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 clathrin-coated 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.

Fig. 1.

Endomembrane targets. (A) Schematic overview of the endomembrane marker proteins used in this study and their localizations. RABD2a/ARA5—post-Golgi/Golgi/TGN/SV, RABF1/ARA6—LE/MVBs, RABF2b/ARA7—LE/MVBs, CLC2—clathrin-coated vesicles, GOT1—Golgi, RABG3f—LE/MVB/vacuole, VAMP711—vacuole. Modified from (16). (B) Localization of fluorescent-tagged marker proteins. Standard confocal micrographs of leaf epidermal cells of A. thaliana transgenic plants stably expressing the indicated recombinant proteins. Scale bars 10 μm. References: pUB:YFP-RabG3f, YFP-RABD2a/ARA5, YFP-VAMP711, YFP-Got1 (26), RABF1/ARA6-RFP, RFP-RABF2b/ARA7 (provided by K. Schumacher, Heidelberg, Germany), p35S:CLC-GFP (provided by S. Bednarek, Madison WI).

Fig. 2.

Proteomic workflow and validation. (A) Workflow for the affinity enrichment and analysis of endomembrane proteins from A. thaliana. (B) Immunoblotting of RFP-RABF2b/ARA7 and YFP-RABG3f proteome enrichments to determine organelle contamination. Total protein extracts, from A. thaliana Col-0 or stably expressing RFP-RABF2b/ARA7 or YFP-RABG3f were subjected to immunoaffinity enrichment of RFP or YFP followed by immunoblotting with αRFP, GFP, BIP2, COXII, RbcL, AHA1 as indicated.

Fig. 3.

Sungear diagram of proteins assigned to the different proteomes. Sungear diagrams generated in virtual plant (http://virtualplant-prod.bio.nyu.edu/cgi-bin/sungear/index.cgi (51)). Groups of proteins are indicated by the black dots, with a size proportional to the number of proteins in the group. The arrows on each dot indicate the proteome assignment of a group of proteins. Enrichments were performed three times for each bait, proteins were accepted with SAINT scores >0.8.

Fig. 4.

Venn diagrams comparing the proteins assigned to YFP-GOT1 (Golgi) YFP-RABD2a/ARA5 (Golgi/TGN/EE), RFP-RABF2b/ARA7 (LE/MVB) with published endomembrane proteomes. The number in each area of the Venn diagram indicate number of proteins assigned to the proteome or proteomes indicated. Venn diagram comparisons of YFP-GOT1 (Golgi), YFP-RABD2a/ARA5 (Golgi/TGN/EE) and RFP-RABF2b/ARA7 (LE/MVB) proteomes. Proteomes are (A) Nikolovski et al. (39), (B) Parsons et al. (37), (C) Sadowski et al./Dunkley et al. (33, 53). (D) Drakakaki et al. (44). (E) Groen et al. (45). (A) Comparison of our YFP-GOT1 proteome (Golgi). (B) Comparison with our RFP-RABF2b/ARA7 (LE/MVB) and YFP-RABD2a/ARA5 (Golgi/TGN/EE enrichments.

Fig. 5.

Venn diagrams comparing the proteins assigned to different endomembrane proteomes. The number in each area of the Venn diagram indicate number of proteins assigned to the proteome or proteomes indicated. (A) Comparison of YFP-GOT1 (Golgi) and RFP-RABF2b/ARA7 (LE/MVB) X² - 78.4, reject H_o (of independent assignment of proteins) p < .001. (B) Comparison of RABF1/ARA6-RFP (LE/MVB, YFP-RABG3f (LE/MVB/vacuole), YFP-VAMP711 (vacuole).

Fig. 6.

Co-localizations of PRA1 family members and RABF1/ARA6 (LE/MVB), RABF2b/ARA7 (LE/MVB) and GOT1 (Golgi). Standard confocal micrographs of leaf epidermis of the indicated A. thaliana transgenic plants stably expressing pUB::YFP-GOT1, RFP-RABF2b/ARA7, or RABF1/ARA6-RFP, transiently transformed using particle bombardment, expressing fluorescent tagged PRA1 family members. Insets show an enlarged section of each image.