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First published online 23 November 2004
doi: 10.1242/jcs.01564

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AtRabF2b (Ara7) acts on the vacuolar trafficking pathway in tobacco leaf epidermal cells

¹ Research School of Biological and Molecular Sciences, Oxford Brookes University, Gipsy Lane, Oxford, OX3 0BP, UK
² Department of Biology, University of Saskatchewan, S7N 5E2, Canada
³ UMR-CNRS 6037, Université de Rouen, 76821 Mont Saint Aignan, France
⁴ Department of Plant Sciences, University of Oxford, South Parks Road, OX1 3RB, UK

View larger version (77K): [in a new window]   Fig. 1. Mutations of AtRabF2b relocalize the chimaera in tobacco leaf epidermal cells. Confocal images 2 days after Agrobacterium inoculation with the indicated mutants at OD600 0.03. (A) YFP-AtRabF2b[wt]. (B) YFP-AtRabF2b[S24N]. (C) GFP-AtRabF2b[Q69L]. (D) GFP-AtRabF2b[wt] (green) and ST-YFP (red). (E) High magnification image of the box in D. Two populations of GFP-AtRabF2b[wt] structures are present: punctate structures that do not colocalize with ST-YFP (arrow) and punctate structures that do (arrowhead). Bar,. (F) GFP-AtRabF2b[S24N] (green), ST-YFP (red). Colocalization is shown in the merged image. (G) GFP-AtRabF2b[Q69L] (green image), ST-YFP (red). GFP-AtRabF2b[Q69L]-positive spherical structures do not colocalize with ST-YFP (arrow, merged image and insert). (H) YFP-AtRabF2b[Q69L] (red) and BobTIP26-1::GFP (green) colocalize with BobTIP26-1::GFP on the tonoplast (arrow, merged image and insert). (I) Cytosolic labelling of YFP-AtRabF2b[N121I]. (J) Cytosolic labelling of YFP-AtRabF2b[C198S,199S]. Bar, 10 µm (A-D and F-J); 5 µm (E); 2 µm (inset G).

View larger version (68K): [in a new window]   Fig. 2. Western blot of AtRabF2b[wt] protein and chimaeras. Proteins were extracted from leaf material at 2 or 4 days after Agrobacterium-mediated transformation. An antibody raised against GFP was used to detect the AtRabF2b fusion protein. (A) Lane 1, protein standard markers; lanes 2-3, AtRabF2b[wt] day 2 and day 4; lanes 4-5, AtRabF2b[S24N] day 2 and day 4; lanes 6-7, AtRabF2b[Q69L] day 2 and day 4; lanes 8-9, AtRabF2b[N123I] day 2 and day 4; lanes 10-11, AtRabF2b[C198,199S] day 2 and day 4; lanes 12-13, PS1-GFP day 2 and day 4; lane 14, untransformed control. (B) Coomassie Blue-stained gel showing equal loading of protein (lanes as above).

View larger version (87K): [in a new window]   Fig. 3. AtRabF2b-tagged chimaeras affect the normal distribution of the pre-vacuolar marker, PS1-GFP. Confocal images of tobacco leaf epidermal cells, 2 days after Agrobacterium inoculation with PS1-GFP (OD600 0.08) and AtRabF2b (OD600 0.03). (A) PS1-GFP (green) and ST-YFP (red). The majority of PS1-GFP does not colocalize with ST-YFP (see inset taken from tangential section of the cell). (B) PS1-GFP (green) and YFP-AtRabF2b[wt] (red) form large aggregates (arrow, merged image). (C) PS1-GFP (green) and YFP-AtRabF2b[S24N] (red) do not form large aggregates (merged image). (D) PS1-GFP (green) and YFP-AtRabF2b[Q69L] (red) form large aggregates (arrow, merged image). (E) PS1-GFP (green) and YFP-AtRabF2b[N123I] (red) do not form large aggregates (merged image). (F) High magnification image of a cell expressing PS1-GFP (green) and YFP-AtRabF2b[wt] (red), showing the nature of the aggregates. (G) High magnification of image in C. Golgi labelling of YFP-AtRabF2b[S24N] colocalizes with a population of PS1-GFP (arrowhead), also present is a population of PS1-GFP that do not colocalize with YFP-AtRabF2b[S24N] (arrow). Bar, 10 µm (A-E); 5 µm (F,G).

View larger version (129K): [in a new window]   Fig. 4. Untagged AtRabF2b chimaeras also affect the normal distribution of the pre-vacuolar marker, PS1-GFP. Confocal images of tobacco leaf epidermal cells, 2 days after Agrobacterium inoculation with PS1-GFP (OD600 0.08) and untagged-AtRabF2b (OD600 0.03). (A) Representative cell transformed with PS1-GFP and untagged AtRabF2b[wt]. Large aggregates are present in the absence of the fluorescent tag (arrow). (B) Representative cell transformed with PS1-GFP and untagged AtRabF2b[Q69L]. Again, large aggregates containing PS1-GFP are present (arrow). Bar, 10 µm.

View larger version (90K): [in a new window]   Fig. 5. AtRabF2b localizes to the aleu-GFP-labelled PVC. Confocal images of tobacco leaf epidermal cells, 2 days after Agrobacterium inoculation with aleu-GFP (OD600 0.1) and AtRabF2b (OD600 0.03). (A) Aleu-GFP faintly fluoresces in the vacuole and also labels small punctate structures. (B) Aleu-GFP (green) and PS1-YFP (red). Aleu-GFP localizes to the PVC compartment also labelled with PS1-YFP (arrow). (C) Aleu-GFP (green), YFP-AtRabF2b[wt]-labelled structures colocalize with PVC-containing aleurain (arrow). Faint YFP-AtRabF2b[wt] structures are still visible (arrowhead). (D) Aleu-GFP (green) and YFP-AtRabF2b[S24N] (red). Bright, punctate aleurain structures do not colocalize with AtRabF2b[S24N] (arrow). (E) Aleu-GFP (green) and YFP-AtRabF2b[Q69L] (red). Aleu-GFP accumulates in AtRabF2b[Q69L] spheres (insert, merged image). Insert image was taken from an additional cell, which clearly showed aleu-GFP accumulating within the spheres. (F) Aleu-GFP (green) and YFP-AtRabF2b[N123I] (red). Aleurain-positive pre-vacuoles are not affected by the presence of YFP-AtRabF2b[N123I]. Bar, 10 µm (A-F); 2 µm in insert (E).

View larger version (140K): [in a new window]   Fig. 6. Untagged dominant-negative mutant causes secretion of vacuole-targeted aleu-GFP, which can be rescued by AtRabF2b[wt]. Confocal images of tobacco leaf epidermal cells, 48-52 hours after Agrobacterium inoculation with aleu-GFP (OD600 0.1) and untagged-AtRabF2b and AtRabD2a (OD600 0.05). (A) With aleu-GFP alone, faint fluorescence is visible in some vacuoles (arrow). (B) In cells expressing aleu-GFP and AtRabF2b[wt], the fluorescent pattern of aleu-GFP is not affected. (C) With aleu-GFP and AtRabF2b[S24N], location of aleu-GFP is drastically altered by the dominant-negative mutant as fluorescence is clearly visible in the apoplast (arrow). (D) With aleu-GFP, AtRabF2b[S24N] and AtRabF2b[wt], AtRabF2b[wt] has rescued the effect of the dominant-negative mutant on the localization of aleu-GFP. Very few cells secreting GFP to the apoplastic space are present. (E) Cells expressing aleu-GFP, AtRabF2b[S24N] and AtRabD2a; expression of another Rab protein does not rescue the phenotype of aleu-GFP induced by AtRabF2b[S24N] as seen with AtRabF2b[wt]. Bar, 50 µm. (F) Analysis of cells with GFP visible in the apoplast. Data represent mean±s.d. of three independent experiments (eight fields of view per construct) of cells secreting to the apoplast. AL, aleu-GFP; WT, untagged AtRabF2b[wt]; SN, untagged AtRabF2b[S24N]; D2aWT, untagged AtRabD2a[wt].

View larger version (143K): [in a new window]   Fig. 7. Effect of fluorescent-tagged AtRabF2b on trafficking of aleu-GFP. Confocal images of tobacco leaf epidermal cells, 48-52 hours after Agrobacterium inoculation with aleu-GFP (OD600 0.1) and tagged-AtRabF2b (OD600 0.05). (A) With aleu-GFP alone, faint fluorescence is visible in some vacuoles. (B) Aleu-GFP and untagged AtRabF2b[S24N]. (C) Untransformed leaf. (D) Aleu-GFP (green) and YFP-AtRabF2b[wt] (red) expression, showing aleu-GFP is secreted to the apoplast. (E) Aleu-GFP (green) and YFP-AtRabF2b[S24N] (red) expression; aleu-GFP is secreted at similar levels to D. (F) aleu-GFP(green) and YFP-AtRabF2b[C198, 199S] (red) expression; aleu-GFP is not secreted to the apoplast. Bar, 50 µm.

View larger version (28K): [in a new window]   Fig. 8. Location and putative function of AtRabF2b in tobacco leaf epidermal cells. The location of AtRabF2b[wt] on the Golgi apparatus, PVC, and the effect of the dominant-negative mutant on the vacuole targeted protein aleurain-GFP suggest its role in GA to PVC trafficking. The tagged GDP locked mutant AtRabF2b[S24N] localizes entirely to the GA whereas the tagged-GTP locked mutant AtRabF2b[Q69L] localizes to spherical structures at the PVC and also to the tonoplast. (1) Aleu-GFP trafficks through the PVC en route to the vacuole. (2) When untagged AtRAbF2b[S24N] is coexpressed with aleu-GFP, a proportion of the aleu-GFP is secreted to the apoplast. Tagged AtRabF2b[C198,199S] and tagged AtRabF2b[N123I] are entirely cytosolic. aleu-GFP, aleurain-GFP; GA, Golgi apparatus; PVC, prevacuole compartment; V, vacuole.