Protein biocargo of citrus fruit-derived vesicles reveals heterogeneous transport and extracellular vesicles populations

Abstract

Cellular vesicles are membrane-enclosed organelles that transport material inside and outside the cell. Plant-derived vesicles are receiving more and more 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. High expression of patellin-3-like, clathrin heavy chain, HSPs, 14-3-3 protein, glyceraldehyde-3-phosphate dehydrogenase and fructose-bisphosphate aldolase 6 could be observed in all the vesicle samples while Aquaporin was highly expressed only in nanovesicles. Bioinformatics revealed more than hundred protein orthologues potentially implicated in vesicular trafficking. In particular, the presence of CCV, COPI and COPII coat proteins indicates the presence of highly heterogeneous populations of intracellular transport vesicles. Moreover, the differential expressions of hydrolases and oxidoreductases enabled us to gain insights into their functions in citrus fruit sac cells.