LAUSR

Deciphering the role of any protein in a cell requires knowledge of the structure, subcellular localization, and the topological orientation of the protein within the relevant cells. The Sigma-1 receptor (S1R) largely localized to the endoplasmic reticulum (ER) serves as a pluripotent intracellular signaling molecule with diverse roles in the cell including ion channel modulation, stress signaling, and transcriptional regulation (reviewed in Su et al., 2016), however, the basic topology of this protein within a cell remains controversial. The topology of the Nand C-termini of the S1R based on the antibody accessibility technique was reported with both Nand C-termini facing the cytosol (Aydar et al., 2002), while a later report (Hayashi and Su, 2007) suggested that both termini were facing the ER lumen. These topological conclusions assumed a protein structural model with two transmembrane (TM) domains based on the hydrophobicity property of the protein. More recently, the study resolving the crystal structure of the S1R reported the protein to possess a single TM domain with a short N-terminus facing the ER lumen, while most of the protein bulk was located on the cytosolic side of the ER membrane (Schmidt et al., 2016) (Fig. 1A). Due to contradictory reports on the exact topology of the S1R within the ER membrane, we applied the ascorbate peroxidase 2 (APEX2) approach to provide a definitive answer to the S1R protein topology in the ER membrane. A unique feature of APEX2 is that it retains robust peroxidase activity even after strong fixation with 2% glutaraldehyde and brings about its utility as a tag for subcellular detection of proteins of interest with electron microscopy (EM) with clarity not attainable by the more conventional immuno-electron microscope technique (Rhee et al., 2013; Lam et al., 2015; Lee et al., 2016). We created constructs encoding the full-length S1R with APEX2 attached at either the Nor C-terminus of the receptor (Fig. 1B). We also created Sec61B constructs, a one TM domain component of the ER translocon with an insertion topology in the ER with the protein N-terminus facing the cytosol (Dudek et al., 2015). Previous reports indicate that the tagging of Sec61B with APEX2 attached either at the Nor C-terminus does not alter the subcellular targeting to the ER or the topology of the protein inserted into the ER membrane (Lee et al., 2016). Since all constructs also contained green fluorescent protein (GFP) positioned between S1R and APEX2, fluorescence microscopy allowed detection of the expression of the fusion protein. Confocal imaging of ND7/23 cells co-transfected with the ER-localizing Sec61B-mCherry and Nor C-tagged S1R confirmed a direct overlap of the GFP and mCherry signal in the ER, indicating no mistargeting due to epitope-tagging (Fig. S1A). When transfected into cells, the control Sec61B protein was detected in the ER as expected. EM imaging confirmed accumulation of the electron-dense precipitate in the cytosol when the APEX2-tag was attached to the N-terminus of Sec61B, while the APEX2-tag attached to the C-terminus of Sec61B resulted in electron density in the ER lumen (Fig. 1C, left two panels). An analogous experiment with fulllength S1R resulted in detection of electron-dense precipitates in the cytosol when APEX2 was attached to the N-terminus of the receptor, while luminal electron-density was detected with APEX2 attached to the C-terminus of the receptor (Fig. 1C, right two panels). This supports the conclusion that the N-terminus of the full-length S1R faces the cytosol while the C-terminus faces the ER lumen. To probe for the possible presence of TM domains within the S1R protein, we created three different S1R truncations with APEX2 attached at the C-terminus. The three truncation constructs encoded for amino acids 1–80, 1–113, and 1–194 demarcated the boundaries of potential TM domains based on the hydrophobicity plot of the protein. If a TM domain is present, the C-terminus of the truncation construct encompassing such a domain should localize to the opposite side of the ER membrane. EM analysis of the cells expressing these truncation constructs demonstrated localization of the C-terminus of all the constructs in the ER lumen consistent with the interpretation that only one TM domain exists between amino acids 1–80 (Fig. 1D). A silver nitrate and gold chloride modification of the technique allows higher resolution detection of the APEX2catalyzed reaction product as electron-dense nanogold precipitates, rather than as diffuse diaminobenzidine