Subcellular localization and oligomeric structure of the yeast putative stretch-activated Ca2+ channel component Mid1.
The yeast Mid1 protein with an apparent molecular mass of 100 kDa is required for Ca2+ influx stimulated by the mating pheromone and by a capacitative calcium entrylike mechanism acting in response to Ca2+ depletion from the endoplasmic reticulum (ER) and functions as a stretch-activated Ca2+ -permeable channel when expressed in mammalian cells. Our previous work with protease protection experiments has indicated that Mid1 is present in the plasma membrane. In this study, we examined a possible intracellular localization of this protein by indirect fluorescence microscopy and found that Mid1 is present in the ER membrane as well as the plasma membrane. Intracellular fluorescence images for Mid1 were the same as those for the ER marker protein Sec71 but quite different from those of the Golgi protein Ypt1. The results were confirmed by membrane fractionation using Angiografin density gradient analysis. We also investigated the oligomeric structures and protein levels of Mid1 and found that Mid1 forms a 200-kDa oligomer by disulfide bonding. The protein level and modification of Mid1 in the plasma membrane and the ER membrane were unchanged by the mating pheromone. These findings provide new insight into the function of Mid1 in relation to localization, modification, and activation mechanisms.
Yoshimura
Hitoshi
H
Department of Biology, Tokyo Gakugei University, 4-1-1 Nukui kita-machi, Koganei-shi, Tokyo 184-8501, Japan.
Tada
Tomoko
T
Iida
Hidetoshi
H
D015220:Calcium Channels / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004220:Disulfides / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D010675:Pheromones / Q000378:metabolism / Q000494:pharmacology; D011108:Polymers / Q000378:metabolism; D020836:Protein Structure, Quaternary / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism
D015220:Calcium Channels; D004220:Disulfides; C090463:MID1 protein, S cerevisiae; D008562:Membrane Glycoproteins; D010675:Pheromones; D011108:Polymers; C084842:SEC66 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C060755:YPT1 protein, S cerevisiae; D020691:rab GTP-Binding Proteins