1. Structural Biology and Molecular Biophysics
Download icon

Auto-regulation of Rab5 GEF activity in Rabex5 by allosteric structural changes, catalytic core dynamics and ubiquitin binding

  1. Janelle Lauer
  2. Sandra Segeletz
  3. Alice Cezanne
  4. Giambattista Guaitoli
  5. Francesco Raimondi
  6. Marc Gentzel
  7. Vikram Alva
  8. Michael Habeck
  9. Yannis Kalaidzidis
  10. Marius Ueffing
  11. Andrei N Lupas
  12. Christian Johannes Gloeckner
  13. Marino Zerial  Is a corresponding author
  1. Max Planck Institute of Molecular Cell Biology and Genetics, Germany
  2. German Center for Neurodegenerative Diseases, Germany
  3. Heidelberg University, Germany
  4. Technical University Dresden, Germany
  5. Max Planck Institute for Developmental Biology, Germany
  6. Max Planck Institute for Biophysical Chemistry, Germany
  7. University of Tübingen, Germany
Research Article
  • Cited 3
  • Views 997
  • Annotations
Cite this article as: eLife 2019;8:e46302 doi: 10.7554/eLife.46302

Abstract

Intracellular trafficking depends on the function of Rab GTPases, whose activation is regulated by guanine exchange factors (GEFs). The Rab5 GEF, Rabex5, was previously proposed to be auto-inhibited by its C-terminus. Here, we studied full-length Rabex5 and Rabaptin5 proteins as well as domain deletion Rabex5 mutants using hydrogen deuterium exchange mass spectrometry. We generated a structural model of Rabex5, using chemical cross-linking mass spectrometry and integrative modeling techniques. By correlating structural changes with nucleotide exchange activity for each construct, we uncovered new auto-regulatory roles for the Ubiquitin binding domains and the Linker connecting those domains to the catalytic core of Rabex5. We further provide evidence that enhanced dynamics in the catalytic core are linked to catalysis. Our results suggest a more complex auto-regulation mechanism than previously thought and imply that Ubiquitin binding serves not only to position Rabex5 but to also control its Rab5 GEF activity through allosteric structural alterations.

Article and author information

Author details

  1. Janelle Lauer

    Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1412-6766

  2. Sandra Segeletz

    Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
    Competing interests
    The authors declare that no competing interests exist.

  3. Alice Cezanne

    Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
    Competing interests
    The authors declare that no competing interests exist.

  4. Giambattista Guaitoli

    Translational Biomarker in Neurodegenerative Diseases, German Center for Neurodegenerative Diseases, Tübingen, Germany
    Competing interests
    The authors declare that no competing interests exist.

  5. Francesco Raimondi

    Bioquant, Heidelberg University, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.

  6. Marc Gentzel

    Molecular Analysis Mass Spectrometry Center for Molecular and Cellular Bioimaging, Technical University Dresden, Dresden, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4482-6010

  7. Vikram Alva

    Department of Protein Evolution, Max Planck Institute for Developmental Biology, Tübingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1188-473X

  8. Michael Habeck

    Statistical Inverse Problems in Biophysics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.

  9. Yannis Kalaidzidis

    Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
    Competing interests
    The authors declare that no competing interests exist.

  10. Marius Ueffing

    Institute for Ophthalmic Research, Center for Ophthalmology, University of Tübingen, Tübingen, Germany
    Competing interests
    The authors declare that no competing interests exist.

  11. Andrei N Lupas

    Department of Protein Evolution, Max Planck Institute for Developmental Biology, Tübingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1959-4836

  12. Christian Johannes Gloeckner

    Translational Biomarker in Neurodegenerative Diseases, German Center for Neurodegenerative Diseases, Tübingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6494-6944

  13. Marino Zerial

    Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
    For correspondence
    zerial@mpi-cbg.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7490-4235

Funding

Max-Planck-Gesellschaft (Open-access funding)

  • Marino Zerial

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Suzanne R Pfeffer, Stanford University School of Medicine, United States

Publication history

  1. Received: February 21, 2019
  2. Accepted: October 22, 2019
  3. Accepted Manuscript published: November 13, 2019 (version 1)
  4. Version of Record published: November 14, 2019 (version 2)

Copyright

© 2019, Lauer 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.

Metrics

  • 997
    Page views
  • 164
    Downloads
  • 3
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Categories and tags

Further reading

    1. Neuroscience
    2. Structural Biology and Molecular Biophysics

    Molecular basis for functional connectivity between the voltage sensor and the selectivity filter gate in Shaker K+ channels

    Carlos A Z Bassetto Jnr et al.
    Research Article

    In Shaker K+ channels, the S4-S5 linker couples the voltage sensor (VSD) and pore domain (PD). Another coupling mechanism is revealed using two W434F-containing channels: L361R:W434F and L366H:W434F. In L361R:W434F, W434F affects the L361R VSD seen as a shallower Q-V curve that crosses the G-V. In L366H:W434F, L366H relieves the W434F effect converting a non-conductive channel in a conductive one. We report a chain of residues connecting the VSD (S4) to the selectivity filter (SF) in the PD of an adjacent subunit as the molecular basis for voltage-sensor selectivity filter gate (VS-SF) coupling. Single alanine substitutions in this region (L409A, S411A, S412A or F433A) are enough to disrupt the VS-SF coupling, shown by the absence of Q-V and G-V crossing in L361R:W434F mutant and by the lack of ionic conduction in the L366H:W434F mutant. This residue chain defines a new coupling between the VSD and the PD in voltage-gated channels.

    1. Structural Biology and Molecular Biophysics

    A critical residue in the α1M2–M3 linker regulating mammalian GABAA receptor pore gating by diazepam

    Joseph W Nors et al.
    Research Article Updated

    Benzodiazepines (BZDs) are a class of widely prescribed psychotropic drugs that modulate activity of GABAA receptors (GABAARs), neurotransmitter-gated ion channels critical for synaptic transmission. However, the physical basis of this modulation is poorly understood. We explore the role of an important gating domain, the α1M2–M3 linker, in linkage between the BZD site and pore gate. To probe energetics of this coupling without complication from bound agonist, we use a gain of function mutant (α1L9'Tβ2γ2L) directly activated by BZDs. We identify a specific residue whose mutation (α1V279A) more than doubles the energetic contribution of the BZD positive modulator diazepam (DZ) to pore opening and also enhances DZ potentiation of GABA-evoked currents in a wild-type background. In contrast, other linker mutations have little effect on DZ efficiency, but generally impair unliganded pore opening. Our observations reveal an important residue regulating BZD-pore linkage, thereby shedding new light on the molecular mechanism of these drugs.

    1. Cell Biology
    2. Structural Biology and Molecular Biophysics

    Pre-existing bilayer stresses modulate triglyceride accumulation in the ER versus lipid droplets

    Valeria Zoni et al.
    Research Article Updated

    Cells store energy in the form of neutral lipids (NLs) packaged into micrometer-sized organelles named lipid droplets (LDs). These structures emerge from the endoplasmic reticulum (ER) at sites marked by the protein seipin, but the mechanisms regulating their biogenesis remain poorly understood. Using a combination of molecular simulations, yeast genetics, and fluorescence microscopy, we show that interactions between lipids’ acyl-chains modulate the propensity of NLs to be stored in LDs, in turn preventing or promoting their accumulation in the ER membrane. Our data suggest that diacylglycerol, which is enriched at sites of LD formation, promotes the packaging of NLs into LDs, together with ER-abundant lipids, such as phosphatidylethanolamine. On the opposite end, short and saturated acyl-chains antagonize fat storage in LDs and promote accumulation of NLs in the ER. Our results provide a new conceptual understanding of LD biogenesis in the context of ER homeostasis and function.