Fig 1.

Activity of the Cch1 channel in C. neoformans is dependent on a C-terminal modulatory region of the subunit, Mid1. (A) Three truncated mutants of Mid1, each lacking a portion of the C terminus, were constructed. Mid1-CA-124 lacked 124 amino acids (aa), Mid1-CB-91 lacked 91 amino acids, and Mid1-CC-24 lacked 24 amino acids. Each mutant was constitutively expressed in the mid1Δ deletion background of C. neoformans under the control of the actin promoter (27). (B) Sensitivity spot assays were used to identify regions of the Mid1 protein critical for Cch1 activity. The Mid1-CC-24 truncated mutant did not rescue the sensitivity of the mid1Δ deletion strain on low, free [Ca²⁺] medium, suggesting that this region is critical for Cch1 activity. (C) A wild-type yeast (Saccharomyces cerevisiae) strain was transformed with full-length CnMid1. Two representative transformants expressing CnMid1 did not restore growth of the Scmid1Δ deletion strain on low, free [Ca²⁺] medium. (B and C) Serially diluted cells (10⁶, 10⁵, 10⁴, 10³, 10²) from wild-type and mid1Δ deletion strains of C. neoformans and yeast were added to YPD agar plates supplemented with BAPTA (cell impermeative), and spot assays were performed as explained above.