Relative abundance of integral plasma membrane proteins in Arabidopsis leaf and root tissue determined by metabolic labeling and mass spectrometry.

Metabolic labeling of proteins with a stable isotope ((15)N) in intact Arabidopsis plants was used for accurate determination by mass spectrometry of differences in protein abundance between plasma membranes isolated from leaves and roots. In total, 703 proteins were identified, of which 188 were predicted to be integral membrane proteins. Major classes were transporters, receptors, proteins involved in membrane trafficking and cell wall-related proteins.

Reconstitution of water channel function and 2D-crystallization of human aquaporin 8.

Among the thirteen human aquaporins (AQP0-12), the primary structure of AQP8 is unique. By sequence alignment it is evident that mammalian AQP8s form a separate subfamily distinct from the other mammalian aquaporins. The constriction region of the pore determining the solute specificity deviates in AQP8 making it permeable to both ammonia and H(2)O(2) in addition to water.

Proteomic and functional analysis of the mitotic Drosophila centrosome.

Regulation of centrosome structure, duplication and segregation is integrated into cellular pathways that control cell cycle progression and growth. As part of these pathways, numerous proteins with well-established non-centrosomal localization and function associate with the centrosome to fulfill regulatory functions. In turn, classical centrosomal components take up functional and structural roles as part of other cellular organelles and compartments.

Characterization of a cytochrome c gene located at the gene cluster for chlorate respiration in Ideonella dechloratans.

Anaerobic chlorate respiration requires electron transport from the bacterial inner membrane to the soluble periplasmic chlorate reductase. We have recently demonstrated that soluble c cytochromes function as electron carriers for chlorate reduction in Ideonella dechloratans (Smedja Bäcklund et al. 2009).

Proteomics of plasma membranes from poplar trees reveals tissue distribution of transporters, receptors, and proteins in cell wall formation.

By exploiting the abundant tissues available from Populus trees, 3-4 m high, we have been able to isolate plasma membranes of high purity from leaves, xylem, and cambium/phloem at a time (4 weeks after bud break) when photosynthesis in the leaves and wood formation in the xylem should have reached a steady state. More than 40% of the 956 proteins identified were found in the plasma membranes of all three tissues and may be classified as "housekeeping" proteins, a typical example being P-type H(+)-ATPases. Among the 213 proteins predicted to be integral membrane proteins, transporters constitute the largest class (41%) followed by receptors (14%) and proteins involved in cell wall and carbohydrate metabolism (8%) and membrane trafficking (8%).

Periplasmic c cytochromes and chlorate reduction in Ideonella dechloratans.

The aim of this study was to clarify the pathway of electron transfer between the inner membrane components and the periplasmic chlorate reductase. Several soluble c-type cytochromes were found in the periplasm. The optical difference spectrum of dithionite-reduced periplasmic extract shows that at least one of these components is capable of acting as an electron donor to the enzyme chlorate reductase.

A facile method for expression and purification of the Alzheimer's disease-associated amyloid beta-peptide.

We report the development of a high-level bacterial expression system for the Alzheimer's disease-associated amyloid beta-peptide (Abeta), together with a scaleable and inexpensive purification procedure. Abeta(1-40) and Abeta(1-42) coding sequences together with added ATG codons were cloned directly into a Pet vector to facilitate production of Met-Abeta(1-40) and Met-Abeta(1-42), referred to as Abeta(M1-40) and Abeta(M1-42), respectively. The expression sequences were designed using codons preferred by Escherichia coli, and the two peptides were expressed in this host in inclusion bodies.

Extraction of yeast mitochondrial membrane proteins by solubilization and detergent/polymer aqueous two-phase partitioning.

Identification and characterization of membrane proteins is of increasing importance in modern proteomic studies. It is of central interest to have access to methods that combine efficient solubilization with enrichment of proteins and intact protein complexes. Separation methods have been developed based on nondenaturing detergent extraction of yeast mitochondrial membrane proteins followed by enrichment of hydrophobic proteins in aqueous two-phase system.

Purification and proteomic analysis of plant plasma membranes.

All techniques needed for proteomic analyses of plant plasma membranes are described in detail, from isolation of plasma membranes to protein identification by mass spectrometry (MS). Plasma membranes are isolated by aqueous two-phase partitioning yielding vesicles with a cytoplasmic side-in orientation and a purity of about 95%. These vesicles are turned inside-out by treatment with Brij 58, which removes soluble contaminating proteins enclosed in the vesicles as well as loosely attached proteins.

Identification of proteins released by mammalian cells that mediate DNA internalization through proteoglycan-dependent macropinocytosis.

Naked DNA plasmid represents the simplest vehicle for gene therapy and DNA-based vaccination purposes; however, the molecular mechanisms of DNA uptake in mammalian cells are poorly understood. Here, we show that naked DNA uptake occurs via proteoglycan-dependent macropinocytosis, thus challenging the concept of a specific DNA-internalizing receptor. Cells genetically deficient in proteoglycans, which constitute a major source of cell-surface polyanions, exhibited substantially decreased uptake of likewise polyanionic DNA.

Small heat shock proteins prevent aggregation of citrate synthase and bind to the N-terminal region which is absent in thermostable forms of citrate synthase.

Citrate synthase (CS) is often used in chaperone assays since this thermosensitive enzyme aggregates at moderately increased temperatures. Small heat shock proteins (sHsps) are molecular chaperones specialized in preventing the aggregation of other proteins, termed substrate proteins, under conditions of transient heat stress. To investigate the mechanism whereby sHsps bind to and stabilize a substrate protein, we here used peptide array screening covering the sequence of porcine CS (P00889).

Efficient and non-denaturing membrane solubilization combined with enrichment of membrane protein complexes by detergent/polymer aqueous two-phase partitioning for proteome analysis.

It is of central interest in membrane proteomics to establish methods that combine efficient solubilization with enrichment of proteins and intact protein complexes. We have investigated the quantitative and qualitative solubilization efficiency of five commercially available detergents using mitochondria from the yeast Saccharomyces cerevisiae as model system. Combining the zwitterionic detergent Zwittergent 3-10 and the non-ionic detergent Triton X-114 resulted in a complementary solubilization of proteins, which was similar to that of the anionic detergent sodium dodecyl sulfate (SDS).

Analysis of NOD2-mediated proteome response to muramyl dipeptide in HEK293 cells.

NOD2, a cytosolic receptor for the bacterial proteoglycan fragment muramyl dipeptide (MDP), plays an important role in the recognition of intracellular pathogens. Variants in the bacterial sensor domain of NOD2 are genetically associated with an increased risk for the development of Crohn disease, a human chronic inflammatory bowel disease. In the present study, global protein expression changes after MDP stimulation were analyzed by two-dimensional PAGE of total protein extracts of human cultured cells stably transfected with expression constructs encoding for wild type NOD2 (NOD2(WT)) or the disease-associated NOD2 L1007fsinsC (NOD2(SNP13)) variant.

Arabidopsis PDK1: identification of sites important for activity and downstream phosphorylation of S6 kinase.

The Arabidopsis thaliana protein kinase AtPDK1 was identified as a homologue of the mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1), which is involved in a number of physiological processes including cell growth and proliferation. We now show that AtPDK1, expressed in E. coli as a recombinant protein, undergoes autophosphorylation at several sites.

Detection of angiotensin II in supernatants of stimulated mononuclear leukocytes by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass analysis.

Angiotensin II (Ang II) is the major vasoactive component of the renin-angiotensin system. Several components of the renin-angiotensin system have been demonstrated in different tissues. Whereas the roles of tissue and renal renin-angiotensin system have been studied in detail, much less is known on whether the corpuscular elements of circulating blood contribute to Ang II production.

A proteomic method for the analysis of changes in protein concentrations in response to systemic perturbations using metabolic incorporation of stable isotopes and mass spectrometry.

While several techniques exist for assessing quantitative differences among proteomes representing different cell states, methods for assessing how these differences are mediated are largely missing. We present a method that allows one to differentiate between cellular processes, such as protein synthesis, degradation and PTMs which affect protein concentrations. An induced systemic perturbation of a cell culture was coupled to a replacement of the growth medium to one highly enriched in the stable isotope 15N.

Conserved methionines in chloroplasts.

Heat shock proteins counteract heat and oxidative stress. In chloroplasts, a small heat shock protein (Hsp21) contains a set of conserved methionines, which date back to early in the emergence of terrestrial plants. Methionines M49, M52, M55, M59, M62, M67 are located on one side of an amphipathic helix, which may fold back over two other conserved methionines (M97 and M101), to form a binding groove lined with methionines, for sequence-independent recognition of peptides with an overall hydrophobic character.

A peptide methionine sulfoxide reductase highly expressed in photosynthetic tissue in Arabidopsis thaliana can protect the chaperone-like activity of a chloroplast-localized small heat shock protein.

The oxidation of methionine residues in proteins to methionine sulfoxides occurs frequently and protein repair by reduction of the methionine sulfoxides is mediated by an enzyme, peptide methionine sulfoxide reductase (PMSR, EC 1.8.4.