Isolation of Tonoplast Vesicles from Tobacco Protoplasts

Vacuoles were isolated from protoplasts of Nicotiana glutinosa by the method of Mettler and Leonard (Plant Physiol 1979 64: 1114-1120) with minor modifications so that the number of intact protoplasts contaminating the vacuole preparation was reduced to less than 1% (by number). Isopycnic centrifugation of a [(3)H]choline-labeled, sonicated vacuole preparation on linear 5 to 40% sucrose gradients indicated that tonoplast vesicles equilibrated at a density of about 1.12 grams per cubic centimeter. When tonoplast vesicles were isolated on discontinuous sucrose density gradients substrate specific ATPase activity was not found to be associated with this membrane fraction. These results are discussed in terms of the energetics of ion transport through the tonoplast membrane.