Mark L. Johnston

Cloning and molecular analyses of the Arabidopsis thaliana plastid pyruvate dehydrogenase subunits

Herein we report the first molecular description of the pyruvate dehydrogenase component of the higher plant plastid pyruvate dehydrogenase complex. The full-length cDNAs for the E1 alpha (1530 bp) and E1 beta (1441 bp) subunits of the Arabidopsis thaliana plastid pyruvate dehydrogenase contain open reading frames that encode polypeptides of 428 and 406 amino acids, respectively, with calculated molecular weight values of 47,120 and 44,208. The deduced amino acid sequences for Arabidopsis plastid E1 alpha and E1 beta have 61% and 68% identity to the odpA and odpB genes of the red alga Porphyra purpurea, respectively, but only 31% and 32% identity to the plant mitochondrial counterparts. Results of Southern analyses suggest that each subunit is encoded by a single gene. Northern blot analyses indicate expression of mRNAs of the appropriate size in Arabidopsis leaves.

Initial description of the developing soybean seed protein Lys-Nε-acetylome

UNLABELLED Characterization of the myriad protein posttranslational modifications (PTM) is a key aspect of proteome profiling. While there have been previous studies of the developing soybean seed phospho-proteome, herein we present the first analysis of non-histone lysine-N(Ɛ)-acetylation in this system. In recent years there have been reports that lysine acetylation is widespread, affecting thousands of proteins in diverse species from bacteria to mammals. Recently preliminary descriptions of the protein lysine acetylome from the plants Arabidopsis thaliana and Vitis vinifera have been reported. Using a combination of immunoenrichment and mass spectrometry-based techniques, we have identified over 400 sites of lysine acetylation in 245 proteins from developing soybean (Glycine max (L.) Merr., cv. Jack) seeds, which substantially increases the number of known plant N(Ɛ)-lysine-acetylation sites. Results of functional annotation indicate acetyl-proteins are involved with a host of cellular activities. In addition to histones, and other proteins involved in RNA synthesis and processing, acetyl-proteins participate in signaling, protein folding, and a plethora of metabolic processes. Results from in silico localization indicate that lysine-acetylated proteins are present in all major subcellular compartments. In toto, our results establish developing soybean seeds as a physiologically distinct addendum to Arabidopsis thaliana seedlings for functional analysis of protein Lys-N(Ɛ)-acetylation. BIOLOGICAL SIGNIFICANCE Several modes of peptide fragmentation and database search algorithms are incorporated to identify, for the first time, sites of lysine acetylation on a plethora of proteins from developing soybean seeds. The contributions of distinct techniques to achieve increased coverage of the lysine acetylome are compared, providing insight to their respective benefits. Acetyl-proteins and specific acetylation sites are characterized, revealing intriguing similarities as well as differences with those previously identified in other plant and non-plant species.

Using multiplex-staining to study changes in the maize leaf phosphoproteome in response to mechanical wounding

Mechanical wounding of 2-week-old maize (Zea mays L.) leaves, one of the first steps in both pathogen infection and herbivore attack, stimulates metabolism and activates signal transduction pathways dedicated to defense and recovery. The signaling pathways include reversible protein phosphorylation which can modulate protein activities, and transmit signals within cellular pathways and networks. We have used multiplex-staining of high-resolution 2D gels for protein (Sypro Ruby) and phosphorylation (Pro-Q Diamond) as a strategy for quantifying changes in the stoichiometry of phosphorylation after wounding for 270 protein spots. Rigorous statistical analysis of the time-index data allowed us to accept patterns of change in 125 of the spots as non-random, and these patterns were assigned to five clusters. A reliable identity was assigned to 21 selected proteins, most of which have been previously described as phospho-proteins. The results suggest that analysis of protein spots from high-resolution 2D gels by multiplex-staining for protein plus phosphorylation is a strategy that can be broadly useful for study of how the phospho-proteome responds to abiotic stress.

Import, processing, and assembly of the α- and β-subunits of chloroplast pyruvate dehydrogenase

Abstract. Sequence comparisons were used to identify cDNAs potentially encoding the α- and β-subunits of chloroplast pyruvate dehydrogenase. Coupled in-vitro transcription plus translation was used to synthesize radiolabeled pyruvate dehydrogenase α- and β-subunit precursor proteins. When the precursors were incubated with intact pea (Pisum sativum L.) seedling chloroplasts in the presence of ATP, they were imported and proteolytically processed. In contrast, there was no import or processing of the precursors by isolated, intact pea seedling mitochondria. Monospecific antibodies to the recombinant pyruvate dehydrogenase α-subunit were additionally able to co-precipitate radiolabeled pyruvate dehydrogenase β-subunit, indicating association between subunits after import and processing. Furthermore, size-exclusion chromatography was used to identify an αβ heterodimer that is an intermediate in the assembly of the native α2β2 heterotetrameric enzyme.

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

A Met residue is located adjacent to phosphorylation site 1 in the sequences of mitochondrial pyruvate dehydrogenase E1α subunits. When synthetic peptides including site 1 were treated with H2O2, the Met residue was oxidized to methionine sulfoxide (MetSO), and the peptides were no longer phosphorylated by E1α-kinase. Isolated mitochondria were incubated under state III or IV conditions, lysed, the pyruvate dehydrogenase complex (PDC) immunoprecipitated, and tryptic peptides analyzed by MALDI-TOF mass spectrometry. In all instances both Met and MetSO site 1 tryptic-peptides were detected. Similar results were obtained when suspension-cultured cells were incubated with chemical agents known to stimulate production of reactive oxygen species within the mitochondria. Treatment with these agents had no effect upon the amount of total PDC, but decreased the proportion of P-PDC. We propose that the redox-state of the Met residue adjacent to phosphorylation site 1 of pyruvate dehydrogenase contributes to overall regulation of PDC activity in vivo.

Chemical Cross‐Linking Immobilized Concanavalin A for use in Proteomic Analyses

Abstract Lectin affinity chromatography was used to reduce the amount of the abundant glycoprotein β‐conglycinin in total protein samples prepared from developing soybean (Glycine max L. Merrill cv. Jack) seeds. Electrophoretic analysis of both the concanavalin A‐Sepharose binding and non‐binding fraction revealed an abundant protein band at Mr 26,000. The amount of this protein was greatly increased when concanavalin A‐Sepharose was used with urea‐containing buffers. Peptide mass fingerprint analysis of this abundant protein band unequivocally identified it as concanavalin A (con A). A simple and gentle method was used to chemically cross‐link the con A subunits so that the lectin‐Sepharose retained the ability to bind high‐mannose type glycoproteins. The chemically cross‐linked con A‐Sepharose was stable in buffers that contained up to 8M urea, making this an affinity matrix suitable for use in electrophoresis‐based proteomic analyses.

Analysis of soybean tissue culture protein dynamics using difference gel electrophoresis.

UNLABELLED Excised hypocotyls from developing soybean (Glycine max (L.) merr. cv. Jack) were cultivated on agar-solidified medium until callus formed. The calli were then propagated in liquid medium until stable, relatively uniform, finely-divided suspension cultures were obtained. Cells were typically transferred to fresh medium at 7-day intervals. Cultures were harvested by filtration five days (early log phase) or eight days (late log phase) after transfer. In order to evaluate dynamic changes, both intracellular and extracellular proteins were analyzed by 2-dimensional difference gel electrophoresis. Selected spots were subjected to in-gel tryptic-digestion and the resultant peptides were analyzed by nLC-MS/MS. In follow-up studies gel-free shot-gun analyses led to identification of 367 intracellular proteins and 188 extracellular proteins. SIGNIFICANCE The significance of the described research is two-fold. First a gel-based proteomics method was applied to the study of the dynamics of the secretome (extracellular proteins). Second, results of a shot-gun non-gel based proteomic survey of both cellular and extracellular proteins are presented.

A novel regulatory mechanism based upon a dynamic core structure for the mitochondrial pyruvate dehydrogenase complex

The Arabidopsis thaliana genome includes three genes for mitochondrial dihydrolipoamide acetyltransferase, the E2-component of the mitochondrial pyruvate dehydrogenase complex (PDC). Two genes encode E2-proteins with a single lipoyl domain, while the third has a two-lipoyl domain structure. Transcripts for each E2 protein were expressed in all plant organs. Each recombinant AtmtE2 can individually form an icosahedral PDC core structure, and results from bimolecular fluorescence complementation assays are consistent with formation of hetero-core structures from all permutations of the AtmtE2 proteins. We propose a unique regulatory mechanism involving dynamic formation of hetero-core complexes that include both mono- and di-lipoyl forms of AtmtE2.

Digging Deeper into the Seed Proteome: Prefractionation of Total Proteins

Seeds are a common experimental system for many reasons. Among these: (i) they occupy a major niche in agriculture and human nutrition; (ii) they are a rich source of critical genetic information; and (iii) they are a near-ideal system for the study of phytohormone action or the transition from either dormancy or quiescence to active growth and development. One important component of all of these considerations is occurrence of the highly-abundant seed storage proteins (SSP). While on the one hand the high levels of proteins present in seeds make them attractive subjects, the SSP themselves are anathema to proteomics analyses. Without some sort of pretreatment removal of SSP, they will be virtually the only proteins identified in shotgun proteomics analyses. Here in, we describe and compare several methods commonly used to deplete samples of SSP, present the relatively recent application of combinatorial-ligand random-peptide libraries to seed proteomics studies, and speculate briefly on the short-term future.