Open Research Online The Open University���s repository of research publications and other research outputs The modifier subunit of drosophila glutamate-cysteine ligase regulates catalytic activity by covalent and non- covalent interactions and influences glutathione Home- ostasis in vivo. Journal Article How to cite: Fraser, Jennifer A. Kansagra, Pushpa Kotecki, Claire Saunders, Robert D.C. and McLellan, Lesley I. (2003). The modifier subunit of drosophila glutamate-cysteine ligase regulates catalytic activity by covalent and noncovalent interactions and influences glutathione Homeostasis in vivo. Journal of Biological Chemistry, 278(47), pp. 46369���46377. For guidance on citations see FAQs. c [not recorded] Version: [not recorded] Link(s) to article on publisher���s website: http://dx.doi.org/doi:10.1074/jbc.M308035200 http://www.jbc.org/cgi/reprint/278/47/46369 Copyright and Moral Rights for the articles on this site are retained by the individual authors and/or other copy- right owners. For more information on Open Research Online���s data policy on reuse of materials please consult the policies page. oro.open.ac.uk

The Modifier Subunit of Drosophila Glutamate-Cysteine Ligase Regulates Catalytic Activity by Covalent and Noncovalent Interactions and Influences Glutathione Homeostasis in Vivo* Received for publication, July 23, 2003, and in revised form, August 21, 2003 Published, JBC Papers in Press, September 3, 2003, DOI 10.1074/jbc.M308035200 Jennifer A. Fraser���, Pushpa Kansagra��, Claire Kotecki��, Robert D. C. Saunders��, and Lesley I. McLellan����� From the ���Biomedical Research Centre, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, United Kingdom and the ��Department of Biological Sciences, Open University, Walton Hall, Milton Keynes MK7 6AA, United Kingdom 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 Drosoph- ila melanogaster GCL modifier subunit (DmGCLM) can form covalent interactions with the catalytic subunit (DmGCLC) and modify its activity. Candidate compo- nents of intersubunit disulfides (Cys213, Cys214, and Cys267) were identified using matrix-assisted laser des- orption ionization time-of-flight spectroscopy of iodoac- etamide-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 intersub- unit 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 holoen- zyme. Notably, human GCLM could substitute for DmGCLM in modification of DmGCLC activity. The role of DmGCLM in vivo was examined by analysis of a Dro- sophila mutant (l(3)L0580) containing a P-element inser- tion in Gclm. We found that the P-element is not respon- sible for the lethal phenotype and separated the recessive lethal mutation from the P-element by recom- bination. This yielded two fully viable and fertile recom- binants bearing the P-element insertion, which Western and Northern blotting indicated is a severely hypomor- phic allele of Gclm. Glutathione levels were 2-fold lower in the GclmL0580 mutants than in control strains, demonstrating the importance of DmGCLM in the regu- lation of glutathione homeostasis in vivo. The intracellular redox environment is of critical importance in cell physiology. It has a major influence on signaling path- ways and cell fate in response to stress (1). Glutathione is one of the key influences on the redox state of the cell and accord- ingly, intracellular glutathione levels are subject to multilat- eral regulatory mechanisms (2, 3). A major player in the regulation of glutathione homeostasis is glutamate-cysteine ligase (GCL),1 which catalyzes the first and rate-limiting step in de novo synthesis of glutathione from precursor amino acids (4). GCL is located in the cytoplasm, and its activity appears to be regulated at several levels by the antioxidant status of the cell. In addition to being subject to transcriptional activation by pro-oxidants (5���7), evidence sug- gests that the catalytic activity is subject to redox regulation by the reversible formation of disulfide bridges between its two subunits (8, 9). Furthermore, GCL is subject to feedback inhi- bition by glutathione at concentrations that are physiologically relevant (10). The complex regulation of GCL activity high- lights its pivotal role in controlling cellular glutathione synthesis. GCL is a heterodimer composed of a catalytic subunit (GCLC) and a modifier subunit (GCLM). The presence of GCLM modulates the catalytic properties of GCLC by lowering its sensitivity to inhibition by glutathione and by increasing its affinity for glutamate (8, 9). The sensitivity of GCLC to inhibi- tion by glutathione is such that it has been proposed that GCLC would function poorly in vivo without the presence of GCLM. The pioneering biochemical studies of Meister and co- workers (9) in the 1990s provided evidence to suggest that GCLM could further enhance the function of GCLC by the formation of intersubunit disulfide bonds. This proposition was based on the observations that treatment of the GCL holoen- zyme with dithiothreitol (DTT) lowered its affinity for gluta- mate and increased its sensitivity to inhibition by the glutathi- one analog ophthalmic acid. The effects of DTT were dependent on the presence of GCLM. The results prompted the hypothesis that intracellular GCL activity could be increased under con- ditions that deplete glutathione, where the oxidizing environ- ment within the cell would promote disulfide bond formation within GCL. More recently, the role of intermolecular disulfide linkages in modifying GCL activity was investigated by mutagenesis of cysteine residues in the catalytic subunit (11). 8 of the 14 cysteine residues in human GCLC were singly altered to gly- cine, and the effects on activity and ability to form disulfide linkages were examined. One of the 8 cysteines (Cys553) was shown to be involved in influencing the ability of GCLM to increase the activity of GCLC. The mutant holoenzyme was, however, still able to form a 114-kDa complex when analyzed by SDS-PAGE under non-reducing conditions. This suggests * This work was supported by Grants 94/G15091 and 108/G15090 from the Biotechnology and Biological Sciences Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ���adver- tisement��� in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. �� To whom correspondence should be addressed. Tel.: 44-1382- 660111 Fax: 44-1382-669993 E-mail: lesley.mclellan@cancer.org.uk. 1 The abbreviations used are: GCL, glutamate-cysteine ligase GCLC, glutamate-cysteine ligase catalytic subunit GCLM, glutamate-cysteine ligase modifier subunit Dm, Drosophila melanogaster Hs, Homo sapi- ens DTT, dithiothreitol MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 47, Issue of November 21, pp. 46369���46377, 2003 �� 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. This paper is available on line at http://www.jbc.org 46369