kassel university press, ISBN: 978-3-933146-69-4, 2001
Zugl.: Kassel, Univ., Diss. 2001
Content: Retrograde membrane-traffic from the Golgi complex to the endoplasmic reticulum (ER) is driven by COP I coated vesicles. Mutations in the COP I coated vesicle machinery lead to the mislocalization of Emp47p to the vacuole where it is rapidly degraded by vacuolar proteases. In my work I used chemical mutagenesis to generate a randomly mutagenized yeast cell population in which I was looking for temperature sensitive cells with reduced levels of Emp47p. One mutant that displayed a reduction of Emp47p due to a missorting of the protein to the vacuole proved to be defective in CYS3, the gene coding for cystathionine-g-lyase (Cys3p). Evidence is presented for the conclusion that it is the inability of cys3 mutant cells to produce cysteine, the primary source of cytosolic reducing power in yeast, that contributes structurally and functionally to the active centers of dithiol- and monothiol-proteins as well as to short tripeptide GSH, which interferes with the steady state localization of Emp47p. I furthermore provide evidence for the general importance of glutathione in the protection against heat- and oxidative-stress and I identified a particular phospholipid, CDP-diacylglycerol, as a GSH-dependent target of the temperature adaptation process defective in cys3-2D mutants.
In the present study I also characterized the intracellular localization and the recycling properties for the hitherto uncharacterized gene-product of YLR080W, a non-essential type I transmembrane protein with 45% sequence identity to Emp47p. I could show that the consensus di-lysine-based ER-retrieval signals at the C-terminus of Ylr080wp was functional and restricted the subcellular steady state distribution of the protein to the ER-Golgi interface. At the early logarithmic growth-phase, localization of Ylr080wp was indistinguishable from that of the Golgi-protein Emp47p. Towards stationary phase however, and in marked contrast to the behavior of Emp47p, expression of Ylr080wp was at least 50-fold enhanced. At the same time the protein redistributed from the Golgi to the ER. Similar to Emp47p, the localisation of Ylr080wp was dependent on the di-lysine motif and on coatomer. Mutations of the di-lysine signal and mutations in genes coding for coatomer-subunits both resulted in mistargeting of Ylr080wp to the vacuole. Blocking protein-exit from the ER by means of a conditional mutation in the gene coding for the COP II component Sec23p and simultaneously blocking protein synthesis lead to the redistribution of Ylr080wp to the ER, providing direct evidence for recycling from the Golgi to the ER.
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