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

  • 2,919
    Page views
  • 511
    Downloads
  • 25
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

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

Further reading

    1. Cell Biology
    Xiang Wang, Vitaliy V Bondar ... Anastasia G Henry
    Short Report Updated

    Leucine-rich repeat kinase 2 (LRRK2) variants associated with Parkinson’s disease (PD) and Crohn’s disease lead to increased phosphorylation of its Rab substrates. While it has been recently shown that perturbations in cellular homeostasis including lysosomal damage can increase LRRK2 activity and localization to lysosomes, the molecular mechanisms by which LRRK2 activity is regulated have remained poorly defined. We performed a targeted siRNA screen to identify regulators of LRRK2 activity and identified Rab12 as a novel modulator of LRRK2-dependent phosphorylation of one of its substrates, Rab10. Using a combination of imaging and immunopurification methods to isolate lysosomes, we demonstrated that Rab12 is actively recruited to damaged lysosomes and leads to a local and LRRK2-dependent increase in Rab10 phosphorylation. PD-linked variants, including LRRK2 R1441G and VPS35 D620N, lead to increased recruitment of LRRK2 to the lysosome and a local elevation in lysosomal levels of pT73 Rab10. Together, these data suggest a conserved mechanism by which Rab12, in response to damage or expression of PD-associated variants, facilitates the recruitment of LRRK2 and phosphorylation of its Rab substrate(s) at the lysosome.

    1. Cell Biology
    Herschel S Dhekne, Francesca Tonelli ... Suzanne R Pfeffer
    Research Advance Updated

    Activating mutations in the leucine-rich repeat kinase 2 (LRRK2) cause Parkinson’s disease. LRRK2 phosphorylates a subset of Rab GTPases, particularly Rab10 and Rab8A, and we showed previously that these phosphoRabs play an important role in LRRK2 membrane recruitment and activation (Vides et al., 2022). To learn more about LRRK2 pathway regulation, we carried out an unbiased, CRISPR-based genome-wide screen to identify modifiers of cellular phosphoRab10 levels. A flow cytometry assay was developed to detect changes in phosphoRab10 levels in pools of mouse NIH-3T3 cells harboring unique CRISPR guide sequences. Multiple negative and positive regulators were identified; surprisingly, knockout of the Rab12 gene was especially effective in decreasing phosphoRab10 levels in multiple cell types and knockout mouse tissues. Rab-driven increases in phosphoRab10 were specific for Rab12, LRRK2-dependent and PPM1H phosphatase-reversible, and did not require Rab12 phosphorylation; they were seen with wild type and pathogenic G2019S and R1441C LRRK2. As expected for a protein that regulates LRRK2 activity, Rab12 also influenced primary cilia formation. AlphaFold modeling revealed a novel Rab12 binding site in the LRRK2 Armadillo domain, and we show that residues predicted to be essential for Rab12 interaction at this site influence phosphoRab10 and phosphoRab12 levels in a manner distinct from Rab29 activation of LRRK2. Our data show that Rab12 binding to a new site in the LRRK2 Armadillo domain activates LRRK2 kinase for Rab phosphorylation and could serve as a new therapeutic target for a novel class of LRRK2 inhibitors that do not target the kinase domain.