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In vivo functional discrimination between plant thioredoxins by heterologous expression in the yeast Saccharomyces cerevisiae.

ABSTRACT: Whereas vertebrates possess only two thioredoxin genes, higher plants present a much greater diversity of thioredoxins. For example, Arabidopsis thaliana has five cytoplasmic thioredoxins (type h) and at least as many chloroplastic thioredoxins. The abundance of plant thioredoxins leads to the question whether the various plant thioredoxins play a similar role or have specific functions. Because most of these proteins display very similar activities on artificial or biological substrates in vitro, we developed an in vivo approach to answer this question. The disruption of both of the two Saccharomyces cerevisiae thioredoxin genes leads to pleiotropic effects including methionine auxotrophy, H2O2 hypersensitivity, altered cell cycle characteristics, and a limited ability to use methionine sulfoxide as source of methionine. We expressed eight plant thioredoxins (six cytoplasmic and two chloroplastic) in yeast trx1, trx2 double mutant cells and analyzed the different phenotypes. Arabidopsis type h thioredoxin 2 efficiently restored sulfate assimilation whereas Arabidopsis type h thioredoxin 3 conferred H2O2 tolerance. All thioredoxins tested could complement for reduction of methionine sulfoxide, whereas only type h thioredoxins were able to complement the cell cycle defect. These findings clearly indicate that specific interactions between plant thioredoxins and their targets occur in vivo.

SUBMITTER: Mouaheb N

PROVIDER: S-EPMC19738 | biostudies-literature | 1998 Mar

REPOSITORIES: biostudies-literature

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In vivo functional discrimination between plant thioredoxins by heterologous expression in the yeast Saccharomyces cerevisiae.

Mouaheb N N Thomas D D Verdoucq L L Monfort P P Meyer Y Y

Proceedings of the National Academy of Sciences of the United States of America 19980301 6

Whereas vertebrates possess only two thioredoxin genes, higher plants present a much greater diversity of thioredoxins. For example, Arabidopsis thaliana has five cytoplasmic thioredoxins (type h) and at least as many chloroplastic thioredoxins. The abundance of plant thioredoxins leads to the question whether the various plant thioredoxins play a similar role or have specific functions. Because most of these proteins display very similar activities on artificial or biological substrates in vitr ...[more]

PMID: 9501259

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Project description:When expressed in Saccharomyces cerevisiae using either of two constitutive yeast promoters (PGK1prom and CCW12prom), the transporters CDT-1 and CDT-2 from the filamentous fungus Neurospora crassa are able to catalyze, respectively, active transport and facilitated diffusion of cellobiose (and, for CDT-2, also xylan and its derivatives). In S. cerevisiae, endogenous permeases are removed from the plasma membrane by clathrin-mediated endocytosis and are marked for internalization through ubiquitinylation catalyzed by Rsp5, a HECT class ubiquitin:protein ligase (E3). Recruitment of Rsp5 to specific targets is mediated by a 14-member family of endocytic adaptor proteins, termed α-arrestins. Here we demonstrate that CDT-1 and CDT-2 are subject to α-arrestin-mediated endocytosis, that four α-arrestins (Rod1, Rog3, Aly1, and Aly2) are primarily responsible for this internalization, that the presence of the transport substrate promotes transporter endocytosis, and that, at least for CDT-2, residues located in its C-terminal cytosolic domain are necessary for its efficient endocytosis. Both α-arrestin-deficient cells expressing CDT-2 and otherwise wild-type cells expressing CDT-2 mutants unresponsive to α-arrestin-driven internalization exhibit an increased level of plasma membrane-localized transporter compared to that of wild-type cells, and they grow, utilize the transport substrate, and generate ethanol anaerobically better than control cells.ImportanceEthanolic fermentation of the breakdown products of plant biomass by budding yeast Saccharomyces cerevisiae remains an attractive biofuel source. To achieve this end, genes for heterologous sugar transporters and the requisite enzyme(s) for subsequent metabolism have been successfully expressed in this yeast. For one of the heterologous transporters examined in this study, we found that the amount of this protein residing in the plasma membrane was the rate-limiting factor for utilization of the cognate carbon source (cellobiose) and its conversion to ethanol.

Dataset Information

In vivo functional discrimination between plant thioredoxins by heterologous expression in the yeast Saccharomyces cerevisiae.

Publications

In vivo functional discrimination between plant thioredoxins by heterologous expression in the yeast Saccharomyces cerevisiae.

Similar Datasets

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