Phosphoproteins and Protein Kinase Activities Intrinsic to Inner Membranes of Potato Tuber Mitochondria

Struglics, A.; Fredlund, Kenneth M.; Allen, John F.

doi:10.1093/oxfordjournals.pcp.a029515

Cited by 10 publications

(8 citation statements)

References 42 publications

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“…Phosphorylation of the E1α subunit of the branched‐chain α‐ketoacid dehydrogenase and the 18 kDa IP subunit of complex I [7] has so far only been detected in mammalian mitochondria. However, there are clearly more phosphoproteins present since labelling of intact mitochondria or submitochondrial fractions gives up to 30 labelled bands on one‐dimensional (1D) gels [8–11].…”

Section: Introductionmentioning

confidence: 99%

Identification of 14 new phosphoproteins involved in important plant mitochondrial processes

2003

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Protein phosphorylation is a very important posttranslational modi¢cation the role of which is practically unexplored in mitochondria. Using two-dimensional gel electrophoresis followed by mass spectrometry, 14 new phosphoproteins are identi¢ed in potato tuber mitochondria, all household proteins also present in mammalian and fungal mitochondria. Seven of the new phosphoproteins are involved in the tricarboxylic acid cycle or associated reactions, four are subunits of respiratory complexes and involved in electron transport, ATP synthesis and protein processing, two are heat shock proteins and one is involved in defence against oxidative stress. These ¢ndings open up entirely new possibilities for the regulation and signal integration of mitochondrial processes.

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Section: Introductionmentioning

confidence: 99%

Identification of 14 new phosphoproteins involved in important plant mitochondrial processes

2003

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“…Incubating intact mitochondria (3)(4)(5) or submitochondrial fractions like the outer membrane (6) or the inner membrane (7)(8)(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).…”

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Phosphorylation of Formate Dehydrogenase in Potato Tuber Mitochondria

Bykova¹,

Stensballe

Egsgaard³

et al. 2003

Journal of Biological Chemistry

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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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“…1B) showed that the polypeptide and phosphoprotein patterns were quite different. The labelling time was only 2 min, the time giving half‐maximal phosphorylation [11] to maximize the sensitivity to inhibition, resulting in somewhat less 32 P incorporation than in subsequent experiments (cf. Fig.…”

Section: Resultsmentioning

confidence: 93%

“…Protein phosphorylation assays were carried out in a volume of 50 μl containing 250 μg IO‐SMP proteins [11]. Final concentrations in the reaction mixture were as follows: 0.3 M sucrose, 50 mM HEPES–KOH (pH 7.5), 5 mM MgCl 2 , 0.1 mM CaCl 2 and 0.2 mM (0.4–1.0 Ci mmol −1 ) [γ‐ 32 P]ATP.…”

Section: Methodsmentioning

confidence: 99%

“…When inside‐out submitochondrial particles (IO‐SMP) from plant mitochondria are incubated with [γ‐ 32 P]ATP, more than 20 proteins are phosphorylated mostly on serine/threonine residues [11]. The basic properties of the phosphorylation, specificity, time course, pH dependence, K m (ATP), have been characterized and two putative kinases/kinase subunits of 16.5 and 30 kDa identified [11]. In the present study, we show that two distinct phosphoprotein phosphatases are involved in the protein phosphorylation/dephosphorylation in IO‐SMP, one bound to the mitochondrial inner membrane and another, soluble, in the matrix.…”

Section: Introductionmentioning

confidence: 99%

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Protein phosphorylation/dephosphorylation in the inner membrane of potato tuber mitochondria

et al. 2000

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Inside-out inner mitochondrial membranes free of matrix proteins were isolated from purified potato tuber (Solanum tuberosum L.) mitochondria and incubated with [Q Q-32 P]ATP. Proteins were separated by SDS^PAGE and visualized by autoradiography. Phosphorylation of inner membrane proteins, including ATPase subunits, was strongly inhibited by the phosphoprotein phosphatase inhibitor NaF. We propose that an inner membrane phosphoprotein phosphatase is required for activation of the inner membrane protein kinase. When prelabelled inner membranes were incubated in the absence of [Q Q-32 P]ATP, there was no phosphoprotein dephosphorylation unless a soluble matrix fraction was added. This dephosphorylation was inhibited by NaF, but not by okadaic acid. We conclude that the mitochondrial matrix contains a phosphoprotein phosphatase that is responsible for dephosphorylation of inner membrane phosphoproteins. ß

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Phosphoproteins and Protein Kinase Activities Intrinsic to Inner Membranes of Potato Tuber Mitochondria

Cited by 10 publications

References 42 publications

Identification of 14 new phosphoproteins involved in important plant mitochondrial processes

Identification of 14 new phosphoproteins involved in important plant mitochondrial processes

Phosphorylation of Formate Dehydrogenase in Potato Tuber Mitochondria

Protein phosphorylation/dephosphorylation in the inner membrane of potato tuber mitochondria

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