A systematic approach to identify recycling endocytic cargo depending on the GARP complex

  1. Sebastian Eising
  2. Lisa Thiele
  3. Florian Fröhlich  Is a corresponding author
  1. University of Osnabrück, Germany

Abstract

Proteins and lipids of the plasma membrane underlie constant remodeling via a combination of the secretory- and the endocytic pathway. In the yeast endocytic pathway, cargo is sorted for recycling to the plasma membrane or degradation in vacuoles. Previously we have shown a role for the GARP complex in sphingolipid sorting and homeostasis (Fröhlich et al. 2015). However, the majority of cargo sorted in a GARP dependent process remain largely unknown. Here we use auxin induced degradation of GARP combined with mass spectrometry based vacuolar proteomics and lipidomics to show that recycling of two specific groups of proteins, the amino-phospholipid flippases and cell wall synthesis proteins depends on a functional GARP complex. Our results suggest that mis-sorting of flippases and remodeling of the lipid composition are the first occurring defects in GARP mutants. Our assay can be adapted to systematically map cargo of the entire endocytic pathway.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 3 and 4.

Article and author information

Author details

  1. Sebastian Eising

    Department of Biology/Chemistry, University of Osnabrück, Osnabrück, Germany
    Competing interests
    The authors declare that no competing interests exist.
  2. Lisa Thiele

    Department of Biology/Chemistry, University of Osnabrück, Osnabrück, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Florian Fröhlich

    Department of Biology/Chemistry, University of Osnabrück, Osnabrück, Germany
    For correspondence
    florian.froehlich@biologie.uni-osnabrueck.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8307-2189

Funding

Deutsche Forschungsgemeinschaft (FR 3647/2-1)

  • Florian Fröhlich

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

Reviewing Editor

  1. Peter Tontonoz, University of California, Los Angeles, United States

Version history

  1. Received: October 19, 2018
  2. Accepted: January 29, 2019
  3. Accepted Manuscript published: January 29, 2019 (version 1)
  4. Version of Record published: February 13, 2019 (version 2)

Copyright

© 2019, Eising 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

  • 3,057
    views
  • 521
    downloads
  • 29
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

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)

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

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

  1. Sebastian Eising
  2. Lisa Thiele
  3. Florian Fröhlich
(2019)
A systematic approach to identify recycling endocytic cargo depending on the GARP complex
eLife 8:e42837.
https://doi.org/10.7554/eLife.42837

Share this article

https://doi.org/10.7554/eLife.42837

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Jiabin Pan, Rui Zhou ... Xiang-dong Li
    Research Article

    Transport and localization of melanosome at the periphery region of melanocyte are depended on myosin-5a (Myo5a), which associates with melanosome by interacting with its adaptor protein melanophilin (Mlph). Mlph contains four functional regions, including Rab27a-binding domain, Myo5a GTD-binding motif (GTBM), Myo5a exon F-binding domain (EFBD), and actin-binding domain (ABD). The association of Myo5a with Mlph is known to be mediated by two specific interactions: the interaction between the exon-F-encoded region of Myo5a and Mlph-EFBD and that between Myo5a-GTD and Mlph-GTBM. Here, we identify a third interaction between Myo5a and Mlph, that is, the interaction between the exon-G-encoded region of Myo5a and Mlph-ABD. The exon-G/ABD interaction is independent from the exon-F/EFBD interaction and is required for the association of Myo5a with melanosome. Moreover, we demonstrate that Mlph-ABD interacts with either the exon-G or actin filament, but cannot interact with both of them simultaneously. Based on above findings, we propose a new model for the Mlph-mediated Myo5a transportation of melanosomes.

    1. Cell Biology
    Yuki Date, Yukiko Sasazawa ... Shinji Saiki
    Research Article

    The autophagy-lysosome pathway plays an indispensable role in the protein quality control by degrading abnormal organelles and proteins including a-synuclein (aSyn) associated with the pathogenesis of Parkinson's disease (PD). However, the activation of this pathway is mainly by targeting lysosomal enzymic activity. Here, we focused on the autophagosome-lysosome fusion process around the microtubule-organizing center (MTOC) regulated by lysosomal positioning. Through high-throughput chemical screening, we identified 6 out of 1,200 clinically approved drugs enabling the lysosomes to accumulate around the MTOC with autophagy flux enhancement. We further demonstrated that these compounds induce the lysosomal clustering through a JIP4-TRPML1-dependent mechanism. Among them, the lysosomal-clustering compound albendazole promoted the autophagy-dependent degradation of Triton-X-insoluble, proteasome inhibitor-induced aggregates. In a cellular PD model, albendazole boosted insoluble aSyn degradation. Our results revealed that lysosomal clustering can facilitate the breakdown of protein aggregates, suggesting that lysosome-clustering compounds may offer a promising therapeutic strategy against neurodegenerative diseases characterized by the presence of aggregate-prone proteins.