Figure 2

Gdt1p is involved in the calcium response to saline stress.

(A) Wild type (WT) or various pmr1 and/or gdt1 yeast mutants expressing apo-aequorin from a plasmid were grown overnight to an OD₆₀₀ of 1.2 in synthetic medium supplemented with coelenterazine (chromophore) to reconstitute the holoenzyme. Afterwards, 200 μL of each culture was transferred to luminometric tubes. After 2 min, NaCl (saline stress) was added at a final concentration of 1.33 M and the signal monitored for 20 min, then the lumimetric units were converted into the [Ca²⁺]_cyt using the equation from Allen et al.⁴². All displayed results are representative of those obtained in at least three replicates. (B) Proteins from the membrane-enriched fractions of exponentially growing cells (OD₆₀₀ = 1.2) of the indicated strains were separated by SDS-PAGE and transferred to nitrocellulose membranes, which were then immunoblotted with antibodies against Gdt1p, Pmc1p, Pmr1p, Vcx1p, or Yvc1p. Coomassie blue-staining of the SDS-polyacrylamide gel indicated equal sample loading (Fig S3). (C) Cultures of the indicated strains were grown in synthetic medium to an OD₆₀₀ of 3, then the cellular Ca²⁺ content was measured by ICP-AES on the dry matter. The data were analyzed by one-way analysis of variance (ANOVA) followed by a post-hoc Tukey-Kramer multiple comparisons test. The values are expressed as the mean ± S.E.M (n = 3). Letters not shared in two bars denote a significant difference (p < 0.05). The pmr1Δ+ GDT1 strain corresponds to the pmr1Δ mutant overexpressing GDT1 under the control of the constitutive TPI1 promoter. Extracellular Ca²⁺ concentration for those three experiments was assessed by ICP-AES to be around 1 mM.