Abstract
(Englisch)
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We have screened the entire collection of viable yeast gene disruptants generated by the EUROFAN consortium for defects in the endocytic pathway. The high through put screening was performed by growing each yeast disruptant individually, then incubating these strains with Lucifer yellow CH. The cells were then visualized using fluorescence microscopy. Wild type cells accumulate Lucifer yellow in the vacuole via fluid phase endocytosis, whereas endocytic mutants do not. All mutants that were defective for endocytosis were then put through a battery of others tests, including test for synthetic lethality with a mutant in the vacuolar ATPase (vma2), secretion of the vacuolar enzyme, carboxypeptidase Y, and secretion. Synthetic lethality with vma2 is a frequent characteristic of endocytosis mutants. Since the vacuole biosynthesis and endocytic pathways overlap many endocytic mutants also show defects in vacuole biogenesis as detected by carboxypeptidase Y secretion. We tested secretion to examine the specificity of the membrane trafficking defect. These results were published and identified a number of new endocytosis mutants (4).
During the course of these experiments we noticed several interesting features of some of these mutants that led us to study them in more detail. One of the mutants, encoding a v-SNARE protein, involved in membrane fusion was shown to be localized to and work at an early stage of endocytosis (1). Another mutant displayed a novel endocytic phenotype with Lucifer yellow because the dye was accumulated inside the cells in a non-vacuolar location. We could show that this gene, RCY1, encoded an F-Box protein that is required for recycling of endocytic content (2). The Rcy1 protein was shown to interact with the ubiquitin ligase Skp1p, which also plays a role in recycling (5). We also identified a novel EH-domain protein, Ede1p, that is required for efficient internalization of receptors and permeases at the plasma membrane (3).
Therefore, we have completed our stated goals of the EUROFAN 2 project because we have screened through the entired collection of viable disruptants generated in the program and have identified those genes required for endocytosis. In fact, we went beyond what was originally proposed because we could further characterize some of the mutants in detail and developed new tools in the study of endocytosis and recycling.
1.Séron, K., V. Tieaho, C. Prescianotto-Baschong, T. Aust, M.O. Blondel, P. Guillaud, G. Devilliers, O. W. Rossanese, B.S. Glick, H. Riezman, S. Keränen and R. Haguenauer-Tsapis (1998) A yeast t-SNARE involved in endocytosis. Mol. Biol. Cell, 9, 2873-2889.
2.Wiederkehr, A., S. Avaro, C. Prescianotto-Baschong, R. Haguenauer-Tsapis and H. Riezman (2000) The F-box protein Rcy1p is involved in endocytic membrane traffic and recycling out of an early endosome in S. cerevisiae. J. Cell Biol., 149, 397-410.
3.Gagny B., A. Wiederkehr, P. Dumoulin, B. Winsor, H. Riezman, and R. Haguenauer-Tsapis (2000) A novel EH domain protein of Saccharomyces cerevisiae, Ede1p, involved in endocytosis. J. Cell Sci. 113, 3309-3319.
4.Wiederkehr, A., K.D. Meier, and H. Riezman (2001) Identification and characterization of eleven S. cerevisiae mutants defective in fluid-phase endocytosis. Yeast, 18, 759-773.
5.Galan, J.M., A. Wiederkehr, J.H. Seol, R. Haguenauer-Tsapis, R.J. Deshaies, H. Riezman, and M. Peter (2001) Skp1p and the F-box protein Rcy1p form a non-SCF complex involved in recycling of the SNARE Snc1p in yeast. Mol. Cell. Biol., 21, 3105-3117.
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