Tespa1 is a novel component of mitochondria-associated endoplasmic reticulum membranes and affects mitochondrial calcium flux
Tespa1 is a novel component of mitochondria-associated endoplasmic reticulum membranes and affects mitochondrial calcium flux
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DOI：
10.1016/j.bbrc.2013.02.099
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发表时间：
2013-04-12
影响因子：
3.1
通讯作者：
Shirasawa, Senji
中科院分区：
文献类型：
--
作者：
Matsuzaki, Hiroshi;Fujimoto, Takahiro;Shirasawa, Senji
细胞内Ca2+浓度的调节在许多生物过程中是至关重要的。肌醇1,4,5-三磷酸受体（IP3R）在内质网（ER）膜上作为Ca2+释放通道发挥作用。通过线粒体相关内质网膜（MAM）参与细胞内Ca2+稳态的线粒体Ca2+摄取受到了广泛的关注；然而，将MAM与线粒体联系起来的分子机制仍然难以捉摸。我们之前报道了淋巴细胞中表达的Tespa1（胸腺细胞表达的阳性选择相关基因I）与IP3R的物理相互作用。在本研究中，我们首先对急性T淋巴细胞白血病细胞系Jurkat细胞分别使用抗atp合酶或抗calnexin抗体对Tespa1进行了线粒体标记物或内质网标记物的双重免疫细胞化学染色，发现Tespa1定位在非常靠近线粒体的位置，并且与内质网限制性部分重叠。接下来，我们通过在Jurkat细胞中使用Ca2+成像来检测Tespa1对T细胞受体（TCR）刺激诱导的Ca2+通量的影响。通过对线粒体Ca2+指示剂（Rhod-2）和细胞质Ca2+指示剂（Fluo-4）的分析，Tespa1特异性siRNA减少Tespa1蛋白减少了TCR刺激诱导的进入线粒体和细胞质的Ca2+通量。此外，HEK293细胞的共免疫沉淀实验显示，外源性Tespa1蛋白与mam相关蛋白GRP75（葡萄糖调节蛋白75）物理相互作用，但与线粒体外膜蛋白VDAC1（电压依赖性阴离子通道1）不相互作用。所有这些结果表明，Tespa1将参与ip3r介导的Ca2+释放和MAM室线粒体Ca2+摄取之间的分子联系。(C) 2013爱思唯尔公司版权所有。
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.