TMEM203 encodes a conserved ER resident protein associated with calcium signaling molecules
TMEM203 cDNAs and gene locus encode a potential 136 amino acid integral membrane protein with four predicted trans-membrane (TM) domains, short N- and C-terminal domains with no obvious functional domains (S2 Fig). The predicted TMEM203 protein is remarkably conserved across vertebrate species while predicted proteins of much lower similarity were identified from Drosophila and C. elegans (39% and 27% identity, respectively; data not shown).
Cellular localization of a TMEM203-GFP fusion protein was examined by imaging with ER, mitochondria and plasma membrane (PM) markers. Based on the linescan co-lozalization tool TMEM203-GFP was predominately co-localized with the ER marker protein in HeLa cells (Fig 2A). Considering the localization of TMEM203 and TMEM203’s effect on cytoplasmic calcium levels, we asked if TMEM203 was associated with calcium modulatory proteins complexed within the ER [38–40]. Endogenous IP3R, SERCA2 and STIM1 proteins were co-precipitated with TMEM203-FLAG expressed in transfected HEK293 cells, whereas an ER resident membrane protein unrelated to the SOCE complex, INSIG-1 was not (Fig 2B). TMEM-203-FLAG protein was also detected after immunoprecipitation with antibody to endogenous STIM1 (S3 Fig). Thus ER expressed TMEM203 was associated with proteins critical for regulation of calcium influx and efflux into the ER as well STIM1, the calcium sensor for SOCE.
10.1371/journal.pone.0127480.g002 Fig 2  TMEM203 interacts with regulators of ER calcium stores and overexpression depletes ER calcium stores.
(A) Confocal analysis of HeLa cells transiently expressing TMEM203-GFP with organelle specific markers for ER (top:Calreticulin-RFP), Mitochondria (middle:BDHA-RFP) or plasma membrane (bottom:LCK-RFP). Separation or colocalization of TMEM203-GFP and organelle marker(s) were visualized by the linescan function of MetaMorph: the fluorescence intensity of each pixel of the line of interest (white lines ~ 75 μm) is shown as a xy-graph for the corresponding green and red channels. The line scan shows that TMEM203-GFP predominately overlapped with the ER marker. (Representative of ~ 50 cells from 2 independent experiments). Note, we cannot rule out that TMEM203 is completely absent from the the mitochondria. (B) Western analysis of complexes immune-precipitated TMEM203-Flag from HEK293 cells with indicated antibodies shows specific interaction with endogenous STIM1, IP3R and SERCA2. (Representative of atleast 2 independent experiments). (C) pTUNE-TMEM203-293cells were treated with the indicated dose of IPTG for 48 hrs to induce TMEM203 expression. Levels of TMEM203-Flag protein were detected by western blot. (D) These IPTG induced cells were subjected to Indo-1 based calcium flux measurements by flow cytometry by first treating with thapsigargin (TG) and EGTA. (E) As in (D) but the cells were treated with Ionomycin. (F) As in (D) but following TG treatment CaCl2 was added to record SOCE.