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

Fig 3. ARF1 but not ARFA regulates early secretion and post-Golgi trafficking.

Fig 3

Expression of activation-impaired, YFP-tagged T31N (TN; green) variants of ARF1 and ARFA was induced by 20μM estradiol for 5-6h, and trafficking markers (magenta) were analyzed in immunostaining. (A-G) The COPI subunit γCOP (A, B, E) was recruited to the Golgi in wild-type control (A; Col) and in ARFA-TN-YFP (E-G) but stayed in the cytosol in ARF1-TN-YFP (B-D) expressing lines. (H-N) Clathrin coat (H, J, M) was recruited to the TGN in wild-type control (H) and in ARFA-TN-YFP (L-N) but stayed in the cytosol in ARF1-TN-YFP (I-K). (O-U) BFA treatment (50μM for 1h) was used to visualize endocytosed FM4-64 (O, P, S) in BFA compartments. Endocytosis was unaffected in wild-type control (O) and in ARFA-TN-YFP (S-U). In contrast, ARF1-TN-YFP (Q) interfered with endocytosis of FM4-64 (P-R). Note, cells expressing ARF1 strongly showed almost no endocytosed FM4-64, whereas cells with no or very low expression of ARF1-TN-YFP showed endocytosis. (V-I1) Recycling to the plasma membrane of RFP-PEN1 expressed from the Histone 4 (H4) promoter after accumulation in BFA compartments. (V-B1) Seedlings were treated with 20μM estradiol and 50μM BFA for 5h. RFP-PEN1 (V, W, Z) localized in BFA compartments of root cells in wild-type (V), ARF1-TN-YFP (W-Y) and ARFA-TN-YFP (Z-B1). (C1-I1) Recycling of RFP-PEN1 was analyzed after BFA wash-out for 2h. RFP-PEN1 (C1, D1, G1) returned to the plasma membrane in wild-type controls (C1) and in ARFA-TN-YFP (G1-I1) but not in ARF1-TN-YFP (D1-F1). (J1-P1) Trafficking of the vacuolar cargo, AFVY-RFP (J1, K1, N1), expressed from the estradiol-inducible system, was inhibited in ARF1-TN-YFP (K1-M1) lines but not in wild-type (J1) or in ARFA-TN-YFP (N1-P1). Scale bar, 10μm.