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P Dupree

Publications and source records attributed to P Dupree.

At least 19 recordsLinked to original sources
PubMed2001

Quantitative and reproducible two-dimensional gel analysis using Phoretix 2D Full.

Quantitative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is used to determine changes in individual protein levels in complex protein mixtures. To provide reliable data, the software used for 2-D gel image analysis must provide a linear response over a wide dynamic range of data output. Here, we show that Phoretix 2D Full analysis of 2-D gels stained with colloidal Coomassie Brilliant Blue G-250 can provide a linear measure of changes in protein quantity. We show using a complex mixture of Arabidopsis thaliana proteins, that this is true for essentially all focused proteins, in a data output range greater than three orders of magnitude. An analysis of the factors that affect errors in the results demonstrated that reproducibility of the data is significantly improved by user seeding, whereas it is reduced by use of the background subtraction algorithms.

Algorithms↗
PubMed2001

Identification and characterization of GONST1, a golgi-localized GDP-mannose transporter in Arabidopsis.

Transport of nucleotide sugars across the Golgi apparatus membrane is required for the luminal synthesis of a variety of plant cell surface components. We identified an Arabidopsis gene encoding a nucleotide sugar transporter (designated GONST1) that we have shown by transient gene expression to be localized to the Golgi. GONST1 complemented a GDP-mannose transport-defective yeast mutant (vrg4-2), and Golgi-rich vesicles from the complemented strain displayed increased GDP-mannose transport activity. GONST1 promoter::beta-glucuronidase studies suggested that this gene is expressed ubiquitously. The identification of a Golgi-localized nucleotide sugar transporter from plants will allow the study of the importance of this class of proteins in the synthesis of plant cell surface components such as cell wall polysaccharides.

Amino Acid Sequence↗
PubMed2000

A proteomic analysis of organelles from Arabidopsis thaliana.

We introduce the use of Arabidopsis thaliana callus culture as a system for proteomic analysis of plant organelles using liquid-grown callus. This callus is relatively homogeneous, reproducible and cytoplasmically rich, and provides organelles in sufficient quantities for proteomic studies. A database was generated of mitochondrial, endoplasmic reticulum (ER), Golgi/prevacuolar compartment and plasma membrane (PM) markers using two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (2-D SDS-PAGE) and peptide sequencing or mass spectrometric methods. The major callus membrane-associated proteins were characterised as being integral or peripheral by Triton X-114 phase partitioning. The database was used to define specific proteins at the Arabidopsis callus plasma membrane. This database of organelle proteins provides the basis for future characterisation of the expression and localisation of novel plant proteins.

Amino Acid Sequence↗
PubMed1999

Glycosylphosphatidylinositol-anchored cell-surface proteins from Arabidopsis.

Remodeling of the plant cell surface occurs during the establishment of cell polarity, cellular differentiation, and organ development. This report demonstrates the existence of multiple glycosylphosphatidylinositol (GPI)-anchored proteins in the model plant Arabidopsis. Using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), we also show that GPI-anchored proteins are a relatively abundant class of protein and that they are present at the plant plasma membrane. Furthermore, some of these proteins are released into the extracellular matrix. At least one of these is an arabinogalactan protein (AGP), a class of proteins known to be associated with cellular differentiation. Analysis of the amino acid sequences of two novel AGP-like proteins from Arabidopsis predicts that these proteins contain consensus signals for GPI-anchor addition. These findings support a model where GPI-anchored proteins are involved in the generation of specialized cell surfaces and extracellular signaling molecules.

Amino Acid Sequence↗
PubMed1999

The golgi bypassed

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Journal Article↗
PubMed1998

The plant Golgi apparatus.

The plant Golgi apparatus has an important role in protein glycosylation and sorting, but is also a major biosynthetic organelle that synthesises large quantities of cell wall polysaccharides. This is reflected in the organisation of the Golgi apparatus as numerous individual stacks of cisternae that are dispersed through the cell. Each stack is polarised: the shape of the cisternae and the staining of the membranes change in a cis to trans direction, and the cisternae on the trans side contain more polysaccharides. Numerous glycosyltransferases are required for the synthesis of the complex cell wall polysaccharides. Microscopy and biochemical fractionation studies suggest that these enzymes are compartmentalised within the stack. Although there is no obvious cis Golgi network, the trans-most cisterna or trans Golgi network often buds clathrin-coated and sometimes smooth dense vesicles as well. Here, vacuolar proteins are sorted from the secreted proteins and polysaccharides. This review highlights unique aspects of the organisation and function of the plant Golgi apparatus. Fundamentally similar processes probably underlie Golgi organisation in all organisms, and consideration of the plant Golgi specialisations can therefore be generally informative, as well as being of central importance to plant cell biology.

Animals↗
PubMed1998

Use of a proteome strategy for tagging proteins present at the plasma membrane.

A plasma membrane (PM) fraction was purified from Arabidopsis thaliana using a standard procedure and analyzed by two-dimensional (2D) gel electrophoresis. The proteins were classified according to their relative abundance in PM or cell membrane supernatant fractions. Eighty-two of the 700 spots detected on the PM 2D gels were microsequenced. More than half showed sequence similarity to proteins of known function. Of these, all the spots in the PM-specific and PM-enriched fractions, together with half of the spots with similar abundance in PM fraction and supernatant, have previously been found at the PM, supporting the validity of this approach. Extrapolation from this analysis indicates that (i) approximately 550 polypeptides found at the PM could be resolved on 2D gels; (ii) that numerous proteins with multiple locations are found at the PM; and (iii) that approximately 80% of PM-specific spots correspond to proteins with unknown function. Among the later, half are represented by ESTs or cDNAs in databases. In this way, several unknown gene products were potentially localized to the PM. These data are discussed with respect to the efficiency of organelle proteome approaches to link systematically genomic data to genome expression. It is concluded that generalized proteomes can constitute a powerful resource, with future completion of Arabidopsis genome sequencing, for genome-wide exploration of plant function.

Amino Acid Sequence↗
PubMed1998

Targeting of active sialyltransferase to the plant Golgi apparatus.

Glycosyltransferases in the Golgi apparatus synthesize cell wall polysaccharides and elaborate the complex glycans of glycoproteins. To investigate the targeting of this type of enzyme to plant Golgi compartments, we generated transgenic Arabidopsis plants expressing alpha-2,6-sialyltransferase, a glycosyltransferase of the mammalian trans-Golgi cisternae and the trans-Golgi network. Biochemical analysis as well as immunolight and immunoelectron microscopy of these plants indicate that the protein is targeted specifically to the Golgi apparatus. Moreover, the protein is predominantly localized to the cisternae and membranes of the trans side of the organelle. When supplied with the appropriate substrates, the enzyme has significant alpha-2,6-sialyltransferase activity. These results indicate a conservation of glycosyltransferase targeting mechanisms between plant and mammalian cells and also demonstrate that glycosyltransferases can be subcompartmentalized to specific cisternae of the plant Golgi apparatus.

Animals↗
PubMed1996

Plant embryogenesis: cell division forms a pattern.

The recent discovery that another gene involved in plant embryogenesis encodes a probable regulator of membrane traffic demonstrates the central role of cell wall synthesis and deposition in plant development.

Arabidopsis↗
PubMed1995

Co-operative regulation of endocytosis by three Rab5 isoforms.

Rab proteins are small GTPases involved in the regulation of membrane traffic. Rab5a has been shown to regulate transport in the early endocytic pathway. Here we report the isolation of cDNA clones encoding two highly related isoforms, Rab5b and Rab5c. The two proteins share with Rab5a all the structural features required for regulation of endocytosis. Rab5b and Rab5c colocalize with the both transferrin receptor and Rab5a, stimulate the homotypic fusion between early endosomes in vitro and increase the rate of endocytosis when overexpressed in vivo. These data demonstrate that three Rab5 isoforms cooperate in the regulation of endocytosis in eukaryotic cells.

Amino Acid Sequence↗
PubMed1995

Isolation of a murine cDNA clone encoding Rab19, a novel tissue-specific small GTPase.

Using a rapid amplification of cDNA ends (RACE) cloning approach, we have isolated a cDNA clone encoding Rab19, a novel small GTPase of the Rab subfamily contained within partial sequences previously described [Chavrier et al., Gene 112 (1992) 261-264]. Northern blot analysis of the distribution of the rab19 mRNA in various adult mouse tissues and NIH 3T3 fibroblasts revealed that rab19 is expressed in a tissue-specific manner. The rab19 transcript was detected at high levels in intestine, lung and spleen, and at a lower level in kidney. In contrast, liver, brain, heart and NIH 3T3 fibroblasts contain only very little or no detectable rab19 mRNA. Therefore, Rab19 is likely to represent a novel tissue- or cell type-specific small GTPase.

Amino Acid Sequence↗
PubMed1995

A deficiency of the small GTPase rab8 inhibits membrane traffic in developing neurons.

One of the major activities of developing neurons is the transport of new membrane to the growing axon. Candidates for playing a key role in the regulation of this intense traffic are the small GTP-binding proteins of the rab family. We have used hippocampal neurons in culture and analyzed membrane traffic activity after suppressing the expression of the small GTP-binding protein rab8. Inhibition of protein expression was accomplished by using sequence-specific antisense oligonucleotides. While rab8 depletion resulted in the blockage of morphological maturation in 95% of the neurons, suppression of expression of another rab protein, rab3a, had no effect, and all neurons developed normal axons and dendrites. The impairment of neuronal maturation by rab8 antisense treatment was due to inhibition of membrane traffic. Thus, by using video-enhanced differential interference contrast microscopy, we observed in the rab8-depleted cells a dramatic reduction in the number of vesicles undergoing anterograde transport. Moreover, by incubating antisense-treated neurons with Bodipy-labeled ceramide, a fluorescent marker for newly formed exocytic vesicles, we observed fluorescence labeling restricted to the Golgi apparatus, whereas in control cells labeling was found also in the neurites. These results show the role of the small GTPase rab8 in membrane traffic during neuronal process outgrowth.

Animals↗
PubMed1994

Mapping of Ras-related GTP-binding proteins by GTP overlay following two-dimensional gel electrophoresis.

For identification of Rab, Rac, Rho, Ral, Rap, and Arf proteins on two-dimensional polyacrylamide gels, we have expressed full-length cDNAs of members of these protein families with the T7 RNA polymerase-recombinant vaccinia virus expression system. Membrane preparations from cells expressing the cDNAs were subjected to high-resolution two-dimensional polyacrylamide gel electrophoresis followed by [alpha-32P]GTP ligand blotting. We have mapped 28 small GTP-binding proteins relative to their isoelectric points and according to their molecular weights and by immunoblotting with specific antibodies. Rab and Rho proteins could be specifically identified by extraction of streptolysin O-permeabilized Madin-Darby canine kidney (MDCK) cells with Rab- and Rho-GDP dissociation inhibitor. We applied the reference mapping to analyze the GTP-binding patterns of synaptosome fractions from rat brain. The purified synaptosomes exhibited specific enrichment of Rab3a, Rab5a, Ral, and several other GTPases. This approach and the map we have produced should provide a useful aid for the analysis of the expression and localization of members of all families of small GTP-binding proteins in various cell types and subcellular fractions.

Animals↗
PubMed1994

VIP21-Caveolin, a protein of the trans-Golgi network and caveolae.

VIP21-Caveolin is a component of the filamentous coat surrounding the invaginations of the plasma membrane called caveolae. Unlike the vesicular coat proteins identified so far, VIP21-Caveolin can be classified as an integral membrane protein. Furthermore, it is found in high molecular mass oligomers. Based on its localisation in specialised membrane subdomains, a role for VIP21-Caveolin in membrane protein sorting has been proposed.

Carrier Proteins↗
PubMed1994

Isolation of a mouse cDNA encoding Rab23, a small novel GTPase expressed predominantly in the brain.

The full-length cDNA encoding Rab23, a novel Ras-related small GTPase, was isolated using the sequence of a previously described [Chavrier et al., Gene 112 (1992) 261-264] short cDNA fragment and the rapid amplification of cDNA ends (RACE) PCR techniques. The deduced amino acid sequence was not very closely related to any previously described small GTPase, but was within the Rab subfamily. A Northern analysis revealed that the rab23 mRNA is predominantly expressed in the brain, which places the protein, together with Rab3a and Rab15, in the group of small GTPases characteristic of the nervous system.

Amino Acid Sequence↗
PubMed1994

Cloning and subcellular localization of novel rab proteins reveals polarized and cell type-specific expression.

Small GTPases of the rab subfamily are specific regulators of vesicular transport. The intracellular localization of these proteins has been mostly investigated in cultured cells where they have been found associated with distinct compartments of the exocytic and endocytic pathways. Using a PCR-based cloning approach we have recently identified several novel rab proteins, extending the total number of this family to more than 30 members. Here, we have investigated the mRNA expression in different tissues and the intracellular localization in organ cryosections of two rab proteins, rab18 and rab20. Both northern blot analysis and confocal immunofluorescence microscopy demonstrated that these proteins are expressed in a tissue- and cell type-dependent manner. Despite their presence in non-polarized cells and polarized cells, both proteins are highly expressed on the apical side of kidney tubule epithelial cells. Electron microscopic studies revealed that rab18 and rab20 are located in apical dense tubules, endocytic structures underlying the apical plasma membrane, suggesting that they play a role in apical endocytosis/recycling. In intestinal epithelial cells as well, both proteins were localized apically, but, in addition, rab18 was found associated with the basolateral domain, suggesting that this protein is not restricted to the apical transport machinery of polarized epithelial cells. The results demonstrate that, depending on the epithelial cell type, rab proteins that are also expressed in non-polarized cells may be enriched in one or both surface domains. Together with the observed tissue- and cell type-dependent variation in the expression of the rab proteins, this suggests that the large number of mammalian rab proteins might reflect the specific requirements in the organization of membrane traffic encountered by different cell types.

Amino Acid Sequence↗