Mastorodemos Vasileios, Kotzamani Dimitra, Zaganas Ioannis, Arianoglou Giovanna, Latsoudis Helen, Plaitakis Andreas
Department of Neurology, School of Health Sciences, Faculty of Medicine, University of Crete, Heraklion, Crete 71003, Greece.
Biochem Cell Biol. 2009 Jun;87(3):505-16. doi: 10.1139/o09-008.
Mammalian glutamate dehydrogenase (GDH), an enzyme central to glutamate metabolism, is thought to localize to the mitochondrial matrix, although there are also suggestions for the extramitochondrial presence of this protein. Whereas GDH in mammals is encoded by the GLUD1 gene, humans and the great apes have, in addition, a GLUD2 gene showing a distinct expression pattern. The encoded hGDH1 and hGDH2 isoenzymes are highly homologous, but their leader sequences are more divergent. To explore their subcellular targeting, we constructed expression vectors in which hGDH1 or hGDH2 was fused with the enhanced green fluorescent protein (EGFP) and used these to transfect COS 7, HeLa, CHO, HEK293, or neuroblastoma SHSY-5Y cells. Confocal microscopy revealed GDH-EGFP fluorescence in the cytoplasm within coarse structures. Cotransfection experiments using organelle-specific markers revealed that hGDH1 or hGDH2 colocalized with the mitochondrial marker DsRed2-Mito and to a lesser extent with the endoplasmic reticulum marker DsRed2-ER. Western blots detected two GDH-EGFP specific bands: a ~90 kDa band and a ~95 kDa band associated with the mitochondria and the endoplasmic reticulum containing cytosol, respectively. Deletion of the signal sequence, while altering drastically the fluoresce distribution within the cell, prevented GDH from entering the mitochondria, with the ~90 kDa band being retained in the cytosol. In addition, the deletion eliminated the ~95 kDa band from cell lysates, thus confirming that it represents the full-length GDH. Hence, while most of the hGDHs translocate into the mitochondria (a process associated with cleavage of the signal sequence), part of the protein localizes to the endoplasmic reticulum, probably serving additional functions.
哺乳动物谷氨酸脱氢酶（GDH）是谷氨酸代谢的关键酶，尽管有研究表明该蛋白也存在于线粒体外，但一般认为它定位于线粒体基质。哺乳动物的GDH由GLUD1基因编码，而人类和大猩猩还拥有一个表达模式不同的GLUD2基因。编码的hGDH1和hGDH2同工酶高度同源，但它们的前导序列差异较大。为了探究它们的亚细胞定位，我们构建了hGDH1或hGDH2与增强型绿色荧光蛋白（EGFP）融合的表达载体，并用于转染COS 7、HeLa、CHO、HEK293或神经母细胞瘤SHSY - 5Y细胞。共聚焦显微镜显示，在粗糙结构的细胞质中有GDH - EGFP荧光。使用细胞器特异性标记物的共转染实验表明，hGDH1或hGDH2与线粒体标记物DsRed2 - Mito共定位，与内质网标记物DsRed2 - ER的共定位程度较低。蛋白质免疫印迹检测到两条GDH - EGFP特异性条带：一条约90 kDa的条带和一条约95 kDa的条带，分别与线粒体和含有内质网的胞质溶胶相关。信号序列的缺失虽然极大地改变了细胞内的荧光分布，但阻止了GDH进入线粒体，约90 kDa的条带保留在胞质溶胶中。此外，缺失使细胞裂解物中约95 kDa的条带消失，从而证实它代表全长GDH。因此，虽然大多数hGDH转运到线粒体中（这一过程与信号序列的切割有关），但部分蛋白质定位于内质网，可能发挥额外的功能。
