Phosphorylation of Formate Dehydrogenase in Potato Tuber Mitochondria

Abstract

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 Reversible protein phosphorylation is one of the most important regulatory mechanisms in living cells (1), yet little is know about protein phosphorylation in mitochondria. The only well studied case is that of phosphorylation of the E1␣-subunit of the pyruvate dehydrogenase (PDH) 1 complex (2). The PDH complex contains both a kinase and a protein phosphatase, and phosphorylation inactivates the complex, which is reactivated by dephosphorylation. This means that the entry of carbon compounds from glycolysis into the citric acid cycle is turned off under conditions of high ATP concentration. Although the PDH kinase and phosphatase system is the only fully characterized system for reversible protein phosphorylation in mitochondria, there are clear indications that others are present. Incubating intact mitochondria (3-5) or submitochondrial fractions like the outer membrane (6) or the inner membrane (7-9) with [␥-32 P]ATP gives labeling of up to 30 proteins (as detected by one-dimensional gel electrophoresis) indicating that kinases are present in these fractions and in contact with target proteins. The matrix appears to contain one of more protein phosphatases responsible for dephosphorylation of inner membrane phosphoproteins (8, 9).Only eight mitochondrial phosphoproteins have been identified to date. In addition to the E1␣-subunit of PDH, these are band ␦Ј-subunits of the ATP synthase (10), HSP70 (11, 12) and MTSHP (13,14), nucleoside diphosphate kinase (15, 16), E1 of the branched-chain ␣-ketoacid dehydrogenase (2), and the 18-kDa iron protein subunit of complex I in mammalian mitochondria (17). In a very recent study (18) a further 14 mitochondrial phosphoproteins were identified, all household proteins involved in the citric acid cycle and associated reactions, the respiratory chain complexes, in heat shock proteins, and the detoxification of reactive oxygen species.Formate dehydrogenase (FDH) catalyzes the oxidation of formate to CO 2 reducing NAD ϩ to NADH in the process. Formate may have a role in biosynthesis of numerous compounds, such as in energetic metabolism and in signal transduction pathways related to stress response. The enzyme is induced in potato leaves by a variety of stresses including hypoxia (19,20). It is one of the most abundant proteins in the matrix of potato tuber mitochondria (19), but its function is unknown partly be...

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“…It is possible that these represent isoforms of FDH with different phosphorylation status (58). However, no significant changes were observed in formate-dependent oxygen consumption or the maximal catalytic FDH activity across the time points examined (Fig.…”

Catalytic mechanism and application of formate dehydrogenase

“…Only in 2003, phosphorylation of the enzyme was detected in mitochondria of potato [60]. FDH was phosphorylated on the residues Thr76 and Thr333 [60], and, depending on the modification degree, multiple isoforms were produced with pI varying from 6.75 to 7.19 [61]. The phosphorylation degree was inversely proportional to oxygen concentration in the cell and strongly decreased in the presence of NAD + , for mate, and pyruvate.…”