NECAPs are negative regulators of the AP2 clathrin adaptor complex

  1. Gwendolyn M Beacham
  2. Edward A Partlow
  3. Jeffrey J Lange
  4. Gunther Hollopeter  Is a corresponding author
  1. Cornell University, United States
  2. Stowers Institute for Medical Research, United States

Abstract

Eukaryotic cells internalize transmembrane receptors via clathrin-mediated endocytosis, but it remains unclear how the machinery underpinning this process is regulated. We recently discovered that membrane-associated muniscin proteins such as FCHo and SGIP initiate endocytosis by converting the AP2 clathrin adaptor complex to an open, active conformation that is then phosphorylated (Hollopeter et al., 2014). Here we report that loss of ncap-1, the sole C. elegans gene encoding an adaptiN Ear-binding Coat-Associated Protein (NECAP), bypasses the requirement for FCHO-1. Biochemical analyses reveal AP2 accumulates in an open, phosphorylated state in ncap-1 mutant worms, suggesting NECAPs promote the closed, inactive conformation of AP2. Consistent with this model, NECAPs preferentially bind open and phosphorylated forms of AP2 in vitro and localize with constitutively open AP2 mutants in vivo. NECAPs do not associate with phosphorylation-defective AP2 mutants, implying that phosphorylation precedes NECAP recruitment. We propose NECAPs function late in endocytosis to inactivate AP2.

Article and author information

Author details

  1. Gwendolyn M Beacham

    Department of Molecular Medicine, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Edward A Partlow

    Department of Molecular Medicine, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Jeffrey J Lange

    Stowers Institute for Medical Research, Kansas City, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Gunther Hollopeter

    Department of Molecular Medicine, Cornell University, Ithaca, United States
    For correspondence
    gh383@cornell.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6409-0530

Funding

National Science Foundation (Graduate Research Fellowship DGE-1650441)

  • Gwendolyn M Beacham

National Institutes of Health (Training Grant GM007273-43)

  • Gwendolyn M Beacham
  • Edward A Partlow

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

Reviewing Editor

  1. Suzanne R Pfeffer, Stanford University School of Medicine, United States

Publication history

  1. Received: September 25, 2017
  2. Accepted: January 17, 2018
  3. Accepted Manuscript published: January 18, 2018 (version 1)
  4. Version of Record published: January 25, 2018 (version 2)

Copyright

© 2018, Beacham 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. Gwendolyn M Beacham
  2. Edward A Partlow
  3. Jeffrey J Lange
  4. Gunther Hollopeter
(2018)
NECAPs are negative regulators of the AP2 clathrin adaptor complex
eLife 7:e32242.
https://doi.org/10.7554/eLife.32242

Further reading

    1. Cell Biology
    Gunther Hollopeter et al.
    Research Article Updated

    The AP2 clathrin adaptor complex links protein cargo to the endocytic machinery but it is unclear how AP2 is activated on the plasma membrane. Here we demonstrate that the membrane-associated proteins FCHo and SGIP1 convert AP2 into an open, active conformation. We screened for Caenorhabditis elegans mutants that phenocopy the loss of AP2 subunits and found that AP2 remains inactive in fcho-1 mutants. A subsequent screen for bypass suppressors of fcho-1 nulls identified 71 compensatory mutations in all four AP2 subunits. Using a protease-sensitivity assay we show that these mutations restore the open conformation in vivo. The domain of FCHo that induces this rearrangement is not the F-BAR domain or the µ-homology domain, but rather is an uncharacterized 90 amino acid motif, found in both FCHo and SGIP proteins, that directly binds AP2. Thus, these proteins stabilize nascent endocytic pits by exposing membrane and cargo binding sites on AP2.

    1. Cell Biology
    2. Developmental Biology
    Anna Keppner et al.
    Research Article Updated

    Spermatogenesis is a highly specialized differentiation process driven by a dynamic gene expression program and ending with the production of mature spermatozoa. Whereas hundreds of genes are known to be essential for male germline proliferation and differentiation, the contribution of several genes remains uncharacterized. The predominant expression of the latest globin family member, androglobin (Adgb), in mammalian testis tissue prompted us to assess its physiological function in spermatogenesis. Adgb knockout mice display male infertility, reduced testis weight, impaired maturation of elongating spermatids, abnormal sperm shape, and ultrastructural defects in microtubule and mitochondrial organization. Epididymal sperm from Adgb knockout animals display multiple flagellar malformations including coiled, bifid or shortened flagella, and erratic acrosomal development. Following immunoprecipitation and mass spectrometry, we could identify septin 10 (Sept10) as interactor of Adgb. The Sept10-Adgb interaction was confirmed both in vivo using testis lysates and in vitro by reciprocal co-immunoprecipitation experiments. Furthermore, the absence of Adgb leads to mislocalization of Sept10 in sperm, indicating defective manchette and sperm annulus formation. Finally, in vitro data suggest that Adgb contributes to Sept10 proteolysis in a calmodulin-dependent manner. Collectively, our results provide evidence that Adgb is essential for murine spermatogenesis and further suggest that Adgb is required for sperm head shaping via the manchette and proper flagellum formation.