1. Biochemistry and Chemical Biology
  2. Microbiology and Infectious Disease
Download icon

Formation of a β-barrel membrane protein is catalyzed by the interior surface of the assembly machine protein BamA

  1. James Lee
  2. David Tomasek
  3. Thiago Santos
  4. Mary D May
  5. Ina Meuskens
  6. Daniel Kahne  Is a corresponding author
  1. Harvard University, United States
Research Article
  • Cited 25
  • Views 2,403
  • Annotations
Cite this article as: eLife 2019;8:e49787 doi: 10.7554/eLife.49787

Abstract

The β-barrel assembly machine (Bam) complex in Gram-negative bacteria and its counterparts in mitochondria and chloroplasts fold and insert outer membrane β-barrel proteins. BamA, an essential component of the complex, is itself a β-barrel and is proposed to play a central role in assembling other barrel substrates. Here, we map the path of substrate insertion by the Bam complex using site-specific crosslinking to understand the molecular mechanisms that control β-barrel folding and release. We find that the C-terminal strand of the substrate is stably held by BamA and that the N-terminal strands of the substrate are assembled inside the BamA β-barrel. Importantly, we identify contacts between the assembling β-sheet and the BamA interior surface that determine the rate of substrate folding. Our results support a model in which the interior wall of BamA acts as a chaperone to catalyze β-barrel assembly.

Data availability

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

Article and author information

Author details

  1. James Lee

    Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. David Tomasek

    Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1212-4601
  3. Thiago Santos

    Department of Chemistry and Chemical Biology, Harvard University, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Mary D May

    Department of Chemistry and Chemical Biology, Harvard University, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Ina Meuskens

    Department of Chemistry and Chemical Biology, Harvard University, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Daniel Kahne

    Department of Chemistry and Chemical Biology, Harvard University, Cambridge, United States
    For correspondence
    kahne@chemistry.harvard.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8296-1424

Funding

National Institute of Allergy and Infectious Diseases (AI081059)

  • James Lee
  • David Tomasek
  • Thiago Santos
  • Mary D May
  • Ina Meuskens
  • Daniel Kahne

National Institute of General Medical Sciences (F31GM116210)

  • James Lee

National Institute of Allergy and Infectious Diseases (T32AI132120)

  • Mary D May

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

Reviewing Editor

  1. Ramanujan S Hegde, MRC Laboratory of Molecular Biology, United Kingdom

Publication history

  1. Received: June 29, 2019
  2. Accepted: November 13, 2019
  3. Accepted Manuscript published: November 14, 2019 (version 1)
  4. Version of Record published: December 2, 2019 (version 2)

Copyright

© 2019, Lee 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

  • 2,403
    Page views
  • 512
    Downloads
  • 25
    Citations

Article citation count generated by polling the highest count across the following sources: Scopus, Crossref, PubMed Central.

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)

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

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

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Rania Elsabrouty et al.
    Research Article Updated

    UbiA prenyltransferase domain-containing protein-1 (UBIAD1) utilizes geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K2 subtype menaquinone-4. The prenyltransferase has emerged as a key regulator of sterol-accelerated, endoplasmic reticulum (ER)-associated degradation (ERAD) of HMG CoA reductase, the rate-limiting enzyme in synthesis of cholesterol and nonsterol isoprenoids including GGpp. Sterols induce binding of UBIAD1 to reductase, inhibiting its ERAD. Geranylgeraniol (GGOH), the alcohol derivative of GGpp, disrupts this binding and thereby stimulates ERAD of reductase and translocation of UBIAD1 to Golgi. We now show that overexpression of Type 1 polyisoprenoid diphosphate phosphatase (PDP1), which dephosphorylates GGpp and other isoprenyl pyrophosphates to corresponding isoprenols, abolishes protein geranylgeranylation as well as GGOH-induced ERAD of reductase and Golgi transport of UBIAD1. Conversely, these reactions are enhanced in the absence of PDP1. Our findings indicate PDP1-mediated hydrolysis of GGpp significantly contributes to a feedback mechanism that maintains optimal intracellular levels of the nonsterol isoprenoid.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Jesse M Hansen et al.
    Research Article Updated

    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.