029

A non-vacuolar compartment maintains Ca ²⁺ homeostasis in a vacuolar biogenesis mutant of S. cerevisiae .

The yeast vacuole is the major site of intracellular Ca ²⁺ storage, and thus plays a key role in maintaining cytosolic Ca ²⁺ levels within a narrow physiological range. In the current study, we compared the Ca ²⁺ storage defects of a pmc1/vcx1 strain (which lacks the two known vacuolar Ca ²⁺ transporters) with a vps33 strain, which contains a severe (class C) defect in vacuolar biogenesis. We found that the vps33 strain is much more sensitive to either high or low extracellular Ca ²⁺ concentrations than a pmc1/vcx1 strain, suggesting that the vacuole has additional capacity for Ca ²⁺ uptake besides that provided by the PMC1 and VCX1 gene products. While the total cellular Ca ²⁺ level found in the pmc1/vcx1 strain was 2-3 fold lower than the wild type strain, the total cellular Ca ²⁺ in the vps33 strain or the vps33/pmc1/vcx1 strain was actually higher than the wild type strain. Most cellular Ca ²⁺ in the vps33 and vps33/pmc1/vcx1 strains was located in a readily exchangeable pool, consistent with its location in a compartment other than the vacuole. Experiments using an aequorin construct to monitor transient changes in the cytosolic concentration of Ca ²⁺ showed that both the vps33 and vps33/pmc1/vcx1 strains were more severely compromised in their ability to control cytosolic Ca ²⁺ levels upon exposure to high extracellular Ca ²⁺ than the pmc1/vcx1 strain. Our results indicate that Ca ²⁺ storage in other organelle(s) can maintain Ca ²⁺ homeostasis in the absence of vacuolar function.

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