As it serves an important function in the compartmentalization of a series of components, from secondary metabolites to proteins, the vacuole is a central organelle in plant cell biology and development. One of the most important mechanisms regulating not only vacuolar biogenesis but also its luminal content, is the vacuolar sorting of proteins. This sorting mechanism is based upon the recognition of specific signal sequences, vacuolar sorting determinants, by sorting receptors, which then act by redirecting vacuolar cargo away from the default secretory pathway, and into the vacuolar lumen. One of the most direct ways of understanding if a given peptide possesses vacuolar sorting determinant capability is to isolate it, fuse it with a marker and express it in a plant system such as Nicotiana tabacum, a pipeline which will be described in more detail in this chapter.
Keywords: Endomembrane system; Fluorescent markers; Nicotiana tabacum; Trafficking; Transient expression; Vacuolar sorting determinants; Vacuole.