Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation

Abstract

The formation of a clathrin-coated vesicle is a major membrane remodeling process that is crucial for membrane traffic in cells. Besides clathrin, these vesicles contain at least 100 different proteins although it is unclear how many are essential for the formation of the vesicle. Here, we show that intracellular clathrin-coated formation can be induced in living cells using minimal machinery and that it can be achieved on various membranes, including the mitochondrial outer membrane. Chemical heterodimerization was used to inducibly attach a clathrin-binding fragment 'hook' to an 'anchor' protein targeted to a specific membrane. Endogenous clathrin assembled to form coated pits on the mitochondria, termed MitoPits, within seconds of induction. MitoPits are double-membraned invaginations that form preferentially on high curvature regions of the mitochondrion. Upon induction, all stages of CCV formation - initiation, invagination, and even fission - were faithfully reconstituted. We found no evidence for the functional involvement of accessory proteins in this process. In addition, fission of MitoPit-derived vesicles was independent of known scission factors including dynamins and dynamin-related protein 1 (Drp1), suggesting that the clathrin cage generates sufficient force to bud intracellular vesicles. Our results suggest that, following its recruitment, clathrin is sufficient for intracellular clathrin-coated vesicle formation.

Data availability

Data and code associated with this study are available at https://github.com/quantixed/p057p034 (Royle, 2022).

The following data sets were generated

Article and author information

Author details

  1. Cansu Küey

    Division of Biomedical Sciences, University of Warwick, Coventry, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  2. Méghane Sittewelle

    Division of Biomedical Sciences, University of Warwick, Coventry, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  3. Gabrielle Larocque

    Division of Biomedical Sciences, University of Warwick, Coventry, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8295-9378
  4. Miguel Hernández-González

    Division of Biomedical Sciences, University of Warwick, Coventry, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  5. Stephen J Royle

    Division of Biomedical Sciences, University of Warwick, Coventry, United Kingdom
    For correspondence
    s.j.royle@warwick.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8927-6967

Funding

UK Research and Innovation (EP/L016494/1)

  • Cansu Küey

UK Research and Innovation (BB/V003062/1)

  • Méghane Sittewelle
  • Stephen J Royle

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

Copyright

© 2022, Küey 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. Cansu Küey
  2. Méghane Sittewelle
  3. Gabrielle Larocque
  4. Miguel Hernández-González
  5. Stephen J Royle
(2022)
Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation
eLife 11:e78929.
https://doi.org/10.7554/eLife.78929

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https://doi.org/10.7554/eLife.78929

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