Localization of RNA-degrading enzyme activity within vacuoles of cultured tomato cells

Protoplasts were prepared from cells of a tomato ( Lycopersicon esculentum Mill. cv. Lukullus) suspension culture and purified to eliminate the highly active exogenous RNase present in the enzyme mixture used for cell wall digestion. The purified protoplasts were used to determine the location of the endogenous RNase activity (measured at pH 5 with yeast RNA as the substrate). Vacuoles were released by shaking the purified protoplasts in alkaline buffer containing EDTA. RNase was unambiguously shown to be located within the vacuoles by (i) its co-purification with the vacuoles in a discontinuous gradient and by (ii) the co-migration of RNase and α-mannosidase (EC 3.2.1.24), a vacuolar marker, during repeated centrifugation of the vacuoles. Vacuolar RNase was insensitive to EDTA, Mg²⁺, Mn²⁺ and Ca²⁺ but was stimulated by citrate or KH₂PO₄. It exhibited a pH-optimum in the range of pH 5–6. Gel electrophoretic analysis revealed one single band for RNase of isolated vacuoles. This activity co-migrated with an RNase from cells extracted under mild conditions. Thus, it was possible to distinguish the vacuolar RNase from the RNase of extracellular origin. RNA-degrading activity was present in vacuoles throughout the growth of the culture. The activity in vacuoles gradually increased during exponential growth followed by a dramatic increase in the stationary phase.