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Drosophila Rae1 controls the abundance of the ubiquitin ligase Highwire in post-mitotic neurons

Nature Neuroscience volume 14, pages 1267–1275 (2011)Cite this article

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Abstract

The evolutionarily conserved Highwire (Hiw)/Drosophila Fsn E3 ubiquitin ligase complex is required for normal synaptic morphology during development and axonal regeneration after injury. However, little is known about the molecular mechanisms that regulate the Hiw E3 ligase complex. Using tandem affinity purification techniques, we identified Drosophila Rae1 as a previously unknown component of the Hiw/Fsn complex. Loss of Rae1 function in neurons results in morphological defects at the neuromuscular junction that are similar to those seen in hiw mutants. We found that Rae1 physically and genetically interacts with Hiw and restrains synaptic terminal growth by regulating the MAP kinase kinase kinase Wallenda. Moreover, we found that the Rae1 is both necessary and sufficient to promote Hiw protein abundance, and it does so by binding to Hiw and protecting Hiw from autophagy-mediated degradation. These results describe a previously unknown mechanism that selectively controls Hiw protein abundance during synaptic development.

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Figure 1: Rae1 and Hiw interact with each other in neurons.
Figure 2: Generation of Rae1 mutant alleles.
Figure 3: Rae1 is required to restrain synaptic terminal growth at the NMJ.
Figure 4: Rae1 genetically interacts with hiw to restrain synaptic terminal growth.
Figure 5: A structure and function analysis of Hiw functional domains.
Figure 6: Rae1 interacts with a fragment in the Hiw C-terminal region, and coexpression of Rae1 with NT-Hiw-CT suppresses the NT-Hiw-CT–induced dominant-negative overgrowth phenotype.
Figure 7: Rae1 promotes Hiw abundance.
Figure 8: Rae1-Hiw interaction prevents autophagy-mediated degradation of Hiw protein.

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References

  1. Po, M.D., Hwang, C. & Zhen, M. PHRs: bridging axon guidance, outgrowth and synapse development. Curr. Opin. Neurobiol. 20, 100–107 (2010).

    Article  CAS  Google Scholar 

  2. DiAntonio, A. et al. Ubiquitination-dependent mechanisms regulate synaptic growth and function. Nature 412, 449–452 (2001).

    Article  CAS  Google Scholar 

  3. Wan, H.I. et al. Highwire regulates synaptic growth in Drosophila. Neuron 26, 313–329 (2000).

    Article  CAS  Google Scholar 

  4. Uthaman, S.B., Godenschwege, T.A. & Murphey, R.K. A mechanism distinct from highwire for the Drosophila ubiquitin conjugase bendless in synaptic growth and maturation. J. Neurosci. 28, 8615–8623 (2008).

    Article  CAS  Google Scholar 

  5. Schaefer, A.M., Hadwiger, G.D. & Nonet, M.L. rpm-1, a conserved neuronal gene that regulates targeting and synaptogenesis in C. elegans. Neuron 26, 345–356 (2000).

    Article  CAS  Google Scholar 

  6. Zhen, M., Huang, X., Bamber, B. & Jin, Y. Regulation of presynaptic terminal organization by C. elegans RPM-1, a putative guanine nucleotide exchanger with a RING-H2 finger domain. Neuron 26, 331–343 (2000).

    Article  CAS  Google Scholar 

  7. Bloom, A.J., Miller, B.R., Sanes, J.R. & DiAntonio, A. The requirement for Phr1 in CNS axon tract formation reveals the corticostriatal boundary as a choice point for cortical axons. Genes Dev. 21, 2593–2606 (2007).

    Article  CAS  Google Scholar 

  8. Burgess, R.W. et al. Evidence for a conserved function in synapse formation reveals Phr1 as a candidate gene for respiratory failure in newborn mice. Mol. Cell Biol. 24, 1096–1105 (2004).

    Article  CAS  Google Scholar 

  9. D'Souza, J. et al. Formation of the retinotectal projection requires Esrom, an ortholog of PAM (protein associated with Myc). Development 132, 247–256 (2005).

    Article  CAS  Google Scholar 

  10. Culican, S.M., Bloom, A.J., Weiner, J.A. & DiAntonio, A. Phr1 regulates retinogeniculate targeting independent of activity and ephrin-A signaling. Mol. Cell. Neurosci. 41, 304–312 (2009).

    Article  CAS  Google Scholar 

  11. Lewcock, J.W., Genoud, N., Lettieri, K. & Pfaff, S.L. The ubiquitin ligase Phr1 regulates axon outgrowth through modulation of microtubule dynamics. Neuron 56, 604–620 (2007).

    Article  CAS  Google Scholar 

  12. Yan, D., Wu, Z., Chisholm, A.D. & Jin, Y. The DLK-1 kinase promotes mRNA stability and local translation in C. elegans synapses and axon regeneration. Cell 138, 1005–1018 (2009).

    Article  CAS  Google Scholar 

  13. Xiong, X. et al. Protein turnover of the Wallenda/DLK kinase regulates a retrograde response to axonal injury. J. Cell Biol. 191, 211–223 (2010).

    Article  CAS  Google Scholar 

  14. Hammarlund, M., Nix, P., Hauth, L., Jorgensen, E.M. & Bastiani, M. Axon regeneration requires a conserved MAP kinase pathway. Science 323, 802–806 (2009).

    Article  CAS  Google Scholar 

  15. Scholich, K., Pierre, S. & Patel, T.B. Protein associated with Myc (PAM) is a potent inhibitor of adenylyl cyclases. J. Biol. Chem. 276, 47583–47589 (2001).

    Article  CAS  Google Scholar 

  16. Guo, Q., Xie, J., Dang, C.V., Liu, E.T. & Bishop, J.M. Identification of a large Myc-binding protein that contains RCC1-like repeats. Proc. Natl. Acad. Sci. USA 95, 9172–9177 (1998).

    Article  CAS  Google Scholar 

  17. Garbarini, N. & Delpire, E. The RCC1 domain of protein associated with Myc (PAM) interacts with and regulates KCC2. Cell Physiol. Biochem. 22, 31–44 (2008).

    Article  CAS  Google Scholar 

  18. Abrams, B., Grill, B., Huang, X. & Jin, Y. Cellular and molecular determinants targeting the Caenorhabditis elegans PHR protein RPM-1 to perisynaptic regions. Dev. Dyn. 237, 630–639 (2008).

    Article  CAS  Google Scholar 

  19. Wu, C., Wairkar, Y.P., Collins, C.A. & DiAntonio, A. Highwire function at the Drosophila neuromuscular junction: spatial, structural, and temporal requirements. J. Neurosci. 25, 9557–9566 (2005).

    Article  CAS  Google Scholar 

  20. Liao, E.H., Hung, W., Abrams, B. & Zhen, M. An SCF-like ubiquitin ligase complex that controls presynaptic differentiation. Nature 430, 345–350 (2004).

    Article  CAS  Google Scholar 

  21. Wu, C., Daniels, R.W. & DiAntonio, A. DFsn collaborates with Highwire to down-regulate the Wallenda/DLK kinase and restrain synaptic terminal growth. Neural Dev. 2, 16 (2007).

    Article  Google Scholar 

  22. Saiga, T. et al. Fbxo45 forms a novel ubiquitin ligase complex and is required for neuronal development. Mol. Cell. Biol. 29, 3529–3543 (2009).

    Article  CAS  Google Scholar 

  23. Collins, C.A., Wairkar, Y.P., Johnson, S.L. & DiAntonio, A. Highwire restrains synaptic growth by attenuating a MAP kinase signal. Neuron 51, 57–69 (2006).

    Article  CAS  Google Scholar 

  24. Nakata, K. et al. Regulation of a DLK-1 and p38 MAP kinase pathway by the ubiquitin ligase RPM-1 is required for presynaptic development. Cell 120, 407–420 (2005).

    Article  CAS  Google Scholar 

  25. Shen, W. & Ganetzky, B. Autophagy promotes synapse development in Drosophila. J. Cell Biol. 187, 71–79 (2009).

    Article  CAS  Google Scholar 

  26. Brown, J.A. et al. A mutation in the Schizosaccharomyces pombe rae1 gene causes defects in poly(A)+ RNA export and in the cytoskeleton. J. Biol. Chem. 270, 7411–7419 (1995).

    Article  CAS  Google Scholar 

  27. Pritchard, C.E., Fornerod, M., Kasper, L.H. & van Deursen, J.M. RAE1 is a shuttling mRNA export factor that binds to a GLEBS-like NUP98 motif at the nuclear pore complex through multiple domains. J. Cell Biol. 145, 237–254 (1999).

    Article  CAS  Google Scholar 

  28. Jeganathan, K.B., Malureanu, L. & van Deursen, J.M. The Rae1-Nup98 complex prevents aneuploidy by inhibiting securin degradation. Nature 438, 1036–1039 (2005).

    Article  CAS  Google Scholar 

  29. Sitterlin, D. Characterization of the Drosophila Rae1 protein as a G1 phase regulator of the cell cycle. Gene 326, 107–116 (2004).

    Article  CAS  Google Scholar 

  30. Kraemer, D., Dresbach, T. & Drenckhahn, D. Mrnp41 (Rae1p) associates with microtubules in HeLa cells and in neurons. Eur. J. Cell Biol. 80, 733–740 (2001).

    Article  CAS  Google Scholar 

  31. Rigaut, G. et al. A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol. 17, 1030–1032 (1999).

    Article  CAS  Google Scholar 

  32. McCabe, B.D. et al. Highwire regulates presynaptic BMP signaling essential for synaptic growth. Neuron 41, 891–905 (2004).

    Article  CAS  Google Scholar 

  33. Conne, B., Stutz, A. & Vassalli, J.D. The 3′ untranslated region of messenger RNA: a molecular 'hotspot' for pathology? Nat. Med. 6, 637–641 (2000).

    Article  CAS  Google Scholar 

  34. Bolognani, F. & Perrone-Bizzozero, N.I. RNA-protein interactions and control of mRNA stability in neurons. J. Neurosci. Res. 86, 481–489 (2008).

    Article  CAS  Google Scholar 

  35. Levine, B. & Kroemer, G. Autophagy in the pathogenesis of disease. Cell 132, 27–42 (2008).

    Article  CAS  Google Scholar 

  36. Pierre, S.C., Hausler, J., Birod, K., Geisslinger, G. & Scholich, K. PAM mediates sustained inhibition of cAMP signaling by sphingosine-1-phosphate. EMBO J. 23, 3031–3040 (2004).

    Article  CAS  Google Scholar 

  37. Murthy, V. et al. Pam and its ortholog highwire interact with and may negatively regulate the TSC1.TSC2 complex. J. Biol. Chem. 279, 1351–1358 (2004).

    Article  CAS  Google Scholar 

  38. Grill, B. et al. C. elegans RPM-1 regulates axon termination and synaptogenesis through the Rab GEF GLO-4 and the Rab GTPase GLO-1. Neuron 55, 587–601 (2007).

    Article  CAS  Google Scholar 

  39. Johansen, T. & Lamark, T. Selective autophagy mediated by autophagic adapter proteins. Autophagy 7, 279–296 (2011).

    Article  CAS  Google Scholar 

  40. Candiano, G. et al. Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 25, 1327–1333 (2004).

    Article  CAS  Google Scholar 

  41. Wairkar, Y.P. et al. Unc-51 controls active zone density and protein composition by downregulating ERK signaling. J. Neurosci. 29, 517–528 (2009).

    Article  CAS  Google Scholar 

  42. Scott, R.C., Juhasz, G. & Neufeld, T.P. Direct induction of autophagy by Atg1 inhibits cell growth and induces apoptotic cell death. Curr. Biol. 17, 1–11 (2007).

    Article  CAS  Google Scholar 

  43. Petersen, S.A., Fetter, R.D., Noordermeer, J.N., Goodman, C.S. & DiAntonio, A. Genetic analysis of glutamate receptors in Drosophila reveals a retrograde signal regulating presynaptic transmitter release. Neuron 19, 1237–1248 (1997).

    Article  CAS  Google Scholar 

  44. Loureiro, J. & Peifer, M. Roles of Armadillo, a Drosophila catenin, during central nervous system development. Curr. Biol. 8, 622–632 (1998).

    Article  CAS  Google Scholar 

  45. Daniels, R.W. et al. A single vesicular glutamate transporter is sufficient to fill a synaptic vesicle. Neuron 49, 11–16 (2006).

    Article  CAS  Google Scholar 

  46. Tian, X. et al. RAB26 and RAB3D are direct transcriptional targets of MIST1 that regulate exocrine granule maturation. Mol. Cell. Biol. 30, 1269–1284 (2010).

    Article  CAS  Google Scholar 

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Acknowledgements

We are particularly grateful to D. Sitterlin for providing the antibody to Rae1. We would like to thank the A.J. Siteman Cancer Center at the Washington University School of Medicine and Barnes-Jewish Hospital in St. Louis, Missouri, and the Department of Physiology at the Louisiana State University Health Sciences Center in New Orleans for the use of their proteomic core facilities. We are grateful to T. Neufeld and the Bloomington Stock Center for fly stocks, and the Developmental Studies Hybridoma Bank for antibodies. We also thank B. Grill and Y. Jin for helpful comments. This work is supported by grants from the US National Institutes of Health (NS070962 to C.W. and DA020812 to A.D.).

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Authors and Affiliations

  1. Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, New Orleans, Louisiana, USA

    Xiaolin Tian, Jing Li & Chunlai Wu

  2. Department of Developmental Biology, Washington University School of Medicine, St. Louis, Missouri, USA

    Vera Valakh & Aaron DiAntonio

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  1. Xiaolin Tian
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Contributions

C.W. and A.D. initiated the project and conducted the Hiw structure and function analysis. C.W. directed the rest of the studies. X.T. and C.W. conceived and designed the experiments. X.T., J.L. and C.W. performed the experiments. V.V. contributed to the characterization of the Rae1 mutant phenotype. C.W. and X.T. wrote the manuscript with input from A.D.

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Correspondence to Chunlai Wu.

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Tian, X., Li, J., Valakh, V. et al. Drosophila Rae1 controls the abundance of the ubiquitin ligase Highwire in post-mitotic neurons. Nat Neurosci 14, 1267–1275 (2011). https://doi.org/10.1038/nn.2922

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