Immunological characterization of two dominant tonoplast polypeptides.

At least 14 distinct polypeptides reside in the tonoplast of barley (Hordeum vulgare) mesophyll vacuoles. Two of the polypeptides were isolated from two-dimensional separations of vacuoplast membrane proteins and used for immunization. With the antisera, the localization on the membrane and the distribution of the polypeptides in the plant kingdom and in various tissues of barley plants was studied. The polypeptides have an apparent molecular mass of 31 and 40 kilodaltons. After freeze-thaw cycles or washing of the membranes with 4.5 millimolar NaCl, the polypeptides were still sedimented with the membranes, suggesting an intrinsic localization. The antiserum against the 31-kilodalton polypeptide bound to the outer surface of isolated intact vacuoles. In chromatographic separations of Triton X-100-solubilized membrane fractions, the residual activities of various acid hydrolases eluted distinct from the 31- and 40-kilodalton polypeptides. Both polypeptides tend to form larger aggregates, however smaller than the tonoplast ATPase. Cross-reactive polypeptides were present in higher and lower plants (the green alga Chara corallina and the liverwort Conocephalum) and in liver tissue from rat and beef, but were not detected in other animal tissues tested so far. The results indicate a wide distribution of these tonoplast polypeptides in vacuole-containing organisms.