Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
권호 표지
DOI QR코드

DOI QR Code

Biochemical Characterization of the Num1-Mdm36 Complex at the Mitochondria-Plasma Membrane Contact Site

  • Won, Jongdae (Department of Chemistry, College of Natural Sciences, Seoul National University) ;
  • Choi, Yuri (Department of Chemistry, College of Natural Sciences, Seoul National University) ;
  • Yun, Yaejin (Department of Chemistry, College of Natural Sciences, Seoul National University) ;
  • Lee, Hyung Ho (Department of Chemistry, College of Natural Sciences, Seoul National University)
  • Received : 2021.01.23
  • Accepted : 2021.03.03
  • Published : 2021.04.30
Download PDF
⟨ Previous Next ⟩

Abstract

In eukaryotic cells, organelles are distributed and positioned in proximity to each other through molecular tether proteins. Among these, the mitochondria-endoplasmic reticulum cortex anchor (MECA) is a well-known tethering complex in Saccharomyces cerevisiae that tethers mitochondria to the plasma membrane and plays a key role in mitochondrial fission. The main components of MECA are Num1 and Mdm36, and it is known that Mdm36 binds to Num1 to enhance mitochondrial tethering. To better understand the biochemical characteristics of the Num1-Mdm36 complex at the molecular level, we purified the coiled-coil domain of Num1, full-length Mdm36, and Num1-Mdm36 complex and identified the oligomeric state and stoichiometric characteristics of the Num1-Mdm36 complex by chemical crosslinking, size-exclusion chromatography coupled with multi-angle light scattering, and isothermal titration calorimetry. Mdm36 exists as a dimer and interacts preferentially with Num1 with a stoichiometry of 2:2, forming a heterotetrameric complex. Furthermore, we narrowed down the specific binding region of Num1, which is essential for interacting with Mdm36, and showed that their binding affinity is strong enough to tether both mitochondrial and plasma membranes. Our biochemical characterizations suggest a stoichiometric model of the Num1-Mdm36 complex at the mitochondria-plasma membrane contact site in budding yeast.

Keywords

Acknowledgement

This study was also supported by a grant from the National Research Foundation (NRF) of Korea, funded by the Korean government (2015R1A5A1008958, 2015M3D3A1A01064919, and 2018R1A2B2008142).

References

  1. Booth, D.M., Enyedi, B., Geiszt, M., Varnai, P., and Hajnoczky, G. (2016). Redox nanodomains are induced by and control calcium signaling at the ER-mitochondrial interface. Mol. Cell 63, 240-248. https://doi.org/10.1016/j.molcel.2016.05.040
  2. Cerveny, K.L., Studer, S.L., Jensen, R.E., and Sesaki, H. (2007). Yeast mitochondrial division and distribution require the cortical num1 protein. Dev. Cell 12, 363-375. https://doi.org/10.1016/j.devcel.2007.01.017
  3. Chen, W., Ping, H.A., and Lackner, L.L. (2018). Direct membrane binding and self-interaction contribute to Mmr1 function in mitochondrial inheritance. Mol. Biol. Cell 29, 2346-2357. https://doi.org/10.1091/mbc.E18-02-0122
  4. de Brito, O.M. and Scorrano, L. (2008). Mitofusin 2 tethers endoplasmic reticulum to mitochondria. Nature 456, 605-610. https://doi.org/10.1038/nature07534
  5. Dong, D., Huang, X., Li, L., Mao, H., Mo, Y., Zhang, G., Zhang, Z., Shen, J., Liu, W., Wu, Z., et al. (2020). Super-resolution fluorescence-assisted diffraction computational tomography reveals the three-dimensional landscape of the cellular organelle interactome. Light Sci. Appl. 9, 11. https://doi.org/10.1038/s41377-020-0249-4
  6. Eisenberg-Bord, M., Shai, N., Schuldiner, M., and Bohnert, M. (2016). A tether is a tether is a tether: tethering at membrane contact sites. Dev. Cell 39, 395-409. https://doi.org/10.1016/j.devcel.2016.10.022
  7. Garcia De La Torre, J., Huertas, M.L., and Carrasco, B. (2000). Calculation of hydrodynamic properties of globular proteins from their atomic-level structure. Biophys. J. 78, 719-730. https://doi.org/10.1016/S0006-3495(00)76630-6
  8. Hammermeister, M., Schodel, K., and Westermann, B. (2010). Mdm36 is a mitochondrial fission-promoting protein in Saccharomyces cerevisiae. Mol. Biol. Cell 21, 2443-2452. https://doi.org/10.1091/mbc.E10-02-0096
  9. Hayashi, T., Rizzuto, R., Hajnoczky, G., and Su, T.P. (2009). MAM: more than just a housekeeper. Trends Cell Biol. 19, 81-88. https://doi.org/10.1016/j.tcb.2008.12.002
  10. Helle, S.C., Kanfer, G., Kolar, K., Lang, A., Michel, A.H., and Kornmann, B. (2013). Organization and function of membrane contact sites. Biochim. Biophys. Acta 1833, 2526-2541. https://doi.org/10.1016/j.bbamcr.2013.01.028
  11. Huang, X., Jiang, C., Yu, L., and Yang, A. (2020). Current and emerging approaches for studying inter-organelle membrane contact sites. Front. Cell Dev. Biol. 8, 195. https://doi.org/10.3389/fcell.2020.00195
  12. Kakimoto, Y., Tashiro, S., Kojima, R., Morozumi, Y., Endo, T., and Tamura, Y. (2018). Visualizing multiple inter-organelle contact sites using the organelle-targeted split-GFP system. Sci. Rep. 8, 6175. https://doi.org/10.1038/s41598-018-24466-0
  13. Kormanec, J., Schaaff-Gerstenschlager, I., Zimmermann, F.K., Perecko, D., and Kuntzel, H. (1991). Nuclear migration in Saccharomyces cerevisiae is controlled by the highly repetitive 313 kDa NUM1 protein. Mol. Gen. Genet. 230, 277-287. https://doi.org/10.1007/BF00290678
  14. Kraft, L.M. and Lackner, L.L. (2018). Mitochondrial anchors: positioning mitochondria and more. Biochem. Biophys. Res. Commun. 500, 2-8. https://doi.org/10.1016/j.bbrc.2017.06.193
  15. Lackner, L.L., Ping, H., Graef, M., Murley, A., and Nunnari, J. (2013). Endoplasmic reticulum-associated mitochondria-cortex tether functions in the distribution and inheritance of mitochondria. Proc. Natl. Acad. Sci. U. S. A. 110, E458-E467. https://doi.org/10.1073/pnas.1215232110
  16. Naon, D. and Scorrano, L. (2014). At the right distance: ER-mitochondria juxtaposition in cell life and death. Biochim. Biophys. Acta 1843, 2184-2194. https://doi.org/10.1016/j.bbamcr.2014.05.011
  17. Nascimbeni, A.C., Giordano, F., Dupont, N., Grasso, D., Vaccaro, M.I., Codogno, P., and Morel, E. (2017). ER-plasma membrane contact sites contribute to autophagosome biogenesis by regulation of local PI3P synthesis. EMBO J. 36, 2018-2033. https://doi.org/10.15252/embj.201797006
  18. Ouasti, S., Matarrese, P., Paddon, R., Khosravi-Far, R., Sorice, M., Tinari, A., Malorni, W., and Degli Esposti, M. (2007). Death receptor ligation triggers membrane scrambling between Golgi and mitochondria. Cell Death Differ. 14, 453-461. https://doi.org/10.1038/sj.cdd.4402043
  19. Ping, H.A., Kraft, L.M., Chen, W., Nilles, A.E., and Lackner, L.L. (2016). Num1 anchors mitochondria to the plasma membrane via two domains with different lipid binding specificities. J. Cell Biol. 213, 513-524. https://doi.org/10.1083/jcb.201511021
  20. Prinz, W.A., Toulmay, A., and Balla, T. (2020). The functional universe of membrane contact sites. Nat. Rev. Mol. Cell Biol. 21, 7-24. https://doi.org/10.1038/s41580-019-0180-9
  21. Rizzuto, R., Pinton, P., Carrington, W., Fay, F.S., Fogarty, K.E., Lifshitz, L.M., Tuft, R.A., and Pozzan, T. (1998). Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses. Science 280, 1763-1766. https://doi.org/10.1126/science.280.5370.1763
  22. Shai, N., Yifrach, E., van Roermund, C.W.T., Cohen, N., Bibi, C., IJlst, L., Cavellini, L., Meurisse, J., Schuster, R., Zada, L., et al. (2018). Systematic mapping of contact sites reveals tethers and a function for the peroxisome-mitochondria contact. Nat. Commun. 9, 1761. https://doi.org/10.1038/s41467-018-03957-8
  23. Tang, X., Germain, B.S., and Lee, W.L. (2012). A novel patch assembly domain in Num1 mediates dynein anchoring at the cortex during spindle positioning. J. Cell Biol. 196, 743-756. https://doi.org/10.1083/jcb.201112017
  24. Travis, S.M., DAmico, K., Yu, I.M., McMahon, C., Hamid, S., Ramirez-Arellano, G., Jeffrey, P.D., and Hughson, F.M. (2020). Structural basis for the binding of SNAREs to the multisubunit tethering complex Dsl1. J. Biol. Chem. 295, 10125-10135. https://doi.org/10.1074/jbc.ra120.013654
  25. Valm, A.M., Cohen, S., Legant, W.R., Melunis, J., Hershberg, U., Wait, E., Cohen, A.R., Davidson, M.W., Betzig, E., and Lippincott-Schwartz, J. (2017). Applying systems-level spectral imaging and analysis to reveal the organelle interactome. Nature 546, 162-167. https://doi.org/10.1038/nature22369
  26. Westermann, B. (2015). The mitochondria-plasma membrane contact site. Curr. Opin. Cell Biol. 35, 1-6. https://doi.org/10.1016/j.ceb.2015.03.001