Figure 1: TGN subdomain labelled by SYP61 is enriched in hVLCFAs and sterols as compared with Golgi or TGN subdomain RAB-A2a enriched for clathrin. | Nature Communications

Figure 1: TGN subdomain labelled by SYP61 is enriched in hVLCFAs and sterols as compared with Golgi or TGN subdomain RAB-A2a enriched for clathrin.

From: Enrichment of hydroxylated C24- and C26-acyl-chain sphingolipids mediates PIN2 apical sorting at trans-Golgi network subdomains

Figure 1

(a–g) Immunolocalization of CHC (b,e) in Arabidopsis root epithelial cells expressing either the TGN marker RAB-A2a–YFP (a) or the TGN marker SYP61–CFP (d). Strong co-localization between RAB-A2a and CHC is detected in merged images (c), whereas weaker co-localization is visualized between SYP61 and CHC (f,g). Statistical analysis show highly significant difference between RAB-A2a/CHC co-localization and SYP61/CHC co-localization (n=50 cells distributed over 10 roots for each experiment, 3 biological replicates). (h) Immunopurifications of SYP61–CFP-, MEMB12–YFP- and RAB-A2a–YFP-labelled compartments were performed by incubating a step-gradient-purified TM fraction with beads coated with anti-CFP/YFP antibodies. (i) Western blottings on IP SYP61–CFP-, RAB-A2a–YFP- and MEM12–YFP-labelled intact vesicles. IP, beads-IP fraction; TM, input, step-gradient-purified TM fraction. As compared with the input (TM), anti-CFP/YFP antibodies revealed that all protein markers (SYP61–CFP, MEMB12–YFP or RAB-A2a–YFP) are enriched in their targeted IP compartments. Sec21p and MEMB11 markers of the Golgi apparatus are enriched in IP MEMB12–YFP-labelled Golgi but not in IP SYP61–CFP-labelled TGN or RAB-A2a–YFP-labelled TGN. The ECHIDNA and SYP61 markers of TGN-associated secretory vesicles are enriched in IP SYP61–CFP-labelled TGN but not in MEMB12–YFP-labelled Golgi or RAB-A2a–YFP-labelled TGN. V-ATPase VHA-E, which traffic through the TGN, is not enriched in SYP61-immunopurified or MEMB12-immunopurified fraction but is slightly enriched in IP RAB-A2a–YFP-labelled TGN. The PMA2 and PM-ATPase markers for PM are not enriched in any IP compartments. (j,k) Acyl-chain composition of the total pool of FAs contained in IP fractions. (j) As compared with MEMB12–YFP-labelled Golgi, SYP61–CFP-labelled TGN shows a significant enrichment (about 2-fold, n=11 IPs for each compartment, 11 biological replicates for each compartment) in hVLCFAs h22:0, h24:1, h24:0, h26:0 and sterols. (k) As compared with MEMB12–YFP-labelled Golgi, RAB-A2a–YFP-labelled TGN does not display any enrichment in hVLCFAs or sterols (n=11 IPs for each compartment, 11 biological replicates for each compartment). Statistics were done by two-sided Wilcoxon’s rank-sum test, **P-value<0.01, ***P-value<0.001. All scale bars, 5 μm.

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