Mitochondrial Glycerol Phosphate Acyltransferase Contains Two Transmembrane Domains with the Active Site in the N-terminal Domain Facing the Cytosol

González-Baró, María R.; Granger, Deborah A.; Coleman, Rosalind A.

doi:10.1074/jbc.m107885200

Cited by 52 publications

(43 citation statements)

References 38 publications

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“…This predicted TMD coincides with the first TMD of mouse GPAT1 (Fig. 1A) [19]. All the algorithms predicted an additional TMD, either between residues 204−226 or 301−329.…”

Section: Gpat2 Is Predicted To Have At Least Two Transmembrane Domainssupporting

confidence: 73%

“…Cysteines near active site motifs and present in GPAT2 only shown in white lettering on black and underlined. The transmembrane domains for GPAT1 [19] and the potential transmembrane domains for GPAT2 are bolded and underlined. Amino acid residues identical for all isoforms are indicated with an asterisk (*); Pieces of rat tissues stored in RNALater were homogenized with a rotor-stator homogenizer and RNA was isolated with the RNeasy kit.…”

Section: Discussionmentioning

confidence: 99%

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Cloning and functional characterization of a novel mitochondrial N-ethylmaleimide-sensitive glycerol-3-phosphate acyltransferase (GPAT2)

Wang

Lee²,

Gong³

et al. 2007

Archives of Biochemistry and Biophysics

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Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and rate-limiting step in glycerolipid synthesis. Several mammalian GPAT activities have been recognized, including Nethylmaleimide (NEM)-sensitive isoforms in microsomes and mitochondria and an NEM-resistant form in mitochondrial outer membrane (GPAT1). We have now cloned a second mitochondrial isoform, GPAT2 from mouse testis. The open reading frame encodes a protein of 798 amino acids with a calculated mass of 88.8 kDa and 27% amino acid identity to GPAT1. Testis mRNA expression was 50-fold higher than in liver or brown adipose tissue, but the specific activity of NEM-sensitive GPAT in testis mitochondria was similar to that in liver. When Cos-7 cells were transiently transfected with GPAT2, NEM-sensitive GPAT activity increased 30%. Confocal microscopy confirmed a mitochondrial location. Incubation of GPAT2-transfected Cos-7 cells with trace (3 μM; 0.25μCi) [1-14 C]oleate for 6 h increased incorporation of [ 14 C]oleate into TAG 84%. In contrast, incorporation into phospholipid species was lower than in control cells. Although a polyclonal antibody raised against full-length GPAT1 detected an ∼89 kDa band in liver and testis from GPAT1 null mice and both 89 and 80 kDa bands in BAT from the knockout animals, the GPAT2 protein expressed in Cos-7 cells was only 80 kDa. In vitro translation showed a single product of 89 kDa. Unlike GPAT1, GPAT2 mRNA abundance in liver was not altered by fasting or refeeding. GPAT2 is likely to have a specialized function in testis.The synthesis of triacylglycerol and all glycerophospholipids begins with the acylation of glycerol-3-phosphate with long-chain fatty acyl-CoA to produce 1-acylglycerol-3-phosphate. This reaction is catalyzed by glycerol-3-phosphate acyltransferase (GPAT 1 ; EC 3.1.3.9) which exhibits the lowest specific activity of all enzymes in the glycerol-3-phosphate pathway, suggesting that it is the rate limiting step [1].Three mammalian GPAT activities have been differentiated based on their subcellular location and biochemical properties [2,3]. GPAT activity is present in microsomal and mitochondrial Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. fractions. In most tissues, 90% of the activity is attributed to a microsomal GPAT which is sensitive to inactivation by sulfhydryl reagents such as NEM. Microsomal GPAT3, prominent in adipose tissue, has been cloned [4]. Microsomal GPAT activity does not appear to be regulated in liver. In contrast, the mitochondrial isoform (GPAT1) 2 that has been purified [5] and cloned [6,7], is highly regulated by i...

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“…This predicted TMD coincides with the first TMD of mouse GPAT1 (Fig. 1A) [19]. All the algorithms predicted an additional TMD, either between residues 204−226 or 301−329.…”

Section: Gpat2 Is Predicted To Have At Least Two Transmembrane Domainssupporting

confidence: 73%

Section: Discussionmentioning

confidence: 99%

Cloning and functional characterization of a novel mitochondrial N-ethylmaleimide-sensitive glycerol-3-phosphate acyltransferase (GPAT2)

Wang

Lee²,

Gong³

et al. 2007

Archives of Biochemistry and Biophysics

Self Cite

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“…1 A), we performed immunocytochemistry on human PINK1-transfected neuroblastoma SH-SY5Y cells treated with either digitonin or Triton X-100 to respectively permeabilize the plasma membrane only or all cellular bilayer membranes (27). SH-SY5Y cells transfected stably with a cDNA plasmid expressing full-length human PINK1-IRES-eGFP were probed with antibodies raised against specific subcellular proteins (Fig.…”

Section: Resultsmentioning

confidence: 99%

The kinase domain of mitochondrial PINK1 faces the cytoplasm

Zhou

Huang

Shao

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

287

259

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Mutations in PTEN-induced putative kinase 1 (PINK1) are a cause of autosomal recessive familial Parkinson's disease (PD). Efforts in deducing the PINK1 signaling pathway have been hindered by controversy around its subcellular and submitochondrial localization and the authenticity of its reported substrates. We show here that this mitochondrial protein exhibits a topology in which the kinase domain faces the cytoplasm and the N-terminal tail is inside the mitochondria. Although deletion of the transmembrane domain disrupts this topology, common PD-linked PINK1 mutations do not. These results are critical in rectifying the location and orientation of PINK1 in mitochondria, and they should help decipher its normal physiological function and potential pathogenic role in PD.parkin ͉ Parkinson's disease ͉ mitochondria ͉ topology P arkinson's disease (PD), the second most common neurodegenerative disorder, is a sporadic condition that can occasionally be inherited (1). The rationale for studying the rare genetic forms of PD is based on the phenotypic similarity between the familial and sporadic forms of the disease, implying that the two share important pathogenic mechanisms. Among the different gene products associated with familial PD (2), PTEN-induced putative kinase-1 (PINK1) is localized to the mitochondria (3-10), an organelle strongly linked to PD pathogenesis. Although recessively inherited PD mutations in PINK1 are found throughout the protein, they are most commonly found in the only recognized functional domain of PINK1 (3,11,12), which is a serine/threonine kinase domain similar to that in the Ca 2ϩ /calmodulin kinase family (13,14). Overexpression of wild-type PINK1 can rescue the phenotype caused by PINK1 mutations in Drosophila (15, 16), supporting the notion that the mutated allele gives rise to a loss-of-function phenotype.Human PINK1 is a 581-aa polypeptide with a predicted N-terminal mitochondrial targeting signal (MTS), consistent with the observation that PINK1 localizes to mitochondria (Fig. 1A) (3-10). Loss-of-function mutations in the gene encoding parkin (an E3 ubiquitin ligase) cause the most frequent forms of recessive familial PD (17). In Drosophila, parkin is thought to operate within the same molecular pathway as PINK1 to modulate mitochondrial morphology (15,16,18), an intriguing observation given the fact that parkin has been reported to essentially be cytosolic (19).Both the subcellular and submitochondrial locations of PINK1 and the authenticity of its reported substrates have been controversial. Although it is agreed that PINK1 is a mitochondrialtargeted protein, PINK1 has been reported to reside in the inner mitochondrial membrane (IMM) (6,7,9,20), the mitochondrial intermembrane space (IMS) (6,8,9), the outer mitochondrial membrane (OMM) (7), and even the cytoplasm (21-24). Furthermore, the tumor necrosis factor type 1 receptor-associated protein (TRAP1) and the serine protease HtrA2/Omi have been identified as putative PINK1 substrates (8, 9). Although TRAP1 is localized mainly i...

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“…4B). To confirm that p66Shc localizes within mitochondria, the mitochondrial and the ER-containing fractions were treated with proteinase K, which can digest only those proteins that are not protected by closed phospholipidic bilayers (20). As expected, proteinase K degraded calnexin in the ER fraction ( Fig.…”

Section: P66shc Localizes In Different Intracellular Compartments Incmentioning

confidence: 99%

The Life Span Determinant p66Shc Localizes to Mitochondria Where It Associates with Mitochondrial Heat Shock Protein 70 and Regulates Trans-membrane Potential

Orsini

Migliaccio

Moroni

et al. 2004

Journal of Biological Chemistry

264

239

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P66Shc regulates life span in mammals and is a critical component of the apoptotic response to oxidative stress. It functions as a downstream target of the tumor suppressor p53 and is indispensable for the ability of oxidative stress-activated p53 to induce apoptosis. The molecular mechanisms underlying the apoptogenic effect of p66Shc are unknown. Here we report the following three findings. (i) The apoptosome can be properly activated in vitro in the absence of p66Shc only if purified cytochrome c is supplied. (ii) Cytochrome c release after oxidative signals is impaired in the absence of p66Shc. (iii) p66Shc induces the collapse of the mitochondrial trans-membrane potential after oxidative stress. Furthermore, we showed that a fraction of cytosolic p66Shc localizes within mitochondria where it forms a complex with mitochondrial Hsp70. Treatment of cells with ultraviolet radiation induced the dissociation of this complex and the release of monomeric p66Shc. We propose that p66Shc regulates the mitochondrial pathway of apoptosis by inducing mitochondrial damage after dissociation from an inhibitory protein complex. Genetic and biochemical evidence suggests that mitochondria regulate life span through their effects on the energetic metabolism (mitochondrial theory of aging). Our data suggest that mitochondrial regulation of apoptosis might also contribute to life span determination.

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Mitochondrial Glycerol Phosphate Acyltransferase Contains Two Transmembrane Domains with the Active Site in the N-terminal Domain Facing the Cytosol

Cited by 52 publications

References 38 publications

Cloning and functional characterization of a novel mitochondrial N-ethylmaleimide-sensitive glycerol-3-phosphate acyltransferase (GPAT2)

Cloning and functional characterization of a novel mitochondrial N-ethylmaleimide-sensitive glycerol-3-phosphate acyltransferase (GPAT2)

The kinase domain of mitochondrial PINK1 faces the cytoplasm

The Life Span Determinant p66Shc Localizes to Mitochondria Where It Associates with Mitochondrial Heat Shock Protein 70 and Regulates Trans-membrane Potential

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