Regeneration of a lytic central vacuole and of neutral peripheral vacuolescan be visualized by green fluorescent proteins targeted to either type ofvacuoles

Protein trafficking to two different types of vacuoles was investigated in tobacco (Nicotiana tabacum cv SR1) mesophyll protoplasts using two differen t vacuolar green fluorescent proteins (GFPs). One GFP is targeted to a pH-n eutral vacuole by the C-terminal vacuolar sorting determinant of tobacco ch itinase A, whereas the other GFP is targeted to an acidic lytic vacuole by the N-terminal propeptide of barley aleurain, which contains a sequence-spe cific vacuolar sorting determinant. The trafficking and final accumulation in the central vacuole (CV) or in smaller peripheral vacuoles differed for the two reporter proteins, depending on the cell type. Within 2 d, evacuola ted (mini-) protoplasts regenerate a large CV. Expression of the two vacuol ar GFPs in miniprotoplasts indicated that the newly formed CV was a lytic v acuole, whereas neutral vacuoles always remained peripheral. Only later, on ce the regeneration of the CV was completed, the content of peripheral stor age vacuoles could be seen to appear in the CV of a third of the cells, app arently by heterotypic fusion.