Identification of functional domains of Mid1, a stretch-activated channel component, necessary for localization to the plasma membrane and Ca2+ permeation

The Saccharomyces cerevisiae MIDI gene product (Mid1) is a stretch-activated Ca2+-permeable channel component required for Ca2+ influx and the maintenance of viability of cells exposed to the mating pheromone, alpha-factor. It is composed of 548-amino-acid (aa) residues with four hydrophobic segments, H1 (aa 2-22), H2 (aa 92-111), H3 (aa 337-356) and H4 (aa 366-388). It also has 16 putative N-glycosylation sites. In this study, sequentially truncated Mid1 proteins conjugated with GFP were expressed in S. cerevisiae cells. The truncated protein containing the region from H I to H3 (Mid(1-360)-GFP) localized normally in the plasma and endoplasmic reticulum (ER) membranes and complemented the low viability and Ca2+-uptake activity of the mid1 mutant, whereas Mid(1-133)-GFP containing the region from H1 to H2 did not. Mid1(Delta 3-22)-GFP lacking the H I region failed to localize in the plasma membrane. Membrane fractionation showed that Mid1(1-22)-GFP containing only H I localized in the plasma membrane in the presence of a-factor, suggesting that H I is a signal sequence responsible for the a-factor-induced Mid1 delivery to the plasma membrane. The region from H1 to H3 is required for the localization of Mid1 in the plasma and ER membranes. Finally, trafficking of Mid1-GFP to the plasma membrane was dependent on the N-glycosylation of Mid1 and the transporter protein Sec12. (c) 2005 Elsevier Inc. All rights reserved.