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Spatially regulated ubiquitin ligation by an ER/nuclear membrane ligase

Nature volume 443, pages 827–831 (2006)Cite this article

Abstract

The ubiquitin system targets many cellular proteins. Doa10 (also known as Ssm4), a yeast transmembrane ubiquitin ligase (E3), resides in the endoplasmic reticulum (ER), but it attaches ubiquitin to soluble proteins that concentrate in the nucleus. A central question is how nuclear substrates gain access to an enzyme in the ER. Here we show that Doa10 reaches the inner nuclear membrane. A subcomplex of nuclear pore subunits is important for this transport. Notably, another ER transmembrane E3, Hrd1 (also known as Der3), cannot localize efficiently to the inner nuclear membrane. Tethering Doa10 at the cell periphery inhibits degradation of soluble nuclear substrates but not cytoplasmic ones. If Doa10 is released from these peripheral sites, localization of Doa10 to the nuclear envelope and degradation of its nuclear substrates are restored in parallel. Thus, localization of Doa10 to the inner nuclear membrane is necessary for nuclear substrate degradation. These data indicate that different membrane ubiquitin ligases are spatially sorted within the ER–nuclear envelope membrane system and that this differential localization contributes to their specificity.

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Figure 1: Localization of a transmembrane ubiquitin ligase to the inner nuclear envelope.
Figure 2: Fluorescence assay of INM protein localization.
Figure 3: Localization of Doa10 to the INM is required for nuclear substrate degradation.
Figure 4: Nuclear substrate degradation and determinants of Doa10 INM localization.

References

  1. Hochstrasser, M. Ubiquitin-dependent protein degradation. Annu. Rev. Genet. 30, 405–439 (1996)

    Article  CAS  Google Scholar 

  2. Hershko, A. & Ciechanover, A. The ubiquitin system. Annu. Rev. Biochem. 67, 425–479 (1998)

    Article  CAS  Google Scholar 

  3. Pickart, C. M. & Eddins, M. J. Ubiquitin: structures, functions, mechanisms. Biochim. Biophys. Acta 1695, 55–72 (2004)

    Article  CAS  Google Scholar 

  4. Varshavsky, A. Regulated protein degradation. Trends Biochem. Sci. 30, 283–286 (2005)

    Article  CAS  Google Scholar 

  5. Pines, J. & Lindon, C. Proteolysis: anytime, any place, anywhere?. Nature Cell Biol. 7, 731–735 (2005)

    Article  CAS  Google Scholar 

  6. Swanson, R., Locher, M. & Hochstrasser, M. A conserved ubiquitin ligase of the nuclear envelope/endoplasmic reticulum that functions in both ER-associated and Matα2 repressor degradation. Genes Dev. 15, 2660–2674 (2001)

    Article  CAS  Google Scholar 

  7. Kreft, S. G., Wang, L. & Hochstrasser, M. Membrane topology of the yeast endoplasmic reticulum-localized ubiquitin ligase Doa10 and comparison with its human ortholog TEB4 (MARCH-VI). J. Biol. Chem. 281, 4646–4653 (2006)

    Article  CAS  Google Scholar 

  8. Chen, P., Johnson, P., Sommer, T., Jentsch, S. & Hochstrasser, M. Multiple ubiquitin-conjugating enzymes participate in the in vivo degradation of the yeast MATα2 repressor. Cell 74, 357–369 (1993)

    Article  CAS  Google Scholar 

  9. Ravid, T., Kreft, S. G. & Hochstrasser, M. Membrane and soluble substrates of the Doa10 ubiquitin ligase are degraded by distinct pathways. EMBO J. 25, 533–543 (2006)

    Article  CAS  Google Scholar 

  10. Johnson, P. R., Swanson, R., Rakhilina, L. & Hochstrasser, M. Degradation signal masking by heterodimerization of MATα2 and MATa1 blocks their mutual destruction by the ubiquitin-proteasome pathway. Cell 94, 217–227 (1998)

    Article  CAS  Google Scholar 

  11. Andrulis, E. D., Neiman, A. M., Zappulla, D. C. & Sternglanz, R. Perinuclear localization of chromatin facilitates transcriptional silencing. Nature 394, 592–595 (1998); erratum. 395, 525 (1998)

    Article  ADS  CAS  Google Scholar 

  12. Taddei, A., Hediger, F., Neumann, F. R. & Gasser, S. M. The function of nuclear architecture: a genetic approach. Annu. Rev. Genet. 38, 305–345 (2004)

    Article  CAS  Google Scholar 

  13. Marelli, M., Lusk, C. P., Chan, H., Aitchison, J. D. & Wozniak, R. W. A link between the synthesis of nucleoporins and the biogenesis of the nuclear envelope. J. Cell Biol. 153, 709–724 (2001)

    Article  CAS  Google Scholar 

  14. Wright, R., Basson, M., D'Ari, L. & Rine, J. Increased amounts of HMG-CoA reductase induce “karmellae”: a proliferation of stacked membrane pairs surrounding the yeast nucleus. J. Cell Biol. 107, 101–114 (1988)

    Article  CAS  Google Scholar 

  15. Isaac, C., Pollard, J. W. & Meier, U. T. Intranuclear endoplasmic reticulum induced by Nopp140 mimics the nucleolar channel system of human endometrium. J. Cell Sci. 114, 4253–4264 (2001)

    CAS  PubMed  Google Scholar 

  16. Hitchcock, A. L. et al. The conserved Npl4 protein complex mediates proteasome-dependent membrane-bound transcription factor activation. Mol. Biol. Cell 12, 3226–3241 (2001)

    Article  CAS  Google Scholar 

  17. Rape, M. et al. Mobilization of processed, membrane-tethered SPT23 transcription factor by CDC48UFD1/NPL4, a ubiquitin-selective chaperone. Cell 107, 667–677 (2001)

    Article  CAS  Google Scholar 

  18. Hampton, R. Y. ER-associated degradation in protein quality control and cellular regulation. Curr. Opin. Cell Biol. 14, 476–482 (2002)

    Article  CAS  Google Scholar 

  19. Hinshaw, J. E., Carragher, B. O. & Milligan, R. A. Architecture and design of the nuclear pore complex. Cell 69, 1133–1141 (1992)

    Article  CAS  Google Scholar 

  20. Suntharalingam, M. & Wente, S. R. Peering through the pore: nuclear pore complex structure, assembly, and function. Dev. Cell 4, 775–789 (2003)

    Article  CAS  Google Scholar 

  21. Worman, H. J. & Courvalin, J. C. The inner nuclear membrane. J. Membr. Biol. 177, 1–11 (2000)

    Article  CAS  Google Scholar 

  22. Ohba, T., Schirmer, E. C., Nishimoto, T. & Gerace, L. Energy- and temperature-dependent transport of integral proteins to the inner nuclear membrane via the nuclear pore. J. Cell Biol. 167, 1051–1062 (2004)

    Article  CAS  Google Scholar 

  23. Nehrbass, U., Rout, M. P., Maguire, S., Blobel, G. & Wozniak, R. W. The yeast nucleoporin Nup188p interacts genetically and physically with the core structures of the nuclear pore complex. J. Cell Biol. 133, 1153–1162 (1996)

    Article  CAS  Google Scholar 

  24. Damelin, M. & Silver, P. A. In situ analysis of spatial relationships between proteins of the nuclear pore complex. Biophys. J. 83, 3626–3636 (2002)

    Article  ADS  CAS  Google Scholar 

  25. Goode, B. L. et al. Coronin promotes the rapid assembly and cross-linking of actin filaments and may link the actin and microtubule cytoskeletons in yeast. J. Cell Biol. 144, 83–98 (1999)

    Article  CAS  Google Scholar 

  26. Hall, M. N., Hereford, L. & Herskowitz, I. Targeting of E. coli β-galactosidase to the nucleus in yeast. Cell 36, 1057–1065 (1984)

    Article  CAS  Google Scholar 

  27. Appleton, B. A., Wu, P. & Wiesmann, C. The crystal structure of murine coronin-1: a regulator of actin cytoskeletal dynamics in lymphocytes. Structure 14, 87–96 (2006)

    Article  CAS  Google Scholar 

  28. Ishii, K., Arib, G., Lin, C., Van Houwe, G. & Laemmli, U. K. Chromatin boundaries in budding yeast: the nuclear pore connection. Cell 109, 551–562 (2002)

    Article  CAS  Google Scholar 

  29. Casolari, J. M. et al. Genome-wide localization of the nuclear transport machinery couples transcriptional status and nuclear organization. Cell 117, 427–439 (2004)

    Article  CAS  Google Scholar 

  30. Brickner, J. H. & Walter, P. Gene recruitment of the activated INO1 locus to the nuclear membrane. PLoS Biol. 2, e342 (2004)

    Article  Google Scholar 

  31. Ma, J. & Ptashne, M. A new class of yeast transcriptional activators. Cell 51, 113–119 (1987)

    Article  CAS  Google Scholar 

  32. Plemper, R. K., Bohmler, S., Bordallo, J., Sommer, T. & Wolf, D. H. Mutant analysis links the translocon and BiP to retrograde protein transport for ER degradation. Nature 388, 891–895 (1997)

    Article  ADS  CAS  Google Scholar 

  33. Giddings, T. H. et al. Using rapid freeze and freeze-substitution for the preparation of yeast cells for electron microscopy and three-dimensional analysis. Methods Cell Biol. 67, 27–42 (2001)

    Article  CAS  Google Scholar 

  34. King, M. C., Lusk, C. P. & Blobel, G. Karyopherin-mediated import of integral inner nuclear membrane proteins. Nature 442, 1003–1007 (2006)

    Article  ADS  CAS  Google Scholar 

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Acknowledgements

We thank R. Sternglanz and R. Wozniak for strains and plasmids, P. Crews for generously providing LTA, O. Kerscher for Supplementary Fig. 1, and J. Wu and J. Wolenski for advice on confocal microscopy. We are grateful to S. Kreft, A. Lewis and T. Ravid for comments on the manuscript. This work was supported by the NIH. Author Contributions M.D. performed all the experiments, and M.D. and M.H. planned the experiments, performed the data analysis and wrote the paper.

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  1. Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, P.O. Box 208114, Connecticut, 06520-8114, New Haven, USA

    Min Deng & Mark Hochstrasser

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  1. Min Deng
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Correspondence to Mark Hochstrasser.

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Deng, M., Hochstrasser, M. Spatially regulated ubiquitin ligation by an ER/nuclear membrane ligase. Nature 443, 827–831 (2006). https://doi.org/10.1038/nature05170

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