Increased expression of TMX1 inhibits ER Ca²⁺ uptake but promotes ER-derived Ca²⁺ flux. (A) Analysis of TMX1-transfected A375P lysates. A375P cells were stably transfected with TMX1 constructs as indicated and probed with our anti-TMX1 antibody. (B) TMX1 redox activity. TMX1 disulfide reduction activity was measured by adding immunoprecipitated TMX1 or its mutants (TMX-SXXS or TMX-CCAA) to 100 nM Di-E-GSSG in the presence of 5 µM DTT. The graph plots the increase in fluorescence versus nontransfected control immunoprecipitate. The gel on top shows lysates from a typical experiment analyzed for the FLAG-TMX1 content via Western blot. **, P < 0.01; standard error indicated. (C) TMX1 and TMX1 mutant distribution among microsomes (containing ER, endosomes, and lysosomes), mitochondria, and the MAM. HeLa homogenates from cells transfected with pcDNA3 or plasmids expressing FLAG-tagged WT TMX1 or the CCAA or SXXS mutants were fractionated via the Percoll fractionation protocol into cytosol, microsomes, pure mitochondria, and MAM, as indicated on the right side. Equal cell equivalents have been loaded, and TMX1 was detected via its FLAG tag. (D) TMX1-SERCA2b interaction depends on TMX1 redox activity and localization. A375P cells were transfected with mycSERCA2b and FLAG-tagged TMX1 mutants as indicated. DSP cross-linked lysates (5% inputs, left) and myc immunoprecipitates were analyzed for coimmunoprecipitating FLAG-tagged TMX1. n = 3; ***, P = 0.000197 for TMX WT versus SXXS. ***, P = 0.000013 and 0.000011 for immunoprecipitation; standard error indicated. (E) Measurement of ER Ca²⁺ content. A375P cells were transfected with TMX1 constructs as indicated and cotransfected with a plasmid encoding ER-targeted aequorin. Luminescence was plotted from three independent experiments. *, P = 0.01; standard error expressed versus A375P. (F) Measurement of mitochondrial Ca²⁺ after histamine-triggered Ca²⁺ release. A375P cells transfected with TMX1 and mitochondrial R-GECO1 were treated with 50 µM histamine, and probe fluorescence was recorded. Results from three independent experiments are summarized as a graph representing maximum response. Experiments were done in the presence and absence of extracellular Ca²⁺. Differences are not statistically significant with Ca²⁺ but significant without Ca²⁺ (*, P = 0.01; standard error expressed vs. A375P). (G) Measurement of mitochondrial Ca²⁺ after histamine-triggered Ca²⁺ release in heterozygous TMX1 KO HeLa cells rescued with TMX1 or its mutants. TMX1 KO HeLa cells rescued with TMX1 or its mutants (identified via fluorescence signal derived from pIRES-EGFP) were transfected with mitochondrial R-GECO1 and treated with 50 µM histamine in the absence of extracellular Ca²⁺, and probe fluorescence was recorded. Results from three independent experiments are summarized as a graph representing maximum response and slope of the signal increase (*, P = 0.03 for FLAG-TMX1; standard error expressed vs. HeLa).