The nematode Caenorhabditis elegans is a powerful model system to study contemporary biological problems. This system would be even more useful if we had mutations in all the genes of this multicellular metazoan. The combined efforts of the C. elegans Deletion Mutant Consortium and individuals within the worm community are moving us ever closer to this goal. At present, of the 20,377 protein-coding genes in this organism, 6764 genes with associated molecular lesions are either deletions or null mutations (WormBase WS220). Our three laboratories have contributed the majority of mutated genes, 6841 mutations in 6013 genes. The principal method we used to detect deletion mutations in the nematode utilizes polymerase chain reaction (PCR). More recently, we have used array comparative genome hybridization (aCGH) to detect deletions across the entire coding part of the genome and massively parallel short-read sequencing to identify nonsense, splicing, and missense defects in open reading frames. As deletion strains can be frozen and then thawed when needed, these strains will be an enduring community resource. Our combined molecular screening strategies have improved the overall throughput of our gene-knockout facilities and have broadened the types of mutations that we and others can identify. These multiple strategies should enable us to eventually identify a mutation in every gene in this multicellular organism. This knowledge will usher in a new age of metazoan genetics in which the contribution to any biological process can be assessed for all genes.

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. INTRODUCTIONVarious kinds of fatty acids are distributed in membrane phospholipids in mammalian cells and tissues (Lands and Crawford, 1976;Holub and Kuksis, 1978;MacDonald and Sprecher, 1991). The fatty acyl residues of individual phospholipids seem to be under strict metabolic regulation. In general, saturated fatty acids are esterified at the sn-1 position, whereas polyunsaturated fatty acids (PUFAs), such as arachidonic acid (AA), are commonly found at the sn-2 position. Three-fourths or more of the phosphatidylinositol (PI) fraction in rat liver and brain constitutes the 1-stearoyl-2-arachidonoyl species (Holub and Kuksis, 1971;Baker and Thompson, 1972). In contrast, the total pool of precursor phosphatidic acid (PA) in rat liver and brain has a fatty acid composition that does not resemble that of PI, showing a low AA content (Possmayer et al., 1969;Akesson et al., 1970;Baker and Thompson, 1972). Selectivity could be expressed during de novo synthesis at the level of formation of cytidine 5Ј-diphosphate (CDP)-diacylglycerol from PA and cytidine 5Ј-triphosphate or in the use of CDP-diacylglycerol in the final reaction. In fact, CDP-diacylglycerol synthase, which prefers 1-stearoyl-2-arachidonoyl PA as a substrate in vitro, has been cloned, although expression is restricted to testis, retina, and brain (Saito et al., 1997).An alternative mechanism for species selection has been proposed on the basis of turnover studies in rat brain in vivo (Baker and Thompson, 1972). [ 3 H]AA and [ 14 C]glycerol injected intracerebrally were incorporated almost exclusively into brain phospholipids. Comparison of PI and PA radioactivity suggest that the initial flux of AA into PI was independent of de novo synthesi...

Phosphatidylinositol (PI) is a relatively minor component of membrane phospholipids but plays important roles in signal transduction through distinct phosphorylated derivatives of the inositol head group ( 1, 2 ). Threefourths or more of membrane PI obtained from mammalian tissues consist of the 1-stearoyl-2-arachidonoyl (18:0/20:4) species ( 3, 4 ), which is thought to be formed by a fatty acid remodeling reaction after the de novo synthesis of PI ( 5-10 ). The remodeling reaction involves the hydrolysis of a fatty acyl ester bond at the sn -1 or sn -2 position of the newly synthesized PI and subsequent incorporation of the appropriate fatty acid into the position. In an RNA interference (RNAi)-based genetic screen using Caenorhabditis elegans , we identifi ed mboa-7/ LPIAT1 as an acyltransferase that selectively incorporates arachidonic acid into the sn-2 position of PI ( 11 ). More recently, we demonstrated that C. elegans acl-8 , acl-9 , and acl-10 , which show signifi cant sequence homology to each other, encode acyltransferases that incorporate stearic acid (18:0) into the sn-1 position of PI ( 12 ). Stearic acid attached at the sn -1 position of PI Abstract 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 identifi ed 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 identifi ed 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-defi cient mice and demonstrated that LYCAT determined the fatty acid composition of PI in vivo. LYCATdefi cient 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. Abbreviations: AGPAT, 1-acylglycerol-3-phosphate O -acyltransferase; A-P axis, anterior-posterio axis; CL, cardiolipin; ER, endoplasmic reticulum; LPCAT, lysophosphatidylcholine acyltransferase; LPEAT, lysophosphatidylethanolamine acyltransferase; LPGAT, lysophosphatidylglycerol acyltransferase; LPIAT, lysophosphatidylinositol acyltransferase; LPSAT, lysophosphatidylserine acyltransferase; LYCAT, lysocardiolipin acyltransferase; MEF, mouse embryonic fi broblast; PC, phosphatidylcholi...

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.