Dwarf open reading frame (DWORF) is a direct activator of the sarcoplasmic reticulum calcium pump SERCA

Abstract

The sarcoplasmic reticulum calcium pump SERCA plays a critical role in the contraction-relaxation cycle of muscle. In cardiac muscle, SERCA is regulated by the inhibitor phospholamban. A new regulator, dwarf open reading frame (DWORF), has been reported to displace phospholamban from SERCA. Here, we show that DWORF is a direct activator of SERCA, increasing its turnover rate in the absence of phospholamban. Measurement of in-cell calcium dynamics supports this observation and demonstrates that DWORF increases SERCA-dependent calcium reuptake. These functional observations reveal opposing effects of DWORF activation and phospholamban inhibition of SERCA. To gain mechanistic insight into SERCA activation, fluorescence resonance energy transfer experiments revealed that DWORF has a higher affinity for SERCA in the presence of calcium. Molecular modeling and molecular dynamics simulations provide a model for DWORF activation of SERCA, where DWORF modulates the membrane bilayer and stabilizes the conformations of SERCA that predominate during elevated cytosolic calcium.

Data availability

All data generated or analyzed during this study are included in the manuscript.

Article and author information

Author details

  1. M'Lynn E Fisher

    Biochemistry, University of Alberta, Edmonton, Canada
    Competing interests
    No competing interests declared.
  2. Elisa Bovo

    Department of Cell and Molecular Physiology, Loyola University Chicago, Chicago, United States
    Competing interests
    No competing interests declared.
  3. Rodrigo Aguayo-Ortiz

    Internal Medicine, University of Michigan, Ann Arbor, United States
    Competing interests
    No competing interests declared.
  4. Ellen E Cho

    Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, United States
    Competing interests
    No competing interests declared.
  5. Marsha P Pribadi

    Department of Cell and Molecular Physiology, Loyola University Chicago, Chicago, United States
    Competing interests
    No competing interests declared.
  6. Michael P Dalton

    Department of Cell and Molecular Physiology, Loyola University Chicago, Chicago, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5296-5099
  7. Nishadh Rathod

    Biochemistry, University of Alberta, Edmonton, Canada
    Competing interests
    No competing interests declared.
  8. M Joanne Lemieux

    Biochemistry, University of Alberta, Edmonton, Canada
    Competing interests
    M Joanne Lemieux, Reviewing editor, eLife.
  9. L Michel Espinoza-Fonseca

    Internal Medicine, University of Michigan, Ann Arbor, United States
    Competing interests
    No competing interests declared.
  10. Seth L Robia

    Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1193-9510
  11. Aleksey V Zima

    Department of Cell and Molecular Physiology, Loyola University Chicago, Chicago, United States
    Competing interests
    No competing interests declared.
  12. Howard S Young

    Biochemistry, University of Alberta, Edmonton, Canada
    For correspondence
    hyoung@ualberta.ca
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5990-8422

Funding

National Institutes of Health (R01HL092321)

  • Seth L Robia

National Institutes of Health (R01HL092321)

  • Howard S Young

National Institutes of Health (R01HL143816)

  • Seth L Robia

National Institutes of Health (R01HL143816)

  • Howard S Young

National Institutes of Health (R01GM120142)

  • L Michel Espinoza-Fonseca

National Institutes of Health (R01HL148068)

  • L Michel Espinoza-Fonseca

National Institutes of Health (R01HL130231)

  • Aleksey V Zima

Natural Sciences and Engineering Research Council of Canada (RGPIN-2016-06478)

  • M Joanne Lemieux

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

Copyright

© 2021, Fisher 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

  • 2,672
    views
  • 347
    downloads
  • 29
    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. M'Lynn E Fisher
  2. Elisa Bovo
  3. Rodrigo Aguayo-Ortiz
  4. Ellen E Cho
  5. Marsha P Pribadi
  6. Michael P Dalton
  7. Nishadh Rathod
  8. M Joanne Lemieux
  9. L Michel Espinoza-Fonseca
  10. Seth L Robia
  11. Aleksey V Zima
  12. Howard S Young
(2021)
Dwarf open reading frame (DWORF) is a direct activator of the sarcoplasmic reticulum calcium pump SERCA
eLife 10:e65545.
https://doi.org/10.7554/eLife.65545

Share this article

https://doi.org/10.7554/eLife.65545

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Santi Mestre-Fos, Lucas Ferguson ... Jamie HD Cate
    Research Article

    Stem cell differentiation involves a global increase in protein synthesis to meet the demands of specialized cell types. However, the molecular mechanisms underlying this translational burst and the involvement of initiation factors remains largely unknown. Here, we investigate the role of eukaryotic initiation factor 3 (eIF3) in early differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPCs). Using Quick-irCLIP and alternative polyadenylation (APA) Seq, we show eIF3 crosslinks predominantly with 3’ untranslated region (3’-UTR) termini of multiple mRNA isoforms, adjacent to the poly(A) tail. Furthermore, we find that eIF3 engagement at 3’-UTR ends is dependent on polyadenylation. High eIF3 crosslinking at 3’-UTR termini of mRNAs correlates with high translational activity, as determined by ribosome profiling, but not with translational efficiency. The results presented here show that eIF3 engages with 3’-UTR termini of highly translated mRNAs, likely reflecting a general rather than specific regulatory function of eIF3, and supporting a role of mRNA circularization in the mechanisms governing mRNA translation.

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Ana Patrícia Graça, Vadim Nikitushkin ... Gerald Lackner
    Research Article

    Mycofactocin is a redox cofactor essential for the alcohol metabolism of mycobacteria. While the biosynthesis of mycofactocin is well established, the gene mftG, which encodes an oxidoreductase of the glucose-methanol-choline superfamily, remained functionally uncharacterized. Here, we show that MftG enzymes are almost exclusively found in genomes containing mycofactocin biosynthetic genes and are present in 75% of organisms harboring these genes. Gene deletion experiments in Mycolicibacterium smegmatis demonstrated a growth defect of the ∆mftG mutant on ethanol as a carbon source, accompanied by an arrest of cell division reminiscent of mild starvation. Investigation of carbon and cofactor metabolism implied a defect in mycofactocin reoxidation. Cell-free enzyme assays and respirometry using isolated cell membranes indicated that MftG acts as a mycofactocin dehydrogenase shuttling electrons toward the respiratory chain. Transcriptomics studies also indicated remodeling of redox metabolism to compensate for a shortage of redox equivalents. In conclusion, this work closes an important knowledge gap concerning the mycofactocin system and adds a new pathway to the intricate web of redox reactions governing the metabolism of mycobacteria.