| Title: | "Identification of functional domains of Mid1, a stretch-activated channel component, necessary for localization to the plasma membrane and Ca2+ permeation" |
| Author(s): | Ozeki-Miyawaki C; Moriya Y; Tatsumi H; Iida H; Sokabe M; |
| Address: | "Department of Physiology, Nagoya University School of Medicine, Nagoya, Aichi 466-8550, Japan" |
| DOI: | 10.1016/j.yexcr.2005.08.014 |
| ISSN/ISBN: | 0014-4827 (Print) 0014-4827 (Linking) |
| Abstract: | "The Saccharomyces cerevisiae MID1 gene product (Mid1) is a stretch-activated Ca(2+)-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 H1 to H3 (Mid1(1-360)-GFP) localized normally in the plasma and endoplasmic reticulum (ER) membranes and complemented the low viability and Ca(2+)-uptake activity of the mid1 mutant, whereas Mid1(1-133)-GFP containing the region from H1 to H2 did not. Mid1(Delta3-22)-GFP lacking the H1 region failed to localize in the plasma membrane. Membrane fractionation showed that Mid1(1-22)-GFP containing only H1 localized in the plasma membrane in the presence of alpha-factor, suggesting that H1 is a signal sequence responsible for the alpha-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" |
| Keywords: | Animals CHO Cells Calcium/*metabolism Cell Membrane/*metabolism Cricetinae Endoplasmic Reticulum/metabolism Genetic Complementation Test Green Fluorescent Proteins/genetics/metabolism Immunoblotting Membrane Glycoproteins/chemistry/*physiology Protein Str; |
| Notes: | "MedlineOzeki-Miyawaki, Chikako Moriya, Yoshie Tatsumi, Hitoshi Iida, Hidetoshi Sokabe, Masahiro eng 2005/10/06 Exp Cell Res. 2005 Nov 15; 311(1):84-95. doi: 10.1016/j.yexcr.2005.08.014. Epub 2005 Oct 3" |
