Protein Urmylierung in Hefe - Mechanismus und funktionelle Signifikanz

Details zum Projekt
Projektlaufzeit: 06/201605/2019


Ubiquitin-related modifier 1 (Urm1) from budding yeast is a ubiquitin family (UbF) member with protein and tRNA modification functions typical of eukaryal UbF modifiers and prokaryal sulphur carriers, respectively. Despite its well documented role in tRNA modification/thiolation, only one protein (Ahp1) conjugated to Urm1 is known to date from yeast stressing that both the significance of urmylation and Urm1 target identity are open issues in Saccharomyces cerevisiae. Nonetheless, based on the recent isolation of various human URM1 targets, assigning biological functions to protein urmylation seems feasible. Therefore, we propose two complementary strategies for studying Urm1 pathways in yeast. In the first, bioinformatics will identify selected yeast homologs of human targets for direct urmylation and cross-complementation assays. This has the potential to identify bona fide Urm1 targets and study conservation within Urm1 modification pathways among eukaryotes. The second uses a TAP-tagged bait of Urm1 for proteome-wide Urm1 target identification in yeast by mass spectrometry. To validate urmylation, candidates from each approach will be assessed by independent Urm1 conjugation assays and bona fide Urm1 interactions are correlated with phenotypic profiles obtained from strains mutated in the appropriate Urm1 target genes. The latter holds promise to ask how urmylation may affect target protein function and importantly, whether protein urmylation and tRNA thiolation represent related or independent branches of Urm1 modification pathways. In sum, by combining molecular, biochemical and bioinformatical methods, the proposal is likely to provide new insights into the biology and significance of the eukaryal Urm1 modification pathway.

Zuletzt aktualisiert 2018-26-04 um 13:40