Inhibition of ESCRT function inhibits vacuolar arrival of ubiquitin-modified reporters. CLSM and western-blot analysis of tobacco mesophyll protoplasts expressing the indicated plasmids. A-F: RFP-TMD23-Ub (A-C, red) or RFP-TMD20-Ub (D-F, green). and the soluble vacuolar marker aleurain-GFP (green) colocalize in the vacuole. (G) The vacuolar GFP core of GFP-sporamin (open arrowhead) and the cellular transit form (closed arrowhead) disappear when coexpressed with 10 μg plasmid DNA encoding for the dominant-negative mutant AtSKD1(AQ). (−) mock-transfected, (+) positive control. H-I: AtSKD1(AQ) expression inhibits vacuolar delivery of RFP-TMD23-Ub (red) and aleurain-GFP (green) (H) or RFP-TMD20-Ub (red) and aleurain-GFP (green) (I). All reporters accumulate in punctae (arrowheads) under these conditions.