Supporting Information
S1 Fig  TMEM203 expression drives calcineurin and calcium dependent NFAT transcription factor activation.
TMEM203-FLAG over-expression in 293 cells for 48 hrs induced NFAT dependent luciferase expression in a dose dependent manner, Treatment with 1uM PMA for 8 hrs enhanced TMEM203 mediated NFAT activation whereas a treatment with 5uM cyclosporine A or 10 nM SKF96365 inhibited TMEM203’s activity. Data is representative of at least 3 independent experiments (mean; +/- SD; n = 3).
(TIF) Click here for additional data file.   S2 Fig  TMEM203 is a predicted integral membrane protein that is highly conserved across vertebrates.
(A)-Trans-membrane domain prediction of the TMEM203 sequence of using the TMHMM algorithm. TMEM203 is predicted to have 4 pass TM domains (red) with 3 loops and both N terminal and C terminal (green) facing the same side. (B)- TMEM203 amino acid sequences from various organisms were analyzed for conservation using PRALINE multiple sequence alignment tool. (http://www.ibi.vu.nl/programs/pralinewww/). The following TMEM203 protein sequences were retrieved from NCBI—Homo sapiens NP_444273; Mus musculus NP_796318; Xenopus laevis NP_001085810; Macaca mulatta NP_001248131; Gallus gallus XP_001234196; Danio rerio NP_001002519.
(TIF) Click here for additional data file.   S3 Fig  TMEM203 complexes with STIM1.
Western analysis of complexes immune-precipitated with endogenous STIM1 from HEK293 cells with indicated antibodies shows specific interaction with overexpressed TMEM203-FLAG. (Representative of atleast 2 independent experiments).
(TIF) Click here for additional data file.   S4 Fig  Tmem203 gene targeting strategy.
(A)- As described in the methods section. WT: wild type allele; KO+neo: knockout allele with neo; KO: knockout allele without neo. B: BamHI. (B). Gene disruption was confirmed by sequencing Tmem203 cDNA from lung tissue. The 102 bp fragment from the vector (marked in bold and underlined) disrupts the adjoining Tmem203 gene sequence in the Tmem203 null genome. The disrupted Tmem203 gene in the Tmem203 null genome lacks the start codon (ATG) for Tmem203 orf. While antibody adequate for detection of TMEM203 protein are not available, sequencing of cDNAs demonstrate that remaining tmem203 mRNA from knockout animals could only encode the last 38 amino acids of the predicted tmem 203 protein.
(TIF) Click here for additional data file.   S5 Fig  Altered calcium/NFAT dependent gene expression in Tmem203 deficient Mouse Embryonic Fibroblast cells.
(A) MEF cells derived from Tmem203—WT and null mice were stimulated with 100 nM TG for 4 hrs and subsequently the expression of calcium/NFAT dependent genes—Calreticulin (Calr) or Cacitonin receptor (Caclr) were determined by quantitative real-time PCR. (Mean; +/-; SD n = 4). Significant difference in Carl expression (P value = 0.0000001) and Caclr expression (P value = 0.0000002) was observed between WT and Tmem203 null MEF cells. (B) As described in (A), the expression of Carl and Caclr was determined in response to stimulation with 1uM ionomycin. Significant difference in Carl expression (P value = 0.000002) and Caclr expression (P value = 0.00000004) was observed between WT and Tmem203 null MEF cells.
(TIF) Click here for additional data file.   S6 Fig  Tmem203 is robustly expressed in mouse testes.
Quantitative real-time PCR analysis of Tmem203 transcript from various mouse tissue types showing abundant expression of Tmem203 in testes.
(TIF) Click here for additional data file.   S7 Fig  Body weight and Testes weight analysis of Tmem203 null male mice.
Compared to wild type control mice, Tmem203 null mice had significantly lower (A) Mean absolute body weights; 32.93 ± 0.80 grams versus 27.66 ± 1.98 grams, respectively; p value = 0.0006. (B) Mean absolute combined left and right testes weights; 0.2216 ± 0.103 grams versus 0.1608 ± 0.0142 grams, respectively; p value < 0.0001. (C) Mean testes to body weight ratios; 0.0067 ± 0.004 versus 0.0058 ± 0.0007 grams, respectively; p value = 0.0458. (D) Mean testes to brain weight ratios; 0.4544 ± 0.0570 grams versus 0.3504 ± 0.0332 grams, respectively; p value = 0.0078.
(TIF) Click here for additional data file.   S1 Table  cDNAs that induce CRTC1 translocation.
List of cDNAs, in addition to TPRV6 and PKA that induced translocation of CRTC1 to the nucleus in a HeLa CRTC1-eGFP expressing cell line. However, only TMEM203, could efficiently translocate TORC1 without inducing gross morphologic and/or apoptotic changes.
(DOCX) Click here for additional data file.   S2 Table  List of genes down-regulated in Tmem203 null mouse testes.
Only genes with Avg Log2 FC < -0.5 are shown.
(DOCX) Click here for additional data file.   S3 Table  List of genes up-regulated in Tmem203 null mouse testes.
Only genes with Avg Log2 FC >0.5 are shown.
(DOCX) Click here for additional data file.