Identification of functional domains of Mid1, a stretch-activated channel component, necessary for localization to the plasma membrane and Ca2+ permeation.
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.
Ozeki-Miyawaki
Chikako
C
Department of Physiology, Nagoya University School of Medicine, Nagoya, Aichi 466-8550, Japan.
Moriya
Yoshie
Y
Tatsumi
Hitoshi
H
Iida
Hidetoshi
H
Sokabe
Masahiro
M
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000502:physiology*; D017434:Protein Structure, Tertiary; D018000:Receptors, Peptide / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000502:physiology*; D017384:Sequence Deletion
C090463:MID1 protein, S cerevisiae; D008562:Membrane Glycoproteins; D018000:Receptors, Peptide; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D002118:Calcium