COPI mediates recycling of an exocytic SNARE by recognition of a ubiquitin sorting signal

Abstract

The COPI coat forms transport vesicles from the Golgi complex and plays a poorly defined role in endocytic trafficking. Here we show that COPI binds K63-linked polyubiquitin and this interaction is crucial for trafficking of a ubiquitinated yeast SNARE (Snc1). Snc1 is a v-SNARE that drives fusion of exocytic vesicles with the plasma membrane, and then recycles through the endocytic pathway to the Golgi for reuse in exocytosis. Removal of ubiquitin from Snc1, or deletion of a β'-COP subunit propeller domain that binds K63-linked polyubiquitin, disrupts Snc1 recycling causing aberrant accumulation in internal compartments. Moreover, replacement of the β'-COP propeller domain with unrelated ubiquitin-binding domains restores Snc1 recycling. These results indicate that ubiquitination, a modification well known to target membrane proteins to the lysosome or vacuole for degradation, can also function as recycling signal to sort a SNARE into COPI vesicles in a non-degradative pathway.

Article and author information

Author details

  1. Peng Xu

    Department of Biological Sciences, Vanderbilt University, Nashville, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Hannah M Hankins

    Department of Biological Sciences, Vanderbilt University, Nashville, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Chris MacDonald

    Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Samuel J Erlinger

    Department of Biological Sciences, Vanderbilt University, Nashville, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8347-2617
  5. Meredith N Frazier

    Department of Biological Sciences, Vanderbilt University, Nashville, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Nicholas S Diab

    Department of Biological Sciences, Vanderbilt University, Nashville, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Robert C Piper

    Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Lauren P Jackson

    Department of Biological Sciences, Vanderbilt University, Nashville, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3705-6126
  9. Jason A MacGurn

    Department of Cell and Developmental Biology, Vanderbilt University, Nashville, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5063-259X
  10. Todd R Graham

    Department of Biological Sciences, Vanderbilt University, Nashville, United States
    For correspondence
    tr.graham@vanderbilt.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3256-2126

Funding

National Institutes of Health (5R01GM118452)

  • Todd R Graham

Pew Charitable Trusts

  • Lauren P Jackson

National Institutes of Health (5R01GM058202)

  • Robert C Piper

National Institutes of Health (1R35GM119525)

  • Lauren P Jackson

National Institutes of Health (1R01GM118491)

  • Jason A MacGurn

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

Copyright

© 2017, Xu 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

  • 3,947
    views
  • 672
    downloads
  • 63
    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. Peng Xu
  2. Hannah M Hankins
  3. Chris MacDonald
  4. Samuel J Erlinger
  5. Meredith N Frazier
  6. Nicholas S Diab
  7. Robert C Piper
  8. Lauren P Jackson
  9. Jason A MacGurn
  10. Todd R Graham
(2017)
COPI mediates recycling of an exocytic SNARE by recognition of a ubiquitin sorting signal
eLife 6:e28342.
https://doi.org/10.7554/eLife.28342

Share this article

https://doi.org/10.7554/eLife.28342

Further reading

    1. Cell Biology
    Dharmendra Kumar Nath, Subash Dhakal, Youngseok Lee
    Research Advance

    Understanding how the brain controls nutrient storage is pivotal. Transient receptor potential (TRP) channels are conserved from insects to humans. They serve in detecting environmental shifts and in acting as internal sensors. Previously, we demonstrated the role of TRPγ in nutrient-sensing behavior (Dhakal et al., 2022). Here, we found that a TRPγ mutant exhibited in Drosophila melanogaster is required for maintaining normal lipid and protein levels. In animals, lipogenesis and lipolysis control lipid levels in response to food availability. Lipids are mostly stored as triacylglycerol in the fat bodies (FBs) of D. melanogaster. Interestingly, trpγ deficient mutants exhibited elevated TAG levels and our genetic data indicated that Dh44 neurons are indispensable for normal lipid storage but not protein storage. The trpγ mutants also exhibited reduced starvation resistance, which was attributed to insufficient lipolysis in the FBs. This could be mitigated by administering lipase or metformin orally, indicating a potential treatment pathway. Gene expression analysis indicated that trpγ knockout downregulated brummer, a key lipolytic gene, resulting in chronic lipolytic deficits in the gut and other fat tissues. The study also highlighted the role of specific proteins, including neuropeptide DH44 and its receptor DH44R2 in lipid regulation. Our findings provide insight into the broader question of how the brain and gut regulate nutrient storage.

    1. Cell Biology
    2. Immunology and Inflammation
    Mykhailo Vladymyrov, Luca Marchetti ... Britta Engelhardt
    Tools and Resources

    The endothelial blood-brain barrier (BBB) strictly controls immune cell trafficking into the central nervous system (CNS). In neuroinflammatory diseases such as multiple sclerosis, this tight control is, however, disturbed, leading to immune cell infiltration into the CNS. The development of in vitro models of the BBB combined with microfluidic devices has advanced our understanding of the cellular and molecular mechanisms mediating the multistep T-cell extravasation across the BBB. A major bottleneck of these in vitro studies is the absence of a robust and automated pipeline suitable for analyzing and quantifying the sequential interaction steps of different immune cell subsets with the BBB under physiological flow in vitro. Here, we present the under-flow migration tracker (UFMTrack) framework for studying immune cell interactions with endothelial monolayers under physiological flow. We then showcase a pipeline built based on it to study the entire multistep extravasation cascade of immune cells across brain microvascular endothelial cells under physiological flow in vitro. UFMTrack achieves 90% track reconstruction efficiency and allows for scaling due to the reduction of the analysis cost and by eliminating experimenter bias. This allowed for an in-depth analysis of all behavioral regimes involved in the multistep immune cell extravasation cascade. The study summarizes how UFMTrack can be employed to delineate the interactions of CD4+ and CD8+ T cells with the BBB under physiological flow. We also demonstrate its applicability to the other BBB models, showcasing broader applicability of the developed framework to a range of immune cell-endothelial monolayer interaction studies. The UFMTrack framework along with the generated datasets is publicly available in the corresponding repositories.