Tespa1 is a novel component of mitochondria-associated endoplasmic reticulum membranes and affects mitochondrial calcium flux.

Regulation of intracellular Ca(2+) concentration is critical in numerous biological processes. Inositol 1,4,5-trisphosphate receptor (IP3R) functions as the Ca(2+) release channel on endoplasmic reticulum (ER) membranes. Much attention has been dedicated to mitochondrial Ca(2+) uptake via mitochondria-associated ER membranes (MAM) which is involved in intracellular Ca(2+) homeostasis; however, the molecular mechanisms that link the MAM to mitochondria still remain elusive. We previously reported that Tespa1 (thymocyte-expressed, positive selection-associated gene 1) expressed in lymphocytes physically interacts with IP3R. In this study, we first performed double-immunocytochemical staining of Tespa1 with a mitochondrial marker or an ER marker on an acute T lymphoblastic leukemia cell line, Jurkat cells, by using anti-ATP synthase or anti-calnexin antibody, respectively, and demonstrated that Tespa1 was localized very close to mitochondria and the Tespa1 localization was overlapped with restricted portion of ER. Next, we examined the effects of Tespa1 on the T cell receptor (TCR) stimulation-induced Ca(2+) flux by using Ca(2+) imaging in Jurkat cells. Reduction of Tespa1 protein by Tespa1-specific siRNA diminished TCR stimulation-induced Ca(2+) flux into both mitochondria and cytoplasm through the analyses of the mitochondrial Ca(2+) indicator (Rhod-2) and the cytoplasmic Ca(2+) indicator (Fluo-4), respectively. Furthermore, co-immunoprecipitation assay in HEK293 cells revealed that exogenous Tespa1 protein physically interacted with a MAM-associated protein, GRP75 (glucose-regulated protein 75), but not with an outer mitochondrial membrane protein, VDAC1 (voltage-dependent anion channel 1). All these results suggested that Tespa1 will participate in the molecular link between IP3R-mediated Ca(2+) release and mitochondrial Ca(2+) uptake in the MAM compartment.