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...
We describe a proteomics method for determining the subcellular localization of membrane proteins. Organelles are partially separated using centrifugation through selfgenerating density gradients. Proteins from each organelle co-fractionate and therefore exhibit similar distributions in the gradient. Protein distributions can be determined through a series of pair-wise comparisons of gradient fractions, using cleavable ICAT to enable relative quantitation of protein levels by MS. The localization of novel proteins is determined using multivariate data analysis techniques to match their distributions to those of proteins that are known to reside in specific organelles. Using this approach, we have simultaneously demonstrated the localization of membrane proteins in both the endoplasmic reticulum and the Golgi apparatus in Arabidopsis. Localization of organelle proteins by isotope tagging is a new tool for high-throughput protein localization, which is applicable to a wide range of research areas such as the study of organelle function and protein trafficking.
Background: Cell growth underlies many key cellular and developmental processes, yet a limited number of studies have been carried out on cell-growth regulation. Comprehensive studies at the transcriptional, proteomic and metabolic levels under defined controlled conditions are currently lacking.
Background: iTRAQ™ technology for protein quantitation using mass spectrometry is a recent, powerful means of determining relative protein levels in up to four samples simultaneously. Although protein identification of samples generated using iTRAQ may be carried out using any current identification software, the quantitation calculations have been restricted to the ProQuant software supplied by Applied Biosciences. i-Tracker software has been developed to extract reporter ion peak ratios from non-centroided tandem MS peak lists in a format easily linked to the results of protein identification tools such as Mascot and Sequest. Such functionality is currently not provided by ProQuant, which is restricted to matching quantitative information to the peptide identifications from Applied Biosciences' Interrogator™ software.
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.
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