Sucrose transport across the vacuolar membrane of Ananas comosus

In Ananas comosus L. (Merr.) (pineapple), a widely cultivated bromeliad that exhibits crassulacean acid metabolism (CAM), much of the carbohydrate synthesized during the daytime appears to accumulate as soluble sugars in the vacuole. To investigate the mechanism of sugar transport into the vacuole, microsomal extracts were prepared from deacidifying leaves harvested during Phase III of the CAM cycle. The vesicle preparations exhibited features expected for a fraction highly enriched in vacuolar membranes (tonoplast), i.e. the ATPase activity of 16 +/- 2 nkat mg(-1) protein was inhibited 96% by 50 mM KNO3, an inhibitor of vacuolar ATPases, and was only 7% inhibited by 100 muM NaN3 plus 100 muM Na3VO4, inhibitors of mitochondrial and plasma membrane ATPases, respectively. Further, the microsomal ATPase activity showed a pH optimum between 7.0 and 8.0, typical of a vacuolar ATPase. When presented with Mg-ATP, vesicles established H+ gradients that could be maintained for at least 25 min. The vesicles were able to take up [C-14] sucrose from an external medium. Sucrose uptake exhibited saturable kinetics with an apparent K-m of 50 +/- 7 mM sucrose and apparent V-max of 171 +/- 5 pkat mg(-1) protein. Sucrose uptake was not dependent upon, nor stimulated by, Mg-ATP, suggesting that the mechanism of sucrose transport into the vacuole in A. comosus does not involve H+-coupled cotransport. However, the initial rates of sucrose uptake from the external medium were stimulated when vesicles were preloaded with sucrose. This trans-stimulation is consistent with characteristics expected for a sucrose uniporter capable of operating in an exchange mode. It is proposed that the accumulation of glucose and fructose in leaf vacuoles of Ananas during the light period involves at least two steps - transport of sucrose into the vacuole by a mechanism exhibiting characteristics of a sucrose uniporter, followed by cleavage of sucrose by a vacuolar acid invertase to form glucose and fructose.