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An inventory of interactors of the human HSP60/HSP10 chaperonin in the mitochondrial matrix space

Abstract

The HSP60/HSP10 chaperonin assists folding of proteins in the mitochondrial matrix space by enclosing them in its central cavity. The chaperonin forms part of the mitochondrial protein quality control system. It is essential for cellular survival and mutations in its subunits are associated with rare neurological disorders. Here we present the first survey of interactors of the human mitochondrial HSP60/HSP10 chaperonin. Using a protocol involving metabolic labeling of HEK293 cells, cross-linking, and immunoprecipitation of HSP60, we identified 323 interacting proteins. As expected, the vast majority of these proteins are localized to the mitochondrial matrix space. We find that approximately half of the proteins annotated as mitochondrial matrix proteins interact with the HSP60/HSP10 chaperonin. They cover a broad spectrum of functions and metabolic pathways including the mitochondrial protein synthesis apparatus, the respiratory chain, and mitochondrial protein quality control. Many of the genes encoding HSP60 interactors are annotated as disease genes. There is a correlation between relative cellular abundance and relative abundance in the HSP60 immunoprecipitates. Nineteen abundant matrix proteins occupy more than 60% of the HSP60/HSP10 chaperonin capacity. The reported inventory of interactors can form the basis for interrogating which proteins are especially dependent on the chaperonin.

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References

  1. Bie AS et al (2016) Effects of a mutation in the HSPE1 gene encoding the mitochondrial co-chaperonin HSP10 and its potential association with a neurological and developmental disorder. Front Mol Biosci 3:65. https://doi.org/10.3389/fmolb.2016.00065

  2. Bross P (2015) The Hsp60 Chaperonin. Springer Briefs in Molecular Science. Springer International Publishing, Cham, Switzerland. https://doi.org/10.1007/978-3-319-26088-4

  3. Bross P et al (2008) The Hsp60-(p.V98I) mutation associated with hereditary spastic paraplegia SPG13 compromises chaperonin function both in vitro and in vivo. JBiolChem 283:15694. https://doi.org/10.1074/jbc.M800548200

  4. Calvo SE, Clauser KR, Mootha VK (2016) MitoCarta2.0: an updated inventory of mammalian mitochondrial proteins nucleic acids res 44:D1251-D1257. https://doi.org/10.1093/nar/gkv1003

  5. Casari G et al (1998) Spastic paraplegia and OXPHOS impairment caused by mutations in paraplegin, a nuclear-encoded mitochondrial metalloprotease. Cell 93:973–983

  6. Chaudhuri TK, Farr GW, Fenton WA, Rospert S, Horwich AL (2001) GroEL/GroES-mediated folding of a protein too large to be encapsulated. Cell 107:235–246

  7. Cheng MY et al (1989) Mitochondrial heat-shock protein hsp60 is essential for assembly of proteins imported into yeast mitochondria. Nature 337:620–625

  8. Christensen JH et al (2010) Inactivation of the hereditary spastic paraplegia-associated Hspd1 gene encoding the Hsp60 chaperone results in early embryonic lethality in mice. Cell Stress Chaperones 15:851–863

  9. Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. NatBiotechnol 26:1367–1372

  10. Di Bella D et al (2010) Mutations in the mitochondrial protease gene AFG3L2 cause dominant hereditary ataxia SCA28. Nat Genet 42:313–321. https://doi.org/10.1038/ng.544

  11. Dikoglu E et al (2015) Mutations in LONP1, a mitochondrial matrix protease, cause CODAS syndrome. Am J Med Genet A 167:1501–1509. https://doi.org/10.1002/ajmg.a.37029

  12. Diodato D et al (2014) VARS2 and TARS2 mutations in patients with mitochondrial encephalomyopathies. Hum Mutat 35:983–989. https://doi.org/10.1002/humu.22590

  13. Dubaquie Y, Looser R, Fünfschilling U, Jenö P, Rospert S (1998) Identification of in vivo substrates of the yeast mitochondrial chaperonins reveals overlapping but non-identical requirement for hsp60 and hsp10. EMBO J 17:5868–5876

  14. Fayet O, Ziegelhoffer T, Georgopoulos C (1989) The groES and groEL heat shock gene products of Escherichia coli are essential for bacterial growth at all temperatures JBacteriol 171:1379-1385

  15. Fujiwara K, Ishihama Y, Nakahigashi K, Soga T, Taguchi H (2010) A systematic survey of in vivo obligate chaperonin-dependent substrates. EMBO J 29:1552–1564

  16. Geiger T, Wehner A, Schaab C, Cox J, Mann M (2012) Comparative proteomic analysis of eleven common cell lines reveals ubiquitous but varying expression of most proteins. Mol Cell Proteomics 11:M111. https://doi.org/10.1074/mcp.M111.014050

  17. Ghezzi D, Zeviani M (2012) Assembly factors of human mitochondrial respiratory chain complexes: physiology and pathophysiology. AdvExpMedBiol 748:65–106

  18. Gottesman S, Wickner S, Maurizi MR (1997) Protein quality control: triage by chaperones and proteases. Genes Dev 11:815–823. https://doi.org/10.1101/gad.11.7.815

  19. Gough J, Karplus K, Hughey R, Chothia C (2001) Assignment of homology to genome sequences using a library of hidden Markov models that represent all proteins of known structure. J Mol Biol 313:903–919. https://doi.org/10.1006/jmbi.2001.5080

  20. Gregersen N, Bross P, Vang S, Christensen JH (2006) Protein misfolding and human disease. Annu Rev Genomics Hum Genet 7:103–124. https://doi.org/10.1146/annurev.genom.7.080505.115737

  21. Hansen JJ et al (2002) Hereditary spastic paraplegia SPG13 is associated with a mutation in the gene encoding the mitochondrial chaperonin Hsp60. Am J Hum Genet 70:1328–1332

  22. Hartl FU, Hayer-Hartl M (2009) Converging concepts of protein folding in vitro and in vivo. Nat Struct Mol Biol 16:574. https://doi.org/10.1038/nsmb.1591

  23. Hayer-Hartl M, Bracher A, Hartl FU (2016) The GroEL-GroES Chaperonin Machine: A Nano-Cage for Protein Folding Trends Biochem Sci 41:62-76. https://doi.org/10.1016/j.tibs.2015.07.009

  24. Hipp MS, Kasturi P, Hartl FU (2019) The proteostasis network and its decline in ageing. Nat Rev Mol Cell Biol 20:421–435. https://doi.org/10.1038/s41580-019-0101-y

  25. Hohfeld J, Hartl FU (1994) Role of the chaperonin cofactor Hsp10 in protein folding and sorting in yeast mitochondria. J Cell Biol 126:305–315

  26. Houry WA, Frishman D, Eckerskorn C, Lottspeich F, Hartl FU (1999) Identification of in vivo substrates of the chaperonin. GroEL Nature 402:147–154. https://doi.org/10.1038/45977

  27. Hung V et al (2014) Proteomic mapping of the human mitochondrial intermembrane space in live cells via ratiometric APEX tagging. Mol Cell 55:332–341. https://doi.org/10.1016/j.molcel.2014.06.003

  28. Jenkinson EM et al (2013) Perrault syndrome is caused by recessive mutations in CLPP, encoding a mitochondrial ATP-dependent chambered protease. Am J Hum Genet 92:605–613

  29. Kao TY et al (2015) Mitochondrial Lon regulates apoptosis through the association with Hsp60-mtHsp70 complex. Cell Death Dis 6:e1642. https://doi.org/10.1038/cddis.2015.9

  30. Kerner MJ, Naylor DJ, Ishihama Y, Maier T, Chang HC, Stines AP, Georgopoulos C, Frishman D, Hayer-Hartl M, Mann M, Hartl FU (2005) Proteome-wide analysis of chaperonin-dependent protein folding in Escherichia coli. Cell 122:209–220

  31. Kim YE, Hipp MS, Bracher A, Hayer-Hartl M, Hartl FU (2013) Molecular chaperone functions in protein folding and proteostasis. Annu Rev Biochem 82:323–355

  32. Levy-Rimler G, Bell R, Ben Tal N, Azem A (2002) Type I chaperonins: not all are created equal FEBS Lett 529:1

  33. Levy-Rimler G et al. (2001) The effect of nucleotides and mitochondrial chaperonin 10 on the structure and chaperone activity of mitochondrial chaperonin 60 EurJBiochem 268:3465-3472

  34. Liu F, Lossl P, Rabbitts BM, Balaban RS, Heck AJR (2018) The interactome of intact mitochondria by cross-linking mass spectrometry provides evidence for coexisting respiratory supercomplexes. Mol Cell Proteomics 17:216–232. https://doi.org/10.1074/mcp.RA117.000470

  35. Magen D et al (2008) Mitochondrial hsp60 chaperonopathy causes an autosomal-recessive neurodegenerative disorder linked to brain hypomyelination and leukodystrophy. Am J Hum Genet 83:30–42

  36. Magnoni R et al (2013) Late onset motoneuron disorder caused by mitochondrial Hsp60 chaperone deficiency in mice. Neurobiol Dis 54:12–23. https://doi.org/10.1016/j.nbd.2013.02.012

  37. Magnoni R, Palmfeldt J, Hansen J, Christensen JH, Corydon TJ, Bross P (2014) The Hsp60 folding machinery is crucial for manganese superoxide dismutase folding and function. Free Radic Res 48:168–179. https://doi.org/10.3109/10715762.2013.858147

  38. Nielsen KL, McLennan N, Masters M, Cowan NJ (1999) A single-ring mitochondrial chaperonin (Hsp60-Hsp10) can substitute for GroEL-GroES in vivo. J Bacteriol 181:5871–5875

  39. Nisemblat S, Yaniv O, Parnas A, Frolow F, Azem A (2015) Crystal structure of the human mitochondrial chaperonin symmetrical football complex Proceedings of the National Academy of Sciences of the United States of America 112:6044-6049. https://doi.org/10.1073/pnas.1411718112

  40. Ong SE, Mann M (2006) A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC). Nature Protocols 1:2650–2660. https://doi.org/10.1038/nprot.2006.427

  41. Ostermann J, Horwich AL, Neupert W, Hartl FU (1989) Protein folding in mitochondria requires complex formation with hsp60 and ATP hydrolysis. Nature 341:125–130

  42. Ott M, Herrmann JM (2010) Co-translational membrane insertion of mitochondrially encoded proteins. Biochim Biophys Acta 1803:767–775. https://doi.org/10.1016/j.bbamcr.2009.11.010

  43. Perezgasga L, Segovia L, Zurita M (1999) Molecular characterization of the 5' control region and of two lethal alleles affecting the hsp60 gene in Drosophila melanogaster FEBS Lett 456:269-273

  44. Pfanner N, Warscheid B, Wiedemann N (2019) Mitochondrial proteins: from biogenesis to functional networks. Nat Rev Mol Cell Biol 20:267–284. https://doi.org/10.1038/s41580-018-0092-0

  45. Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2:1896. https://doi.org/10.1038/nprot.2007.261

  46. Rhee HW, Zou P, Udeshi ND, Martell JD, Mootha VK, Carr SA, Ting AY (2013) Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. Science (New York, NY) 339:1328–1331

  47. Royer-Bertrand B et al (2015) Mutations in the heat-shock protein A9 (HSPA9) gene cause the EVEN-PLUS syndrome of congenital malformations and skeletal dysplasia. Sci Rep 5:17154. https://doi.org/10.1038/srep17154

  48. Saijo T, Welch WJ, Tanaka K (1994) Intramitochondrial folding and assembly of medium-chain acyl-CoA dehydrogenase (MCAD) - demonstration of impaired transfer of K304E-variant MCAD from its complex with Hsp60 to the native tetramer. JBiolChem 269:4401–4408

  49. Saisawat P et al (2014) Whole-exome resequencing reveals recessive mutations in TRAP1 in individuals with CAKUT and VACTERL association. Kidney Int 85:1310–1317. https://doi.org/10.1038/ki.2013.417

  50. Sakikawa C, Taguchi H, Makino Y, Yoshida M (1999) On the maximum size of proteins to stay and fold in the cavity of GroEL underneath GroES. J Biol Chem 274:21251–21256

  51. Sissler M, Gonzalez-Serrano LE, Westhof E (2017) Recent advances in mitochondrial aminoacyl-trna synthetases and disease. Trends Mol Med 23:693–708. https://doi.org/10.1016/j.molmed.2017.06.002

  52. Szegő ÉM et al (2019) Cytosolic trapping of a mitochondrial heat shock protein is an early pathological event in synucleinopathies. Cell Rep 28:65–77.e66. https://doi.org/10.1016/j.celrep.2019.06.009

  53. Taylor RW et al (2014) Use of whole-exome sequencing to determine the genetic basis of multiple mitochondrial respiratory chain complex deficiencies. Jama 312:68–77. https://doi.org/10.1001/jama.2014.7184

  54. Wasilewski M, Chojnacka K, Chacinska A (2017) Protein trafficking at the crossroads to mitochondria. Biochim Biophys Acta 1864:125–137. https://doi.org/10.1016/j.bbamcr.2016.10.019

  55. Wisniewski JR, Zougman A, Nagaraj N, Mann M (2009) Universal sample preparation method for proteome analysis. Nat Methods 6:359–362. https://doi.org/10.1038/nmeth.1322

  56. Xu ZH, Horwich AL, Sigler PB (1997) The crystal structure of the asymmetric GroEL-GroES- (ADP)(7) chaperonin complex. Nature 388:741–750

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Funding

We thank the HEALTH graduate school, Aarhus University for support (PhD scholarship and travel and material support to A.B.), and the Hede Nielsens Fond, and the Deutscher Akademischer Austauschdienst (DAAD) for funding of materials, travel and accommodation.

Author information

Anne Bie, Peter Bross, Ulrich Hartl, Roman Körner, Thomas Corydon, Johan Palmfeldt, and Mark Hipp contributed to the study conception and design. Data collection and analysis were performed by Anne Bie, Roman Körner, Johan Palmfeldt, Mark Hipp, Cagla Cömert, and Peter Bross. The first draft of the manuscript was written by Anne Bie and Peter Bross. All authors commented on previous versions of the manuscript, and all authors read and approved the final manuscript. Ulrich Hartl and Peter Bross provided resources and lab-facilities.

Correspondence to Peter Bross.

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Bie, A.S., Cömert, C., Körner, R. et al. An inventory of interactors of the human HSP60/HSP10 chaperonin in the mitochondrial matrix space. Cell Stress and Chaperones (2020). https://doi.org/10.1007/s12192-020-01080-6

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Keywords

  • HSP60
  • HSP10
  • Molecular chaperone
  • Chaperonin
  • Mitochondrial protein quality control
  • Protein folding