Abstract
The endoplasmic reticulum (ER) contains various enzymes that metabolize fatty acids (FAs). Given that FAs are the components of membranes, FA metabolic enzymes might be associated with regulation of ER membrane functions. However, it remains unclear whether there is the interplay between FA metabolic enzymes and ER membrane proteins. Trans-2-enoyl-CoA reductase (TER) is an FA reductase present in the ER membrane and catalyzes the last step in the FA elongation cycle and sphingosine degradation pathway. Here we identify sarco(endo)plasmic reticulum Ca2+-ATPase 2b (SERCA2b), an ER Ca2+ pump responsible for Ca2+ accumulation in the ER, as a TER-binding protein by affinity purification from HEK293 cell lysates. We show that TER directly binds to SERCA2b by in vitro assays using recombinant proteins. Thapsigargin, a specific SERCA inhibitor, inhibits this binding. TER binds to SERCA2b through its conserved C-terminal region. TER overexpression suppresses SERCA2b ATPase activity in microsomal membranes of HEK293 cells. Depletion of TER increases Ca2+ storage in the ER and accelerates SERCA2b-dependent Ca2+ uptake to the ER after ligand-induced Ca2+ release. Moreover, depletion of TER reduces the Ca2+-dependent nuclear translocation of nuclear factor of activated T cells 4. These results demonstrate that TER is a negative regulator of SERCA2b, implying the direct linkage of FA metabolism and Ca2+ accumulation in the ER.
Highlights
Fatty acids (FAs) are essential constituents of biological membranes, energy storage, and precursor of signaling molecules
Because sarco(endo)plasmic reticulum Ca2+-ATPase 2b (SERCA2b) is the only protein localized to the endoplasmic reticulum (ER) in the identified proteins, and expected to be abundant in the Strep-Trans-2-enoyl-CoA reductase (TER) pull-down fraction, we characterized SERCA2b as a potential binding partner of TER in this study
TER is an FA reductase in the ER membrane, which plays an essential role in the FA elongation cycle and sphingosine 1phosphate (S1P) metabolism
Summary
We first sought to identify ER protein(s) that bind to TER by affinity purification/mass spectrometry. In addition to StrepTER, bands at 55, 95, 170, 180, and 400 kDa were detected in the pull-down fraction from Strep-TER– expressing HEK293 cells (Fig. 1A). SERCA2b was coimmunoprecipitated with TER (Fig. 1D), indicating the binding of endogenous TER to endogenous SERCA2b. FLAG-tagged full-length TER, ΔN, ΔC, and N-term along with 3xHA-tagged SERCA2b were expressed in HEK293 cells, followed by immunoprecipitation with the anti-FLAG antibody. Not GST or GST-N-term, pulled down 3xHA-SERCA2b (Fig. 3C), indicating that the C-terminal region of TER binds to SERCA2b. These results show that TER directly binds to SERCA2b through its C-terminal region. Tg treatment reduced the amount of 3xHA-SERCA2b pulled down with GST-C-term (Fig. 3D)
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