Identification of Regulatory and Cargo Proteins of Endosomal and Secretory Pathways in Arabidopsis thaliana by Proteomic Dissection*

Abstract

The cell's endomembranes comprise an intricate, highly dynamic and well-organized system. In plants, the proteins that regulate function of the various endomembrane compartments and their cargo remain largely unknown. Our aim was to dissect subcellular trafficking routes by enriching for partially overlapping subpopulations of endosomal proteomes associated with endomembrane markers. We selected RABD2a/ARA5, RABF2b/ARA7, RABF1/ARA6, and RABG3f as markers for combinations of the Golgi, trans-Golgi network (TGN), early endosomes (EE), secretory vesicles, late endosomes (LE), multivesicular bodies (MVB), and the tonoplast. As comparisons we used Golgi transport 1 (GOT1), which localizes to the Golgi, clathrin light chain 2 (CLC2) labeling clathrincoated vesicles and pits and the vesicle-associated membrane protein 711 (VAMP711) present at the tonoplast. We developed an easy-to-use method by refining published protocols based on affinity purification of fluorescent fusion constructs to these seven subcellular marker proteins in Arabidopsis thaliana seedlings. We present a total of 433 proteins, only five of which were shared among all enrichments, while many proteins were common between endomembrane compartments of the same trafficking route. Approximately half, 251 proteins, were assigned to one enrichment only. Our dataset contains known regulators of endosome functions including small GTPases, SNAREs, and tethering complexes. We identify known cargo proteins such as PIN3, PEN3, CESA, and the recently defined TPLATE complex. The subcellular localization of two GTPase regulators predicted from our enrichments was validated using live-cell imaging. This is the first proteomic dataset to discriminate between such highly overlapping endomembrane compartments in plants and can be used as a general proteomic resource to predict the localization of proteins and identify the components of regulatory complexes and provides a useful tool for the identification of new protein markers of the endomembrane system. Molecular & Cellular

VPS9a Activates the Rab5 GTPase ARA7 to Confer Distinct Pre- and Postinvasive Plant Innate Immunity

“…We speculate that this redundancy extends to also cover innate immunity. Interestingly, recent reports point to a clear difference in the composition of ARA6-and ARA7-positive endosomes (Heard et al, 2015) and that plant Rab5 GEFs seem to have evolved a specific domain that regulates ARA6 only (Sunada et al, 2016). Combined, these findings underline a likely difference in ARA6 and ARA7 functionality that we find extends to innate immunity.…”

Extracellular Vesicles Isolated from the Leaf Apoplast Carry Stress-Response Proteins

“…complex, TGN/EE, MVBs/late endosomes, tonoplast, and clathrin-coated vesicle pits (Alexandersson et al, 2004;Fernandez-Calvino et al, 2011;Floerl et al, 2012;Heard et al, 2015). Overall, the EV proteome was most similar to compartments associated with RABF2b/ ARA7, a RAB GTPase distributed between the TGN/ EE and MVBs.…”

An Aphid Effector Targets Trafficking Protein VPS52 in a Host-Specific Manner to Promote Virulence

“…A recent proteomics study to identify proteins associated with endosomal and secretory pathways in Arabidopsis revealed the enrichment of VPS52 in pull-downs of RABF1/ARA6, RABG3f, CLC2, RABD2a/ARA5, and RABF2b/ARA7 (Heard et al, 2015). This suggests that VPS52 may associate with a variety of endomembrane vesicles, perhaps through binding GTPases (Heard et al, 2015). How exactly aphids impact vesicle trafficking via VPS52 remains to be investigated and will require a better understanding of VPS52 function inside plant cells.…”

“…VPS52 interacts with at least one other component of the GARP-complex in plants, VPS51, which is involved in the maintenance of vacuolar morphology as well as leaf shape and vein patterning (Pahari et al, 2014). A recent proteomics study to identify proteins associated with endosomal and secretory pathways in Arabidopsis revealed the enrichment of VPS52 in pull-downs of RABF1/ARA6, RABG3f, CLC2, RABD2a/ARA5, and RABF2b/ARA7 (Heard et al, 2015). This suggests that VPS52 may associate with a variety of endomembrane vesicles, perhaps through binding GTPases (Heard et al, 2015).…”