Fig 1.
Phylogenetic tree of ARF GTPases and ARF-like proteins, and the subcellular localization of Arabidopsis ARF GTPases.
(A) Left: Phylogenetic tree of ARFs and ARF-likes from Arabidopsis (At; green), Human (Hs; magenta) and Yeast (Sc; grey). Class I ARFs, ARL1/2/5/8 and ARFRP1 are conserved among eukaryotes, whereas ARF classes A, B and D are plant-specific. Class II and class III ARFs are present in human and yeast but not in plants. Right: List of Arabidopsis ARF and ARL GTPases with their gene identifiers. (B-Y) Subcellular localization by CLSM of YFP-tagged ARF-GTPases of classes I, A and B expressed from the RPS5a promoter (green channel). (B-I) Class-I ARF (ARFA1c; B; F) co-localized partially with TGN-marker VHA-a1-RFP (magenta; C; D) and Golgi-marker Wave22-mCherry (magenta; G; H). Co-localization in two regions of interest (ROI) in line intensity profiles (E, I). (J-Q) Class-A ARF (ARFB1b; J; N) co-localized partially with TGN-marker VHA-a1-RFP (magenta; K; L) and the Golgi-marker Wave22-mCherry (magenta; O; P). Co-localization in two regions of interest (ROI) in line intensity profiles (M, Q). (R-Y) Class-B ARF (R, V) localized at the plasma membrane and in intracellular punctae. ARFB co-localized partially with TGN-marker VHA-a1-RFP (magenta; S; T) but not with Golgi-marker Wave22-mCherry (magenta; W; X). Co-localization in two regions of interest (ROI) in line intensity profiles (U, Y). Scale bar, 10 μm. (Z-B1) Ultrastructural localization of YFP-tagged ARF GTPases ARF1 (Z, A1) and ARFA (B1) with gold-labelled anti-GFP antibodies. Silver-enhanced 1 nm gold (Z), 6 nm gold (A1-B1). Scale bar (Z-B1), 500 nm.
Fig 2.
ARF1 but not ARFA or ARFB is crucial for primary root growth and seed germination.
(A-P) Activation-impaired (T31N; TN) or hydrolysis-impaired (Q71L; QL) variants of ARF1, ARFA and ARFB were expressed from the estradiol-inducible system. (A) Primary root growth was severely affected by ARF1-TN-YFP, ARF1-QL-YFP and ARFA-QL-YFP but not by ARFA-TN-YFP, ARFB-TN-YFP or ARFB-QL-YFP. Number of seedlings analyzed is indicated above each column. (+), 20μM estradiol; (-), without estradiol. See also S1 Data. (B-N) Seed germination on estradiol-containing medium is inhibited by ARF1-TN-YFP (C, D), ARF1-QL-YFP (I, J) and ARFA-QL-YFP (K, L) but not by ARFA-TN-YFP (E, F), ARFB-TN-YFP (G, H) or ARFB-QL-YFP (M, N). Scale bar, 5 mm (B-N). (O, P) Western blot of estradiol-induced expression of activation-impaired (TN) or hydrolysis-impaired (QL) variants of ARF1, ARFA and ARFB in seedlings (O). Asterisk, expected size of approx. 47kDa. (P) γCOP as loading control; two asterisks, expected size of approx. 100kDa. COL, wild-type control. (Q-S) Quantitative secretion assay. (Q) Co-expression of the secretory reporter α-amylase with wild type (WT), activation-impaired (TN) or hydrolysis-impaired (QL) ARFA variants in tobacco protoplasts. Secretion index, ratio of extracellular to intracellular reporter activity; control, expression of α-amylase alone. (R) Co-expression of α-amylase and ARFA-QL with rising concentrations of ARFA-WT, ARFA-TN or ARF1-WT. Numbers below indicate the amount of respective plasmid used for protoplast transformation relative to ARFA-QL; constant amounts of α-amylase and ARFA-QL were transformed. (S) Co-expression of α-amylase and ARFA-QL with rising concentrations of ARFA-TN,QL. Numbers below indicate the amount of respective plasmid used for protoplast transformation relative to ARFA-QL; constant amounts of α-amylase and ARFA-QL were transformed. Panels below (Q-S): ARF protein expression levels evidenced indirectly by detection of GFP. Both ARF and GFP coding sequences are under control of the bidirectional mas promoter (consisting of a mas1’ and a mas2’ part) on the same plasmid, with mas1’ directing GFP expression and mas2’ directing ARF expression in a ratio of 1 to 10 [65]. Note full recovery of secretion by ARFA-TN (R) and ARFA-TN,QL (S) overexpression. See also S2, S3 and S4 Data.
Fig 3.
ARF1 but not ARFA regulates early secretion and post-Golgi trafficking.
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.
Fig 4.
Ultrastructural defects of endomembrane compartments caused by interference with ARF1 or ARFA function.
Seedling root cells were ultrastructurally analyzed after high-pressure freezing, freeze-substitution and embedding in epoxy resin. (A-E) ARF1-TN-YFP. (A) Tubular ER (er) connected to large compartments (erc) and circular structures (*). (B, C) Circular structures (*) connected to ER contain several membranes including ER membranes. (D) Circular structures are heavily labeled with gold-conjugated anti-ARF1 antibodies (arrows). (E) Disintegrated Golgi stack (arrowhead). (F, G) ARF1-QL-YFP. (F) Large vesicle aggregates with Golgi(-derived) cisternae (arrowheads). (G) Golgi cisternae (arrowheads) producing unusually large (secretory) vesicles. (H, I) ARFA-TN-YFP. (H) Golgi stacks (arrowheads) and TGN structures (t). (I) Golgi stack (arrowhead) with slightly enlarged TGN compartment (t) at higher magnification. (K-M) ARFA-QL-YFP. (K) Aberrant Golgi stacks (arrowheads) and TGN structures (t) in two adjacent cells. (L) Cell-wall stubs (cw) and aberrant Golgi stack (arrowhead) and TGN structures (t). (M) Aberrant rounded Golgi stacks (arrowheads) with budding vesicles and large TGN structures (t) at higher magnification. Abbreviations: cw, cell wall; er, endoplasmic reticulum (ER); erc, ER-connected compartment; m, mitochondrion; n, nucleus; t, trans-Golgi network (TGN); v, vacuole. Scale bars, 500 nm.
Fig 5.
Hydrolysis-impaired forms of ARF1 and ARFA block early secretion, endosomal recycling as well as post-Golgi and vacuolar trafficking.
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.
Fig 6.
Interaction of ARF1 and ARFA with different ARF-GEFs and localization of ARFA in big5 mutant.
(A-F) Co-immunoprecipitation (Co-IP) studies. (A) Co-IP of GNOM-Myc with ARF1-YFP, using GFP-Trap-agarose beads (IP: α-GFP) followed by immunoblot analysis (IB) with α-Myc antibody. Seedlings expressing only GNOM-Myc (GN-Myc) were used as control. (B) Co-IP of endogenous and YFP-tagged ARF1 with GNOM-Myc, using α-Myc-agarose beads (IP) followed by IB with α-GFP antibody and α-ARF1 antibody to detect YFP-tagged and endogenous ARF1. (C) Co-IP of endogenous and YFP-tagged ARF1 with GNL1-Myc, using α-Myc-agarose beads (IP) followed by IB analysis with α-GFP antibody and α-ARF1 antibody. (D) Co-IP of endogenous ARF1 with BIG3-YFP, using GFP-Trap-agarose beads (IP: α-GFP) followed by IB with α-ARF1 antibody. Col, wild-type control. (E) Co-IP of ARFA (ARFB1c)-RFP and endogenous ARF1 with BIG5-YFP, using GFP-Trap-agarose beads (IP: α-GFP) followed by IB analysis with α-RFP antibody and α-ARF1 antibody. (F) Co-IP of ARFA (ARFB1c)-RFP and endogenous ARF1 with BIG4-YFP, using GFP-Trap-agarose beads (IP: α-GFP) followed by IB analysis using α-RFP antibody and α-ARF1 antibody. IN, input; IP; immunoprecipitate; IB, immunoblot; kDa, kilodalton. (G-J) ARFA-YFP localization in wild-type (WT) (G-H) and big5 mutant (I-J). Note the highly cytosolic signal of ARFA-YFP in big5. Scale bars, 10μm. (K) In-vitro GDP-GTP exchange activity of the catalytic SEC7 domain of BIG3 (blue) and BIG5 (red) on ARFA. Negative control, ARFA alone (black). See also S5 Data.
Fig 7.
Swapping of catalytic domains between Golgi-localized and TGN-localized ARF-GEFs did not impair ARF-GEF function.
(A) Left panel: Expression of GNL1SEC7(BIG3):myc transgene complements the germination defect of gnl1 mutant seeds on agar plates containing 7μM BFA. Col-0, wild-type control; T/T, homozygous for transgene; T/-, hemizygous for transgene. Right panel: Expression of GNL1SEC7(BIG3):myc transgene rescues the stunted growth phenotype of gnl1 mutant plants. (B) Left panel: Expression of BIG3SEC7(GNL1):YFP transgene complements the germination defect of big3 mutant seeds on agar plates containing 5μM BFA. All BIG3SEC7(GNL1):YFP lines are hemizygous for the transgene. UBQ10::BIG3:YFP, positive control. Right panel: Phenotype of big3 mutant seedlings rescued by expression of BIG3SEC7(GNL1):YFP transgene compared to big3 mutant, wild-type (Col-0) control, and BIG3:YFP transgenic seedlings (positive control); on agar plates containing 5μM BFA. See also S6 Data.
Fig 8.
Interactions between ARFs and ARF-GEFs in various trafficking pathways in Arabidopsis (model).
Class-I ARFs (ARF1) are recruited to Golgi stack and TGN. Class-A ARFs (ARFA) are mainly present at TGN, a minor fraction localizing at the Golgi stack. Class-B ARF (ARFB) localized at PM and at TGN. GNOM and GNL1 activate class-I ARFs (ARF1) at the Golgi to carry out COPI-mediated Golgi-ER retrograde trafficking. Activation of class-I ARFs (ARF1) at the TGN by BIG1-4 occurs in secretory pathways to PM, cell plate and vacuole. ARF1 is also activated by BIG5 during endocytosis and by GNOM during recycling to the PM. In addition to ARF1, BIG5 and a subset of BIG1-4 could also activate ARFA (and possibly, ARFB), which plays no essential role in any trafficking pathway. AP1/CCV, adaptor protein complex 1/clathrin-coated vesicles; MVB, multivesicular body; PM, plasma membrane; RE, recycling endosomes; TGN, trans-Golgi network. The asterisk indicates localization of individual ARF GTPases whereas unmarked ARFs represent probable functional involvement in the respective transport steps.