Light-dependent inhibition of clathrin-mediated endocytosis in yeast unveils conserved functions of the AP2 complex

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Summary

Clathrin-mediated endocytosis (CME) is an essential cellular process, conserved among eukaryotes. Yeast constitutes a powerful genetic model to dissect the complex endocytic machinery, yet there is a lack of specific pharmacological agents to interfere with CME in these organisms. TL2 is a light-regulated peptide inhibitor targeting the AP2-β-adaptin/β-arrestin interaction and that can photocontrol CME with high spatiotemporal precision in mammalian cells. Here, we study endocytic protein dynamics by live-cell imaging of the fluorescently tagged coat-associated protein Sla1-GFP, demonstrating that TL2 retains its inhibitory activity in S. cerevisiae spheroplasts. This is despite the β-adaptin/β-arrestin interaction not being conserved in yeast. Our data indicate that the AP2 α-adaptin is the functional target of activated TL2. We identified as interacting partners for the α-appendage, the Eps15 and epsin homologues Ede1 and Ent1. This demonstrates that endocytic cargo loading and sensing can be executed by conserved molecular interfaces, regardless of the proteins involved.

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Highlights

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Traffic Light peptide TL2 is a photoswitchable inhibitor of CME in yeast
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TL2 targets yeast α-adaptin, unveiling relevant Eps15 and epsin/AP2 interactions
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Acute inhibition of yeast AP2 uncovers previously unnoticed endocytic functions
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Conserved molecular interfaces execute similar endocytic functions

Abstract

Biochemistry; Biological sciences; Cell biology; Molecular biology; Natural sciences

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Most cited references 58

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NIH Image to ImageJ: 25 years of image analysis

Kevin W Eliceiri, Caroline A Schneider, Wayne S Rasband (2019)

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Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4

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Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae.

M Longtine, A. McKenzie, D DeMarini … (1998)

An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes. This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest. We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications. Using as selectable marker the S. cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5+ or Escherichia coli kan(r) gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging). Because of the modular nature of the plasmids, they allow efficient and economical use of a small number of PCR primers for a wide variety of gene manipulations. Thus, these plasmids should further facilitate the rapid analysis of gene function in S. cerevisiae.

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Author and article information

Contributors

María Isabel Geli

Pau Gorostiza

Journal

Journal ID (nlm-ta): iScience

Journal ID (iso-abbrev): iScience

Title: iScience

Publisher: Elsevier

ISSN (Electronic): 2589-0042

Publication date PMC-release: 12 September 2023

Publication date Collection: 20 October 2023

Publication date (Electronic): 12 September 2023

Volume: 26

Issue: 10

Electronic Location Identifier: 107899

Affiliations

[1 ]Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology, Barcelona, Spain

[2 ]Centro de Investigación Biomédica en Red – Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain

[3 ]Department of Cell Biology, Institute for Molecular Biology of Barcelona (IBMB-CSIC), Barcelona, Spain

[4 ]Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain

[5 ]Molecular Imaging Platform, Institute for Molecular Biology of Barcelona (IBMB-CSIC), Barcelona, Spain

[6 ]Department of Inorganic and Organic Chemistry, University of Barcelona (UB), Barcelona, Spain

[7 ]Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain

Author notes

[∗ ]Corresponding author mgfbmc@ 123456ibmb.csic.es

[∗∗ ]Corresponding author pau@ 123456icrea.cat

[8]

Present address: Department of Chemistry, Molecular Sciences Research Hub, Imperial College London, London, United Kingdom

[9]

Present address: Institute of Functional Biology and Genomics, Salamanca, Spain

[10]

Present address: IDP Discovery Pharma, Parc Científic de Barcelona (PCB), Barcelona, Spain

[11]

Present address: Ernst & Young, Barcelona, Spain

[12]

Lead contact

Article

Publisher Item ID: S2589-0042(23)01976-4 Publisher ID: 107899

DOI: 10.1016/j.isci.2023.107899

PMC ID: 10520943

PubMed ID: 37766990

SO-VID: 6869ea08-c898-460f-8420-e485761a9048

License:

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

History

Date received : 19 July 2023

Date revision received : 4 August 2023

Date accepted : 8 September 2023

Light-dependent inhibition of clathrin-mediated endocytosis in yeast unveils conserved functions of the AP2 complex

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Most cited references 58

NIH Image to ImageJ: 25 years of image analysis

Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4

Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae.

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