Unconventional Protein Secretion

Christopher Stanly, Immacolata Fiume, Giovambattista Capasso, Gabriella Pocsfalvi, Stanly, Christopher, Fiume, Immacolata, Capasso, Giovambattista, Pocsfalvi, Gabriella

Andrea Pompa, Francesca De Marchis

Exosomes are nanovesicles of endocytic origin that are about 30-100 nm in diameter, surrounded by a lipid bilayer membrane, and contain proteins, nucleic acids, and other molecules. Mammalian cells- and biological fluids-derived exosomes have become the subject for a wide range of investigations in biological and biomedical sciences. More recently, a new interest is on the verge of rising: the presence of nanovesicles in plants. Lipoprotein vesicles from apoplastic fluid and exosome-like vesicles (ELVs) from fruit juice have been isolated and shown that they could be loaded with drugs and uptaken by recipient cells. In order to explore and analyze the contents and functions of ELVs, they must be isolated and purified with intense care. Isolation of ELVs can be a tedious process and often characterized by the co-purification of undesired contaminants. Here we describe a method which isolates ELVs based on their buoyant density. The method utilizes differential centrifugation in step 1 and 1 and 2 M sucrose/deuterium oxide double-cushion ultracentrifugation in step 2, to purify two diverse ELV subpopulations. In this method fruit juice is used as an example of starting material, although this protocol can be used for the isolation of vesicles from apoplastic fluid too. The quality and the quantity of ELV preparations have been found appropriate for downstream biological and structural studies, like proteomics, transcriptomics, and lipidomics.