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. 2007 Apr 28;12(4):509–522. doi: 10.2478/s11658-007-0020-3

Procaspase-9 is attached to the mitochondrial outer membrane in the early stages of apoptosis

Irina Milisav 1,✉, Dušan Šuput 1
PMCID: PMC6275611  PMID: 17468838

Abstract

Procaspase-9 is the zymogen form of one of the apoptosis initiators, caspase-9. Its cellular location may differ depending on the cell type; it is found throughout the cytosol, although some of it may be associated with the mitochondria. Procaspase-9 relocates from the cytosol to the mitochondria shortly after the triggering of apoptosis in rat hepatocytes. We investigated whether the mitochondrial protein import machineries import procaspase-9. The combined results of protein import analyses, mitochondrial fractionation and protease treatments of intact and swollen mitochondria imply that procaspase-9 attaches to the outer surface of the mitochondrial outer membrane.

Key words: Caspase-9, Procaspase-9, Mitochondria, Apoptosis, Protein import, Rotenone, Localization

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Abbreviations used

DHFR

dihidrofolate-reductase

MPP

mitochondrial processing peptidase

OXA

oxidase assembly complex

PK

proteinase K

PMSF

phenylmethyl sulfonyl fluoride

pSu9

targeting sequence of subunit 9 of F0F1-ATPase

SAM

sorting and assembly machinery

SDS-PAGE

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

TIM

translocase of the inner membrane

TOB

topogenesis of the outer membrane

TOM

translocase of the outer membrane

Try

trypsin

References

  • 1.Kerr J.F.R., Wyllie H., Currie A.R. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer. 1972;26:239–257. doi: 10.1038/bjc.1972.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Cohen G.M. Caspases: the executioners of apoptosis. Biochem. J. 1997;326:1–16. doi: 10.1042/bj3260001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Salvesen G.S., Dixit V.M. Caspases: intracellular signaling by proteolysis. Cell. 1997;91:443–446. doi: 10.1016/S0092-8674(00)80430-4. [DOI] [PubMed] [Google Scholar]
  • 4.Slee E.A., Harte M.T., Kluck R.M., Wolf B.B., Casiano C.A., Newmeyer D.D., Wang H.G., Reed J.C., Nicholson D.W., Alnemri E.S., Green D.R., Martin S.J. Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2,-3,-6,-7,-8, and-10 in a caspase-9-dependent manner. J. Cell Biol. 1999;144:281–292. doi: 10.1083/jcb.144.2.281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Robertson J.D., Enoksson M., Suomela M., Zhivotovsky B., Orrenius S. Caspase-2 acts upstream of mitochondria to promote cytochrome c release during etoposide-induced apoptosis. J. Biol. Chem. 2002;277:29803–29809. doi: 10.1074/jbc.M204185200. [DOI] [PubMed] [Google Scholar]
  • 6.Morishima N., Nakanishi K., Takenouchi H., Shibata T., Yasuhiko Y. An endoplasmic reticulum stress-specific caspase cascade in apoptosis. Cytochrome c-independent activation of caspase-9 by caspase-12. J. Biol. Chem. 2002;277:34287–34294. doi: 10.1074/jbc.M204973200. [DOI] [PubMed] [Google Scholar]
  • 7.Luo X., Budihardjo I., Zou H., Slaughter C., Wang X. Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell. 1998;94:481–490. doi: 10.1016/S0092-8674(00)81589-5. [DOI] [PubMed] [Google Scholar]
  • 8.Li H., Zhu H., Xu C., Yuan J. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell. 1998;94:491–501. doi: 10.1016/S0092-8674(00)81590-1. [DOI] [PubMed] [Google Scholar]
  • 9.Li P., Nijhawan D., Budihardjo I., Srinivasula S.M., Ahmad M., Alnemri E.S., Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 1997;91:479–489. doi: 10.1016/S0092-8674(00)80434-1. [DOI] [PubMed] [Google Scholar]
  • 10.Cardone M.H., Roy N., Stennicke H.R., Salvesen G.S., Franke T.F., Stanbridge E., Frisch S., Reed J.C. Regulation of cell death protease caspase-9 by phosphorylation. Science. 1998;13:1318–1321. doi: 10.1126/science.282.5392.1318. [DOI] [PubMed] [Google Scholar]
  • 11.Shikama Y.U.M., Miyashita T., Yamada M. Comprehensive studies on subcellular localizations and cell death-inducing activities of eight GFPtagged apoptosis-related caspases. Exp. Cell Res. 2001;264:315–325. doi: 10.1006/excr.2000.5153. [DOI] [PubMed] [Google Scholar]
  • 12.Shimohama S., Tanino H., Fujimoto S. Differential subcellular localization of caspase family proteins in the adult rat brain. Neuroscience Lett. 2001;315:125–128. doi: 10.1016/S0304-3940(01)02336-9. [DOI] [PubMed] [Google Scholar]
  • 13.van Loo G., Saelens X., Matthijssens F., Schotte P., Beyaert R., Declercq W., Vandenabeele P. Caspases are not localized in mitochondria during life or death. Cell Death Diff. 2002;9:1207–1211. doi: 10.1038/sj.cdd.4401101. [DOI] [PubMed] [Google Scholar]
  • 14.Potokar M., Milisav I., Kreft M., Stenovec M., Zorec R. Apoptosis triggered redistribution of caspase-9 from cytoplasm to mitochondria. FEBS Lett. 2003;544:153–159. doi: 10.1016/S0014-5793(03)00494-0. [DOI] [PubMed] [Google Scholar]
  • 15.Zhivotovsky B., Samali A., Gahm A., Orrenius S. Caspases: their intracellular localization and translocation during apoptosis. Cell Death Differ. 1999;6:644–651. doi: 10.1038/sj.cdd.4400536. [DOI] [PubMed] [Google Scholar]
  • 16.Yuan H., Mutomba M., Prinz I., Gottlieb R.A. Differential processing of cytosolic and mitochondrial caspases. Mitochondrion. 2001;1:61–69. doi: 10.1016/S1567-7249(01)00002-2. [DOI] [PubMed] [Google Scholar]
  • 17.Susin S.A., Lorenzo H.K., Zamzami N., Marzo I., Brenner C., Larochette N., Prévost M.-C., Alzari P.M., Kroemer G. Mitochondrial release of caspase-2 and-9 during the apoptotic process. J. Exp. Med. 1999;189:381–393. doi: 10.1084/jem.189.2.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Krajewski S., Krajewska M., Ellerby L.M., Welsh K., Xie Z., Deveraux Q.L., Salvesen G.S., Bredesen D.E., Rosenthal R.E., Fiskum G., Reed J. Release of caspase-9 from mitochondria during neuronal apoptosis and cerebral ischemia. Proc. Natl. Acad. Sci. USA. 1999;96:5752–5757. doi: 10.1073/pnas.96.10.5752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Chandra D., Tang D.G. Mitochondrially localized active caspase-9 and caspase-3 result mostly from translocation from the cytosol and partly from caspase-mediated activation in the organelle. J. Biol. Chem. 2003;278:17408–17420. doi: 10.1074/jbc.M300750200. [DOI] [PubMed] [Google Scholar]
  • 20.Saleh A., Srinivasula S.M., Acharya S., Fishel R., Alnemri E.S. Cytochrome c and dATP-mediated oligomerization of Apaf-1 is a prerequisite for procaspase-9 activation. J. Biol. Chem. 1999;274:17941–17945. doi: 10.1074/jbc.274.25.17941. [DOI] [PubMed] [Google Scholar]
  • 21.Cain K., Brown D.G., Langlais C., Cohen G.M. Caspase activation involves the formation of the aposome, a large (approximately 700 kDa) caspase-activating complex. J. Biol. Chem. 1999;274:22686–22692. doi: 10.1074/jbc.274.32.22686. [DOI] [PubMed] [Google Scholar]
  • 22.Ritter P.M., Marti A., Blanc C., Baltzer A., Krajewski S., Reed J.C., Jaggi R. Nuclear localization of procaspase-9 and processing by a caspase-3-like activity in mammary epithelial cells. Eur. J. Cell Biol. 2001;79:358–364. doi: 10.1078/S0171-9335(04)70040-0. [DOI] [PubMed] [Google Scholar]
  • 23.Bitzer M., Armeanu S., Prinz F., Ungerechts G., Wybranietz W., Spiegel M., Bernlöhr C., Cecconi F., Gregor M., Neubert W.J., Schulze-Osthoff K., Lauer U.M. Caspase-8 and Apaf-1-independent caspase-9 activation in Sendai virus-infected cells. J. Biol. Chem. 2002;277:29817–29824. doi: 10.1074/jbc.M111898200. [DOI] [PubMed] [Google Scholar]
  • 24.Mokranjac D., Neupert W. Protein import into mitochondria. Biochem. Soc. Trans. 2005;33:1019–1023. doi: 10.1042/BST20051019. [DOI] [PubMed] [Google Scholar]
  • 25.Hoogenraad N.J., Ward L.A., Ryan M.T. Import and assembly of proteins into mitochondria of mammalian cells. Biochim. Biophys. Acta. 2002;1592:97–105. doi: 10.1016/S0167-4889(02)00268-9. [DOI] [PubMed] [Google Scholar]
  • 26.Stojanovski D., Rissler M., Pfanner N., Meisinger C. Mitochondrial morphology and protein import — a tight connection? Biochim. Biophys. Acta. 2006;1763:414–421. doi: 10.1016/j.bbamcr.2006.02.008. [DOI] [PubMed] [Google Scholar]
  • 27.Hartl F.-U., Pfanner N., Nicholson D.W., Neupert W. Mitochondrial protein import. Biochim. Biophys. Acta. 1989;988:1–45. doi: 10.1016/0304-4157(89)90002-6. [DOI] [PubMed] [Google Scholar]
  • 28.Matocha M.F., Waterman M.R. Discriminatory processing of the precursor forms of cytochrome P-450SCC and adrenodoxin by adrenocortical and heart mitochondria. J. Biol. Chem. 1984;259:8672–8678. [PubMed] [Google Scholar]
  • 29.Matocha M.F., Waterman M.R. Synthesis and processing of mitochondrial steroid hydroxylases. J. Biol. Chem. 1985;260:12259–12265. [PubMed] [Google Scholar]
  • 30.DeSousa G., Dou M., Barbe D., Lacarelle B., Placidi M., Rahmani R. Freshli isolated or cryopreserved human hepatocytes in primary culture: influence of drug metabolism on hepatotoxicity. Toxicol. in vitro. 1991;5:483–486. doi: 10.1016/0887-2333(91)90077-Q. [DOI] [PubMed] [Google Scholar]
  • 31.Daum G., Böhni P. C., Schatz G. Import of proteins into mitochondria. Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria. J. Biol. Chem. 1982;257:13028–13033. [PubMed] [Google Scholar]
  • 32.Werner S., Neupert W. Functional and biogenetical heterogeneity of the inner membrane of rat-liver mitochondria. Eur. J. Biochem. 1972;25:379–396. doi: 10.1111/j.1432-1033.1972.tb01707.x. [DOI] [PubMed] [Google Scholar]
  • 33.Bauer M.F., Gempel K., Reichert A.S., Rappold G.A., Lichtner P., Gerbitz K.-D., Neupert W., Brunner M., Hofmann S. Genetic and structural characterization of the human mitochondrial inner membrane translocase. J. Mol. Biol. 1999;289:69–82. doi: 10.1006/jmbi.1999.2751. [DOI] [PubMed] [Google Scholar]
  • 34.Duan H., Orth K., Chinnaiyan A.M., Poirier G.G., Froelich C.J., He W.W., Dixit V.M. ICE-LAP-6, a novel member of the ICE/Ced-3 gene family, is activated by the cytotoxic T cell protease granzyme B. J. Biol. Chem. 1996;271:16720–16724. doi: 10.1074/jbc.271.44.27863. [DOI] [PubMed] [Google Scholar]
  • 35.Rothbauer U., Hofmann S., Mühlenbein N., Paschen S.A., Gerbitz K.-D., Neupert W., Brunner M., Bauer M.F. Role of the Deafness Dystonia Peptide 1 (DDP1) in Import of Human Tim23 into the Inner Membrane of Mitochondria. J. Biol. Chem. 2001;276:37327–37334. doi: 10.1074/jbc.M105313200. [DOI] [PubMed] [Google Scholar]
  • 36.Ungermann C., Neupert W., Cyr D.M. The role of Hsp70 in conferring unidirectionality on protein translocation into mitochondria. Science. 1994;266:1250–1253. doi: 10.1126/science.7973708. [DOI] [PubMed] [Google Scholar]
  • 37.Pelham H.R., Jackson R.J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur. J. Biochem. 1976;67:247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  • 38.Luciano P., Geoffroy S., Brandt A., Hernandez J.-F., Géli V. Functional cooperation of the mitochondrial processing peptidase subunits. J. Mol. Biol. 1997;272:213–225. doi: 10.1006/jmbi.1997.1231. [DOI] [PubMed] [Google Scholar]
  • 39.Kaldi K., Bauer M.F., Sirrenberg C., Neupert W., Brunner M. Biogenesis of Tim23 and Tim17, integral components of the TIM machinery for matrix-targeted preproteins. EMBO J. 1998;17:1569–1576. doi: 10.1093/emboj/17.6.1569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Gordon D.M., Shi Q., Dancis A., Pain D. Maturation of frataxin within mammalian and yeast mitochondria: one-step processing by matrix processing peptidase. Hum. Mol. Genet. 1999;8:2255–2262. doi: 10.1093/hmg/8.12.2255. [DOI] [PubMed] [Google Scholar]

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