8] Isolation of vacuoles and tonoplast from protoplasts

Abstract

This chapter describes the polybascinduced lysis of protoplasts originally designed for yeast and modified for plant vacuoles. When vacuoles are released from protoplasts, whatever the specific procedure used, certain principles must be observed: (1) rapid protoplast isolation procedure to avoid prolonged exposure to wall-degrading enzymes, (2) gentle lysis of the protoplasts in order to avoid rupture of the released vacuoles, (3) efficient lysis in order to limit the occurrence of residual protoplasts, which exhibit similar size and density to the vacuoles and are very difficult to discard by centrifugation methods, and (4) rapid vacuole isolation procedure and limitation of the purification steps in order to reduce bursting of the released vacuoles and leakage of solutes through the tonoplast. The isolation of the tonoplast from vacuoles is a small-scale procedure for the following reasons: (1) the isolation of vacuoles from protoplasts cannot be extended to a very large number of vacuoles and (2) the tonoplast represents a small proportion of the total membrane fraction of the cell.