Cryo-EM structures of CTP synthase filaments reveal mechanism of pH-sensitive assembly during budding yeast starvation

Abstract

Many metabolic enzymes self-assemble into micron-scale filaments to organize and regulate metabolism. The appearance of these assemblies often coincides with large metabolic changes as in development, cancer, and stress. Yeast undergo cytoplasmic acidification upon starvation, triggering the assembly of many metabolic enzymes into filaments. However, it is unclear how these filaments assemble at the molecular level and what their role is in the yeast starvation response. CTP Synthase (CTPS) assembles into metabolic filaments across many species. Here, we characterize in vitro polymerization and investigate in vivo consequences of CTPS assembly in yeast. Cryo-EM structures reveal a pH-sensitive assembly mechanism and highly ordered filament bundles that stabilize an inactive state of the enzyme, features unique to yeast CTPS. Disruption of filaments in cells with non-assembly or pH-insensitive mutations decreases growth rate, reflecting the importance of regulated CTPS filament assembly in homeotstasis.

Data availability

models deposited to PDB as: 7RL0,7RNR,7RKH,7RL5,7RNL,7RMF,7RMK,7RMC,7RMO,7RMVmaps deposited to EMDB as: EMD-24512,EMD-24581,EMD-24497,EMD-24516,EMD-24579,EMD-24566,EMD-24575,EMD-24560,EMD-24576,EMD-24578

Article and author information

Author details

  1. Jesse M Hansen

    University of Washington, Seattle, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Avital Horowitz

    University of Washington, Seattle, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Eric M Lynch

    University of Washington, Seattle, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5897-5167
  4. Daniel P Farrell

    University of Washington, Seattle, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Joel Quispe

    University of Washington, Seattle, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Frank DiMaio

    University of Washington, Seattle, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7524-8938
  7. Justin M Kollman

    Department of Biochemistry, University of Washington, seattle, United States
    For correspondence
    jkoll@uw.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0350-5827

Funding

National Institutes of Health (R01 grant,GM118396)

  • Justin M Kollman

National Institutes of Health (Graduate Student Training Grant,T32 GM007270)

  • Jesse M Hansen

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

Copyright

© 2021, Hansen 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

  • 2,920
    views
  • 378
    downloads
  • 38
    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. Jesse M Hansen
  2. Avital Horowitz
  3. Eric M Lynch
  4. Daniel P Farrell
  5. Joel Quispe
  6. Frank DiMaio
  7. Justin M Kollman
(2021)
Cryo-EM structures of CTP synthase filaments reveal mechanism of pH-sensitive assembly during budding yeast starvation
eLife 10:e73368.
https://doi.org/10.7554/eLife.73368

Share this article

https://doi.org/10.7554/eLife.73368

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Joar Esteban Pinto Torres, Mathieu Claes ... Yann G-J Sterckx
    Research Article

    African trypanosomes are the causative agents of neglected tropical diseases affecting both humans and livestock. Disease control is highly challenging due to an increasing number of drug treatment failures. African trypanosomes are extracellular, blood-borne parasites that mainly rely on glycolysis for their energy metabolism within the mammalian host. Trypanosomal glycolytic enzymes are therefore of interest for the development of trypanocidal drugs. Here, we report the serendipitous discovery of a camelid single-domain antibody (sdAb aka Nanobody) that selectively inhibits the enzymatic activity of trypanosomatid (but not host) pyruvate kinases through an allosteric mechanism. By combining enzyme kinetics, biophysics, structural biology, and transgenic parasite survival assays, we provide a proof-of-principle that the sdAb-mediated enzyme inhibition negatively impacts parasite fitness and growth.

    1. Biochemistry and Chemical Biology
    Jianheng Fox Liu, Ben R Hawley ... Samie R Jaffrey
    Tools and Resources

    N 6,2’-O-dimethyladenosine (m6Am) is a modified nucleotide located at the first transcribed position in mRNA and snRNA that is essential for diverse physiological processes. m6Am mapping methods assume each gene uses a single start nucleotide. However, gene transcription usually involves multiple start sites, generating numerous 5’ isoforms. Thus, gene-level annotations cannot capture the diversity of m6Am modification in the transcriptome. Here, we describe CROWN-seq, which simultaneously identifies transcription-start nucleotides and quantifies m6Am stoichiometry for each 5’ isoform that initiates with adenosine. Using CROWN-seq, we map the m6Am landscape in nine human cell lines. Our findings reveal that m6Am is nearly always a high stoichiometry modification, with only a small subset of cellular mRNAs showing lower m6Am stoichiometry. We find that m6Am is associated with increased transcript expression and provide evidence that m6Am may be linked to transcription initiation associated with specific promoter sequences and initiation mechanisms. These data suggest a potential new function for m6Am in influencing transcription.