ANTH domains within CALM, HIP1R, and Sla2 recognize ubiquitin internalization signals

Abstract

Attachment of ubiquitin (Ub) to cell surface proteins serves as a signal for internalization via clathrin-mediated endocytosis (CME). How ubiquitinated membrane proteins engage the internalization apparatus remains unclear. The internalization apparatus contains proteins such as Epsin and Eps15, which bind Ub, potentially acting as adaptors for Ub-based internalization signals. Here we show that additional components of the endocytic machinery including CALM, HIP1R, and Sla2 bind Ub via their N-terminal ANTH domain, a domain belonging to the superfamily of ENTH and VHS domains. Structural studies revealed that Ub binds with µM affinity to a unique C-terminal region within the ANTH domain not found in ENTH domains. Functional studies showed that combined loss of Ub-binding by ANTH-domain proteins and other Ub-binding domains within the yeast internalization apparatus caused defects in the Ub-dependent internalization of the GPCR Ste2 that was engineered to rely exclusively on Ub as an internalization signal. In contrast, these mutations had no effect on the internalization of Ste2 engineered to use an alternate Ub-independent internalization signal. These studies define new components of the internalization machinery that work collectively with Epsin and Eps15 to specify recognition of Ub as an internalization signal.

Data availability

Diffraction data have been deposited in PDB under the accession code 7JXV.

The following data sets were generated

Article and author information

Author details

  1. Natalya Pashkova

    Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Lokesh Gakhar

    Carver College of Medicine Protein Crystallography Core, University of Iowa, Iowa City, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Liping Yu

    Carver College of Medicine NMR Core, University of Iowa, Iowa City, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Nicholas J Schnicker

    Carver College of Medicine NMR Core, University of Iowa, Iowa City, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5189-4943
  5. Annabel Y Minard

    Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Stanley Winistorfer

    Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Ivan E Johnson

    Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Robert C Piper

    Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, United States
    For correspondence
    robert-piper@uiowa.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9995-5699

Funding

National Institutes of Health (GM058202)

  • Robert C Piper

National Institutes of Health (GM058202)

  • Robert C Piper

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

Reviewing Editor

  1. Wade Harper, Harvard Medical School, United States

Publication history

  1. Received: July 28, 2021
  2. Accepted: November 24, 2021
  3. Accepted Manuscript published: November 25, 2021 (version 1)
  4. Version of Record published: December 6, 2021 (version 2)
  5. Version of Record updated: December 16, 2021 (version 3)

Copyright

© 2021, Pashkova 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. Natalya Pashkova
  2. Lokesh Gakhar
  3. Liping Yu
  4. Nicholas J Schnicker
  5. Annabel Y Minard
  6. Stanley Winistorfer
  7. Ivan E Johnson
  8. Robert C Piper
(2021)
ANTH domains within CALM, HIP1R, and Sla2 recognize ubiquitin internalization signals
eLife 10:e72583.
https://doi.org/10.7554/eLife.72583
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