Figure 3. Cna1^NIR-AAA Is Nonfunctional Due to a Defect in Substrate Interaction, and Cells Expressing cna1^NIR-AAA Show Reduced Activity of Crz1-PxIxIT Proteins.

(A) Serial dilutions of yeast strains lacking CN A (cna1 Δ cna2 Δ) and transformed with a plasmid expressing CNA1, cna1^NIR-AAA, or vector were spotted on the following growth media: YPD, YPD-pH 8.0 (high pH), YPD containing 4 mM MnCl₂, and YPD containing 500 mM NaCl. Growth was visualized after incubation at 30°C for 5 days (high pH) or 3 days (other media).

(B) Two-hybrid assay assessing the interactions of Cna1^NIR-AAA with its substrates Hph1, Slm1, and Slm2; the Rcn1 regulator; and the B subunit Cnb1. Serial dilutions of yeast strains were spotted on medium lacking tryptophan and leucine to select for both Gal4-fusion plasmids and on medium lacking histidine, as indicated, to assay for reporter activation. DBD, Gal4-DNA binding domain fusion of Cna1^NIR-AAA. AD, Gal4-activation domain fusions constructed in the plasmid pACT2 containing the indicated ORF. All plasmids were transformed into strain PJ69-4A. Cell growth was determined after 5 days incubation at 30°C.

(C) β-galactosidase activity of a cna1 Δ cna2 Δ crz1 Δ 4×-CDRE-lacZ strain transformed with plasmids expressing CNA1, cna1^NIR-AAA, or vector and cotransformed with plasmids expressing Crz1-PxIxIT proteins as indicated. CaCl₂ (100 mM) was added 1 hr prior to sampling, where indicated. Error bars indicate standard deviation (see Experimental Procedures for details).