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First published online 3 February 2004
doi: 10.1242/jcs.00920

Journal of Cell Science 117, 943-954 (2004)
Published by The Company of Biologists 2004
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The N-myristoylated Rab-GTPase m-Rabmc is involved in post-Golgi trafficking events to the lytic vacuole in plant cells

Susanne Bolte*, Spencer Brown and Béatrice Satiat-Jeunemaitre

Laboratoire de Dynamique de la Compartimentation Cellulaire, Institut des Sciences du Végétal, CNRS, UPR2355, Avenue de la Terrasse, Bâtiment 23-24, 91198 Gif-sur-Yvette Cedex, France



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  		Fig. 1. Characterisation of the m-Rabmc antiserum by two-dimensional gel-electrophoresis. (A) Western blot of Mesembryanthemum crystallinum protein extracts incubated with the m-Rabmc antiserum. The arrow indicates the single polypeptide recognized by the m-Rabmc antiserum, with a molecular mass of approximately 21 kDa and an isoelectric point near pH 7. (B) Silver-stained gel of the western blot in A. The arrow indicates the putative position of the m-Rabmc-labelled polypeptide. (C) Western blots of protein extracts of M. crystallinum leaf extracts (a), BY-2 cells (b), A. thaliana suspension cells (c) and tobacco leaves (d). m-Rabmc antiserum stains a single band of approximately 21 kDa.

 


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  		Fig. 2. Confocal microscopy of immunofluorescence staining with m-Rabmc antiserum in various plant tissues. m-Rabmc antiserum reveals a punctuate pattern throughout the cytoplasm, some of the structures being typically organised in a ring-like pattern (arrows, also see insets). (A) m-Rabmc labelling of upper leaf epidermal cells of M. crystallinum. Single optical section of the upper epidermal layer with stomatal guard cells (s) surrounded by pavement cells. (B) M. crystallinum root cell projected from 15 single optical sections of 0.5 µm. (C) Image stack of 20 optical sections of 0.5 µm of a BY-2 cell. n, nucleus. Scale bars: 5 µm.

 


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  		Fig. 3. Co-localisation of m-Rabmc with markers for the prevacuolar compartment and Golgi apparatus in BY-2 cells and A. thaliana protoplasts. Antibody staining was done in BY-2 cells using FITC-labelled or Cy3-labelled secondary antibodies and in A. thaliana protoplasts expressing sialyl transferase-YFP (ST-YFP) using an Alexa568-labelled secondary antibody. Co-expression of m-Rabmc-CFP and ST-YFP was performed in A. thaliana protoplasts. Images were colour-coded using Adobe Photoshop. Confocal images represent single images of BY-2 cells or Arabidopsis protoplasts as stated. Scale bars: 5 µm. (A-C) m-Rabmc-Cy3/BP80-FITC dual labelling of a BY-2 cell. (A) Single image of a cell stained with m-Rabmc antiserum. (B) Prevacuolar staining by BP80 of the same cell. (C) Merged image of the dual labelling reveals an almost complete co-localisation of the two proteins (yellow). (D-F) m-Rabmc-Cy3/Pep12-FITC dual labelling of a BY-2 cell. (D) Single image of a cell stained with m-Rabmc antiserum. (E) The same cell stained with Pep12. (F) Merged image shows a high level of co-localisation of the two proteins (yellow). (G-I) Single image of a BY2 cell showing m-Rabmc-FITC/JIM84-Cy3 dual labelling. (G) m-Rabmc-FITC; (H) Golgi-marker JIM84-Cy3; (I) merged image of the dual labelling. Note co-localisation (yellow arrowheads, inset). (J-L) Single image of an A. thaliana protoplast co-transfected with m-Rabmc-CFP and the trans-Golgi marker ST-YFP. (J) m-Rabmc CFP staining; (K) ST-YFP staining; (L) the merged image shows the single labelling of m-Rabmc-stained prevacuoles (green arrowheads), ST-YFP-stained GA (red arrowheads) and co-localisation of the two proteins (yellow arrowheads). (M-O) Single image of a fixed protoplast expressing ST-YFP and labelled with m-Rabmc antiserum. (M) m-Rabmc staining; (N) ST-YFP staining; (O) the merged image shows the single labelling of m-Rabmc-stained prevacuoles (green arrowheads) and ST-YFP-labelled GA (red arrowhead) and co-localisation of both proteins on some Golgi structures (yellow arrowheads).

 


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  		Fig. 4. Co-localisation of the Ca2+-ATPase ACA4 and m-Rabmc with the PVC and the GA. Secondary antibodies used were as described in Fig. 3. Images were colour-coded with Adobe Photoshop. Single confocal images of isolated BY-2 cells. Scale bars: 5 µm. (A-C) Single image of a cell co-labelled with ACA4-Cy3/BP80-FITC. (A) ACA4 staining; (B) BP80 staining; (C) merged image reveals an almost complete co-localisation of ACA4 and BP80 (yellow). (D-F) Single image of a cell co-labelled with ACA4-FITC/JIM84-Cy3. (D) ACA4 labelling; (E) labelling of the GA recognised by JIM84; (F) merged image of the dual labelling. Note some co-localisation (yellow arrowheads, inset). (G-I) Image stack of a single cell labelled with ACA4-FITC/m-Rabmc-Cy3. The secondary Cy3-coupled antibody was a F(ab')2 fragment. (G) ACA4-labelling; (H) m-Rabmc labelling; (I) merged images. Note the almost complete co-localisation of ACA4 and m-Rabmc (yellow).

 


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  		Fig. 5. Effects of the m-Rabmc(N147I) mutant. A. thaliana protoplasts were transformed with either wild-type m-Rabmc-CFP (A,F,H), the dominant negative mutant m-Rabmc(N147I)-CFP (B,G,I), untagged wild-type m-Rab (C) or m-Rabmc(N147I) (D,E). Fluorescent markers for the different cellular destinations were pseudocoloured in red (aleurain-GFP, chitinase-YFP, FM4-64) with the exception of ST-YFP (C-E, green). Expression of the wild-type and mutant m-Rabmc protein was assessed by monitoring the CFP fluorescence (A,B and F-I). Confocal analysis was performed 16 hours after transformation. Scale bars: 5 µm. The white arrowhead indicates a ring-like structure. Lv, lytic vacuole. (A,B) Wild-type m-Rabmc-CFP punctuate pattern (A, green) and aleurain-GFP vacuolar signal (A, red) in A. thaliana protoplasts. The expression pattern of the dominant negative mutant m-Rabmc(N147I)-CFP is cytosolic (B, green) and causes a punctuate aleurain-GFP pattern (B, red). (C-E) ST-YFP-labelled Golgi stacks (green) and aleurain-GFP labelling (red) in protoplasts expressing wild-type m-Rabmc (C) or mutant-expressing protoplasts (D,E). (D) Top view of a cell and (E) an image taken from the middle of the cell. Note the lack of aleurain-GFP staining in the lytic vacuole in the mutant-expressing protoplasts (D,E). (F,G) Wild-type punctuate m-Rabmc-CFP staining (F, green) or cytosolic mutant m-Rabmc(N147I)-CFP pattern (G, green) and chitinase-YFP vacuolar pattern (F,G, red). The mutant has no effect on the transport of chitinase-YFP. (H,I) Internalisation of FM4-64 (H,I, red) in sub-micron structures in wild-type (H, green) or mutant-expressing protoplasts (I, green). The staining pattern is not affected by the expression of the mutant m-Rabmc(N147I).

 


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  		Fig. 6. Location and putative function of m-Rabmc in plant cells. The compartment marked by BP80, m-Rabmc and the calcium ATPase ACA4 is named the secretory prevacuolar compartment (PVCs). The location of m-Rabmc on the PVC, its partial association with the GA and its function in aleurain transport to the lytic vacuole suggest an involvement in GA/PVC trafficking. m-Rabmc might regulate the transport of vacuolar proteins from the GA to the PVC (1) that in turn would fuse with the lytic vacuole (LV) to deliver cargo and presumably tonoplast proteins (2). Dotted arrows with open arrowheads indicate a putative recycling event of m-Rabmc between the GA and the prevacuolar compartment. Ara6, the A. thaliana homologue of m-Rabmc, has been assigned to FM4-64-labelled sub-micron structures called endosomes (Ueda et al., 2001Go) (3). These structures are named endocytic prevacuolar compartments (PVCe) in this model. The broken double-headed arrow respects the possibility that the PVCs and PVCe may be the same compartment.

 

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© The Company of Biologists Ltd 2004