Dissecting the phase separation and oligomerization activities of the carboxysome positioning protein McdB

Abstract

Across bacteria, protein-based organelles called bacterial microcompartments (BMCs) encapsulate key enzymes to regulate their activities. The model BMC is the carboxysome that encapsulates enzymes for CO2 fixation to increase efficiency and is found in many autotrophic bacteria, such as cyanobacteria. Despite their importance in the global carbon cycle, little is known about how carboxysomes are spatially regulated. We recently identified the two-factor system required for the maintenance of carboxysome distribution (McdAB). McdA drives the equal spacing of carboxysomes via interactions with McdB, which associates with carboxysomes. McdA is a ParA/MinD ATPase, a protein family well-studied in positioning diverse cellular structures in bacteria. However, the adaptor proteins like McdB that connect these ATPases to their cargos are extremely diverse. In fact, McdB represents a completely unstudied class of proteins. Despite the diversity, many adaptor proteins undergo phase separation, but functional roles remain unclear. Here, we define the domain architecture of McdB from the model cyanobacterium Synechococcus elongatus PCC 7942, and dissect its mode of biomolecular condensate formation. We identify an N-terminal intrinsically disordered region (IDR) that modulates condensate solubility, a central coiled-coil dimerizing domain that drives condensate formation, and a C-terminal domain that trimerizes McdB dimers and provides increased valency for condensate formation. We then identify critical basic residues in the IDR, which we mutate to glutamines to solubilize condensates. Finally, we find that a condensate-defective mutant of McdB has altered association with carboxysomes and influences carboxysome enzyme content. The results have broad implications for understanding spatial organization of BMCs and the molecular grammar of protein condensates.

Data availability

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

Article and author information

Author details

  1. Joseph L Basalla

    Department of Molecular, Cellular, and Developmental Biology, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Claudia A Mak

    Department of Biological Chemistry, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5903-1766
  3. Jordan Byrne

    Department of Molecular, Cellular, and Developmental Biology, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Maria Ghalmi

    Department of Molecular, Cellular, and Developmental Biology, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Y Hoang

    Department of Biological Chemistry, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Anthony G Vecchiarelli

    Department of Molecular, Cellular, and Developmental Biology, University of Michigan-Ann Arbor, Ann Arbor, United States
    For correspondence
    ave@umich.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6198-3245

Funding

National Science Foundation (1941966)

  • Anthony G Vecchiarelli

National Science Foundation (1817478)

  • Anthony G Vecchiarelli

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

Reviewing Editor

  1. Rohit V Pappu, Washington University in St Louis, United States

Version history

  1. Received: June 24, 2022
  2. Accepted: September 1, 2023
  3. Accepted Manuscript published: September 5, 2023 (version 1)

Copyright

© 2023, Basalla 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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  1. Joseph L Basalla
  2. Claudia A Mak
  3. Jordan Byrne
  4. Maria Ghalmi
  5. Y Hoang
  6. Anthony G Vecchiarelli
(2023)
Dissecting the phase separation and oligomerization activities of the carboxysome positioning protein McdB
eLife 12:e81362.
https://doi.org/10.7554/eLife.81362

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