Ubiquitin-dependent transport of an integral PM protein to the vacuole. A-B: Schematic representation of the constructs RFP-TMD23-Ub (A) and RFP-TMD23 (B). Localization of the constructs in tobacco mesophyll protoplasts after 36 h (C-E, to allow for vacuolar accumulation) or 24 h (I-N, for colocalization analysis) and leaf epidermal cells (F-H). C: RFP-TMD23-Ub is targeted into the vacuolar lumen. (D) Isolated vacuole. (E) RFP-TMD23 localizes to the PM. F-H: Localization of the reporters given above in planta, confirming the results from C-E. I-K: RFP-TMD23-Ub (red) and YFP-SYP61 (green) partially colocalize (arrowheads). L-N: RFP-TMD23-Ub (red) and MVB/LE marker GFP-BP80 (green) overlap in punctate structures (arrowheads). RFP-TMD23-Ub localizes to ring-like MVB structures after wortmannin-treatment (+WM; inset in L). Scale bars = 5 μm. For quantitative analysis of colocalization see Additional file 2.