Cellular and Molecular Life Sciences

, Volume 72, Issue 17, pp 3401–3409 | Cite as

Direct visualization of vaults within intact cells by electron cryo-tomography

  • Cora L. Woodward
  • Luiza M. Mendonça
  • Grant J. JensenEmail author
Research Article


The vault complex is the largest cellular ribonucleoprotein complex ever characterized and is present across diverse Eukarya. Despite significant information regarding the structure, composition and evolutionary conservation of the vault, little is know about the complex’s actual biological function. To determine if intracellular vaults are morphologically similar to previously studied purified and recombinant vaults, we have used electron cryo-tomography to characterize the vault complexes found in the thin edges of primary human cells growing in tissue culture. Our studies confirm that intracellular vaults are similar in overall size and shape to purified and recombinant vaults previously analyzed. Results from subtomogram averaging indicate that densities within the vault lumen are not ordered, but randomly distributed. We also observe that vaults located in the extreme periphery of the cytoplasm predominately associate with granule-like structures and actin. Our ultrastructure studies augment existing biochemical, structural and genetic information on the vault, and provide important intracellular context for the ongoing efforts to understand the biological function of the native cytoplasmic vault.


Vault Ribonucleoprotein complex Granules RNA Electron cryo-tomography 



We thank Zhiheng Yu and M. Jason de la Cruz of the Howard Hughes Medical Institute CryoEM Shared Resource at Janelia Farm for assistance with data collection. This work was supported by NIH Grant 2P50GM082545 (to GJJ).

Supplementary material

Supplementary material 1 (MOV 2069 kb)


  1. 1.
    Mikyas Y, Makabi M, Raval-Fernandes S, Harrington L, Kickhoefer VA, Rome LH, Stewart PL (2004) Cryoelectron microscopy imaging of recombinant and tissue derived vaults: localization of the MVP N termini and VPARP. J Mol Biol 344(1):91–105. doi: 10.1016/j.jmb.2004.09.021 PubMedCrossRefGoogle Scholar
  2. 2.
    Kedersha NL, Heuser JE, Chugani DC, Rome LH (1991) Vaults. III. Vault ribonucleoprotein particles open into flower-like structures with octagonal symmetry. J Cell Biol 112(2):225–235PubMedCrossRefGoogle Scholar
  3. 3.
    Kedersha NL, Rome LH (1986) Isolation and characterization of a novel ribonucleoprotein particle: large structures contain a single species of small RNA. J Cell Biol 103(3):699–709PubMedCrossRefGoogle Scholar
  4. 4.
    Kickhoefer VA, Vasu SK, Rome LH (1996) Vaults are the answer, what is the question? Trends Cell Biol 6(5):174–178PubMedCrossRefGoogle Scholar
  5. 5.
    Kedersha NL, Miquel MC, Bittner D, Rome LH (1990) Vaults. II. Ribonucleoprotein structures are highly conserved among higher and lower eukaryotes. J Cell Biol 110(4):895–901PubMedCrossRefGoogle Scholar
  6. 6.
    Stephen AG, Raval-Fernandes S, Huynh T, Torres M, Kickhoefer VA, Rome LH (2001) Assembly of vault-like particles in insect cells expressing only the major vault protein. J Biol Chem 276(26):23217–23220. doi: 10.1074/jbc.C100226200 PubMedCrossRefGoogle Scholar
  7. 7.
    Anderson DH, Kickhoefer VA, Sievers SA, Rome LH, Eisenberg D (2007) Draft crystal structure of the vault shell at 9-A resolution. PLoS Biol 5(11):e318. doi: 10.1371/journal.pbio.0050318 PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Casanas A, Querol-Audi J, Guerra P, Pous J, Tanaka H, Tsukihara T, Verdaguer N, Fita I (2013) New features of vault architecture and dynamics revealed by novel refinement using the deformable elastic network approach. Acta Crystallogr D Biol Crystallogr 69(Pt 6):1054–1061. doi: 10.1107/S0907444913004472 PubMedCrossRefGoogle Scholar
  9. 9.
    Tanaka H, Kato K, Yamashita E, Sumizawa T, Zhou Y, Yao M, Iwasaki K, Yoshimura M, Tsukihara T (2009) The structure of rat liver vault at 3.5 angstrom resolution. Science (New York, NY) 323(5912):384–388. doi: 10.1126/science.1164975 CrossRefGoogle Scholar
  10. 10.
    Kong LB, Siva AC, Kickhoefer VA, Rome LH, Stewart PL (2000) RNA location and modeling of a WD40 repeat domain within the vault. RNA (New York, NY) 6(6):890–900CrossRefGoogle Scholar
  11. 11.
    Kickhoefer VA, Rajavel KS, Scheffer GL, Dalton WS, Scheper RJ, Rome LH (1998) Vaults are up-regulated in multidrug-resistant cancer cell lines. J Biol Chem 273(15):8971–8974PubMedCrossRefGoogle Scholar
  12. 12.
    Berger W, Steiner E, Grusch M, Elbling L, Micksche M (2008) Vaults and the major vault protein: novel roles in signal pathway regulation and immunity. Cell Mol Life Sci CMLS 66(1):43–61. doi: 10.1007/s00018-008-8364-z CrossRefGoogle Scholar
  13. 13.
    Mossink MH, van Zon A, Fränzel-Luiten E, Schoester M, Kickhoefer VA, Scheffer GL, Scheper RJ, Sonneveld P, Wiemer EAC (2002) Disruption of the murine major vault protein (MVP/LRP) gene does not induce hypersensitivity to cytostatics. Cancer Res 62(24):7298–7304PubMedGoogle Scholar
  14. 14.
    Dortet L, Mostowy S, Samba-Louaka A, Louaka AS, Gouin E, Nahori M-A, Wiemer EAC, Dussurget O, Cossart P (2011) Recruitment of the major vault protein by InlK: a Listeria monocytogenes strategy to avoid autophagy. PLoS Pathog 7(8):e1002168. doi: 10.1371/journal.ppat.1002168 PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Mrazek J, Kreutmayer SB, Grasser FA, Polacek N, Huttenhofer A (2007) Subtractive hybridization identifies novel differentially expressed ncRNA species in EBV-infected human B cells. Nucleic Acids Res 35(10):e73. doi: 10.1093/nar/gkm244 PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Liu S, Hao Q, Peng N, Yue X, Wang Y, Chen Y, Wu J, Zhu Y (2012) Major vault protein: a virus-induced host factor against viral replication through the induction of type-I interferon. Hepatology 56(1):57–66. doi: 10.1002/hep.25642 PubMedCrossRefGoogle Scholar
  17. 17.
    Daugherty MD, Young JM, Kerns JA, Malik HS (2014) Rapid evolution of PARP genes suggests a broad role for ADP-ribosylation in host-virus conflicts. PLoS Genet 10(5):e1004403. doi: 10.1371/journal.pgen.1004403.s018 PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Tanaka H, Tsukihara T (2012) Structural studies of large nucleoprotein particles, vaults. Proc Jpn Acad Ser B Phys Biol Sci 88(8):416–433PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Eichenmüller B, Kedersha N, Solovyeva E, Everley P, Lang J, Himes RH, Suprenant KA (2003) Vaults bind directly to microtubules via their caps and not their barrels. Cell Motil Cytoskelet 56(4):225–236CrossRefGoogle Scholar
  20. 20.
    Ando D, Mattson MK, Xu J, Gopinathan A (2014) Cooperative protofilament switching emerges from inter-motor interference in multiple-motor transport. Sci Rep 4:7255. doi: 10.1038/srep07255 PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Nicastro D, Schwartz C, Pierson J, Gaudette R, Porter ME, McIntosh JR (2006) The molecular architecture of axonemes revealed by cryoelectron tomography. Science 313(5789):944–948. doi: 10.1126/science.1128618 PubMedCrossRefGoogle Scholar
  22. 22.
    Gan L, Jensen GJ (2012) Electron tomography of cells. Q Rev Biophys 45(1):27–56. doi: 10.1017/S0033583511000102 PubMedCrossRefGoogle Scholar
  23. 23.
    Kong LB, Siva AC, Rome LH, Stewart PL (1999) Structure of the vault, a ubiquitous cellular component. Structure 7(4):371–379PubMedCrossRefGoogle Scholar
  24. 24.
    Pilhofer M, Ladinsky MS, McDowall AW, Petroni G, Jensen GJ (2011) Microtubules in bacteria: ancient tubulins build a five-protofilament homolog of the eukaryotic cytoskeleton. PLoS Biol 9(12):e1001213. doi: 10.1371/journal.pbio.1001213 PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Kickhoefer VA, Liu Y, Kong LB, Snow BE, Stewart PL, Harrington L, Rome LH (2001) The Telomerase/vault-associated protein TEP1 is required for vault RNA stability and its association with the vault particle. J Cell Biol 152(1):157–164PubMedCentralPubMedCrossRefGoogle Scholar
  26. 26.
    Querol-Audi J, Casanas A, Uson I, Luque D, Caston JR, Fita I, Verdaguer N (2009) The mechanism of vault opening from the high resolution structure of the N-terminal repeats of MVP. EMBO J 28(21):3450–3457. doi: 10.1038/emboj.2009.274 PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Yang J, Kickhoefer VA, Ng BC, Gopal A, Bentolila LA, John S, Tolbert SH, Rome LH (2010) Vaults are dynamically unconstrained cytoplasmic nanoparticles capable of half vault exchange. ACS Nano 4(12):7229–7240. doi: 10.1021/nn102051r PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Kickhoefer VA, Siva AC, Kedersha NL, Inman EM, Ruland C, Streuli M, Rome LH (1999) The 193-kD vault protein, VPARP, is a novel poly(ADP-ribose) polymerase. J Cell Biol 146(5):917–928PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Vasu SK, Rome LH (1995) Dictyostelium vaults: disruption of the major proteins reveals growth and morphological defects and uncovers a new associated protein. J Biol Chem 270(28):16588–16594PubMedCrossRefGoogle Scholar
  30. 30.
    Mrazek J, Toso D, Ryazantsev S, Zhang X, Zhou ZH, Fernandez BC, Kickhoefer VA, Rome LH (2014) Polyribosomes are molecular 3D nanoprinters that orchestrate the assembly of vault particles. ACS Nano. doi: 10.1021/nn504778h PubMedCentralPubMedGoogle Scholar
  31. 31.
    Weber SC, Brangwynne CP (2012) Getting RNA and protein in phase. Cell 149 (6):1188–1191. doi: 10.1016/j.cell.2012.05.022 PubMedCrossRefGoogle Scholar
  32. 32.
    Hyman AA, Brangwynne CP (2011) Beyond stereospecificity: liquids and mesoscale organization of cytoplasm. Dev Cell 21(1):14–16. doi: 10.1016/j.devcel.2011.06.013 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Basel 2015

Authors and Affiliations

  • Cora L. Woodward
    • 1
  • Luiza M. Mendonça
    • 1
    • 3
  • Grant J. Jensen
    • 1
    • 2
    Email author
  1. 1.Division of BiologyCalifornia Institute of TechnologyPasadenaUSA
  2. 2.Howard Hughes Medical InstituteCalifornia Institute of TechnologyPasadenaUSA
  3. 3.Departamento de VirologiaUniversidade Federal do Rio de JaneiroRio de JaneiroBrazil

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