FIGURE 1.

Functionality of co-expressed CAX half proteins in yeast. A, suppression of Ca²⁺ sensitivity in K667 yeast through co-expression of various combinations of sCAX1 N-terminal half protein (N-sCAX1), sCAX3 N-terminal half protein (N-sCAX3), CAX1 C-terminal half protein (C-CAX1), and CAX3 C-terminal half protein (C-CAX3), plus epitope-tagged HA-N-sCAX1+C-CAX3-GFP. Saturated liquid cultures of K667 yeast cells were diluted in stepwise 5-fold dilutions and spotted onto selection medium and YPD medium containing either 100 mm or 200 mm CaCl₂. Yeast cells expressing sCAX1 and empty vector were used as controls. The plates were incubated and photographed after 2 days at 30 °C. B, time course of ⁴⁵Ca²⁺ uptake into endomembrane vesicles prepared from K667 yeast co-expressing sCAX1+empty vector, N-sCAX1+C-CAX1, N-sCAX1+C-CAX3, and empty vector alone. Solid circle, pH-dependent ⁴⁵Ca²⁺ uptake; open square, uptake in the presence of the protonophore gramicidin. The Ca²⁺ ionophore A23187 (5 μm) was added at 12 min. The reduction in Ca²⁺ uptake following A23187 addition demonstrated that accumulation into the vesicles occurred. The data represent the means ±S.E. of three independent experiments.