The pyruvate dehydrogenase complex (PDC) is subjected to multiple interacting levels of control in plant cells. The first level is subcellular compartmentation. Plant cells are unique in having two distinct, spatially separated forms of the PDC; mitochondrial (mtPDC) and plastidial (plPDC). The mtPDC is the site of carbon entry into the tricarboxylic acid cycle, while the plPDC provides acetyl-CoA and NADH for de novo fatty acid biosynthesis. The second level of regulation of PDC activity is the control of gene expression. The genes encoding the subunits of the mt-and plPDCs are expressed following developmental programs, and are additionally subject to physiological and environmental cues. Thirdly, both the mt-and plPDCs are sensitive to product inhibition, and, potentially, to metabolite effectors. Finally, the two different forms of the complex are regulated by distinct organelle-specific mechanisms. Activity of the mtPDC is regulated by reversible phosphorylation catalyzed by intrinsic kinase and phosphatase components. An additional level of sensitivity is provided by metabolite control of the kinase activity. The plPDC is not regulated by reversible phosphorylation. Instead, activity is controlled to a large extent by the physical environment that exists in the plastid stroma.

Plant cells are unique in that they contain four species of alpha-ketoacid dehydrogenase complex: plastidial pyruvate dehydrogenase, mitochondrial pyruvate dehydrogenase, alpha-ketoglutarate (2-oxoglutarate) dehydrogenase, and branched-chain alpha-ketoacid dehydrogenase. All complexes include multiple copies of three components: an alpha-ketoacid dehydrogenase/decarboxylase, a dihydrolipoyl acyltransferase, and a dihydrolipoyl dehydrogenase. The mitochondrial pyruvate dehydrogenase complex additionally includes intrinsic regulatory protein-kinase and -phosphatase enzymes. The acyltransferases form the intricate geometric core structures of the complexes. Substrate channeling plus active-site coupling combine to greatly enhance the catalytic efficiency of these complexes. These alpha-ketoacid dehydrogenase complexes occupy key positions in intermediary metabolism, and a basic understanding of their properties is critical to genetic and metabolic engineering. The current status of knowledge of the biochemical, regulatory, structural, genomic, and evolutionary aspects of these fascinating multienzyme complexes are reviewed.

Previous systems analyses in plants have focused on a single developmental stage or time point, although it is often important to additionally consider time-index changes. During seed development a cascade of events occurs within a relatively brief time scale. We have collected protein and transcript expression data from five sequential stages of Arabidopsis (Arabidopsis thaliana) seed development encompassing the period of reserve polymer accumulation. Protein expression profiling employed twodimensional gel electrophoresis coupled with tandem mass spectrometry, while transcript profiling used oligonucleotide microarrays. Analyses in biological triplicate yielded robust expression information for 523 proteins and 22,746 genes across the five developmental stages, and established 319 protein/transcript pairs for subsequent pattern analysis. General linear modeling was used to evaluate the protein/transcript expression patterns. Overall, application of this statistical assessment technique showed concurrence for a slight majority (56%) of expression pairs. Many specific examples of discordant protein/ transcript expression patterns were detected, suggesting that this approach will be useful in revealing examples of posttranscriptional regulation.

A total of 89 J-domain proteins were identified in the genome of the model flowering plant Arabidopsis thaliana. The deduced amino acid sequences of the J-domain proteins were analyzed for an assortment of structural features and motifs. Based on the results of sequence comparisons and structure and function predictions, 51 distinct families were identified. The families ranged in size from 1 to 6 members. Subcellular localizations of the A thaliana J-domain proteins were predicted; species were found in both the soluble and membrane compartments of all cellular organelles. Based on digital Northern analysis, the J-domain proteins could be separated into groups of low, medium, and moderate expression levels. This genomics-based analysis of the A thaliana J-domain proteins establishes a framework for detailed studies of biological function and specificity. It additionally provides a comprehensive basis for evolutionary comparisons.

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The peroxisomal isoform of ascorbate peroxidase (APX) is a novel membrane isoform that functions in the regeneration of NAD ؉ and protection against toxic reactive oxygen species. The intracellular localization and sorting of peroxisomal APX were examined both in vivo and in vitro. Epitope-tagged peroxisomal APX, which was expressed transiently in tobacco BY-2 cells, localized to a reticular/circular network that resembled endoplasmic reticulum (ER; 3,3 -dihexyloxacarbocyanine iodide-stained membranes) and to peroxisomes. The reticular network did not colocalize with other organelle marker proteins, including three ER reticuloplasmins. However, in vitro, peroxisomal APX inserted posttranslationally into the ER but not into other purified organelle membranes (including peroxisomal membranes). Insertion into the ER depended on the presence of molecular chaperones and ATP. These results suggest that regions of the ER serve as a possible intermediate in the sorting pathway of peroxisomal APX. Insight into this hypothesis was obtained from in vivo experiments with brefeldin A (BFA), a toxin that blocks vesicle-mediated protein export from ER. A transiently expressed chloramphenicol acetyltransferase-peroxisomal APX (CAT-pAPX) fusion protein accumulated only in the reticular/circular network in BFA-treated cells; after subsequent removal of BFA from these cells, the CATpAPX was distributed to preexisting peroxisomes. Thus, plant peroxisomal APX, a representative enzymatic peroxisomal membrane protein, is sorted to peroxisomes through an indirect pathway involving a preperoxisomal compartment with characteristics of a distinct subdomain of the ER, possibly a peroxisomal ER subdomain. INTRODUCTIONPeroxisomes are found in virtually all eukaryotic cells and are delineated by a single boundary membrane. These organelles typically are involved in the generation and degradation of toxic hydrogen peroxide and the ␤ oxidation of fatty acids; they also house a diversity of enzymes that participate in various other metabolic processes specific to the organism, cell/tissue type, or environmental conditions (Huang et al., 1983;Baker, 1996;Gietl, 1996).Nuclear genes encode all of the protein and enzyme constituents of the peroxisomal matrix. After synthesis on polysomes in the cytosol, these proteins are targeted posttranslationally to the organelle in a regulated manner. At least two types of evolutionarily conserved peroxisomal targeting signals (PTSs) are capable of directing proteins to the peroxisomal matrix (Olsen, 1998;Subramani, 1998). The type 1 PTS (PTS1) is an uncleaved C-terminal tripeptide motif-that is, small basic hydrophobic residues-or variants thereof (Mullen et al., 1997a(Mullen et al., , 1997b, that is found in the many peroxisomal matrix-destined proteins. The type 2 PTS (PTS2) is a nonapeptide motif (R/K-X 6 -H/Q-A/L/F [where X indicates any amino acid]) (Flynn et al., 1998) located in the N terminus of another set of matrix proteins that are proteolytically processed after import into peroxisomes. Proteinaceo...

SummaryProtein-protein interactions or protein complexes are integral in nearly all cellular processes, ranging from metabolism to structure. Elucidating both individual protein associations and complex protein interaction networks, while challenging, is an essential goal of functional genomics. For example, discovering interacting partners for a 'protein of unknown function' can provide insight into actual function far beyond what is possible with sequence-based predictions, and provide a platform for future research. Synthetic genetic approaches such as two-hybrid screening often reveal a perplexing array of potential interacting partners for any given target protein. It is now known, however, that this type of anonymous screening approach can yield high levels of false-positive results, and therefore putative interactors must be confirmed by independent methods. In vitro biochemical strategies for identifying interacting proteins are varied and time-honored, some being as old as the field of protein chemistry itself. Herein we discuss five biochemical approaches for isolating and characterizing protein-protein interactions in vitro: co-immunoprecipitation, blue native gel electrophoresis, in vitro binding assays, protein cross-linking, and rate-zonal centrifugation. A perspective is provided for each method, and where appropriate specific, trial-tested methods are included.

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