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Open AccessArticle

The GABARAP Co-Secretome Identified by APEX2-GABARAP Proximity Labelling of Extracellular Vesicles

1
Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany
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Institute of Biological Information Processing (IBI-7: Structural Biochemistry), Forschungszentrum Jülich, Leo-Brandt-Straße, 52428 Jülich, Germany
3
Institute of Molecular Medicine I, Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany
4
Molecular Proteomics Laboratory, Biologisch-Medizinisches Forschungszentrum (BMFZ), Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany
5
Munich Cluster for Systems Neurology (SyNergy), Ludwig-Maximilians-Universität München, Feodor-Lynen-Straße 17, 81377 München, Germany
*
Authors to whom correspondence should be addressed.
Cells 2020, 9(6), 1468; https://doi.org/10.3390/cells9061468
Received: 4 March 2020 / Revised: 5 June 2020 / Accepted: 12 June 2020 / Published: 16 June 2020
The autophagy-related ATG8 protein GABARAP has not only been shown to be involved in the cellular self-degradation process called autophagy but also fulfils functions in intracellular trafficking processes such as receptor transport to the plasma membrane. Notably, available mass spectrometry data suggest that GABARAP is also secreted into extracellular vesicles (EVs). Here, we confirm this finding by the immunoblotting of EVs isolated from cell culture supernatants and human blood serum using specific anti-GABARAP antibodies. To investigate the mechanism by which GABARAP is secreted, we applied proximity labelling, a method for studying the direct environment of a protein of interest in a confined cellular compartment. By expressing an engineered peroxidase (APEX2)-tagged variant of GABARAP—which, like endogenous GABARAP, was present in EVs prepared from HEK293 cells—we demonstrate the applicability of APEX2-based proximity labelling to EVs. The biotinylated protein pool which contains the APEX2-GABARAP co-secretome contained not only known GABARAP interaction partners but also proteins that were found in APEX2-GABARAP’s proximity inside of autophagosomes in an independent study. All in all, we not only introduce a versatile tool for co-secretome analysis in general but also uncover the first details about autophagy-based pathways as possible biogenesis mechanisms of GABARAP-containing EVs. View Full-Text
Keywords: ATG8; GABARAP; autophagy; APEX2; proximity; extracellular vesicle; exosome; secretion ATG8; GABARAP; autophagy; APEX2; proximity; extracellular vesicle; exosome; secretion
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Sanwald, J.L.; Poschmann, G.; Stühler, K.; Behrends, C.; Hoffmann, S.; Willbold, D. The GABARAP Co-Secretome Identified by APEX2-GABARAP Proximity Labelling of Extracellular Vesicles. Cells 2020, 9, 1468.

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