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KIF1Bβ- and KIF1A-mediated axonal transport of presynaptic regulator Rab3 occurs in a GTP-dependent manner through DENN/MADD

Abstract

Synaptic proteins are synthesized in the cell body and transported down the axon by microtubule-dependent motors. We previously reported that KIF1Bβ and KIF1A motors are essential for transporting synaptic vesicle precursors; however the mechanisms that regulate transport, as well as cargo recognition and control of cargo loading and unloading remain largely unknown. Here, we show that DENN/MADD (Rab3-GEP) is an essential part of the regulation mechanism through direct interaction with the stalk domain of KIF1Bβ and KIF1A. We also show that DENN/MADD binds preferentially to GTP–Rab3 and acts as a Rab3 effector. These molecular interactions are fundamental as sequential genetic perturbations revealed that KIF1Bβ and KIF1A are essential for the transport of DENN/MADD and Rab3, whereas DENN/MADD is essential for the transport of Rab3. GTP–Rab3 was more effectively transported than GDP–Rab3, suggesting that the nucleotide state of Rab3 regulates axonal transport of Rab3-carrying vesicles through preferential interaction with DENN/MADD.

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Figure 1: The DENN/MADD death domain binds to the KIF1Bβ- stalk domain.
Figure 2: Reduction of Rab3 and DENN/MADD in Kif1b knockout distal axons.
Figure 3: Dominant-negative DENN/MADD–DD affects Rab3 distribution.
Figure 4: Knockdown of DENN/MADD affects Rab3 distribution.
Figure 5: DENN/MADD knockdown inhibits axonal transport of GTP–Rab3.
Figure 6: The MADD domain binds preferentially to GTP–Rab3.
Figure 7: Rab3 is transported in its GTP form.

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References

  1. Kandel, E.R. The molecular biology of memory storage: a dialogue between genes and synapses. Science 294, 1030–1038 (2001).

    Article  CAS  PubMed  Google Scholar 

  2. Nakata, T., Terada, S. & Hirokawa, N. Visualization of the dynamics of synaptic vesicle and plasma membrane proteins in living axons. J. Cell Biol. 140, 659–674 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Mundigl, O. et al. Synaptic vesicle proteins and early endosomes in cultured hippocampal neurons: differential effects of Brefeldin A in axon and dendrites. J. Cell Biol. 122, 1207–1221 (1993).

    Article  CAS  PubMed  Google Scholar 

  4. Hirokawa, N. Kinesin and dynein superfamily proteins and the mechanism of organelle transport. Science 279, 519–526 (1998).

    Article  CAS  PubMed  Google Scholar 

  5. Hirokawa, N. & Takemura, R. Molecular motors and mechanisms of directional transport in neurons. Nature Rev. Neurosci. 6, 201–214 (2005).

    Article  CAS  Google Scholar 

  6. Aizawa, H. et al. Kinesin family in murine central nervous system. J. Cell Biol. 119, 1287–1296 (1992).

    Article  CAS  PubMed  Google Scholar 

  7. Okada, Y., Yamazaki, H., Sekine-Aizawa, Y. & Hirokawa, N. The neuron-specific kinesin superfamily protein KIF1A is a unique monomeric motor for anterograde axonal transport of synaptic vesicle precursors. Cell 81, 769–780 (1995).

    Article  CAS  PubMed  Google Scholar 

  8. Zhao, C. et al. Charcot-Marie-Tooth disease type 2A caused by mutation in a microtubule motor KIF1Bβ. Cell 105, 587–597 (2001).

    Article  CAS  PubMed  Google Scholar 

  9. Yonekawa, Y. et al. Defect in synaptic vesicle precursor transport and neuronal cell death in KIF1A motor protein-deficient mice. J. Cell Biol. 141, 431–441 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Hall, D. H. & Hedgecock E. M. Kinesin-related gene unc-104 is required for axonal transport of synaptic vesicles in C. elegans. Cell 65, 837–847 (1991).

    Article  CAS  PubMed  Google Scholar 

  11. Klopfenstein, D. R. & Vale, R. D. The lipid binding pleckstrin homology domain in UNC-104 kinesin is necessary for synaptic vesicle transport in Caenorhabditis elegans. Mol. Biol. Cell 15, 3729–3739 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Schluter, O.M., Schmitz, R., Jahn, C., Rosenmund, C. & Sudhof T.C. A complete genetic analysis of neuronal Rab3 function. J. Neurosci. 24, 6629–6637 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Fischer von Mollard, G., Sudhof, T.C. & Jahn, R. A small GTP-binding protein dissociates from synaptic vesicles during exocytosis. Nature 349, 79–81 (1991).

    Article  CAS  PubMed  Google Scholar 

  14. Stettler, O., Moya, K. L., Zahraoui, A. & Tavitian, B. Developmental changes in the localization of the synaptic vesicle protein rab3A in rat brain. Neuroscience 62, 587–600 (1994).

    Article  CAS  PubMed  Google Scholar 

  15. Geppert, M., Goda, Y., Stevens, C. F. & Sudhof, T. C. The small GTP-binding protein Rab3A regulates a late step in synaptic vesicle fusion. Nature 387, 810–814 (1997).

    Article  CAS  PubMed  Google Scholar 

  16. Ngsee, J. K., Fleming, A. M. & Scheller, R. H. A rab protein regulates the localization of secretory granules in AtT-20 cells. Mol. Biol. Cell 7, 747–756 (1993).

    Article  Google Scholar 

  17. Yamamoto, Y. et al. Distinct roles of Rab3B and Rab13 in the polarized transport of apical, basolateral, and tight junctional membrane proteins to the plasma membrane. Biochem. Biophys. Res. Commun. 308, 270–275 (2003).

    Article  CAS  PubMed  Google Scholar 

  18. Baldini, G., Hohl, T., Lin, H. Y. & Lodish, H. F. Cloning of a Rab3 isotype predominantly expressed in adipocytes. Proc. Natl Acad. Sci. USA 89, 5049–5052 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Roa, M., Paumet, F., Le Mao, J., David, B. & Blank, U. Involvement of the ras-like GTPase rab3d in RBL-2H3 mast cell exocytosis following stimulation via high affinity IgE receptors (FcɛRI). J. Immunol. 159, 2815–2823 (1997).

    CAS  PubMed  Google Scholar 

  20. Li, J.Y., Jahn, R. & Dahlstrom, A. Rab3a, a small GTP-binding protein, undergoes fast anterograde transport but not retrograde transport in neurons. Eur. J. Cell Biol. 67, 297–307 (1995).

    CAS  PubMed  Google Scholar 

  21. Miyoshi, J., Takai, Y. Dual role of DENN/MADD (Rab3GEP) in neurotransmission and neuroprotection. Trends Mol. Med. 10, 476–480 (2004).

    Article  CAS  PubMed  Google Scholar 

  22. Schievella. A. R., Chen, J. H., Graham, J. R. & Lin, L. L. MADD, a novel death domain protein that interacts with the type 1 tumor necrosis factor receptor and activates mitogen-activated protein kinase. J. Biol. Chem. 272, 12069–12075 (1997).

    Article  CAS  PubMed  Google Scholar 

  23. Wada, M. et al. Isolation and characterization of a GDP/GTP exchange protein specific for the Rab3 subfamily small G proteins. J. Biol. Chem. 272, 3875–3878 (1997).

    Article  CAS  PubMed  Google Scholar 

  24. Yamaguchi K. et al. A GDP/GTP exchange protein for the Rab3 small G protein family up-regulates a postdocking step of synaptic exocytosis in central synapses. Proc. Natl Acad. Sci. USA 99, 14536–14541 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Tanaka, M. et al. Role of GDP–Rab3/GTP exchange protein in synaptic vesicle trafficking at the mouse neuromuscular junction. Mol. Biol. Cell 12, 1421–1430 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Iwasaki, K., Staunton, J., Saifee, O., Nonet, M. & Thomas, J. H. aex-3 encodes a novel regulator of presynaptic activity in C. elegans. Neuron 18, 613–622 (1997).

    Article  CAS  PubMed  Google Scholar 

  27. Klopfenstein, D. R., Tomishige, M., Stuurman, N. & Vale, R. D. Role of phosphatidylinositol(4,5)bisphosphate organization in membrane transport by the Unc104 kinesin motor .Cell 109, 347–358 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Weber, C. H. and Vincenz, C. The death domain superfamily: a tale of two interfaces? Trends Biochem. Sci. 26, 475–481 (2001).

    Article  CAS  PubMed  Google Scholar 

  29. Krichevsky, A. M. & Kosik, K., S. RNAi functions in cultured mammalian neurons. Proc. Natl. Acad Sci. USA 99, 11926–11929 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Coppola, T. et al. The death domain of Rab3 guanine nucleotide exchange protein in GDP/GTP exchange activity in living cells. Biochem. J. 362, 273–279 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Luo H. R. et al. GRAB: a physiologic guanine nucleotide exchange factor for Rab3A, which interacts with inositol hexakisphosphate kinase. Neuron 31, 439–451 (2001).

    Article  CAS  PubMed  Google Scholar 

  32. Zerial, M. & McBride, H. Rab proteins as membrane organizers. Nature Rev. Mol. Cell Biol. 2, 107–117 (2001).

    Article  CAS  Google Scholar 

  33. Burstein, E. S., Brondyk, W. H. & Macara, I. G. Amino acid residues in the Ras-like GTPase Rab3A that specify sensitivity to factors that regulate the GTP/GDP cycling of Rab3A. J. Biol. Chem. 267, 22715–22718 (1992).

    CAS  PubMed  Google Scholar 

  34. Callaghan, J., Nixon, S., Bucci, C., Toh, B. H. & Stenmark, H. Direct interaction of EEA1 with Rab5b. Eur. J. Biochem. 265, 361–366 (1999).

    Article  CAS  PubMed  Google Scholar 

  35. Hoepfner, S. et al. Modulation of receptor recycling and degradation by the endosomal kinesin KIF16B. Cell 121, 437–450 (2005).

    Article  CAS  PubMed  Google Scholar 

  36. Jordens, I. et al. The Rab7 effector protein RILP controls lysosomal transport by inducing the recruitment of dynein–dynactin motors. Curr. Biol. 11, 1680–1685 (2001).

    Article  CAS  PubMed  Google Scholar 

  37. Hume, A. N. et al. The leaden gene product is required with Rab27a to recruit myosin Va to melanosomes in melanocytes. Traffic 3, 193–202 (2002).

    Article  CAS  PubMed  Google Scholar 

  38. Fukuda, M., Kuroda, TS. & Mikoshiba, K. Slac2–a/melanophilin, the missing link between Rab27 and myosin Va: implications of a tripartite protein complex for melanosome transport. J. Biol. Chem. 277, 12432–12436 (2002).

    Article  CAS  PubMed  Google Scholar 

  39. Wu X. S. et al. Identification of an organelle receptor for myosin-Va. Nature Cell Biol. 4, 271–278 (2002).

    Article  CAS  PubMed  Google Scholar 

  40. Mahoney, T. R. et al. Regulation of synaptic transmission by RAB-3 and RAB-27 in Caenorhabditis elegans. Mol. Biol. Cell 17, 2617–2625 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Gomi, H., Mori, K., Itohara, S. & Izumi, T. Rab27b is expressed in a wide range of exocytic cells and involved in the delivery of secretory granules near the plasma membrane. Mol. Biol. Cell 18, 4377–4378 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Iwasaki, K. & Toyonaga, R. The Rab3 GDP/GTP exchange factor homolog AEX-3 has a dual function in synaptic transmission. EMBO J. 19, 4806–4816 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Byrd, D. T. et al. UNC-16, a JNK-signaling scaffold protein, regulates vesicle transport in C. elegans. Neuron 32, 787–800 (2001).

    Article  CAS  PubMed  Google Scholar 

  44. Watt, S. A., Kular, G., Fleming, I. N., Downes, C. P. & Lucocq, J. M. Subcellular localization of phosphatidylinositol 4,5-bisphosphate using the pleckstrin homology domain of phospholipase C delta1. Biochem. J. 363, 657–666 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Kanai, Y. et al. KIF5C, a novel neuronal kinesin enriched in motor neurons. J. Neurosci. 20, 6374–84 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Teng, J. et al. The KIF3 motor transports N-cadherin and organizes the developing neuroepithelium. Nature Cell Biol. 7, 474–482 (2005).

    Article  CAS  PubMed  Google Scholar 

  47. Nakata, T. & Hirokawa, N. Microtubules provide directional cues for polarized axonal transport through interaction with kinesin motor head. J. Cell Biol. 162, 1045–1055 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors thank H. Fukuda, H. Sato, T. Aizawa and others from the Hirokawa laboratory for discussions and technical assistance. We are also grateful to the Kazusa DNA Institute (Chiba, Japan) for providing KIAA clones. This study was supported by a Grant-in-Aid for Specially Promoted Research to N.H. and Global COE programme to University of Tokyo from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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Correspondence to Nobutaka Hirokawa.

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Niwa, S., Tanaka, Y. & Hirokawa, N. KIF1Bβ- and KIF1A-mediated axonal transport of presynaptic regulator Rab3 occurs in a GTP-dependent manner through DENN/MADD. Nat Cell Biol 10, 1269–1279 (2008). https://doi.org/10.1038/ncb1785

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