Abstract
Glutamate-cysteine ligase (GCL) has a key influence on glutathione homeostasis. It has been proposed that mammalian GCL is regulated by the redox environment, and we show here that cysteine residues in the Drosophila melanogaster GCL modifier subunit (DmGCLM) can form covalent interactions with the catalytic subunit (DmGCLC) and modify its activity. Candidate components of intersubunit disulfides (Cys213, Cys214, and Cys267) were identified using matrix-assisted laser desorption ionization time-of-flight spectroscopy of iodoacetamide-modified DmGCLM as well as examination of the evolutionary conservation of cysteines. Mutation of the 3 cysteine residues allowed DmGCLM to associate with DmGCLC, but inhibited the formation of intersubunit disulfides. This caused a 2-fold reduction in the catalytic efficiency of Drosophila GCL, although activity remained significantly higher than the catalytic subunit alone. The cysteine mutant was also more sensitive to inhibition by glutathione than the unmodified holoenzyme. Notably, human GCLM could substitute for DmGCLM in modification of DmGCLC activity. The role of DmGCLM in vivo was examined by analysis of a Drosophila mutant (l(3)L0580) containing a P-element insertion in Gclm. We found that the P-element is not responsible for the lethal phenotype and separated the recessive lethal mutation from the P-element by recombination. This yielded two fully viable and fertile recombinants bearing the P-element insertion, which Western and Northern blotting indicated is a severely hypomorphic allele of Gclm. Glutathione levels were approximately 2-fold lower in the GclmL0580 mutants than in control strains, demonstrating the importance of DmGCLM in the regulation of glutathione homeostasis in vivo.
Highlights
The intracellular redox environment is of critical importance in cell physiology
We have examined the role of intermolecular disulfide linkages in regulating Drosophila glutamate-cysteine ligase (GCL) activity and showed that abrogation of the ability to form disulfide bridges between the catalytic and modifier subunits has a significant impact on the catalytic efficiency of the holoenzyme as well as sensitivity to feedback inhibition by glutathione
Our kinetic analyses showed that the mutant DmGCLC/DmGCLMABD holoenzyme was less active than the unmodified holoenzyme, but significantly more active than DmGCLC, supporting the notion that both covalent and non-covalent interactions are important in regulating DmGCL activity
Summary
Cys213 Cys214 Cys224 Cys267 Cys139 that covalent interactions could still occur between the mutated catalytic subunit and GCLM, indicating that Cys553 may not be the only cysteine involved in GCLC association with GCLM. It is possible that more than one disulfide linkage may form between the subunits. We reasoned that the most straightforward method of investigating the importance of intermolecular disulfide linkages in GCL catalysis would be to target the cysteine residues in the modifier subunit; GCLM has fewer cysteines, and interpretation of data would not be confounded by mutation of cysteine residues with a role in catalysis [12]. We have shown previously that GCL from Drosophila melanogaster contains an ϳ31-kDa modifier subunit (DmGCLM) that is capable of forming disulfide linkages with the ϳ80-kDa catalytic subunit (DmGCLC) in vitro [13]. We have investigated the importance of intermolecular disulfide linkages in DmGCL activity and show that the ability to form disulfide bonds impinges on both activity and sensitivity to feedback inhibition by glutathione. We show that Drosophila Gclm mutants, viable, have approximately half the levels of glutathione compared with the control strains, demonstrating the importance of DmGCLM in glutathione homeostasis in vivo
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