A challenging task in the study of the secretory pathway is the identification and localization of new proteins to increase our understanding of the functions of different organelles. Previous proteomic studies of the endomembrane system have been hindered by contaminating proteins, making it impossible to assign proteins to organelles. Here we have used the localization of organelle proteins by the isotope tagging technique in conjunction with isotope tags for relative and absolute quantitation and 2D liquid chromatography for the simultaneous assignment of proteins to multiple subcellular compartments. With this approach, the density gradient distributions of 689 proteins from Arabidopsis thaliana were determined, enabling confident and simultaneous localization of 527 proteins to the endoplasmic reticulum, Golgi apparatus, vacuolar membrane, plasma membrane, or mitochondria and plastids. This parallel analysis of endomembrane components has enabled protein steady-state distributions to be determined. Consequently, genuine organelle residents have been distinguished from contaminating proteins and proteins in transit through the secretory pathway.endomembrane ͉ localization of organelle proteins by isotope tagging ͉ isotope tags for relative and absolute quantitation ͉ organelle proteomics P roteins are spatially organized according to their functions within the eukaryotic cell. Therefore, protein localization is an important step toward assigning functions to the thousands of uncharacterized proteins predicted by the genome-sequencing projects. Proteomics provides powerful tools for characterizing the protein contents of organelles. Confident protein localization, however, requires that either organelle preparations are free of contaminants or that techniques are used to discriminate between genuine organelle residents and contaminating proteins (1). Although reasonably pure preparations of some organelles, such as mitochondria, can be achieved, the components of the endomembrane system so far have proved recalcitrant to purification (2, 3). The constituent organelles of the endomembrane system have similar sizes and densities, making them difficult to separate. In addition, the proteins that reside within this system are in a constant state of flux. Endomembrane proteins traffic through the system en route to their final destination; for example, plasma membrane (PM) proteins travel although the endoplasmic reticulum (ER) and the Golgi apparatus before reaching the cell surface. Proteins within the endomembrane system also cycle between compartments; for example, ER residents continuously escape to the Golgi apparatus and are retrieved in COPI vesicles (4). Consequently, it is not sufficient merely to identify the proteins within a single organelle-enriched fraction. Instead, the steady-state distributions of proteins within the whole endomembrane system must be determined if a realistic insight into the subcellular localization of endomembrane proteins is to be achieved.Localization of organelle proteins by...

The trafficking and function of cell surface proteins in eukaryotic cells may require association with detergent-resistant sphingolipid-and sterol-rich membrane domains. The aim of this work was to obtain evidence for lipid domain phenomena in plant membranes. A protocol to prepare Triton X-100 detergent-resistant membranes (DRMs) was developed using Arabidopsis (Arabidopsis thaliana) callus membranes. A comparative proteomics approach using two-dimensional difference gel electrophoresis and liquid chromatography-tandem mass spectrometry revealed that the DRMs were highly enriched in specific proteins. They included eight glycosylphosphatidylinositol-anchored proteins, several plasma membrane (PM) ATPases, multidrug resistance proteins, and proteins of the stomatin/prohibitin/hypersensitive response family, suggesting that the DRMs originated from PM domains. We also identified a plant homolog of flotillin, a major mammalian DRM protein, suggesting a conserved role for this protein in lipid domain phenomena in eukaryotic cells. Lipid analysis by gas chromatography-mass spectrometry showed that the DRMs had a 4-fold higher sterol-to-protein content than the average for Arabidopsis membranes. The DRMs were also 5-fold increased in sphingolipid-to-protein ratio. Our results indicate that the preparation of DRMs can yield a very specific set of membrane proteins and suggest that the PM contains phytosterol and sphingolipid-rich lipid domains with a specialized protein composition. Our results also suggest a conserved role of lipid modification in targeting proteins to both the intracellular and extracellular leaflet of these domains. The proteins associated with these domains provide important new experimental avenues into understanding plant cell polarity and cell surface processes.Biological membranes consist of a perplexing number of lipids (Edidin, 2003a). The classical model of membranes assumes that these lipids form a homogeneous fluid-like or liquid-disordered (l d ) phase, which allows free diffusion of individual molecules and resident proteins (Edidin, 2003b). However, numerous recent studies on model membranes have demonstrated that certain lipids, in particular sphingolipids and cholesterol, may form relatively stable clusters by tight self-association, thus segregating them from surrounding phospholipids (Schroeder et al., 1994; Ahmed et al., 1997;Dietrich et al., 2001;Silvius, 2003). The association of rigid sterol molecules with the long and saturated acyl chains of sphingolipids results in the formation of a more organized, liquidordered (l o ) phase; l o and l d phases can coexist in the same membrane (Brown and London, 1998;Edidin, 2003b). The lipid raft hypothesis postulates that a sterol-and sphingolipid-rich l o phase is also present in cell membranes and that it forms discrete microdomains or lipid rafts that diffuse in the bulk of the l d phospholipid phase (Simons and Ikonen, 1997;Mayor and Rao, 2004).There is substantial evidence supporting the existence of plasma membrane (PM) domains in ...

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 β(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.bioenergy | glucuronoxylan | glycosyltransferase | plant cell wall | polysaccharide

Xylan, a hemicellulosic component of the plant cell wall, is one of the most abundant polysaccharides in nature. In contrast to dicots, xylan in grasses is extensively modified by α-(1,2)-and α-(1,3)-linked arabinofuranose. Despite the importance of grass arabinoxylan in human and animal nutrition and for bioenergy, the enzymes adding the arabinosyl substitutions are unknown. Here we demonstrate that knocking-down glycosyltransferase (GT) 61 expression in wheat endosperm strongly decreases α-(1,3)-linked arabinosyl substitution of xylan. Moreover, heterologous expression of wheat and rice GT61s in Arabidopsis leads to arabinosylation of the xylan, and therefore provides gain-of-function evidence for α-(1,3)-arabinosyltransferase activity. Thus, GT61 proteins play a key role in arabinoxylan biosynthesis and therefore in the evolutionary divergence of grass cell walls.type II cell walls | second-generation biofuels | dietary fiber C ell walls provide shape and strength to different plant cell types and, moreover, constitute the majority of plant biomass. The cell wall composition of grasses, including the three most productive food crops, rice, wheat, and maize, and the energy crops miscanthus and sugarcane, diverged during evolution from dicots. A major distinguishing feature of grass cell walls is the prevalence and structure of the hemicellulosic component xylan (1). Xylan consists of a linear β-(1,4)-D-xylopyranose (Xylp) chain. It is most commonly substituted by arabinofuranose (Araf) on the C2-or C3-position in arabinoxylan (AX), and (4-O-methyl-) glucuronosyl side chains on the C2-position in glucuronoarabinoxylan (GAX) and glucuronoxylan (GX). The primary and secondary cell walls of grasses contain substantial amounts of GAX, which is also found in primary cell walls of dicots, but at much lower abundance (1, 2). In contrast, xylan in secondary cell walls of dicots is relatively abundant but devoid of arabinosyl side chains (2). The functional significance of the different side chains in planta is largely unknown. In grasses α-(1-3)-linked arabinofuranosyl substitutions can be esterified with p-coumaric or ferulic acid, the latter forming cross-links with other (G)AX chains (3) or with lignin (4). Cross-linking of cell-wall polymers is critical in limiting the digestibility of polysaccharides for bioenergy production and animal feed. In addition, AX has a role as dietary fiber in human foods, particularly in wheat flour, where it constitutes 65-70% of the nonstarch polysaccharide (5). The degree of arabinosylation and feruloylation of AX also determines whether it occurs as soluble or insoluble dietary fiber, which confer different benefits to human health (6).In Arabidopsis thaliana (Arabidopsis), several glycosyltransferases of the GT43 and GT47 families have been shown to be involved in the biosynthesis of the xylan backbone, including IRX9, IRX10, and IRX14 (2). The only enzymes characterized so far that decorate the xylan backbone are members of the GT8 family, GUX1 and GUX2, which are required for gl...