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Visualization and Manipulation of Plasma Membrane-Endoplasmic Reticulum Contact Sites Indicates the Presence of Additional Molecular Components within the STIM1-Orai1 Complex

Semmelweis University, Budapeŝto, Budapest, Hungary
Journal of Biological Chemistry (Impact Factor: 4.57). 11/2007; 282(40):29678-90. DOI: 10.1074/jbc.M704339200
Source: PubMed

ABSTRACT 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.

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    • "Research article (Liou et al., 2005; Zhang et al., 2005; Stathopulos et al., 2006; Wu et al., 2006; Liou et al., 2007; Varnai et al., 2007; Luik et al., 2008; Stathopulos et al., 2008), we hypothesized that this increase in cluster size could result from an increase in the density of Stim1 in the junctional ER or could reflect a specific defect in the ability of Stim1 to efficiently co-cluster Orai1. To distinguish between these possibilities, we first quantified the average intensity of CFP-Stim1 in individual Stim1–Orai1 clusters. "
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    • "The fact that overexpression of the two proteins results in reconstituted CRAC currents has enabled us to address questions about their overall communication [54, 68, 107]. In principle, two pathways are possible as to how STIM transmits the signal of store depletion to the activation of Orai channels in the plasma membrane: either by a simple, direct interaction or via a further molecule [11, 22, 95, 105]. Meanwhile, several independent studies, however, have proven direct binding of soluble STIM1 fragments to Orai, thereby inducing constitutive CRAC currents [32, 34, 57, 58, 66, 106]. "
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