Abstract

Interactions between membrane protein interfaces in lipid bilayers play an important role in membrane protein folding but quantification of the strength of these interactions has been challenging. Studying dimerization of ClC-type transporters offers a new approach to the problem, as individual subunits adopt a stable and functionally verifiable fold that constrains the system to two states - monomer or dimer. Here, we use single-molecule photobleaching analysis to measure the probability of ClC-ec1 subunit capture into liposomes during extrusion of large, multilamellar membranes. The capture statistics describe a monomer to dimer transition that is dependent on the subunit/lipid mole fraction density and follows an equilibrium dimerization isotherm. This allows for the measurement of the free energy of ClC-ec1 dimerization in lipid bilayers, revealing that it is one of the strongest membrane protein complexes measured so far, and introduces it as new type of dimerization model to investigate the physical forces that drive membrane protein association in membranes.

Article and author information

Author details

  1. Rahul Chadda

    Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Venkatramanan Krishnamani

    Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Kacey Mersch

    Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Jason Wong

    Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Marley Brimberry

    Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Ankita Chadda

    Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Ludmila Kolmakova-Partensky

    Department of Biochemistry, Brandeis University, Waltham, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Larry J Friedman

    Department of Biochemistry, Brandeis University, Waltham, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4946-8731
  9. Jeff Gelles

    Department of Biochemistry, Brandeis University, Waltham, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7910-3421
  10. Janice L Robertson

    Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, United States
    For correspondence
    janice-robertson@uiowa.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5499-9943

Funding

National Institutes of Health (R00GM101016)

  • Venkatramanan Krishnamani
  • Kacey Mersch
  • Janice L Robertson

Roy J. Carver Charitable Trust (Early Investigator Award)

  • Rahul Chadda
  • Marley Brimberry
  • Ankita Chadda
  • Janice L Robertson

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

Reviewing Editor

  1. Olga Boudker, Weill Cornell Medical College, United States

Version history

  1. Received: May 1, 2016
  2. Accepted: July 29, 2016
  3. Accepted Manuscript published: August 3, 2016 (version 1)
  4. Version of Record published: September 2, 2016 (version 2)

Copyright

© 2016, Chadda 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

  • 3,744
    views
  • 709
    downloads
  • 49
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

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)

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

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

  1. Rahul Chadda
  2. Venkatramanan Krishnamani
  3. Kacey Mersch
  4. Jason Wong
  5. Marley Brimberry
  6. Ankita Chadda
  7. Ludmila Kolmakova-Partensky
  8. Larry J Friedman
  9. Jeff Gelles
  10. Janice L Robertson
(2016)
The dimerization equilibrium of a ClC Cl-/H+ antiporter in lipid bilayers
eLife 5:e17438.
https://doi.org/10.7554/eLife.17438

Share this article

https://doi.org/10.7554/eLife.17438

Further reading

    1. Biochemistry and Chemical Biology
    2. Neuroscience
    Katarzyna Marta Zoltowska, Utpal Das ... Lucía Chávez-Gutiérrez
    Research Article

    Amyloid β (Aβ) peptides accumulating in the brain are proposed to trigger Alzheimer’s disease (AD). However, molecular cascades underlying their toxicity are poorly defined. Here, we explored a novel hypothesis for Aβ42 toxicity that arises from its proven affinity for γ-secretases. We hypothesized that the reported increases in Aβ42, particularly in the endolysosomal compartment, promote the establishment of a product feedback inhibitory mechanism on γ-secretases, and thereby impair downstream signaling events. We conducted kinetic analyses of γ-secretase activity in cell-free systems in the presence of Aβ, as well as cell-based and ex vivo assays in neuronal cell lines, neurons, and brain synaptosomes to assess the impact of Aβ on γ-secretases. We show that human Aβ42 peptides, but neither murine Aβ42 nor human Aβ17–42 (p3), inhibit γ-secretases and trigger accumulation of unprocessed substrates in neurons, including C-terminal fragments (CTFs) of APP, p75, and pan-cadherin. Moreover, Aβ42 treatment dysregulated cellular homeostasis, as shown by the induction of p75-dependent neuronal death in two distinct cellular systems. Our findings raise the possibility that pathological elevations in Aβ42 contribute to cellular toxicity via the γ-secretase inhibition, and provide a novel conceptual framework to address Aβ toxicity in the context of γ-secretase-dependent homeostatic signaling.

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Ya-Juan Wang, Xiao-Jing Di ... Ting-Wei Mu
    Research Article Updated

    Protein homeostasis (proteostasis) deficiency is an important contributing factor to neurological and metabolic diseases. However, how the proteostasis network orchestrates the folding and assembly of multi-subunit membrane proteins is poorly understood. Previous proteomics studies identified Hsp47 (Gene: SERPINH1), a heat shock protein in the endoplasmic reticulum lumen, as the most enriched interacting chaperone for gamma-aminobutyric acid type A (GABAA) receptors. Here, we show that Hsp47 enhances the functional surface expression of GABAA receptors in rat neurons and human HEK293T cells. Furthermore, molecular mechanism study demonstrates that Hsp47 acts after BiP (Gene: HSPA5) and preferentially binds the folded conformation of GABAA receptors without inducing the unfolded protein response in HEK293T cells. Therefore, Hsp47 promotes the subunit-subunit interaction, the receptor assembly process, and the anterograde trafficking of GABAA receptors. Overexpressing Hsp47 is sufficient to correct the surface expression and function of epilepsy-associated GABAA receptor variants in HEK293T cells. Hsp47 also promotes the surface trafficking of other Cys-loop receptors, including nicotinic acetylcholine receptors and serotonin type 3 receptors in HEK293T cells. Therefore, in addition to its known function as a collagen chaperone, this work establishes that Hsp47 plays a critical and general role in the maturation of multi-subunit Cys-loop neuroreceptors.