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J. Biol. Chem., Vol. 278, Issue 28, 26021-26030, July 11, 2003

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Phosphorylation of Formate Dehydrogenase in Potato Tuber Mitochondria^*

Natalia V. Bykova  , Allan Stensballe ¶, Helge Egsgaard , Ole N. Jensen ¶ and Ian M. Møller ||

From the Plant Research Department, Risø National Laboratory, P. O. Box 49, DK-4000 Roskilde and the ^¶Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark

Two highly phosphorylated proteins were detected after two-dimensional (blue native/SDS-PAGE) gel electrophoretic separation of the matrix fraction isolated from potato tuber mitochondria. These two phosphoproteins were identified by mass spectrometry as formate dehydrogenase (FDH) and the E1-subunit of pyruvate dehydrogenase (PDH). Isoelectric focusing/SDS-PAGE two-dimensional gels separated FDH and PDH and resolved several different phosphorylated forms of FDH. By using combinations of matrix-assisted laser desorption/ionization mass spectrometry and electrospray ionization tandem mass spectrometry, several phosphorylation sites were identified for the first time in FDH and PDH. FDH was phosphorylated on Thr⁷⁶ and Thr³³³, whereas PDH was phosphorylated on Ser²⁹⁴. Both Thr⁷⁶ and Thr³³³ in FDH were accessible to protein kinases, as demonstrated by protein structure homology modeling. The extent of phosphorylation of both FDH and PDH was strongly decreased by NAD⁺, formate, and pyruvate, indicating that reversible phosphorylation of FDH and PDHs was regulated in a similar fashion. At low oxygen concentrations inside the intact potato tubers, FDH activity was strongly increased relative to cytochrome c oxidase activity pointing to a possible involvement of FDH in hypoxic metabolism. Computational sequence analysis indicated that a conserved local sequence motif of pyruvate formate-lyase is found in the Arabidopsis thaliana genome, and this enzyme might be the source of formate for FDH in plants.

Received for publication, January 9, 2003 , and in revised form, March 7, 2003.

^* This work was supported by grants from the Danish Agricultural and Veterinary Research Council (to N. V. B. and I. M. M.) and from the Danish Natural Science Research Council (to O. N. J.). 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.

Present address: Dept. of Physics and Astronomy, University of Manitoba, 301 Allen Bldg., Winnipeg, Manitoba R3T 2N2, Canada.

^|| To whom correspondence should be addressed: Plant Research Dept., Risø National Laboratory, Bldg. 301, P. O. Box 49, DK-4000 Roskilde, Denmark. Tel.: 45-46-77-42-13; Fax: 45-46-77-41-22; E-mail: ian.max.moller{at}risoe.dk.

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