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Fig. 5. Effects of the m-Rab_mc(N147I) mutant. A. thaliana protoplasts were transformed with either wild-type m-Rab_mc-CFP (A,F,H), the dominant negative mutant m-Rab_mc(N147I)-CFP (B,G,I), untagged wild-type m-Rab (C) or m-Rab_mc(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-Rab_mc 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-Rab_mc-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-Rab_mc(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-Rab_mc (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-Rab_mc-CFP staining (F, green) or cytosolic mutant m-Rab_mc(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-Rab_mc(N147I).

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