Cellular and Molecular Life Sciences

, Volume 67, Issue 3, pp 433–443 | Cite as

Visualization of subcellular NAD pools and intra-organellar protein localization by poly-ADP-ribose formation

  • Christian Dölle
  • Marc Niere
  • Emilia Lohndal
  • Mathias Ziegler
Research Article

Abstract

Poly-ADP-ribose polymerases (PARPs) use NAD+ as substrate to generate polymers of ADP-ribose. We targeted the catalytic domain of human PARP1 as molecular NAD+ detector into cellular organelles. Immunochemical detection of polymers demonstrated distinct subcellular NAD+ pools in mitochondria, peroxisomes and, surprisingly, in the endoplasmic reticulum and the Golgi complex. Polymers did not accumulate within the mitochondrial intermembrane space or the cytosol. We demonstrate the suitability of this compartment-specific NAD+ and poly-ADP-ribose turnover to establish intra-organellar protein localization. For overexpressed proteins, genetically endowed with PARP activity, detection of polymers indicates segregation from the cytosol and consequently intra-organellar residence. In mitochondria, polymer build-up reveals matrix localization of the PARP fusion protein. Compared to presently used fusion tags for subcellular protein localization, these are substantial improvements in resolution. We thus established a novel molecular tool applicable for studies of subcellular NAD metabolism and protein localization.

Keywords

Compartmentation ADP-ribosylation NAD metabolism Mitochondria Protein import 

References

  1. 1.
    Simpson JC, Pepperkok R (2006) The subcellular localization of the mammalian proteome comes a fraction closer. Genome Biol 7:222CrossRefPubMedGoogle Scholar
  2. 2.
    Huh WK, Falvo JV, Gerke LC, Carroll AS, Howson RW, Weissman JS, O’Shea EK (2003) Global analysis of protein localization in budding yeast. Nature 425:686–691CrossRefPubMedGoogle Scholar
  3. 3.
    Foster LJ, de Hoog CL, Zhang Y, Zhang Y, Xie X, Mootha VK, Mann M (2006) A mammalian organelle map by protein correlation profiling. Cell 125:187–199CrossRefPubMedGoogle Scholar
  4. 4.
    O’Rourke NA, Meyer T, Chandy G (2005) Protein localization studies in the age of ‘Omics’. Curr Opin Chem Biol 9:82–87CrossRefPubMedGoogle Scholar
  5. 5.
    Neupert W, Herrmann JM (2007) Translocation of proteins into mitochondria. Annu Rev Biochem 76:723–749CrossRefPubMedGoogle Scholar
  6. 6.
    Bürkle A (2005) Poly(ADP-ribose). The most elaborate metabolite of NAD+. FEBS J 272:4576–4589CrossRefPubMedGoogle Scholar
  7. 7.
    Hassa PO, Haenni SS, Elser M, Hottiger MO (2006) Nuclear ADP-ribosylation reactions in mammalian cells: where are we today and where are we going? Microbiol Mol Biol Rev 70:789–829CrossRefPubMedGoogle Scholar
  8. 8.
    Schreiber V, Dantzer F, Ame JC, de Murcia G (2006) Poly(ADP-ribose): novel functions for an old molecule. Nat Rev Mol Cell Biol 7:517–528CrossRefPubMedGoogle Scholar
  9. 9.
    Kawamitsu H, Hoshino H, Okada H, Miwa M, Momoi H, Sugimura T (1984) Monoclonal antibodies to poly(adenosine diphosphate ribose) recognize different structures. Biochemistry 23:3771–3777CrossRefPubMedGoogle Scholar
  10. 10.
    Simonin F, Menissier-de Murcia J, Poch O, Muller S, Gradwohl G, Molinete M, Penning C, Keith G, de Murcia G (1990) Expression and site-directed mutagenesis of the catalytic domain of human poly(ADP-ribose)polymerase in Escherichia coli. Lysine 893 is critical for activity. J Biol Chem 265:19249–19256PubMedGoogle Scholar
  11. 11.
    Meyer-Ficca ML, Meyer RG, Coyle DL, Jacobson EL, Jacobson MK (2004) Human poly(ADP-ribose) glycohydrolase is expressed in alternative splice variants yielding isoforms that localize to different cell compartments. Exp Cell Res 297:521–532CrossRefPubMedGoogle Scholar
  12. 12.
    Antonenkov VD, Sormunen RT, Hiltunen JK (2004) The rat liver peroxisomal membrane forms a permeability barrier for cofactors but not for small metabolites in vitro. J Cell Sci 117:5633–5642CrossRefPubMedGoogle Scholar
  13. 13.
    Berger F, Ramirez-Hernandez MH, Ziegler M (2004) The new life of a centenarian: signalling functions of NAD(P). Trends Biochem Sci. 29:111–118CrossRefPubMedGoogle Scholar
  14. 14.
    Blander G, Guarente L (2004) The Sir2 family of protein deacetylases. Annu Rev Biochem 73:417–435CrossRefPubMedGoogle Scholar
  15. 15.
    Taylor DM, Maxwell MM, Luthi-Carther R, Kazantsev AG (2008) Biological and potential therapeutic roles of sirtuin deacetylases. Cell Mol Life Sci 65:4000–4018CrossRefPubMedGoogle Scholar
  16. 16.
    Hallows WC, Lee S, Denu JM (2006) Sirtuins deacetylate and activate mammalian acetyl-CoA synthetases. Proc Natl Acad Sci USA 103:10230–10235CrossRefPubMedGoogle Scholar
  17. 17.
    Nakagawa T, Lomb DJ, Haigis MC, Guarente L (2009) SIRT5 deacetylates carbamoyl phosphate synthetase 1 and regulates the urea cycle. Cell 137:560–570CrossRefPubMedGoogle Scholar
  18. 18.
    Smith S, Giriat I, Schmitt A, de Lange T (1998) Tankyrase, a poly(ADP-ribose) polymerase at human telomeres. Science 282:1484–1487CrossRefPubMedGoogle Scholar
  19. 19.
    Dynek JN, Smith S (2004) Resolution of sister telomere association is required for progression through mitosis. Science 304:97–100CrossRefPubMedGoogle Scholar
  20. 20.
    Yu SW, Wang H, Poitras MF, Coombs C, Bowers WJ, Federoff HJ, Poirier GG, Dawson TM, Dawson VL (2002) Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor. Science 297:259–263CrossRefPubMedGoogle Scholar
  21. 21.
    Fliegert R, Gasser A, Guse AH (2007) Regulation of calcium signalling by adenine-based second messengers. Biochem Soc Trans 35:109–114CrossRefPubMedGoogle Scholar
  22. 22.
    Guse AH, Lee HC (2008) NAADP: a universal Ca2+ trigger. Sci Signal 1:re10Google Scholar
  23. 23.
    Mayevsky A, Rogatsky GG (2007) Mitochondrial function in vivo evaluated by NADH fluorescence: from animal models to human studies. Am J Physiol Cell Physiol 292:C615–C640CrossRefPubMedGoogle Scholar
  24. 24.
    Niere M, Kernstock S, Koch-Nolte F, Ziegler M (2008) Functional localization of two poly(ADP-ribose)-degrading enzymes to the mitochondrial matrix. Mol Cell Biol 28:814–824CrossRefPubMedGoogle Scholar
  25. 25.
    Yu SW, Andrabi SA, Wang H, Kim NS, Poirier GG, Dawson TM, Dawson VL (2006) Apoptosis-inducing factor mediates poly(ADP-ribose) (PAR) polymer-induced cell death. Proc Natl Acad Sci USA 103:18314–18319CrossRefPubMedGoogle Scholar
  26. 26.
    Kawajiri K, Harano T, Omura T (1977) Biogenesis of the mitochondrial matrix enzyme, glutamate dehydrogenase, in rat liver cells. I. Subcellular localization, biosynthesis, and intracellular translocation of glutamate dehydrogenase. J Biochem 82:1403–1416Google Scholar
  27. 27.
    Rosso L, Marques AC, Reichert AS, Kaessmann H (2008) Mitochondrial targeting adaptation of the hominoid-specific glutamate dehydrogenase driven by positive Darwinian selection. PLoS Genet. 4:e1000150Google Scholar
  28. 28.
    Loeffler M, Daugas E, Susin SA, Zamzami N, Metivier D, Nieminen AL, Brothers G, Penninger JM, Kroemer G (2001) Dominant cell death induction by extramitochondrially targeted apoptosis-inducing factor. FASEB J 15:758–767CrossRefPubMedGoogle Scholar
  29. 29.
    Otera H, Ohsakaya S, Nagaura Z, Ishihara N, Mihara K (2005) Export of mitochondrial AIF in response to proapoptotic stimuli depends on processing at the intermembrane space. EMBO J 24:1375–1386CrossRefPubMedGoogle Scholar
  30. 30.
    Abdelkarim GE, Gertz K, Harms C, Katchanov J, Dirnagl U, Szabo C, Endres M (2001) Protective effects of PJ34, a novel, potent inhibitor of poly(ADP-ribose) polymerase (PARP) in in vitro and in vivo models of stroke. Int J Mol Med 7:255–260PubMedGoogle Scholar
  31. 31.
    Bublitz C, Lawler CA (1987) The levels of nicotinamide nucleotides in liver microsomes and their possible significance to the function of hexose phosphate dehydrogenase. Biochem J 245:263–267PubMedGoogle Scholar
  32. 32.
    Haas IG (1994) BiP (GRP78), an essential hsp70 resident protein in the endoplasmic reticulum. Experientia 50:1012–1020CrossRefPubMedGoogle Scholar
  33. 33.
    Llopis J, McCaffery JM, Miyawaki A, Farquhar MG, Tsien RY (1998) Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins. Proc Natl Acad Sci USA 95:6803–6808CrossRefPubMedGoogle Scholar
  34. 34.
    Subramani S, Koller A, Snyder WB (2000) Import of peroxisomal matrix and membrane proteins. Annu Rev Biochem 69:399–418CrossRefPubMedGoogle Scholar
  35. 35.
    Kamijo K, Taketani S, Yokota S, Osumi T, Hashimoto T (1990) The 70-kDa peroxisomal membrane protein is a member of the Mdr (P-glycoprotein)-related ATP-binding protein superfamily. J Biol Chem 265:4534–4540PubMedGoogle Scholar

Copyright information

© Birkhäuser Verlag, Basel/Switzerland 2009

Authors and Affiliations

  • Christian Dölle
    • 1
  • Marc Niere
    • 1
  • Emilia Lohndal
    • 1
  • Mathias Ziegler
    • 1
  1. 1.Department of Molecular BiologyUniversity of BergenBergenNorway

Personalised recommendations