Human GLUD1 and GLUD2 glutamate dehydrogenase localize to mitochondria and endoplasmic reticulum.
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.
Mastorodemos
Vasileios
V
Department of Neurology, School of Health Sciences, Faculty of Medicine, University of Crete, Heraklion, Crete 71003, Greece.
Kotzamani
Dimitra
D
Zaganas
Ioannis
I
Arianoglou
Giovanna
G
Latsoudis
Helen
H
Plaitakis
Andreas
A
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005969:Glutamate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000201:enzymology*; D010957:Plasmids; D014162:Transfection
D017931:DNA Primers; D007527:Isoenzymes; D005969:Glutamate Dehydrogenase