Dissection of protein cargo of citrus fruit juice sac cells-derived vesicles reveals heterogeneous transport and extracellular vesicles subpopulations

Cellular vesicles are membrane-enclosed organelles that transport material inside and outside the cell. Plant-derived vesicles are receiving increasing attention due to their potential as nanovectors for the delivery of biologically active substances. We aimed to expand our understanding about the heterogeneity and the protein biocargo of citrus fruit juice sac cell-derived vesicles. Micro- and nano-sized vesicle fractions were isolated from four citrus species, C. sinensis, C. limon, C. paradisi and C. aurantium, characterized using physicochemical methods and protein cargos were compared using label-free quantitative shotgun proteomics. In each sample approximately 600-800 proteins were identified. Orthologues of most of the top-ranking proteins have previously been reported in extracellular vesicles of mammalian origin. Patellin-3-like, clathrin heavy chain, heat shock proteins, 14-3-3 protein, glyceraldehyde-3-phosphate dehydrogenase and fructose-bisphosphate aldolase 6 were highly expressed in all citrus vesicle fractions. The presence of membrane channel aquaporin on the other hand characteristic of the nanovesicle fractions. Bioinformatics revealed more than hundred protein orthologues potentially implicated in vesicular trafficking. In particular, CCV, COPI and COPII coat proteins indicates the presence of highly heterogeneous populations of intracellular transport vesicles. Moreover, the different hydrolases and oxidoreductases transported within the citrus fruit-derived vesicles can be responsible for the various biological activities possessed by the preparations.