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. 2018 Nov 15;14(11):e1007795. doi: 10.1371/journal.pgen.1007795

Fig 5. Hydrolysis-impaired forms of ARF1 and ARFA block early secretion, endosomal recycling as well as post-Golgi and vacuolar trafficking.

Fig 5

Expression of YFP-tagged hydrolysis-impaired Q71L (QL; green) variants of ARF1 and ARFA was induced by 20μM estradiol for 5-6h and trafficking markers (magenta) were analyzed in immunostaining (A-N) or live-cell imaging (O-I1). (A-G) The COPI subunit γCOP (A, B, E) was recruited to the Golgi in wild-type control (A; Col). Expression of ARF1-QL-YFP (C) and ARFA-QL-YFP (F) induced aggregation of γCOP (B, E) co-localizing with ARF1 and ARFA (D, G). (H-N) Clathrin coat (H, I, L) was recruited to the TGN in wild-type control (H). In contrast, clathrin formed aggregates and co-localized with ARF1-QL-YFP (I-K) and ARFA-QL-YFP (L-N). (O-U) BFA treatment (50μM for 1h) was used to visualize endocytosed FM4-64 (O, P, S) in BFA-compartments. Endocytosis of FM4-64 in ARF1-QL-YFP (P-R) and ARFA-QL-YFP (S-U) was comparable to wild-type control (O). (V-B1) Seedlings were treated with 20μM estradiol and 50μM BFA for 5h. Recycling of H4::RFP-PEN1 was analyzed after BFA wash-out for 2h. H4::RFP-PEN1 (V, W, Z) returned to the plasma membrane in wild-type control (V). ARF1-QL-YFP (W-Y) and ARF A-QL-YFP (Z-B1) interfered with recycling. (C1-I1) Trafficking of the vacuolar cargo, AFVY-RFP (C1, D1, G1), expressed from the estradiol-inducible system, was inhibited in ARF1-QL-YFP (D1-F1) and in ARFA-QL-YFP (G1-I1) expressing lines but not in wild-type (C1). Scale bar, 10μm. The same wild-type controls were used as in Figs 3 and S3 and S5.