Regulation of cellular Ca²⁺ by yeast vacuoles

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 from 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 UM as the extracellular Ca²⁺ concentration 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.