Biomedical subjects

Anne Marmagne

Publications and source records attributed to Anne Marmagne.

4 recordsLinked to original sources

PubMed2006

A versatile method for deciphering plant membrane proteomes.

Proteomics is a very powerful approach to link the information contained in sequenced genomes, like that of Arabidopsis, to the functional knowledge provided by studies of plant cell compartments. This article summarizes the different steps of a versatile strategy that has been developed to decipher plant membrane proteomes. Initiated with envelope membranes from spinach chloroplasts, this strategy has been adapted to thylakoids, and further extended to a series of membranes from the model plant Arabidopsis: chloroplast envelope membranes, plasma membrane, and mitochondrial membranes. The first step is the preparation of highly purified membrane fractions from plant tissues. The second step in the strategy is the fractionation of membrane proteins on the basis of their physico-chemical properties. Chloroform/methanol extraction and washing of membranes with NaOH, NaCl or any other agent led to the simplification of the protein content of the fraction to be analysed. The next step is the genuine proteomic step, i.e. the separation of proteins by 1D-gel electrophoresis followed by in-gel proteolytic digestion of the polypeptides, analysis of the proteolytic peptides using mass spectrometry, and protein identification by searching through databases. The last step is the validation of the procedure by checking the subcellular location. The results obtained by using this strategy demonstrate that a combination of different proteomics approaches, together with bioinformatics, indeed provide a better understanding of the biochemical machinery of the different plant membranes at the molecular level.

Alkalies↗

PubMed2006

Purification and fractionation of membranes for proteomic analyses.

Proteomics is a very powerful approach to link the information contained in sequenced genomes, such as Arabidopsis, to the functional knowledge provided by studies of plant cell compartments. However, membrane proteomics remains a challenge. One way to bring into view the complex mixture of proteins present in a membrane is to develop proteomic analyses based on (1) the use of highly purified membrane fractions and (2) fractionation of membrane proteins to retrieve as many proteins as possible (from the most to the less hydrophobic ones). To illustrate such strategies, we choose two types of membranes, the plasma membrane and the chloroplast envelope membranes. Both types of membranes can be prepared in a reasonable degree of purity from different types of tissues: the plasma membrane from cultured cells and the chloroplast envelope membrane from whole plants. This article is restricted to the description of methods for the preparation of highly purified and characterized plant membrane fractions and the subsequent fractionation of these membrane proteins according to simple physicochemical criteria (i.e., chloroform/methanol extraction, alkaline or saline treatments) for further analyses using modern proteomic methodologies.

Arabidopsis↗

PubMed2004

Arabidopsis formin AtFH6 is a plasma membrane-associated protein upregulated in giant cells induced by parasitic nematodes.

Plant-parasitic nematodes Meloidogyne spp induce an elaborate permanent feeding site characterized by the redifferentiation of root cells into multinucleate and hypertrophied giant cells. We have isolated by a promoter trap strategy an Arabidopsis thaliana formin gene, AtFH6, which is upregulated during giant cell formation. Formins are actin-nucleating proteins that stimulate de novo polymerization of actin filaments. We show here that three type-I formins were upregulated in giant cells and that the AtFH6 protein was anchored to the plasma membrane and uniformly distributed. Suppression of the budding defect of the Saccharomyces cerevisiae bni1Delta bnr1Delta mutant showed that AtFH6 regulates polarized growth by controlling the assembly of actin cables. Our results suggest that AtFH6 might be involved in the isotropic growth of hypertrophied feeding cells via the reorganization of the actin cytoskeleton. The actin cables would serve as tracks for vesicle trafficking needed for extensive plasma membrane and cell wall biogenesis. Therefore, determining how plant parasitic nematodes modify root cells into giant cells represents an attractive system to identify genes that regulate cell growth and morphogenesis.

Actins↗

PubMed2004

Identification of new intrinsic proteins in Arabidopsis plasma membrane proteome.

Identification and characterization of anion channel genes in plants represent a goal for a better understanding of their central role in cell signaling, osmoregulation, nutrition, and metabolism. Though channel activities have been well characterized in plasma membrane by electrophysiology, the corresponding molecular entities are little documented. Indeed, the hydrophobic protein equipment of plant plasma membrane still remains largely unknown, though several proteomic approaches have been reported. To identify new putative transport systems, we developed a new proteomic strategy based on mass spectrometry analyses of a plasma membrane fraction enriched in hydrophobic proteins. We produced from Arabidopsis cell suspensions a highly purified plasma membrane fraction and characterized it in detail by immunological and enzymatic tests. Using complementary methods for the extraction of hydrophobic proteins and mass spectrometry analyses on mono-dimensional gels, about 100 proteins have been identified, 95% of which had never been found in previous proteomic studies. The inventory of the plasma membrane proteome generated by this approach contains numerous plasma membrane integral proteins, one-third displaying at least four transmembrane segments. The plasma membrane localization was confirmed for several proteins, therefore validating such proteomic strategy. An in silico analysis shows a correlation between the putative functions of the identified proteins and the expected roles for plasma membrane in transport, signaling, cellular traffic, and metabolism. This analysis also reveals 10 proteins that display structural properties compatible with transport functions and will constitute interesting targets for further functional studies.

Arabidopsis↗