Visualization and manipulation of plasma membrane-endoplasmic reticulum contact sites indicates the presence of additional molecular components within the STIM1-Orai1 Complex.
STIM1, a recently identified endoplasmic reticulum (ER) protein, rapidly translocates to a plasma membrane-adjacent ER compartment upon depletion of the ER Ca(2+) stores. Here we use a novel means, namely a chemically inducible bridge formation between the plasma and ER membranes, to highlight the plasma membrane-adjacent ER compartment and show that this is the site where STIM1 and its Ca(2+) channel partner, Orai1, form a productive interaction upon store depletion. By changing the length of the linkers connecting the plasma and ER membranes, we show that Orai1 requires a larger space than STIM1 between the two membranes. This finding suggests that Orai1 is part of a larger macromolecular cluster with an estimated 11-14-nm protrusion to the cytoplasm, whereas the cytoplasmic domain of STIM1 fits in a space calculated to be less than 6 nm. We finally show that agonist-induced translocation of STIM1 is rapidly reversible and only partially affects STIM1 in the juxtanuclear ER compartment. These studies are the first to detect juxtaposed areas between the ER and the plasma membrane in live cells, revealing novel details of STIM1-Orai1 interactions.
Section on Molecular Signal Transduction, NICHD, National Institutes of Health, Bethesda, Maryland 20892-4510, USA.
Péter
P
Várnai
Balázs
B
Tóth
Dániel J
DJ
Tóth
László
L
Hunyady
Tamas
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Balla
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000502:physiology*; D008954:Models, Biological; D009363:Neoplasm Proteins / Q000502:physiology*; D000071740:ORAI1 Protein; D011485:Protein Binding; D000071737:Stromal Interaction Molecule 1; D013997:Time Factors
D001426:Bacterial Proteins; D015220:Calcium Channels; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; C054562:yellow fluorescent protein, Bacteria; D002118:Calcium