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doi: 10.1242/10.1242/jcs.00372

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Extracellular Ca²⁺ sensing contributes to excess Ca²⁺ accumulation and vacuolar fragmentation in a pmr1 mutant of S. cerevisiae

¹ Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
² Department of Clinical Chemistry, University Medical School, 7624 Peçs, Hungary

View larger version (35K): [in a new window]   Fig. 1. Pmc1p and calcineurin activity are required for growth of the pmr1 mutant in a medium containing a reduced level of divalent cations. The indicated strains were streaked on YPD plates containing the following supplements: (A) YPD alone, (B) YPD plus 1mM BAPTA or (C) YPD plus 1mM BAPTA and 10 µg/ml CsA.

View larger version (16K): [in a new window]   Fig. 2. Ca2+ accumulation and PMC1 transcription are induced when the pmr1 mutant is grown in a medium containing a reduced level of divalent cations. (A) Relative PMC1 mRNA levels in strains grown in the presence of different levels of divalent cations. (B) Total cell Ca2+ levels in strains grown in the presence of different levels of divalent cations.

View larger version (12K): [in a new window]   Fig. 3. Ca2+ uptake and accumulation are increased in the pmr1/pmc1 strain relative to the pmr1 strain in YPD medium. (A) Relative high-affinity 45Ca2+ uptake during a 30 minute time period. (B) Total cell Ca2+ levels measured by flame photometry.

View larger version (17K): [in a new window]   Fig. 4. VCX1 plays an important role in Ca2+ homeostasis in the pmc1 and pmr1/pmc1 strains. (A) Total cellular Ca2+ levels measured with flame photometry. (B) Cytosolic free Ca2+ levels measured with an aequorin Ca2+ reporter system. The arrow represents the addition of 100 mM CaCl2 (see Materials and Methods for further details).

View larger version (61K): [in a new window]   Fig. 5. Vacuoles are fragmented in the pmr1 and pmr1/pmc1 strains when grown in YPD medium. Vacuolar morphology was monitored using the vital fluorescent dye FM 4-64 or the Pmc1p-GFP fusion protein as indicated.

View larger version (110K): [in a new window]   Fig. 6. Vacuolar fragmentation correlates with cellular Ca2+ stress in the WT and pmr1 strains. Vacuolar morphology was monitored by fluorescence of a Pmc1p-GFP fusion. The WT strain (A) and pmr1 strain (B) were grown under the indicated environmental conditions.

View larger version (32K): [in a new window]   Fig. 7. Model showing how cellular Ca2+ uptake in yeast is coordinately regulated by two distinct mechanisms. The first mechanism is a CCE-like response that couples cellular Ca2+ uptake to Ca2+ store depletion in the ER/Golgi apparatus. The second mechanism couples cellular Ca2+ uptake to the level of Ca2+ in the extracellular environment.

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