The solid blackish ER lumen indicates the presence of the APEX2-tag facing the ER lumen for all truncation constructs. S1R 1–80aa spans the presumed first TM domain (left), S1R 1–113aa extends to just beyond the putative second TM domain (middle), and S1R 1–194aa spans the putative third hydrophobic domain (right) (see Fig. (D) Similar experiment with expression of C-terminus-tagged S1R-truncation constructs. The presence of “open” whitish appearance of the ER lumen indicates the lack of electron-dense reaction product in the ER lumen consistent with the APEX-tag facing the cytosol, while the “solid” blackish ER appearance inside the ER lumen indicates localization of the APEX-tag facing the ER lumen. The images are EM photomicrographs of the respective constructs expressed in ND9/27 cells. (C) Cartoon of the ER membrane spanning control Sec61B with the N-terminus facing the cytosol or the S1R indicating the location of the APEX2 tag.

The truncation constructs separate the major hydrophobic potential TM domains of the S1R protein. The cartoons below are the five GFP-APEX2 fusion S1R constructs (one N-tagged and four C-tagged) examined in the current study. Positive score values are increasingly hydrophobic ( ).

The third relatively hydrophobic stretch of residues thought to possibly dip into the membrane spans aa 176–194. The putative first TM spans aa 11–29 and the second TM spans aa 91–109. (B) Hydrophobicity plot of the 223-residue human S1R protein (top) with a cartoon of the hydrophobic potential TM domains indicated above. A novel topology with N-terminus in the cytosol with one TM domain (current study). N-terminus in the lumen with one TM domain (Schmidt et al., 2016), and 4. Both N- and C-termini are luminal with two TM domains (Hayashi and Su 2007), 3. Both N- and C-termini are cytosolic with two TM domains (Aydar et al., 2002), 2. (A) Three topologies of S1R in the ER membrane have been proposed. 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).ĭetermination of S1R topology in the ER by APEX2-tagging. 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. 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. These topological conclusions assumed a protein structural model with two transmembrane (TM) domains based on the hydrophobicity property of the protein. The topology of the N- and C-termini of the S1R based on the antibody accessibility technique was reported with both N- and 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. 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. 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.

Osmosis in er lumen