Membrane Transporters in Citrus clementina Fruit Juice-Derived Nanovesicles

Abstract

The cellular vesicle is a fluid-filled structure separated from the surrounding environment by a biological membrane. Here, we isolated nanovesicles (NVs) from the juice of clementines using a discontinuous density gradient ultracentrifugation method. To gain information about the protein content of vesicles, mass spectrometry-based organelle proteomics and bioinformatics were applied to the exosome-like vesicle fraction isolated in the 1 mol/L sucrose/D₂O cushion. Analysis of 1018 identified proteins revealed a highly complex mixture of different intra, extracellular and artificially-formed vesicle populations. In particular, clathrin-coated vesicles were significantly expressed in this sample. Membrane transporters are significantly represented in clementines nanovesicles. We have found 162 proteins associated with the transport Gene Ontology term (GO: 0006810) which includes; 71 transmembrane transport related, 53 vesicle mediated and 50 intracellular transporters. Platellin-3 like carrier protein containing a Sec14 domain is known to have a role in plant-virus interaction and that is one of the most abundant proteins in our dataset. The presence of transmembrane transporters like ATPases, aquaporins, ATP Binding Cassette (ABC) transporters and tetraspanins, regulators of protein trafficking suggests that nanovesicles of clementines can actively interact with their environment in a controlled way.

Keywords: ATPases; Citrus clementina; biocargo; bioinformatics; citrus; intercellular communication; mass spectrometry; plant-derived vesicles; proteomics; tetraspanins; transporters.

Figure 1

Schematic chart of the experimental work performed to isolate, characterize and analyze C. clementine fruit juice-derived exosome-like nanovesicles. (A) Lower left image shows the pellets obtained after diffferential ultracentrifugation (UC) lower right image shows the separation obtained by sucrose/D₂O double cushion UC. The vesicles floating above the 1M sucrose/D₂O cushion were found to be similar in density to mammalian extracellular vesicles. (B) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) protein profiles (right) and of vesicle populations in the 1 M and 2 M sucrose/D₂O cushions and the particle-size distributions of vesicles isolated in the 1M sucrose/D₂O cushion and measured using nanoparticle tracking analysis (NTA). (C). Venn diagram generated by FunRich software [1] shows the numbers of unique and common Orthologous Groups (OGs) of the identified protein. OGs of Citrus clementina (azure) were compared to four citrus species (C. sinensis, C. limon, C. paradise and C. aurantium) (yellow) [13] and EVpedia (red) [14].

Figure 2

Enrichment and distribution of (A) transport-related Gene Ontology (GO): 0006810 and (B) vesicle mediated transport-related GO: 16192 terms in the protein data set of nanovesicles isolated from the juice of C. clementine.