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Systematic proteomics of the VCP–UBXD adaptor network identifies a role for UBXN10 in regulating ciliogenesis

Nature Cell Biology volume 17, pages 13561369 (2015) | Download Citation

  • A Corrigendum to this article was published on 25 February 2016

This article has been updated

Abstract

The AAA-ATPase VCP (also known as p97 or CDC48) uses ATP hydrolysis to ‘segregate’ ubiquitylated proteins from their binding partners. VCP acts through UBX-domain-containing adaptors that provide target specificity, but the targets and functions of UBXD proteins remain poorly understood. Through systematic proteomic analysis of UBXD proteins in human cells, we reveal a network of over 195 interacting proteins, implicating VCP in diverse cellular pathways. We have explored one such complex between an unstudied adaptor UBXN10 and the intraflagellar transport B (IFT-B) complex, which regulates anterograde transport into cilia. UBXN10 localizes to cilia in a VCP-dependent manner and both VCP and UBXN10 are required for ciliogenesis. Pharmacological inhibition of VCP destabilized the IFT-B complex and increased trafficking rates. Depletion of UBXN10 in zebrafish embryos causes defects in left–right asymmetry, which depends on functional cilia. This study provides a resource for exploring the landscape of UBXD proteins in biology and identifies an unexpected requirement for VCP–UBXN10 in ciliogenesis.

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Change history

  • 01 February 2016

    In the version of this Resource originally published, the sequence for the UBXN7 morpholino was reported incorrectly in the Methods section as 5′-TTTTGGATTCTCCACCCGAAGCCAT-3′. The correct sequence is 5′-ATGCGTCTCCGAGAGTCGCCATCTT-3′. This has been corrected in all online versions of the Resource.

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Acknowledgements

This work was supported by NIH grants R37-NS083524 and RO1-AG011085 to J.W.H. We would like to thank the Nikon Imaging Center (Harvard Medical School) for microscopy assistance. We would like to thank J. Wallingford (UT Austin) for the Xenopus laevis IFT-43 construct and M. Nachury and A. Nager (Stanford) for helpful discussions.

Author information

Affiliations

  1. Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA

    • Malavika Raman
    • , John R. Lydeard
    • , Edward L. Huttlin
    •  & J. Wade Harper
  2. Department of Systems Biology, Harvard Institutes of Medicine, 4 Blackfan Circle, Boston, Massachusetts 02115, USA

    • Mikhail Sergeev
    •  & Jagesh V. Shah
  3. Renal Division, Brigham and Women’s Hospital, Harvard Institutes of Medicine, 4 Blackfan Circle, Boston, Massachusetts 02115, USA

    • Mikhail Sergeev
    •  & Jagesh V. Shah
  4. Department of Medicine, New Research Building, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA

    • Maija Garnaas
    •  & Wolfram Goessling
  5. Department of Health Science and Technology, New Research Building, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA

    • Maija Garnaas
    •  & Wolfram Goessling
  6. Harvard-MIT, Division of Health Sciences and Technology, Harvard Institutes of Medicine, 4 Blackfan Circle, Boston, Massachusetts 02115, USA

    • Jagesh V. Shah

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Contributions

J.W.H. and M.R. conceived the project. M.R. prepared cell lines, performed immunofluorescence in cells and zebrafish, carried out cilia length measurements, performed and analysed AP–MS experiments (with the assistance of J.R.L.), and performed biochemical studies. M.S. performed cilia trafficking experiments under the direction of J.V.S. M.G. performed zebrafish experiments under the direction of W.G. E.L.H. performed the statistical analysis to determine P values for the MS data set. M.R. and J.W.H. wrote the paper with input from all authors.

Competing interests

J.W.H. is a consultant for Biogen-Idec and Millennium: The Takeda Oncology Company.

Corresponding author

Correspondence to J. Wade Harper.

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Videos

  1. 1.

    UBXN10 exhibits IFT-like transport within cilia.

    Live cell imaging of GFP-UBXN10 demonstrating IFT-like particle motility within cilia in hTERT-RPE1 cells.

  2. 2.

    UBXN10 and IFT-B migrate as part of the same complex within cilia.

    Dual colour TIRF microscopy of IMCD cells expressing EYFP-IFT88 and UBXN10-mCHERRY to visualize co-migration within cilia.

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DOI

https://doi.org/10.1038/ncb3238

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