Elongation Factor 3, EF3, Associates with the Calcium Channel Cch1 and Targets Cch1 to the Plasma Membrane in Cryptococcus neoformans

Abstract

ABSTRACT Ca 2+ -mediated signaling events in eukaryotic cells are initiated by Ca 2+ channels located in the plasma membranes and endomembranes. Cch1, a high-affinity Ca 2+ channel in the plasma membranes of Cryptococcus neoformans and other fungi, plays a role in many different cellular processes, but the mechanisms that regulate Cch1 are not well understood. A Ras recruitment two-hybrid screen was used to identify protein partners of Cch1 as a means of identifying possible mechanisms of channel regulation. Here, we show that Cch1 specifically associates with a cytoplasmic protein known as elongation factor 3 (EF3). The robust interaction between the cytosolic C terminus of the Cch1 protein and EF3 shown here was confirmed by demonstrating that Cch1 could coimmunoprecipitate with EF3 in yeast lysates. To examine the effects of EF3 on Cch1 behavior, we altered the EF3 gene function by constructing a C. neoformans antisense EF3 repression strain. Our results show that the repression of EF3 led to the mislocalization of Cch1, suggesting a role for EF3 in targeting Cch1 to the plasma membrane of C. neoformans . Consistent with this notion, the antisense EF3 repression strain displayed a growth defect under conditions of limited extracellular Ca 2+ . Collectively, these results suggest that EF3 and Cch1 are functionally coupled and that EF3 has a function apart from its role in the protein translation cycle.