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Subcellular localization of fumarase in mammalian cells and tissues

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Abstract

Fumarase, a mitochondrial matrix protein, is previously indicated to be present in substantial amounts in the cytosol as well. However, recent studies show that newly synthesized human fumarase is efficiently imported into mitochondria with no detectable amount in the cytosol. To clarify its subcellular localization, the subcellular distribution of fumarase in mammalian cells/tissues was examined by a number of different methods. Cell fractionation using either a mitochondria fraction kit or extraction with low concentrations of digitonin, detected no fumarase in a 100,000 g supernatant fraction. Immunoflourescence labeling with an affinity-purified antibody to fumarase and an antibody to the mitochondrial Hsp60 protein showed identical labeling pattern with labeling seen mainly in mitochondria. Detailed studies were performed using high-resolution immunogold electron microscopy to determine the subcellular localization of fumarase in rat tissues, embedded in LR White resin. In thin sections from kidney, liver, heart, adrenal gland and anterior pituitary, strong and specific labeling due to fumarase antibody was only detected in mitochondria. However, in the pancreatic acinar cells, in addition to mitochondria, highly significant labeling was also observed in the zymogen granules and endoplasmic reticulum. The observed labeling in all cases was completely abolished upon omission of the primary antibody indicating that it was specific. In a western blot of purified zymogen granules, a fumarase-antibody cross-reactive protein of the same molecular mass as seen in the mitochondria was present. These results provide evidence that fumarase in mammalian cells/tissues is mainly localized in mitochondria and significant amounts of this protein are not present in the cytosol. However, these studies also reveal that in certain tissues, in addition to mitochondria, this protein is also present at specific extramitochondrial sites. Although the cellular function of fumarase at these extramitochondrial locations is not known, the appearance/localization of fumarase outside mitochondria may help explain how mutations in this mitochondrial protein can give rise to a number of different types of cancers.

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References

  • Akiba T, Hiraga K, Tuboi S (1984) Intracellular distribution of fumarase in various animals. J Biochem (Tokyo) 96:189–195

    CAS  Google Scholar 

  • Bradbury MW, Berk PD (2000) Mitochondrial aspartate aminotransferase: direction of a single protein with two distinct functions to two subcellular sites does not require alternative splicing of the mRNA. Biochem J 345(Pt 3):423–427

    Article  PubMed  CAS  Google Scholar 

  • Brokstad KA, Kalland KH, Russell WC, Matthews DA (2001) Mitochondrial protein p32 can accumulate in the nucleus. Biochem Biophys Res Commun 281:1161–1169

    Article  PubMed  CAS  Google Scholar 

  • Bruschi SA, West KA, Crabb JW, Gupta RS, Stevens JL (1993) Mitochondrial HSP60 (P1 protein) and a HSP70-like protein (mortalin) are major targets for modification during S-(1,1,2,2-tetrafluoroethyl)-l-cysteine-induced nephrotoxicity. J Biol Chem 268:23157–23161

    PubMed  CAS  Google Scholar 

  • Buchwald P, Krummeck G, Rodel G (1991) Immunological identification of yeast SCO1 protein as a component of the inner mitochondrial membrane. Mol Gen Genet 229:413–420

    Article  PubMed  CAS  Google Scholar 

  • Cavanagh AC (1996) Identification of early pregnancy factor as chaperonin 10: implications for understanding its role. Rev Reprod 1:28–32

    Article  PubMed  CAS  Google Scholar 

  • Cavanagh AC (1997) An update on the identity of early pregnancy factor and its role in early pregnancy. J Assist Reprod Genet 14:492–495

    Article  PubMed  CAS  Google Scholar 

  • Cavanagh AC, Morton H (1994) The purification of early-pregnancy factor to homogeneity from human platelets and identification as chaperonin 10. Eur J Biochem 222:551–560

    Article  PubMed  CAS  Google Scholar 

  • Cechetto JD, Gupta RS (2000) Immunoelectron microscopy provides evidence that tumor necrosis factor receptor-associated protein 1 (TRAP-1) is a mitochondrial protein which also localizes at specific extramitochondrial sites. Exp Cell Res 260:30–39

    Article  PubMed  CAS  Google Scholar 

  • Cechetto JD, Soltys BJ, Gupta RS (2000) Localization of mitochondrial 60-kDa heat shock chaperonin protein (Hsp60) in pituitary growth hormone secretory granules and pancreatic zymogen granules. J Histochem Cytochem 48:45–56

    PubMed  CAS  Google Scholar 

  • Cechetto JD, Sadacharan SK, Berk PD, Gupta RS (2002) Immunogold localization of mitochondrial aspartate aminotransferase in mitochondria and on the cell surface in normal rat tissues. Histol Histopathol 17:353–364

    PubMed  CAS  Google Scholar 

  • Chen X, Walker AK, Strahler JR, Simon ES, Tomanicek-Volk SL, Nelson BB, Hurley MC, Ernst SA, Williams JA, Andrews PC (2006) Organellar proteomics: analysis of pancreatic zymogen granule membranes. Mol Cell Proteomics 5:306–312

    Article  PubMed  CAS  Google Scholar 

  • Coughlin EM, Christensen E, Kunz PL, Krishnamoorthy KS, Walker V, Dennis NR, Chalmers RA, Elpeleg ON, Whelan D, Pollitt RJ, Ramesh V, Mandell R, Shih VE (1998) Molecular analysis and prenatal diagnosis of human fumarase deficiency. Mol Genet Metab 63:254–262

    Article  PubMed  CAS  Google Scholar 

  • Deocaris CC, Kaul SC, Wadhwa R (2006) On the brotherhood of the mitochondrial chaperones mortalin and heat shock protein 60. Cell Stress Chaperones 11:116–128

    Article  PubMed  CAS  Google Scholar 

  • Eng C, Kiuru M, Fernandez MJ, Aaltonen LA (2003) A role for mitochondrial enzymes in inherited neoplasia and beyond. Nat Rev Cancer 3:193–202

    Article  PubMed  CAS  Google Scholar 

  • Felts SJ, Owen BA, Nguyen P, Trepel J, Donner DB, Toft DO (2000) The hsp90-related protein TRAP1 is a mitochondrial protein with distinct functional properties. J Biol Chem 275:3305–3312

    Article  PubMed  CAS  Google Scholar 

  • Fiskum G, Craig SW, Decker GL, Lehninger AL (1980) The cytoskeleton of digitonin-treated rat hepatocytes. Proc Natl Acad Sci USA 77:3430–3434

    Article  PubMed  CAS  Google Scholar 

  • Ghebrehiwet B, Lim BL, Kumar R, Feng X, Peerschke EI (2001) gC1q-R/p33, a member of a new class of multifunctional and multicompartmental cellular proteins, is involved in inflammation and infection. Immunol Rev 180:65–77

    Article  PubMed  CAS  Google Scholar 

  • Gingrich JR, Pelkey KA, Fam SR, Huang Y, Petralia RS, Wenthold RJ, Salter MW (2004) Unique domain anchoring of Src to synaptic NMDA receptors via the mitochondrial protein NADH dehydrogenase subunit 2. Proc Natl Acad Sci USA 101:6237–6242

    Article  PubMed  CAS  Google Scholar 

  • Goetz JG, Nabi IR (2006) Interaction of the smooth endoplasmic reticulum and mitochondria. Biochem Soc Trans 34:370–373

    Article  PubMed  CAS  Google Scholar 

  • Gupta RS, Bowes T, Sadacharan SK, Singh B (2005) Intracellular disposition of mitochondrial molecular chaperones: Hsp60, mHsp70, Cpn10 and TRAP-1. In: Henderson B, Pockley AG (eds) Molecular chaperones and cell signalling. Cambridge University Press, New York, pp 22–41

    Google Scholar 

  • Haraguchi CM, Mabuchi T, Yokota S (2003) Localization of a mitochondrial type of NADP-dependent isocitrate dehydrogenase in kidney and heart of rat: an immunocytochemical and biochemical study. J Histochem Cytochem 51:215–226

    PubMed  CAS  Google Scholar 

  • Henderson B, Pockley AG (2005) Molecular chaperones and cell signalling. Cambridge University Press, Cambridge

    Google Scholar 

  • Hubner S, Bahr C, Gossmann H, Efthymiadis A, Drenckhahn D (2003) Mitochondrial and nuclear localization of kanadaptin. Eur J Cell Biol 82:240–252

    Article  PubMed  Google Scholar 

  • Isola LM, Zhou SL, Kiang CL, Stump DD, Bradbury MW, Berk PD (1995) 3T3 fibroblasts transfected with a cDNA for mitochondrial aspartate aminotransferase express plasma membrane fatty acid-binding protein and saturable fatty acid uptake. Proc Natl Acad Sci USA 92:9866–9870

    Article  PubMed  CAS  Google Scholar 

  • Jamieson JD, Palade GE (1967) Intracellular transport of secretory proteins in the pancreatic exocrine cell. II. Transport to condensing vacuoles and zymogen granules. J Cell Biol 34:597–615

    Article  PubMed  CAS  Google Scholar 

  • Jones M, Gupta RS, Englesberg E (1994) Enhancement in amount of P1 (hsp60) in mutants of Chinese hamster ovary (CHO-K1) cells exhibiting increases in the A system of amino acid transport. Proc Natl Acad Sci USA 91:858–862

    Article  PubMed  CAS  Google Scholar 

  • Kaul SC, Yaguchi T, Taira K, Reddel RR, Wadhwa R (2003) Overexpressed mortalin (mot-2)/mthsp70/GRP75 and hTERT cooperate to extend the in vitro lifespan of human fibroblasts. Exp Cell Res 286:96–101

    Article  PubMed  CAS  Google Scholar 

  • Khan IU, Wallin R, Gupta RS, Kammer GM (1998) Protein kinase A-catalyzed phosphorylation of heat shock protein 60 chaperone regulates its attachment to histone 2B in the T lymphocyte plasma membrane. Proc Natl Acad Sci USA 95:10425–10430

    Article  PubMed  CAS  Google Scholar 

  • Knox C, Sass E, Neupert W, Pines O (1998) Import into mitochondria, folding and retrograde movement of fumarase in yeast. J Biol Chem 273:25587–25593

    Article  PubMed  CAS  Google Scholar 

  • Kotti TJ, Savolainen K, Helander HM, Yagi A, Novikov DK, Kalkkinen N, Conzelmann E, Hiltunen JK, Schmitz W (2000) In mouse alpha-methylacyl-CoA racemase, the same gene product is simultaneously located in mitochondria and peroxisomes. J Biol Chem 275:20887–20895

    Article  PubMed  CAS  Google Scholar 

  • Lakshmipathy U, Campbell C (1999) The human DNA ligase III gene encodes nuclear and mitochondrial proteins. Mol Cell Biol 19:3869–3876

    PubMed  CAS  Google Scholar 

  • Le Gall IM, Bendayan M (1996) Possible association of chaperonin 60 with secretory proteins in pancreatic acinar cells. J Histochem Cytochem 44:743–749

    PubMed  CAS  Google Scholar 

  • Lehtonen R, Kiuru M, Vanharanta S, Sjoberg J, Aaltonen LM, Aittomaki K, Arola J, Butzow R, Eng C, Husgafvel-Pursiainen K, Isola J, Jarvinen H, Koivisto P, Mecklin JP, Peltomaki P, Salovaara R, Wasenius VM, Karhu A, Launonen V, Nupponen NN, Aaltonen LA (2004) Biallelic inactivation of fumarate hydratase (FH) occurs in nonsyndromic uterine leiomyomas but is rare in other tumors. Am J Pathol 164:17–22

    PubMed  CAS  Google Scholar 

  • Loveland B, Wang CR, Yonekawa H, Hermel E, Lindahl KF (1990) Maternally transmitted histocompatibility antigen of mice: a hydrophobic peptide of a mitochondrially encoded protein. Cell 60:971–980

    Article  PubMed  CAS  Google Scholar 

  • Maguire M, Coates AR, Henderson B (2002) Chaperonin 60 unfolds its secrets of cellular communication. Cell Stress Chaperones 7:317–329

    Article  PubMed  CAS  Google Scholar 

  • Meenakshi J, Anupama, Goswami SK, Datta K (2003) Constitutive expression of hyaluronan binding protein 1 (HABP1/p32/gC1qR) in normal fibroblast cells perturbs its growth characteristics and induces apoptosis. Biochem Biophys Res Commun 300:686–693

    Article  PubMed  CAS  Google Scholar 

  • Meertens LM, Miyata KS, Cechetto JD, Rachubinski RA, Capone JP (1998) A mitochondrial ketogenic enzyme regulates its gene expression by association with the nuclear hormone receptor PPARalpha. EMBO J 17:6972–6978

    Article  PubMed  CAS  Google Scholar 

  • Mueller JC, Andreoli C, Prokisch H, Meitinger T (2004) Mechanisms for multiple intracellular localization of human mitochondrial proteins. Mitochondrion 3:315–325

    Article  PubMed  CAS  Google Scholar 

  • Nagy A, Wilhelm M, Kovacs G (2003) Mutations of mtDNA in renal cell tumours arising in end-stage renal disease. J Pathol 199:237–242

    Article  PubMed  CAS  Google Scholar 

  • Poyton RO, Duhl DMJ, Clarkson GHD (1992) Protein export from the mitochondrial matrix. Trends Cell Biol 2:369–375

    Article  PubMed  CAS  Google Scholar 

  • Ran Q, Wadhwa R, Kawai R, Kaul SC, Sifers RN, Bick RJ, Smith JR, Pereira-Smith OM (2000) Extramitochondrial localization of mortalin/mthsp70/PBP74/GRP75. Biochem Biophys Res Commun 275:174–179

    Article  PubMed  CAS  Google Scholar 

  • Ranford JC, Coates AR, Henderson B (2000) Chaperonins are cell-signalling proteins: the unfolding biology of molecular chaperones. Expert Rev Mol Med 2000:1–17

    Article  PubMed  CAS  Google Scholar 

  • Rhodin JAG (1974) Histology. Oxford University Press, New York

    Google Scholar 

  • Sadacharan SK, Cavanagh AC, Gupta RS (2001) Immunoelectron microscopy provides evidence for the presence of mitochondrial heat shock 10-kDa protein (chaperonin 10) in red blood cells and a variety of secretory granules. Histochem Cell Biol 116:507–517

    Article  PubMed  CAS  Google Scholar 

  • Sadacharan SK, Singh B, Bowes T, Gupta RS (2005) Localization of mitochondrial DNA encoded cytochrome c oxidase subunits I and II in rat pancreatic zymogen granules and pituitary growth hormone granules. Histochem Cell Biol 124:409–421

    Article  PubMed  CAS  Google Scholar 

  • Sass E, Blachinsky E, Karniely S, Pines O (2001) Mitochondrial and cytosolic isoforms of yeast fumarase are derivatives of a single translation product and have identical amino termini. J Biol Chem 276:46111–46117

    Article  PubMed  CAS  Google Scholar 

  • Sass E, Karniely S, Pines O (2003) Folding of fumarase during mitochondrial import determines its dual targeting in yeast. J Biol Chem 278:45109–45116

    Article  PubMed  CAS  Google Scholar 

  • Schon EA (2000) Mitochondrial genetics and disease. Trends Biochem Sci 25:555–560

    Article  PubMed  CAS  Google Scholar 

  • Singh B, Gupta RS (1992) Expression of human 60-kDa heat shock protein (HSP60 or P1) in Escherichia coli and the development and characterization of corresponding monoclonal antibodies. DNA Cell Biol 11:489–496

    Article  PubMed  CAS  Google Scholar 

  • Singh B, Gupta RS (2006) Mitochondrial import of human and yeast fumarase in live mammalian cells: retrograde translocation of the yeast enzyme is mainly caused by its poor targeting sequence. Biochem Biophys Res Commun 346:911–918

    Article  PubMed  CAS  Google Scholar 

  • Singh B, Soltys BJ, Wu ZC, Patel HV, Freeman KB, Gupta RS (1997) Cloning and some novel characteristics of mitochondrial Hsp70 from Chinese hamster cells. Exp Cell Res 234:205–216

    Article  PubMed  CAS  Google Scholar 

  • Smalheiser NR (1996) Proteins in unexpected locations. Mol Biol Cell 7:1003–1014

    PubMed  CAS  Google Scholar 

  • Soltys BJ, Gupta RS (1996) Immunoelectron microscopic localization of the 60-kDa heat shock chaperonin protein (Hsp60) in mammalian cells. Exp Cell Res 222:16–27

    Article  PubMed  CAS  Google Scholar 

  • Soltys BJ, Gupta RS (1999) Mitochondrial-matrix proteins at unexpected locations: are they exported? Trends Biochem Sci 24:174–177

    Article  PubMed  CAS  Google Scholar 

  • Soltys BJ, Gupta RS (2000) Mitochondrial proteins at unexpected cellular locations: export of proteins from mitochondria from an evolutionary perspective. Int Rev Cytol 194:133–196

    Article  PubMed  CAS  Google Scholar 

  • Soltys BJ, Kang D, Gupta RS (2000) Localization of P32 protein (gC1q-R) in mitochondria and at specific extramitochondrial locations in normal tissues. Histochem Cell Biol 114:245–255

    PubMed  CAS  Google Scholar 

  • Soltys BJ, Andrews DA, Jemmerson R, Gupta RS (2001) Cytochrome c localizes in secretory granules in pancreas and anterior pituitary. Cell Biol Int 25:331–338

    Article  PubMed  CAS  Google Scholar 

  • Stein I, Peleg Y, Even-Ram S, Pines O (1994) The single translation product of the FUM1 gene (fumarase) is processed in mitochondria before being distributed between the cytosol and mitochondria in Saccharomyces cerevisiae. Mol Cell Biol 14:4770–4778

    PubMed  CAS  Google Scholar 

  • Strobel G, Zollner A, Angermayr M, Bandlow W (2002) Competition of spontaneous protein folding and mitochondrial import causes dual subcellular location of major adenylate kinase. Mol Biol Cell 13:1439–1448

    Article  PubMed  CAS  Google Scholar 

  • Stump DD, Zhou SL, Berk PD (1993) Comparison of plasma membrane FABP and mitochondrial isoform of aspartate aminotransferase from rat liver. Am J Physiol 265:G894–G902

    PubMed  CAS  Google Scholar 

  • Suzuki T, Sato M, Yoshida T, Tuboi S (1989) Rat liver mitochondrial and cytosolic fumarases with identical amino acid sequences are encoded from a single gene. J Biol Chem 264:2581–2586

    PubMed  CAS  Google Scholar 

  • Suzuki T, Yoshida T, Tuboi S (1992) Evidence that rat liver mitochondrial and cytosolic fumarases are synthesized from one species of mRNA by alternative translational initiation at two in-phase AUG codons. Eur J Biochem 207:767–772

    Article  PubMed  CAS  Google Scholar 

  • Thevenod F, Gasser KW, Hopfer U (1990) Dual modulation of chloride conductance by nucleotides in pancreatic and parotid zymogen granules. Biochem J 272:119–126

    PubMed  CAS  Google Scholar 

  • Tondera D, Santel A, Schwarzer R, Dames S, Giese K, Klippel A, Kaufmann J (2004) Knockdown of MTP18, a novel phosphatidylinositol 3-kinase-dependent protein, affects mitochondrial morphology and induces apoptosis. J Biol Chem 279:31544–31555

    Article  PubMed  CAS  Google Scholar 

  • Valgardsdottir R, Brede G, Eide LG, Frengen E, Prydz H (2001) Cloning and characterization of MDDX28, a putative dead-box helicase with mitochondrial and nuclear localization. J Biol Chem 276:32056–32063

    Article  PubMed  CAS  Google Scholar 

  • Velez-Granell CS, Arias AE, Torres-Ruiz JA, Bendayan M (1994) Molecular chaperones in pancreatic tissue: the presence of cpn10, cpn60 and hsp70 in distinct compartments along the secretory pathway of the acinar cells. J Cell Sci 107(Pt 3):539–549

    PubMed  CAS  Google Scholar 

  • Xu Q, Schett G, Seitz CS, Hu Y, Gupta RS, Wick G (1994) Surface staining and cytotoxic activity of heat-shock protein 60 antibody in stressed aortic endothelial cells. Circ Res 75:1078–1085

    PubMed  CAS  Google Scholar 

  • Yang SH, Liu R, Perez EJ, Wen Y, Stevens SM Jr, Valencia T, Brun-Zinkernagel AM, Prokai L, Will Y, Dykens J, Koulen P, Simpkins JW (2004) Mitochondrial localization of estrogen receptor beta. Proc Natl Acad Sci USA 101:4130–4135

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported by a research grant from the Canadian Institute of Health Research. We thank Dr. Paul Berk for the gift of antibody to mit-AspAT.

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  1. Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada, L8N 3Z5

    Timothy Bowes, Bhag Singh & Radhey S. Gupta

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  1. Timothy Bowes
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Correspondence to Radhey S. Gupta.

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Bowes, T., Singh, B. & Gupta, R.S. Subcellular localization of fumarase in mammalian cells and tissues. Histochem Cell Biol 127, 335–346 (2007). https://doi.org/10.1007/s00418-006-0249-3

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