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.

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.

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.

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.

Outcome of treatment in alcoholic women.

One hundred and seventy-eight females (mean age 40.6 +/- 10.2 years) were retrospectively studied by questionnaires for a mean duration of 46 (17-75) months. Sixty-two percent were married or living maritally. One third were working. The mean alcohol intake was 157 +/- 76 g/day and 57.3% had alcohol dependence for less than 5 years. Twenty-seven patients (15%) were lost to follow-up; out of the 151 remaining patients, 7 (4%) refused to answer and 18 (12%) died. Suicide and alcoholism complications were a frequent cause of death. One hundred and twenty-six questionnaires were obtained. Twenty-eight women (22%) were abstinent. A good outcome determined by the state of alcoholization (abstinence or moderate consumption) and the improvement of quality of life, was found in 44% of patients. Absence of marital life and greater alcohol intake were related to a poor outcome, whereas enrollment in a fellowship of recovering alcoholics was more frequent in abstinent patients. The mortality rate was important in alcoholic females. A number of factors were related to the outcome.