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Detailed Analysis of Protein Topology of Extracellular Vesicles–Evidence of Unconventional Membrane Protein Orientation

Journal article
Authors Aleksander Cvjetkovic
Su Chul Jang
Barbora Konečná
Johanna L Höög
Carina Sihlbom
Cecilia Lässer
Jan Lötvall
Published in Scientific Reports
Volume 6
Pages Article number: 36338
ISSN 2045-2322
Publication year 2016
Published at Krefting Research Centre
Core Facilities, Proteomics
Department of Chemistry and Molecular Biology
Pages Article number: 36338
Language en
Keywords exosome, proteomics, electron microscopy, vesicle
Subject categories Cell Biology


Extracellular vesicles (EVs) are important mediators of intercellular communication that change the recipient cell by shuttling lipids, RNA, or protein cargo between cells. Here, we investigate the topology of the protein cargo found in EVs, as this topology can fundamentally influence the biological effects of EVs. A multiple proteomics approach, combining proteinase treatment and biotin tagging, shows that many proteins of cytosolic origin are localized on the surface of EVs. A detailed analysis of the EV proteome at the peptide level revealed that a number of EV membrane proteins are present in a topologically reversed orientation compared to what is annotated. Two examples of such proteins, SCAMP3 and STX4, were confirmed to have a reversed topology. This reversed typology was determined using flow cytometry and fluorescent microscopy with antibodies directed toward their cytoplasmic epitopes. These results describe a novel workflow to define the EV proteome and the orientation of each protein, including membrane protein topology. These data are fundamentally important to understanding the EV proteome and required to fully explain EV biogenesis as well as biological function in recipient cells.

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