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

Péter Várnai,Balázs Tóth,Dániel J Tóth,László Hunyady,Tamas Balla

doi:10.1074/jbc.m704339200

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.

Highlights

It has long been known that Ca2ϩ-mobilizing agonists activate a Ca2ϩ entry pathway subsequent to their mobilization of intracellular Ca2ϩ stores by a mechanism that has eluded identification until most recently
Since STIM1 can be glycosylated and is found in the plasma membrane (PM), it was of great importance to determine whether the rapid appearance of STIM1 in the form of numerous puncta at the PM upon store depletion represents a translocation of the protein within the endoplasmic reticulum (ER) from the reticulo-tubular to a membrane-adjacent region or whether it involves the incorporation of the molecule into the PM
This, does not rule out that STIM1 in the PM plays additional roles related to some forms of Ca2ϩ entry process [18] or that it can cluster at the PM after store depletion [19]

Summary

EXPERIMENTAL PROCEDURES

Materials—Rapamycin and thapsigargin were purchased from Calbiochem. Apyrase and ATP were obtained from Sigma. The human Orai was obtained as an expressed sequence tag clone (id: 3914595, Open Biosystems) and was tagged with CFP, YFP, or mRFP at its C terminus with a linker (AGANSGAGAGAGAILSRGAAAGAAGPVAT) inserted between Orai and the fluorescent protein based on the pEGFP-N1 vector in which the starting Met of GFP was changed to Leu. The cytomegalovirus promoter in some of the STIM1 and Orai constructs was replaced by the thymidine kinase (TK) promoter amplified from the pRL-TK vector of Promega (nucleotides 7–1029) using the VspI and NheI restriction sites. For targeting of the FRB protein to the cytoplasmic surface of the ER, the C-terminal localization sequence (residues 521– 587) of the human SacI phosphatase (obtained as an expressed sequence tag clone: 3049075 from Open Biosystems) was added to the C terminus of the FRB fragment with a linker of GSGAGAGAGAILNSRV between the two proteins This sequence was placed behind CFP, mRFP, or GFP using the pEGFP-C1 plasmid backbone. The difference in response ware was used for data acquisition

RESULTS

DISCUSSION