The Golgi Apparatus Plays a Significant Role in the Maintenance of Ca2+ Homeostasis in the vps33ΔVacuolar Biogenesis Mutant of Saccharomyces cerevisiae

Abstract

The vacuole is the major site of intracellular Ca 2؉ storage in yeast and functions to maintain cytosolic Ca 2؉ levels within a narrow physiological range. In this study, we examined how cellular Ca 2؉ homeostasis is maintained in a vps33⌬ vacuolar biogenesis mutant. We found that growth of the vps33⌬ strain was sensitive to high or low extracellular Ca 2؉ . This strain could not properly regulate cytosolic Ca 2؉ levels and was able to retain only a small fraction of its total cellular Ca 2؉ in a nonexchangeable intracellular pool. Surprisingly, the vps33⌬ strain contained more total cellular Ca 2؉ than the wild type strain. Because most cellular Ca 2؉ is normally found within the vacuole, this suggested that other intracellular compartments compensated for the reduced capacity to store Ca 2؉ within the vacuole of this strain. To test this hypothesis, we examined the contribution of the Golgi-localized Ca 2؉ ATPase Pmr1p in the maintenance of cellular Ca 2؉ homeostasis. We found that a vps33⌬/pmr1⌬ strain was hypersensitive to high extracellular Ca 2؉ . In addition, certain combinations of mutations effecting both vacuolar and Golgi Ca 2؉ transport resulted in synthetic lethality. These results indicate that the Golgi apparatus plays a significant role in maintaining Ca 2؉ homeostasis when vacuolar biogenesis is compromised.

The Ca2+ Homeostasis Defects in a pgm2Δ Strain of Saccharomyces cerevisiae Are Caused by Excessive Vacuolar Ca2+ Uptake Mediated by the Ca2+-ATPase Pmc1p

“…This indicates that the loss of Pmc1p function can largely suppress the UPR induction associated with the pgm2⌬ mutation. Given the well characterized role of Pmc1p as a vacuolar Ca 2ϩ transporter (3,16,39), these results suggest that this suppression of the UPR results from a specific reduction in Ca 2ϩ sequestration into the vacuole.…”

“…Measurement of total cell Ca 2ϩ by flame photometry was also carried out as previously described (3,29).…”

“…The cells were then harvested by centrifugation at 4000 ϫ g for 5 min, washed, and resuspended in fresh YPGal supplemented with 50 mM CaCl 2 . At the indicated times, aliquots were removed, filtered, washed, and processed for scintillation counting as previously described (3).…”

“…Ca 2ϩ transport across the plasma membrane and its intracellular sequestration is tightly regulated such that the resting cytosolic Ca 2ϩ concentration is maintained in a range of 50 -200 nM (1)(2)(3)(4). Small variations in cytosolic Ca 2ϩ that occur in response to a number of stimuli are sufficient to activate a variety of Ca 2ϩ -sensing proteins, such as calmodulin and calcineurin.…”

“…The transporters responsible for maintaining proper Ca 2ϩ levels in the secretory pathway include the Ca 2ϩ -ATPases Pmr1p (3,(13)(14)(15)(16) and Cod1p/Spf1p (17)(18)(19)(20). Pmr1p is localized primarily to the Golgi apparatus, where it plays an essential role in maintaining the lumenal Ca 2ϩ concentration required for the proper glycosylation and processing of proteins in this compartment (13,14).…”