Mitochondria-type GPAT is required for mitochondrial fusion
2013; Springer Nature; Volume: 32; Issue: 9 Linguagem: Inglês
10.1038/emboj.2013.77
ISSN1460-2075
AutoresYohsuke Ohba, Takeshi Sakuragi, Eriko Kage‐Nakadai, Naoko H. Tomioka, Nozomu Kono, Rieko Imae, Asuka Inoue, Junken Aoki, Naotada Ishihara, Takao Inoué, Shohei Mitani, Hiroyuki Arai,
Tópico(s)Metabolism and Genetic Disorders
ResumoArticle9 April 2013free access Mitochondria-type GPAT is required for mitochondrial fusion Yohsuke Ohba Yohsuke Ohba Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan Search for more papers by this author Takeshi Sakuragi Takeshi Sakuragi Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan Search for more papers by this author Eriko Kage-Nakadai Eriko Kage-Nakadai Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan CREST, Japan Science and Technology Agency (JST), Tokyo, Japan Search for more papers by this author Naoko H Tomioka Naoko H Tomioka Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan Search for more papers by this author Nozomu Kono Nozomu Kono Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan CREST, Japan Science and Technology Agency (JST), Tokyo, Japan Search for more papers by this author Rieko Imae Rieko Imae Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan CREST, Japan Science and Technology Agency (JST), Tokyo, Japan Search for more papers by this author Asuka Inoue Asuka Inoue Laboratory of Molecular and Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan Search for more papers by this author Junken Aoki Junken Aoki Laboratory of Molecular and Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan Search for more papers by this author Naotada Ishihara Naotada Ishihara Department of Protein Biochemistry, Institute of Life Science, Kurume University, Kurume, Japan Search for more papers by this author Takao Inoue Takao Inoue Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan CREST, Japan Science and Technology Agency (JST), Tokyo, JapanPresent address: Division of Cellular and Gene Therapy Products, National Institute of Health Sciences, Tokyo 158-8501, Japan Search for more papers by this author Shohei Mitani Shohei Mitani Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan CREST, Japan Science and Technology Agency (JST), Tokyo, Japan Search for more papers by this author Hiroyuki Arai Corresponding Author Hiroyuki Arai Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan CREST, Japan Science and Technology Agency (JST), Tokyo, Japan Search for more papers by this author Yohsuke Ohba Yohsuke Ohba Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan Search for more papers by this author Takeshi Sakuragi Takeshi Sakuragi Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan Search for more papers by this author Eriko Kage-Nakadai Eriko Kage-Nakadai Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan CREST, Japan Science and Technology Agency (JST), Tokyo, Japan Search for more papers by this author Naoko H Tomioka Naoko H Tomioka Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan Search for more papers by this author Nozomu Kono Nozomu Kono Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan CREST, Japan Science and Technology Agency (JST), Tokyo, Japan Search for more papers by this author Rieko Imae Rieko Imae Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan CREST, Japan Science and Technology Agency (JST), Tokyo, Japan Search for more papers by this author Asuka Inoue Asuka Inoue Laboratory of Molecular and Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan Search for more papers by this author Junken Aoki Junken Aoki Laboratory of Molecular and Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan Search for more papers by this author Naotada Ishihara Naotada Ishihara Department of Protein Biochemistry, Institute of Life Science, Kurume University, Kurume, Japan Search for more papers by this author Takao Inoue Takao Inoue Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan CREST, Japan Science and Technology Agency (JST), Tokyo, JapanPresent address: Division of Cellular and Gene Therapy Products, National Institute of Health Sciences, Tokyo 158-8501, Japan Search for more papers by this author Shohei Mitani Shohei Mitani Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan CREST, Japan Science and Technology Agency (JST), Tokyo, Japan Search for more papers by this author Hiroyuki Arai Corresponding Author Hiroyuki Arai Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan CREST, Japan Science and Technology Agency (JST), Tokyo, Japan Search for more papers by this author Author Information Yohsuke Ohba1, Takeshi Sakuragi1,‡, Eriko Kage-Nakadai2,3, Naoko H Tomioka1, Nozomu Kono1,3, Rieko Imae1,2,3, Asuka Inoue4, Junken Aoki4, Naotada Ishihara5, Takao Inoue1,3, Shohei Mitani2,3 and Hiroyuki Arai 1,3 1Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan 2Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan 3CREST, Japan Science and Technology Agency (JST), Tokyo, Japan 4Laboratory of Molecular and Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan 5Department of Protein Biochemistry, Institute of Life Science, Kurume University, Kurume, Japan ‡These authors contributed to equally to this work *Corresponding author. Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.:+81 3 5841 4720; Fax:+81 3 3818 3173; E-mail: [email protected] The EMBO Journal (2013)32:1265-1279https://doi.org/10.1038/emboj.2013.77 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction Glycerolipids, which include phospholipids and triacylglycerol, are ubiquitous and important biological components. Phospholipids are the major constituents of biological membranes, playing important roles in multiple cellular processes including maintenance of the cellular permeability barrier, regulation of the activities of proteins associated with the membrane, and regulation of intracellular signalling by serving as precursors of signalling molecules (Dowhan, 1997). Triacylglycerol is a major storage form of energy, as well as being a major component of secreted lipoproteins. The biosynthetic pathways for these glycerolipids have been well established (Kent, 1995; Dowhan, 1997). The initial and rate-limiting step of glycerolipid synthesis is the acylation of glycerol-3-phosphate (G3P) with long-chain fatty acyl-CoA to form lysophosphatidic acid (LPA). This reaction is catalysed by glycerol-3-phosphate acyltransferase (GPAT) (Coleman et al, 2000; Wendel et al, 2009). LPA is further acylated by LPA acyltransferase (LPAAT) located at the ER to form phosphatidic acid (PA), a common precursor for phospholipid and triacylglycerol synthesis (Coleman and Lee, 2004; Takeuchi and Reue, 2009). To date, four mammalian GPATs have been identified and classified into two groups based on sequence homology and subcellular localization (Figure 1A; Gimeno and Cao, 2008; Wendel et al, 2009). GPAT1 and GPAT2 are mitochondrial GPATs that are localized to the mitochondrial outer membrane, and GPAT3 and GPAT4 are microsomal GPATs that are localized to the endoplasmic reticulum (ER) membrane (Gimeno and Cao, 2008; Wendel et al, 2009). All four of these GPATs are members of the AGPAT (1-acyl-sn-glycerol-3-phosphate acyltransferase) family that contain four conserved AGPAT motifs (Neuwald, 1997). Mitochondrial GPAT1 knockout (GPAT1−/−) mice (Hammond et al, 2002) and microsomal GPAT4 knockout (GPAT4−/−) mice (Vergnes et al, 2006) exhibited reduced triacylglycerol content in the liver. GPAT4−/− mice also showed large reductions in the triacylglycerol contents in adipose tissue, subdermal fat, and milk (Beigneux et al, 2006; Vergnes et al, 2006), while GPAT1−/− mice showed increased hepatic fatty acid oxidation and altered insulin resistance when fed a high fat diet (Hammond et al, 2005; Neschen et al, 2005; Yazdi et al, 2008). Significant GPAT activity remains in the mitochondrial and microsomal fractions in the GPAT1−/− and GPAT4−/− mice, respectively, suggesting that GPAT2 and GPAT3 compensate for the functions of mitochondrial and microsomal GPATs in mice (Lewin et al, 2004; Chen et al, 2008; Nagle et al, 2008). However, the effects of knocking out both mitochondrial GPATs (GPAT1 and GPAT2) or both microsomal GPATs (GPAT3 and GPAT4) have not yet been examined. Figure 1.C. elegans GPATs, acl-4, acl-5, and acl-6, contribute to triacylglycerol synthesis. (A) In mammals and C. elegans, glycerol-3-phosphate acyltransferases (GPATs) are located on both the outer mitochondrial membrane (mitochondrial GPAT) and endoplasmic reticulum (ER) (microsomal GPAT) to produce lysophosphatidic acid (LPA). LPA is further converted into phosphatidic acid (PA), a precursor of triacylglycerol and membrane phospholipids, by LPA acyltransferases (LPAATs) which are localized in the ER membranes. Mammalian mitochondrial GPATs, GPAT1 and GPAT2, correspond to C. elegans ACL-6. Mammalian microsomal GPATs, GPAT3 and GPAT4, correspond to C. elegans ACL-4 and ACL-5. C. elegans LPAATs are ACL-1 and ACL-2. (B–D) Genomic structures of ER-GPAT (acl-4 and acl-5) (B), Mt-GPAT (acl-6) (C) and LPAAT (acl-1 and acl-2) (D). Grey boxes indicate exons and white boxes indicate 5′ and 3′ untranslated regions. The extent of deletion in acl-4(xh10), acl-5(xh19), acl-6(tm3396 and tm3452), acl-1(tm3289), and acl-2(tm3246) are indicated by horizontal double arrows. The positions of the predicted conserved lysophospholipid acyltransferase motifs, characteristic of AGPAT family members, are shown in yellow lines. (E) GPAT activity in the membrane fractions of wild-type and the indicated acl mutants. [14C]Palmitoyl-CoA (40 μM) and glycerol-3-phosphate (G3P) (800 μM) were used. We could not measure GPAT activity of the worms lacking both ER-GPAT and Mt-GPAT double mutants (acl-6;acl-4 acl-5 triple mutants) because they were lethal. (F) Triacylglycerol contents of wild-type and the indicated acl mutants. (G) Incorporation of [14C]palmitic acids into neutral lipid fractions of the living worms. The amount of incorporation was expressed as the percentage of radioactivity incorporated into total lipids. (H) Phospholipids contents of wild-type and the indicated acl mutants. Each bar represents the mean±s.e.m. of at least three independent experiments. *P<0.05, **P<0.01, ***P<0.001 (Student's t test). Download figure Download PowerPoint Most of the enzymes of glycerolipid biosynthesis reside in the ER membrane, but GPATs are also located on the mitochondrial outer membrane, which raises a question: Why is LPA synthesized in both mitochondria and ER? To answer this question, a comprehensive mutational analysis of GPATs would be useful. The C. elegans genome contains two microsomal GPATs (acl-4, acl-5), and one mitochondrial GPAT (acl-6) (Figure 1A). To understand the functional differences of the two types of GPAT, we generated C. elegans deletion mutants of mitochondrial and microsomal GPATs. Mitochondria are essential organelles in most eukaryotic cells that are involved in several metabolic pathways, cell signalling, and apoptosis. Mitochondria undergo dynamic changes in morphology through continual fission and fusion, which is essential for maintenance of mitochondrial function and to adapt mitochondria to cellular needs (Detmer and Chan, 2007). The balance of fission and fusion determines the overall mitochondrial morphology. When mitochondrial fusion is reduced, mitochondria fragment due to ongoing fission; conversely, mitochondria are long and overly interconnected when this balance shifts towards fusion (Smirnova et al, 1998, 2001; Chen et al, 2003; Eura et al, 2003; Santel et al, 2003). Genetic studies in D. melanogaster and S. cerevisiae have led to the identification of components that regulate mitochondrial dynamics (Okamoto and Shaw, 2005; Westermann, 2008; Hoppins and Nunnari, 2009). These include the mitofusin (Mfn), which is involved in mitochondrial fusion, and Drp1, which is involved in mitochondrial fission. Mfn is a large transmembrane GTPase localized in the mitochondrial outer membrane (Santel and Fuller, 2001; Rojo et al, 2002; Chen et al, 2003; Eura et al, 2003; Santel et al, 2003; Ishihara et al, 2004), whereas Drp1 is a cytosolic dynamin-related GTPase that is associated with the mitochondrial outer membrane at sites of fission (Smirnova et al, 1998, 2001). These molecules are evolutionarily conserved in eukaryotic organisms including human and C. elegans. Here, we show that depletion of mitochondrial GPAT in C. elegans causes mitochondrial fragmentation. This defect is most probably induced by impaired mitochondrial fusion. We also demonstrate that injection of LPA, a direct product of GPAT, into cells rescues the mitochondrial defect in the mitochondrial GPAT mutants. We propose, from these results, that in addition to functioning as a precursor of glycerolipid synthesis, LPA produced by mitochondrial GPAT plays an important role in regulating mitochondrial dynamics. Results Identification of microsomal and mitochondrial GPAT in C. elegans A search of the C. elegans genome for proteins with four conserved AGPAT motifs yielded 14 proteins (acl-1-14) (Imae et al, 2010). Among these genes, acl-4 and acl-5 are homologous to microsomal GPATs (GPAT3 and GPAT4) and show 45–60% identity to human GPAT3 and GPAT4 (Figure 1A; Supplementary Figure S1A). acl-6 is a sole C. elegans homologue of mitochondrial GPATs (GPAT1 and GPAT2) and shows about 30% identity to human GPAT1 and GPAT2 (Figure 1A; Supplementary Figure S1B). As expected from the sequence homology, acl-4 and acl-5 gene products colocalized with an ER marker (calnexin) (Supplementary Figure S2A–F), but not with a mitochondrial marker (MitoTracker) (Supplementary Figure S2J–O) when expressed in mammalian cells. In contrast, the acl-6 gene product colocalized with a mitochondrial marker in mammalian cells (Supplementary Figure S2P–R) and also in C. elegans muscle cells (Supplementary Figure S2S–U). Generation of GPAT mutants To address the in vivo functions of LPA-producing enzymes, we isolated deletion mutants of all GPATs by PCR-based screening of UV-TMP-mutagenized libraries. The acl-4(xh10) allele had a deletion that causes a frameshift resulting in a premature stop codon (Figure 1B). The acl-5(xh19) allele lacked the translation initiation codon (Figure 1B). Both alleles lacked the conserved AGPAT motifs that are essential for enzyme activity. Two deletion alleles of acl-6, tm3396, and tm3452, caused a frameshift leading to a premature stop codon (Figure 1C). Both tm3396 and tm3452 appeared to be null or strong loss-of-function alleles because inhibition of acl-6 by RNAi failed to enhance the acl-6 mutant phenotypes described below. Because acl-6(tm3396) and acl-6(tm3452) were phenotypically indistinguishable, we used acl-6(tm3396) mutants in subsequent experiments. In acl-4 acl-5 double mutants (hereafter, referred to as 'ER-GPAT mutants'), GPAT activity in the membrane fraction was reduced to 25% of the level in the wild type (Figure 1E) and the triacylglycerol level was reduced to 46% of the level in the wild type (Figure 1F). Incorporation of supplemented [14C]palmitic acid into the triacylglycerol fraction was also reduced in ER-GPAT mutants (Figure 1G). In contrast, no reductions in GPAT activity, the triacylglycerol level, or incorporation of [14C]palmitic acid into triacylglycerol were observed in acl-6 mutants ('Mt (mitochondrial)-GPAT mutants') (Figure 1E–G). However, in the ER-GPAT mutant background, knockdown of Mt-GPAT significantly reduced the incorporation of [14C]palmitic acid into triacylglycerol (Figure 1G). These data indicate that ER-GPAT and Mt-GPAT complement each other to maintain triacylglycerol synthesis in C. elegans. On the other hand, the amount of phospholipids, the other final products of glycerolipid synthesis, was not affected in either ER-GPAT mutants or Mt-GPAT mutants (Figure 1H). Mutants lacking both mitochondrial and microsomal GPATs (acl-6; acl-4 acl-5 triple mutants) did not survive (Table I; Supplementary Table S1A, see Materials and methods for details). Table 1. Phenotypic consequences of C. elegans GPAT and LPAAT mutants Strain Emb (%) Larval arrest (%) Ste (%)a Brood size Wild type <1 <1 0 326±22 (n=20) ER-GPAT mutants acl-4(xh10) <1 <1 0 286±29 (n=20) acl-5(xh19) <1 <1 0 323±24 (n=20) acl-4(xh10) acl-5(xh19) <1 <1 0 303±21 (n=20) acl-6(tm3396) 18.0 (n=649) <1 74.0 (n=169) 141±80b (n=20) acl-6(tm3452) 16.9 (n=767) <1 64.3 (n=185) 66±39b (n=20) acl-6(tm3396); Ex[Pacl-6::acl-6::gfp] 3.8 (n=394) <1 2.7 (n=74) 172±70b (n=20) acl-6(tm3396); acl-4(xh10) acl-5(xh19) 100c(Emb+larval arrest) ND ND acl-1(tm3289) <1 <1 0 309±32 (n=20) acl-2(tm3246) <1 <1 0 298±50 (n=18) acl-2(tm3246); acl-1(tm3289) 100c(Emb+larval arrest) ND ND acl-4(xh10) acl-5(xh19) acl-1(tm3289) 6.4 (n=299) <1 0 293±30 (n=20) acl-2(tm3246); acl-4(xh10) acl-5(xh19) 4.2 (n=528) <1 0 304±45 (n=19) acl-6(tm3396); acl-1(tm3289) 13.1 (n=727) <1 31.7 (n=145) 160±101b (n=20) acl-2(tm3246) acl-6(tm3396) 19.2 (n=266) 50 for each experiment). *P<0.05, **P<0.01 (Student's t test). (D) LPA acyltransferase activity in the membrane fractions of wild-type, acl-1 mutants, and acl-2 mutants. 1-oleoyl lysophosphatidic acid (40 μM) and [14C]oleoyl-CoA (12.5 μM) were used. Each bar represents the mean±s.e.m. of at least three independent experiments. *P 20 for each experiment). Bars, mean±s.e.m. (J) The bar graph shows the relative frequencies of worms with fragmented, partially fragmented or tubular mitochondria in each strain. The experiment was repeated three times (n>20 for each experiment). Bars, mean±s.e.m. Download figure Download PowerPoint In the wild type, muscle cell mitochondria were tubular and elongated and ran parallel to the myofibrils as previously reported (Figure 3C; Labrousse et al, 1999), while in the Mt-GPAT mutants, they were fragmented and disorganized (Figures 3D and 4J). We also examined the mitochondrial morphology in Mt-GPAT mutants by expressing mitoGFP in the wall muscle cells. Mitochondria observed with mitoGFP, like those observed with MitoTracker, were significantly fragmented in Mt-GPAT mutants (Supplementary Figure S3). The mitoGFP fluorescence mostly matched the Mitotracker stain, indicating that fragmented mitochondria still maintain membrane potential. MitoGFP also detected very small portions of mitochondria that were not detected by Mitotracker (Supplementary Figure S3, arrowhead). TEM analysis showed that mitochondria of the Mt-GPAT mutants were short and round (Figure 3H, arrowheads), whereas those of wild-type muscle were long and tubular (Figure 3G, arrows). We measured the rates of thrashing, readouts for muscle activity, and found that the Mt-GPAT mutation reduced thrashing rates by 78% compared to the wild type (Supplementary Figure S4). We conducted cell-specific rescue experiments to confirm the cell autonomy of Mt-GPAT (acl-6) function. The fragmented mitochondria in muscle cells were rescued by expression of acl-6 in the muscle cells (under myo-3 promoter), but not by expression in the intestine cells (under ges-1 promoter) (Supplementary Figure S5A–C), indicating that acl-6 functions cell autonomously to maintain mitochondrial morphology. Next, we determined whether GPAT activity of Mt-GPAT is required for maintaining mitochondrial morphology. Essential amino-acid residues for the catalytic activity have been identified in mammalian GPAT1 (Dircks et al, 1999). Then, we generated transgenic worms expressing GFP-tagged human GPAT1 (acl-6 [Pmyo-3::hgpat1 (WT)::GFP]) or catalytically inactive hGPAT1 (arginine 318 to alanine; R318A) (acl-6 [Pmyo-3::hgpat1 (R318A)::GFP]). As shown in Figure 3K, the membrane fractions of HEK293 cells expressing hGPAT1 (R318A)-FLAG did not show increased GPAT activity compared with those of hGPAT1 (WT). Mitochondrial fragmentation in Mt-GPAT mutants was rescued in hGPAT1 (WT)-expressing cells (Figure 3I), but not in hGPAT1 (R318A)-expressing cells (Figure 3J). Thus, GPAT activity of Mt-GPAT is required for normal mitochondrial morphology. LPA is required for normal mitochondrial morphology The finding that GPAT activity was required for normal mitochondrial morphology indicated that LPA, a product of GPAT, or the downstream metabolites in glycerolipids synthesis pathway is involved in mitochondrial morphology. Then, we conducted RNAi knockdown of 80 genes whose homologues are reported to be involved in phospholipid s
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