Is there a signal transduction pathway that links events at the plasma membrane to the phosphorylation state of the mitochondrial pyruvate dehydrogenase complex?

Abstract

Monoclonal antibodies against the E1a subunit of pyruvate dehydrogenase (PDH) were used to quantify the mitochondrial pyruvate dehydrogenase complex (mtPDC) by enzyme-linked immunosorbent assay (ELISA). Recombinant Arabidopsis thaliana PDH (E1) was used to calibrate the ELISA. Antibodies against a synthetic phosphopeptide corresponding to phosphorylation site one of E1a were used in an ELISA to quantify phospho-PDC (P-PDC). For calibration of the second ELISA, recombinant E1 was phosphorylated in vitro with recombinant A. thaliana E1-kinase. The two ELISA were used to quantify mitochondrial total-and P-PDC in clarified homogenates from Nicotiana tabacum BY-2 suspension cells. The level of mtPDC remained constant throughout the 7-day growth cycle at 25.1 g 21 FW. During the lag (days 0-2) and stationary (day 7) stages of the growth cycle, the mtPDC was completely phosphorylated (inactive), whereas during the log-growth stage it was completely dephosphorylated (active). Exposure of 3-or 7-day posttransfer suspension cells to osmotic stress significantly decreased proportion of P-PDC. A series of pharmacological studies were undertaken to gain insight into the signal transduction pathways coupling osmotic stress perception with control of mitochondrial respiration. Results from these studies indicate a signal transduction pathway linking stress perception to control of mitochondrial respiration that includes protein kinases and phosphoprotein phosphatases.Abbreviations -E1 or PDH, pyruvate dehydrogenase; E2, dihydrolipoyl acetyltransferase; ELISA, enzyme-linked immunosorbent assay; L2, the E2 inner lipoyl domain; mt, mitochondrial; P-, phospho; PDC, pyruvate dehydrogenase complex; PDK, pyruvate dehydrogenase kinase; PDP, pyruvate dehydrogenase phosphatase.

Oxidation of an Adjacent Methionine Residue Inhibits Regulatory Seryl-Phosphorylation of Pyruvate Dehydrogenase

“…The clarified homogenates were assayed for total and P-PDC by eLIsA. 19 Control cells were mock manipulated. the enzyme systems necessary to reduce MetSO are ubiquitous.…”

“…The method employs specific anti-E1α- 18 and anti-phosphorylation site 1 antibodies. 19 For immunoprecipitation of the PDC prior to MS analysis, the anti-E1α antibodies were cross-linked to POROS 20 protein G-beads (Applied Biosystems, Foster City, CA) essentially as previously described for cross-linking concanavalin A. 15 The POROS protein G-beads were incubated with 10 mM dimethyl pimelimidate (DMP) and 10 mM dimethyl suberimidate (DMS) in 20 mL phosphate-buffered saline (PBS), pH 8.5.…”

“…18 The Nicotiana tabacum BY-2 suspension cells have been previously described. 19 Cells were grown under ambient light at 24 o C and 125 rpm, and transferred weekly to 100 mL of fresh medium in a 250 mL Erlenmeyer flask. For experimental manipulations, 20 mL of cells were taken 4 days after transfer and placed into a 50 mL Erlenmeyer flask.…”

Protein phosphorylation in plant mitochondria

“…The properties and regulation of the complex have been reviewed many times so here we will only give a very brief outline, which allows a comparison with the phosphorylation of FDH discussed later. See Miernyk et al (2007) and Tovar‐Méndes et al (2003) in this issue for more information about PDC phosphorylation.…”

Plant mitochondria—more active than ever!

“…About 20 phosphoproteins have been identified in plant mitochondria to date (Juszczuk et al 2007) and one of them is the E1α‐subunit of the pyruvate dehydrogenase complex, where phosphorylation inhibits the activity. This phosphorylation is strongly induced by hyperosmotic stress indicating that there is a signal transduction pathway linking stress perception to control of mitochondrial respiration that includes mitochondrial protein kinases and phosphatases (Miernyk et al 2007).…”