FAD oxidizes the ERO1-PDI electron transfer chain: The role of membrane integrity q,qq Eszter Papp a , Ga ´bor Nardai a , Jo ´ zsef Mandl b , Ga ´bor Ba ´nhegyi b , Pe ´ter Csermely a, * a Department of Medical Chemistry, Semmelweis University, H-1088 Budapest, Hungary b Endoplasmic Reticulum Research Group of the Hungarian Academy of Sciences and Department of Medical Chemistry, Semmelweis University, H-1088 Budapest, Hungary Received 17 September 2005 Available online 17 October 2005 Abstract The molecular steps of the electron transfer in the endoplasmic reticulum from the secreted proteins during their oxidation are rel- atively unknown. We present here that flavine adenine dinucleotide (FAD) is a powerful oxidizer of the oxidoreductase system, Ero1 and PDI, besides the proteins of rat liver microsomes and HepG2 hepatoma cells. Inhibition of FAD transport hindered the action of FAD. Microsomal membrane integrity was mandatory for all FAD-related oxidation steps downstream of Ero1. The PDI inhibitor bacitracin could inhibit FAD-mediated oxidation of microsomal proteins and PDI, but did not hinder the FAD-driven oxidation of Ero1. Our data demonstrated that Ero1 can utilize FAD as an electron acceptor and that FAD-driven protein oxidation goes through the Ero1-PDI pathway and requires the integrity of the endoplasmic reticulum membrane. Our findings prompt further studies to elu- cidate the membrane-dependent steps of PDI oxidation and the role of FAD in redox folding. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Redox folding; Membrane integrity; Nutritional regulation; Electron transfer chain; FAD Oxidative protein folding is necessary for the maturation of most secreted and plasma membrane proteins. The cor- rect formation of disulfide bonds needs a sensitively regu- lated redox environment. In eukaryotes, this environment is provided by the endoplasmic reticulum (ER) [1,2]. Protein disulfide isomerases help the formation of disul- fide bonds in the ER [3,4]. The major, 58 kDa protein disulfide isomerase of the ER (PDI) constitutes 2% of ER proteins [5]. PDI has disulfide isomerase and chaperone activity also [6]. Later, several other protein disulfide isomerases have been identified, which participate in spe- cialized protein folding pathways in the ER [7,8]. Ero1, a conserved, ER-resident enzyme was identified as an electron acceptor of PDI [9–11]. Ero1 interacts with PDI in eukaryotes initiating the transfer of the oxidizing equiv- alents to folding proteins [12,13]. In yeast, Ero1 association to the membranes required to this action [14]. Human Ero1s also behave as peripheral membrane proteins [15,16]. Molecular oxygen was found to be an electron acceptor for yeast Ero1 [17]. Since yeast Ero1 can enhance disulfide bridge formation under anaerobic conditions [18], the exis- tence of an alternative electron acceptor molecule is possible. In yeast FAD accelerated the disulfide bridge formation by Ero1 [17] and riboflavin depletion resulted in a defective oxidative folding [19]. Furthermore, over-expression of the 0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.10.027 q This work was supported by research grants from the EU (FP6- 506850, FP6-016003), Hungarian Science Foundation (OTKA T37357, T48939, TS40865, and F47281), Hungarian Ministry of Social Welfare (ETT 32/03, 613/03), and by the Hungarian National Research Initiative (NKFP-1A/056/2004 and KKK-0015/3.0). qq Abbreviations: AMS, 4-acetamido-4 0 maleimidylstilbene 2,2 0 -disulfonic acid; DIDS, 4,4 0 (diisothiocyanato)stilbene-2-2 0 -disulfonic acid; DOC, deoxycholic acid; ER, endoplasmic reticulum; Ero1, ER-oxidizing protein 1; ERp44 and ERp72, members of the ER protein disulfide isomerase family; FAD, flavine adenine dinucleotide; Grp94, 94 kDa glucose regulated protein, a major ER chaperone; GSH, glutathione-reduced form; GSSG, oxidized glutathione; PDI, the 58 kDa major protein disulfide isomerase of the endoplasmic reticulum. * Corresponding author. Fax: +36 1 2662755/4131. E-mail address: csermely@puskin.sote.hu (P. Csermely). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 338 (2005) 938–945 BBRC