Detailed Analysis of Protein Topology of Extracellular Vesicles-Evidence of Unconventional Membrane Protein Orientation.
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.
Krefting Research Center, Institute of Medicine, University of Gothenburg, Gothenburg, Sweden.
Aleksander
A
Cvjetkovic
Krefting Research Center, Institute of Medicine, University of Gothenburg, Gothenburg, Sweden.
Su Chul
SC
Jang
Krefting Research Center, Institute of Medicine, University of Gothenburg, Gothenburg, Sweden.|Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University, Bratislava, Slovakia.
Barbora
B
Konečná
Krefting Research Center, Institute of Medicine, University of Gothenburg, Gothenburg, Sweden.|Department of Chemistry and Molecular Biology, Faculty of Natural Sciences, University of Gothenburg, Gothenburg, Sweden.
Johanna L
JL
Höög
Proteomics Core Facility, University of Gothenburg, Gothenburg, Sweden.
Carina
C
Sihlbom
Krefting Research Center, Institute of Medicine, University of Gothenburg, Gothenburg, Sweden.
Cecilia
C
Lässer
Krefting Research Center, Institute of Medicine, University of Gothenburg, Gothenburg, Sweden.
Jan
J
Lötvall
D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D000067128:Extracellular Vesicles / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D050765:Qa-SNARE Proteins / Q000378:metabolism
D002352:Carrier Proteins; D008565:Membrane Proteins; D050765:Qa-SNARE Proteins; C109303:SCAMP3 protein, human; C587292:syntaxin 4, human