Regulation of intracellular Ca2+ concentration is critical in numerous biological processes. Inositol 1,4,5-trisphosphate receptor (IP3R) functions as the Ca2+ release channel on endoplasmic reticulum (ER) membranes. Much attention has been dedicated to mitochondrial Ca2+ uptake via mitochondria-associated ER membranes (MAM) which is involved in intracellular Ca2+ 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 I) 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 Ca2+ flux by using Ca2+ imaging in Jurkat cells. Reduction of Tespa1 protein by Tespa1-specific siRNA diminished TCR stimulation-induced Ca2+ flux into both mitochondria and cytoplasm through the analyses of the mitochondrial Ca2+ indicator (Rhod-2) and the cytoplasmic Ca2+ 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 Ca2+ release and mitochondrial Ca2+ uptake in the MAM compartment. (C) 2013 Elsevier Inc. All rights reserved.