Putative glycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics.

The Golgi apparatus is the central organelle in the secretory pathway and plays key roles in glycosylation, protein sorting, and secretion in plants. Enzymes involved in the biosynthesis of complex polysaccharides, glycoproteins, and glycolipids are located in this organelle, but the majority of them remain uncharacterized. Here, we studied the Arabidopsis (Arabidopsis thaliana) membrane proteome with a focus on the Golgi apparatus using localization of organelle proteins by isotope tagging. By applying multivariate data analysis to a combined data set of two new and two previously published localization of organelle proteins by isotope tagging experiments, we identified the subcellular localization of 1,110 proteins with high confidence. These include 197 Golgi apparatus proteins, 79 of which have not been localized previously by a high-confidence method, as well as the localization of 304 endoplasmic reticulum and 208 plasma membrane proteins. Comparison of the hydrophobic domains of the localized proteins showed that the single-span transmembrane domains have unique properties in each organelle. Many of the novel Golgi-localized proteins belong to uncharacterized protein families. Structure-based homology analysis identified 12 putative Golgi glycosyltransferase (GT) families that have no functionally characterized members and, therefore, are not yet assigned to a Carbohydrate-Active Enzymes database GT family. The substantial numbers of these putative GTs lead us to estimate that the true number of plant Golgi GTs might be one-third above those currently annotated. Other newly identified proteins are likely to be involved in the transport and interconversion of nucleotide sugar substrates as well as polysaccharide and protein modification.

Absence of branches from xylan in Arabidopsis gux mutants reveals potential for simplification of lignocellulosic biomass.

As one of the most abundant polysaccharides on Earth, xylan will provide more than a third of the sugars for lignocellulosic biofuel production when using grass or hardwood feedstocks. Xylan is characterized by a linear ß(1,4)-linked backbone of xylosyl residues substituted by glucuronic acid, 4-O-methylglucuronic acid or arabinose, depending on plant species and cell types. The biological role of these decorations is unclear, but they have a major influence on the properties of the polysaccharide. Despite the recent isolation of several mutants with reduced backbone, the mechanisms of xylan synthesis and substitution are unclear. We identified two Golgi-localized putative glycosyltransferases, GlucUronic acid substitution of Xylan (GUX)-1 and GUX2 that are required for the addition of both glucuronic acid and 4-O-methylglucuronic acid branches to xylan in Arabidopsis stem cell walls. The gux1 gux2 double mutants show loss of xylan glucuronyltransferase activity and lack almost all detectable xylan substitution. Unexpectedly, they show no change in xylan backbone quantity, indicating that backbone synthesis and substitution can be uncoupled. Although the stems are weakened, the xylem vessels are not collapsed, and the plants grow to normal size. The xylan in these plants shows improved extractability from the cell wall, is composed of a single monosaccharide, and requires fewer enzymes for complete hydrolysis. These findings have implications for our understanding of the synthesis and function of xylan in plants. The results also demonstrate the potential for manipulating and simplifying the structure of xylan to improve the properties of lignocellulose for bioenergy and other uses.

Proteomic complex detection using sedimentation (ProCoDeS): screening for proteins in stable complexes and their candidate interaction partners.

Over the last few years, our view of cellular organization has changed from one in which enzymes and proteins usually act independently to the situation at present where we commonly accept that many, if not all, enzymes act in close association with others. Co-precipitation using an antibody against a test protein is the standard assay for the identification of members of protein complexes [Musso, Zhang and Emili (2007) Chem. Rev. 107, 3585-3600]. The introduction of TAP (tandem affinity purification) tagging enhanced original approaches in order to analyse protein complexes on a larger scale with reduced false discoveries of interacting partners due to more efficient purification of complexes. However, this technique has some limitations as a high-throughput tool for systems biology: the requirement for genetic manipulation to express the tagged protein excludes studies of non-transformable organisms and intact tissue. In those cases where TAP is applicable, a considerable amount of work is required to generate the baits and to optimize experimental conditions. A technique developed in our laboratories, ProCoDeS (Proteomic Complex Detection using Sedimentation), focuses on the detection of endogenous complexes. Protein samples are separated by centrifugation and then different fractions from the resulting gradient are analysed using quantitative MS. The identification of possible protein partners is based on statistical analysis of the co-fractionation of proteins, without any need for purification of individual complexes. The prospects of ProCoDeS and similar techniques based on quantitative MS for measurement of protein complex composition are reviewed in the present article.