The conserved aspartate ring of MCU mediates MICU1 binding and regulation in the mitochondrial calcium uniporter complex

  1. Charles B Phillips
  2. Chen-Wei Tsai
  3. Ming-Feng Tsai  Is a corresponding author
  1. Brandeis University, United States

Abstract

The mitochondrial calcium uniporter is a Ca2+ channel that regulates intracellular Ca2+ signaling, oxidative phosphorylation, and apoptosis. It contains the pore-forming MCU protein, which possesses a DIME sequence thought to form a Ca2+ selectivity filter, and also regulatory EMRE, MICU1, and MICU2 subunits. To properly carry out physiological functions, the uniporter must stay closed in resting conditions, becoming open only when stimulated by intracellular Ca2+ signals. This Ca2+-dependent activation, known to be mediated by MICU subunits, is not well understood. Here, we demonstrate that the DIME-aspartate mediates a Ca2+-modulated electrostatic interaction with MICU1, forming an MICU1 contact interface with a nearby Ser residue at the cytoplasmic entrance of the MCU pore. A mutagenesis screen of MICU1 identifies two highly-conserved Arg residues that might contact the DIME-Asp. Perturbing MCU-MICU1 interactions elicits unregulated, constitutive Ca2+ flux into mitochondria. These results indicate that MICU1 confers Ca2+-dependent gating of the uniporter by blocking/unblocking MCU.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. Source data for calcium-45 flux experiments are available via Dryad.

The following data sets were generated

Article and author information

Author details

  1. Charles B Phillips

    Department of Biochemistry, Brandeis University, Waltham, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Chen-Wei Tsai

    Department of Biochemistry, Brandeis University, Waltham, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Ming-Feng Tsai

    Department of Biochemistry, Brandeis University, Waltham, United States
    For correspondence
    mftsai@brandeis.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4277-1885

Funding

Howard Hughes Medical Institute

  • Ming-Feng Tsai

National Institute of General Medical Sciences (R01-GM129345)

  • Chen-Wei Tsai
  • Ming-Feng Tsai

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. The funders pay for the authors' salary and other research expenses.

Copyright

© 2019, Phillips 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

  • 2,531
    views
  • 399
    downloads
  • 61
    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. Charles B Phillips
  2. Chen-Wei Tsai
  3. Ming-Feng Tsai
(2019)
The conserved aspartate ring of MCU mediates MICU1 binding and regulation in the mitochondrial calcium uniporter complex
eLife 8:e41112.
https://doi.org/10.7554/eLife.41112

Share this article

https://doi.org/10.7554/eLife.41112

Further reading

    1. Biochemistry and Chemical Biology
    2. Computational and Systems Biology
    Shinichi Kawaguchi, Xin Xu ... Toshie Kai
    Research Article

    Protein–protein interactions are fundamental to understanding the molecular functions and regulation of proteins. Despite the availability of extensive databases, many interactions remain uncharacterized due to the labor-intensive nature of experimental validation. In this study, we utilized the AlphaFold2 program to predict interactions among proteins localized in the nuage, a germline-specific non-membrane organelle essential for piRNA biogenesis in Drosophila. We screened 20 nuage proteins for 1:1 interactions and predicted dimer structures. Among these, five represented novel interaction candidates. Three pairs, including Spn-E_Squ, were verified by co-immunoprecipitation. Disruption of the salt bridges at the Spn-E_Squ interface confirmed their functional importance, underscoring the predictive model’s accuracy. We extended our analysis to include interactions between three representative nuage components—Vas, Squ, and Tej—and approximately 430 oogenesis-related proteins. Co-immunoprecipitation verified interactions for three pairs: Mei-W68_Squ, CSN3_Squ, and Pka-C1_Tej. Furthermore, we screened the majority of Drosophila proteins (~12,000) for potential interaction with the Piwi protein, a central player in the piRNA pathway, identifying 164 pairs as potential binding partners. This in silico approach not only efficiently identifies potential interaction partners but also significantly bridges the gap by facilitating the integration of bioinformatics and experimental biology.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Yamato Niitani, Kohei Matsuzaki ... Michio Tomishige
    Research Article

    The two identical motor domains (heads) of dimeric kinesin-1 move in a hand-over-hand process along a microtubule, coordinating their ATPase cycles such that each ATP hydrolysis is tightly coupled to a step and enabling the motor to take many steps without dissociating. The neck linker, a structural element that connects the two heads, has been shown to be essential for head–head coordination; however, which kinetic step(s) in the chemomechanical cycle is ‘gated’ by the neck linker remains unresolved. Here, we employed pre-steady-state kinetics and single-molecule assays to investigate how the neck-linker conformation affects kinesin’s motility cycle. We show that the backward-pointing configuration of the neck linker in the front kinesin head confers higher affinity for microtubule, but does not change ATP binding and dissociation rates. In contrast, the forward-pointing configuration of the neck linker in the rear kinesin head decreases the ATP dissociation rate but has little effect on microtubule dissociation. In combination, these conformation-specific effects of the neck linker favor ATP hydrolysis and dissociation of the rear head prior to microtubule detachment of the front head, thereby providing a kinetic explanation for the coordinated walking mechanism of dimeric kinesin.