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The role of vacuolar Ca2+ transport systems in regulating cellular Ca2+ was investigated by measuring the vacuolar Ca2+ transport rate, the free energy available to drive vacuolar Ca2+ transport, the ability of the vacuole to buffer lumenal Ca2+, and the vacuolar Ca2+ efflux rate. The magnitude of the Ca2+ gradient generated by the vacuolar H+gradient best supports a 1 Ca2+:2 H+ coupling ratio for the vacuolar Ca2+/H+ exchanger. This coupling ratio along with a cytosolic Ca2+ concentration of 125 nM would give a vacuolar free Ca2+ concentration of ~30 μM. The total vacuolar Ca2+ concentration is ~2 mM due to Ca2+ binding to vacuolar polyphosphate. The Ca2+ efflux rate form the vacuole is less than the growth rate indicating that the steady-state Ca2+ loading level of the vacuole is dependent mainly on the Ca2+ transport rate and the rate that vacuolar Ca2+ is diluted by growth. Based on the kinetic parameters of vacuolar Ca2+ accumulation in vitro, the maximum rate of Ca2+accumulation in vivo is expected to be ~0.2 nmol of Ca2+ min-1 mg protein-1, a rate that is similar to the cellular Ca2+ accumulation rate. The cytosolic Ca2+ concentration increases from 0.1 μM to 1-2 μM as the extracellular Ca2+ concentrarion is raised from 0.3 mM to 50 mM. The rise in cytosolic Ca2+ concentration increases cellular Ca2+ from 10 to 300 nmol Ca2+/mg by increasing the rate of vacuolar Ca2+ accumulation but does not significantly alter the cellular growth rate.