The Catalytic Subunit of Drosophila Glutamate-Cysteine Ligase Is a Nucleocytoplasmic Shuttling Protein

• Published in: The Journal of biological chemistry 2009-01, Vol.284 (4), p.2266-2274
• Main Authors: Radyuk, Svetlana N., Rebrin, Igor, Luchak, James M., Michalak, Katarzyna, Klichko, Vladimir I., Sohal, Rajindar S., Orr, William C.
• Format: Article
• Language: English
• Subjects: Active Transport, Cell Nucleus; Amino Acid Sequence; Animals; Base Sequence; Catalytic Domain; Cell Cycle; Cell Nucleus - enzymology; Computational Biology; Cytoplasm - enzymology; Drosophila; Drosophila melanogaster - chemistry; Drosophila melanogaster - genetics; Drosophila melanogaster - metabolism; Gene Expression Regulation, Enzymologic; Glutamate-Cysteine Ligase - chemistry; Glutamate-Cysteine Ligase - genetics; Glutamate-Cysteine Ligase - metabolism; Glutathione - metabolism; Humans; Metabolism and Bioenergetics; Molecular Sequence Data; Oxidation-Reduction; Sequence Alignment
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M805913200

GSH concentration is considerably lower in the nucleus than in the cytoplasm; however, it is significantly elevated during active cell proliferation. The main purpose of this study was to understand the mechanism underlying these variations in nuclear/cytoplasmic distribution of GSH. The rate-limiting step in the de novo GSH biosynthesis pathway is catalyzed by glutamate cysteine ligase (GCL), a heterodimer, composed of a catalytic subunit (GCLc) and a modulatory subunit (GCLm). In Drosophila, GCLc, but not GCLm, contains a nuclear localization signal (NLS). Drosophila S2 cells, constitutively expressing regular GCLc protein or expressing GCLc protein with a mutated NLS motif, were generated by transfection. In quiescent S2 cells, GCLc is aggregated in the perinuclear cytosol and the nucleus, whereas GLCm resides solely in the cytosol. In actively proliferating S2 cells, expressing the normal NLS motif, GCLc migrates from the perinuclear cytoplasm into the nucleus, and the nuclear GSH level becomes elevated; in contrast, in proliferating cells, expressing the mutated NLS motif, neither does the GCLc migrate into the nucleus nor does the nuclear GSH amount rise. In S2 cells expressing wild type GCLc, perturbation of cellular redox state by exposure to cadmium resulted in the migration of GCLc into the nucleus but not in cells expressing GCLc with the mutated NLS motif. Overall, results indicated that GSH biosynthesis in the nucleus is associated with migration of only the GCLc subunit from the cytoplasm into the nucleus, and this migration requires the presence of an intact NLS.