remodeling by sequential deacylation and reacylation. We recently identified three Caenorhabditis elegans acyltransferases (ACL-8, ACL-9, and ACL-10) that incorporate stearic acid into the sn-1 position of PI. Mammalian LYCAT, which is the closest homolog of ACL-8, ACL-9, and ACL-10, was originally identified as a lysocardiolipin acyltransferase by an in vitro assay and was subsequently reported to possess acyltransferase activity toward various anionic lysophospholipids. However, the in vivo role of mammalian LYCAT in phospholipid fatty acid metabolism has not been well elucidated. In this study, we generated LYCAT-deficient mice and demonstrated that LYCAT determined the fatty acid composition of PI in vivo. LYCAT-deficient mice were outwardly healthy and fertile. In the mice, stearoyl-CoA acyltransferase activity toward the sn-1 position of PI was reduced, and the fatty acid composition of PI, but not those of other major phospholipids, was altered. Furthermore, expression of mouse LYCAT rescued the phenotype of C. elegans acl-8 acl-9 acl-10 triple mutants. Our data indicate that LYCAT is a determinant of PI molecular species and its function is conserved in C. elegans and mammals.

Intracellular phospholipase A1 and acyltransferase, which are involved in Caenorhabditis elegans stem cell divisions, determine the sn-1 fatty acyl chain of phosphatidylinositol.

Phosphatidylinositol (PI), an important constituent of membranes, contains stearic acid as the major fatty acid at the sn-1 position. This fatty acid is thought to be incorporated into PI through fatty acid remodeling by sequential deacylation and reacylation. However, the genes responsible for the reaction are unknown, and consequently, the physiological significance of the sn-1 fatty acid remains to be elucidated. Here, we identified acl-8, -9, and -10, which are closely related to each other, and ipla-1 as strong candidates for genes involved in fatty acid remodeling at the sn-1 position of PI. In both ipla-1 mutants and acl-8 acl-9 acl-10 triple mutants of Caenorhabditis elegans, the stearic acid content of PI is reduced, and asymmetric division of stem cell-like epithelial cells is defective. The defects in asymmetric division of these mutants are suppressed by a mutation of the same genes involved in intracellular retrograde transport, suggesting that ipla-1 and acl genes act in the same pathway. IPLA-1 and ACL-10 have phospholipase A(1) and acyltransferase activity, respectively, both of which recognize the sn-1 position of PI as their substrate. We propose that the sn-1 fatty acid of PI is determined by ipla-1 and acl-8, -9, -10 and crucial for asymmetric divisions.

Caenorhabditis elegans mboa-7, a member of the MBOAT family, is required for selective incorporation of polyunsaturated fatty acids into phosphatidylinositol.

Phosphatidylinositol (PI) is a component of membrane phospholipids, and it functions both as a signaling molecule and as a compartment-specific localization signal in the form of polyphosphoinositides. Arachidonic acid (AA) is the predominant fatty acid in the sn-2 position of PI in mammals. LysoPI acyltransferase (LPIAT) is thought to catalyze formation of AA-containing PI; however, the gene that encodes this enzyme has not yet been identified. In this study, we established a screening system to identify genes required for use of exogenous polyunsaturated fatty acids (PUFAs) in Caenorhabditis elegans. In C. elegans, eicosapentaenoic acid (EPA) instead of AA is the predominant fatty acid in PI. We showed that an uncharacterized gene, which we named mboa-7, is required for incorporation of PUFAs into PI. Incorporation of exogenous PUFA into PI of the living worms and LPIAT activity in the microsomes were greatly reduced in mboa-7 mutants. Furthermore, the membrane fractions of transgenic worms expressing recombinant MBOA-7 and its human homologue exhibited remarkably increased LPIAT activity. mboa-7 encodes a member of the membrane-bound O-acyltransferase family, suggesting that mboa-7 is LPIAT. Finally, mboa-7 mutants had significantly lower EPA levels in PI, and they exhibited larval arrest and egg-laying defects.