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Expression and Characterization of MICU2, a Ca2+ Sensor Protein

  • Wenping Wu
  • Jimin ZhengEmail author
  • Zongchao JiaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1929)

Abstract

MICU2 is a Ca2+ sensor protein of mitochondrial uniporter which is a highly selective Ca2+ channel mediating mitochondrial Ca2+ uptake to regulate cell death, metabolism, and cytoplasmic Ca2+ signaling. Here we describe the procedures for protein preparation of various MICU2 constructs, which have enabled successful in vitro characterizations of MICU2 including interaction with MICU1 using pull-down assays and oligomerization using multi-angle laser light scattering.

Key words

MICU2 Ca2+ sensor EF hand Mitochondrial SEC MALLS Pull-down Purification 

References

  1. 1.
    Rizzuto R, De Stefani D, Raffaello A, Mammucari C (2012) Mitochondria as sensors and regulators of calcium signalling. Nat Rev Mol Cell Biol 13(9):566–578.  https://doi.org/10.1038/nrm3412 CrossRefPubMedGoogle Scholar
  2. 2.
    O'Rourke B (2007) Mitochondrial ion channels. Annu Rev Physiol 69:19–49.  https://doi.org/10.1146/annurev.physiol.69.031905.163804 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Santo-Domingo J, Demaurex N (2010) Calcium uptake mechanisms of mitochondria. Biochim Biophys Acta 1797(6–7):907–912.  https://doi.org/10.1016/j.bbabio.2010.01.005 CrossRefPubMedGoogle Scholar
  4. 4.
    Drago I, Pizzo P, Pozzan T (2011) After half a century mitochondrial calcium in- and efflux machineries reveal themselves. EMBO J 30(20):4119–4125.  https://doi.org/10.1038/emboj.2011.337 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    De Stefani D, Raffaello A, Teardo E, Szabo I, Rizzuto R (2011) A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter. Nature 476(7360):336–340.  https://doi.org/10.1038/nature10230 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Raffaello A, De Stefani D, Sabbadin D, Teardo E, Merli G, Picard A, Checchetto V, Moro S, Szabo I, Rizzuto R (2013) The mitochondrial calcium uniporter is a multimer that can include a dominant-negative pore-forming subunit. EMBO J 32(17):2362–2376.  https://doi.org/10.1038/emboj.2013.157 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Sancak Y, Markhard AL (2013) EMRE is an essential component of the mitochondrial calcium uniporter complex. Science 342(6164):1379–1382.  https://doi.org/10.1126/science.1242993 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Perocchi F, Gohil VM, Girgis HS, Bao XR, McCombs JE, Palmer AE, Mootha VK (2010) MICU1 encodes a mitochondrial EF hand protein required for Ca(2+) uptake. Nature 467(7313):291–296.  https://doi.org/10.1038/nature09358 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Plovanich M, Bogorad RL, Sancak Y, Kamer KJ, Strittmatter L, Li AA, Girgis HS, Kuchimanchi S, De Groot J, Speciner L, Taneja N, Oshea J, Koteliansky V, Mootha VK (2013) MICU2, a paralog of MICU1, resides within the mitochondrial uniporter complex to regulate calcium handling. PLoS One 8(2):e55785.  https://doi.org/10.1371/journal.pone.0055785 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Oxenoid K, Dong Y, Cao C, Cui T, Sancak Y, Markhard AL, Grabarek Z, Kong L, Liu Z, Ouyang B, Cong Y, Mootha VK, Chou JJ (2016) Architecture of the mitochondrial calcium uniporter. Nature 533(7602):269–273.  https://doi.org/10.1038/nature17656 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Tsai MF, Phillips CB, Ranaghan M, Tsai CW, Wu Y, Willliams C, Miller C (2016) Dual functions of a small regulatory subunit in the mitochondrial calcium uniporter complex. elife 5:e15545.  https://doi.org/10.7554/eLife.15545 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Patron M, Checchetto V, Raffaello A, Teardo E, Vecellio Reane D, Mantoan M, Granatiero V, Szabo I, De Stefani D, Rizzuto R (2014) MICU1 and MICU2 finely tune the mitochondrial Ca2+ uniporter by exerting opposite effects on MCU activity. Mol Cell 53(5):726–737.  https://doi.org/10.1016/j.molcel.2014.01.013 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Kamer KJ, Mootha VK (2014) MICU1 and MICU2 play nonredundant roles in the regulation of the mitochondrial calcium uniporter. EMBO Rep 15(3):299–307.  https://doi.org/10.1002/embr.201337946 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Li D, Wu W, Pei H, Wei Q, Yang Q, Zheng J, Jia Z (2016) Expression and preliminary characterization of human MICU2. Biol Open 5(7):962–969.  https://doi.org/10.1242/bio.018572 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Kamer KJ, Grabarek Z, Mootha VK (2017) High-affinity cooperative Ca2+ binding by MICU1-MICU2 serves as an on-off switch for the uniporter. EMBO Rep 18(8):1397–1411.  https://doi.org/10.15252/embr.201643748 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.College of ChemistryBeijing Normal UniversityBeijingChina
  2. 2.Department of Biochemical and Molecular ScienceQueen UniversityKingstonCanada

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