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J. Biol. Chem., Vol. 279, Issue 1, 95-102, January 2, 2004

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Riboflavin Uptake and FAD Synthesis in Saccharomyces cerevisiae Mitochondria

INVOLVEMENT OF THE Flx1p CARRIER IN FAD EXPORT^*

Valeria Bafunno¶, Teresa Anna Giancaspero||, Carmen Brizio**, Daniela Bufano||, Salvatore Passarella, Eckhard Boles, and Maria Barile¶¶

From the Dipartimento di Biochimica e Biologia Molecolare, Università di Bari, Via Orabona 4, and the Istituto di Biomembrane e Bioenergetica, Consiglio Nazionale delle Ricerche (CNR), Via Amendola 165/A, 70126 Bari, Italy, the Dipartimento di Scienze Animali, Vegetali e dell'Ambiente, Università del Molise, Via De Sanctis, 86100 Campobasso, Italy, and the Institut fuer Mikrobiologie, Goethe-Universitaet Frankfurt, Marie-Curie-Str. 9, D-60439 Frankfurt, Germany

We have studied the functional steps by which Saccharomyces cerevisiae mitochondria can synthesize FAD from cytosolic riboflavin (Rf). Riboflavin uptake into mitochondria took place via a mechanism that is consistent with the existence of (at least two) carrier systems. FAD was synthesized inside mitochondria by a mitochondrial FAD synthetase (EC 2.7.7.2), and it was exported into the cytosol via an export system that was inhibited by lumiflavin, and which was different from the riboflavin uptake system. To understand the role of the putative mitochondrial FAD carrier, Flx1p, in this pathway, an flx1 mutant strain was constructed. Coupled mitochondria isolated from flx1 mutant cells were compared with wild-type mitochondria with respect to the capability to take up Rf, to synthesize FAD from it, and to export FAD into the extramitochondrial phase. Mitochondria isolated from flx1 mutant cells specifically lost the ability to export FAD, but did not lose the ability to take up Rf, FAD, or FMN and to synthesize FAD from Rf. Hence, Flx1p is proposed to be the mitochondrial FAD export carrier. Moreover, deletion of the FLX1 gene resulted in a specific reduction of the activities of mitochondrial lipoamide dehydrogenase and succinate dehydrogenase, which are FAD-binding enzymes. For the flavoprotein subunit of succinate dehydrogenase we could demonstrate that this was not due to a changed level of mitochondrial FAD or to a change in the degree of flavinylation of the protein. Instead, the amount of the flavoprotein subunit of succinate dehydrogenase was strongly reduced, indicating an additional regulatory role for Flx1p in protein synthesis or degradation.

Received for publication, July 28, 2003 , and in revised form, October 3, 2003.

^* This work was supported by grants from Telethon (1205) and the University of Bari (Fondi di Ateneo per la Ricerca) (to M. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this work.

^¶ Supported by a fellowship from European Social Fund (P.O.P. 1994–1999).

^|| Supported by a fellowship from ESF (P.O.P. 2000–2006).

^** A recipient of a post-graduate research fellowship (Assegno di Ricerca) financed by Telethon and by the University of Bari.

^¶¶ To whom correspondence should be addressed: Dipartimento di Biochimica e Biologia Molecolare, Università degli Studi di Bari, Via Orabona 4, 70126 Bari, Italy. Tel.: 39-080-5443364; Fax: 39-080-5443317; E-mail: m.barile{at}biologia.uniba.it.

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