1. Structural Biology and Molecular Biophysics
  2. Cell Biology
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The Sec7 N-terminal regulatory domains facilitate membrane-proximal activation of the Arf1 GTPase

Research Article
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Cite this article as: eLife 2016;5:e12411 doi: 10.7554/eLife.12411

Abstract

The Golgi complex is the central sorting compartment of eukaryotic cells. Arf guanine nucleotide exchange factors (Arf-GEFs) regulate virtually all traffic through the Golgi by activating Arf GTPase trafficking pathways. The Golgi Arf-GEFs contain multiple autoregulatory domains, but the precise mechanisms underlying their function remain largely undefined. We report a crystal structure revealing that the N-terminal DCB and HUS regulatory domains of the Arf-GEF Sec7 form a single structural unit. We demonstrate that the established role of the N-terminal region in dimerization is not conserved; instead, a C-terminal autoinhibitory domain is responsible for dimerization of Sec7. We find that the DCB/HUS domain amplifies the ability of Sec7 to activate Arf1 on the membrane surface by facilitating membrane insertion of the Arf1 amphipathic helix. This enhancing function of the Sec7 N-terminal domains is consistent with the high rate of Arf1-dependent trafficking to the plasma membrane necessary for maximal cell growth.

Article and author information

Author details

  1. Brian C Richardson

    Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Steve L Halaby

    Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Margaret A Gustafson

    Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. J Christopher Fromme

    Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, United States
    For correspondence
    jcf14@cornell.edu
    Competing interests
    The authors declare that no competing interests exist.

Reviewing Editor

  1. Chris G Burd, Yale University, United States

Publication history

  1. Received: October 20, 2015
  2. Accepted: January 13, 2016
  3. Accepted Manuscript published: January 14, 2016 (version 1)
  4. Version of Record published: February 12, 2016 (version 2)
  5. Version of Record updated: June 9, 2016 (version 3)

Copyright

© 2016, Richardson et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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Further reading

    1. Microbiology and Infectious Disease
    2. Structural Biology and Molecular Biophysics
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    Cyclic-di-guanosine monophosphate (c-di-GMP) is an important effector associated with acute-chronic infection transition in Pseudomonas aeruginosa. Previously, we reported a signaling network SiaABCD, which regulates biofilm formation by modulating c-di-GMP level. However, the mechanism for SiaD activation by SiaC remains elusive. Here we determine the crystal structure of SiaC-SiaD-GpCpp complex and revealed a unique mirror symmetric conformation: two SiaD form a dimer with long stalk domains, while four SiaC bind to the conserved motifs on the stalks of SiaD and stabilize the conformation for further enzymatic catalysis. Furthermore, SiaD alone exhibits an inactive pentamer conformation in solution, demonstrating that SiaC activates SiaD through a dynamic mechanism of promoting the formation of active SiaD dimers. Mutagenesis assay confirmed that the stalks of SiaD are necessary for its activation. Together, we reveal a novel mechanism for DGC activation, which clarifies the regulatory networks of c-di-GMP signaling.