Roles of the N- and C-Termini in the Function of the Yvc1p Saccharomyces Cerevisiae TRP Channel

Abstract

In the budding yeast Saccharomyces cerevisiae, cytoplasic calcium levels increase in response to hyperosmotic stress. The Yvc1p TRP channel is responsible for for this response: stress activates Yvc1p, and the channel releases Ca2+ from the vacuolar storage compartment into the cytoplasm. Changes in cytoplasmic Ca2+ concentrations in Saccharomyces are easily observed by monitoring the luminescence of cytoplasmic aequorin (Batiza et al, 1996, JBC). We have explored the utility of this assay for examining Yvc1p function, and have used the assay to study the consequence of truncation mutations of Yvc1p.From the N-terminus, the deletion constructs sequentially remove residues from the start up to the first putative transmembrane domain. We find that the first 30 amino acids are not essential for basic Yvc1p function and response to hyperosmotic shock. At the C-terminus, the panel of deletion constructs spans the segment after TM6 through the last residue. Here we find a more complicated pattern, but Yvc1p function, as judged by aequorin luminescence, is clearly adversely affected by deletions of more than the last 12 residues.