Mechanism and significance of ubiquitin-like protein urmylation in yeast
General Genetics and Functional Genome Biology
Biochemistry
Metabolism, Biochemistry and Genetics of Microorganisms
Final Report Abstract
Using yeast as an experimental model for higher organisms including humans, we address here unclear biological aspects of ubiquitin-related modifier 1 (Urm1). Urm1 is placed at the junction of bacterial sulfur-transfer and eukaryotic conjugations. Hence, it plays dual roles in sulfuration of tRNA (i.e., tRNA thiolation) and protein conjugation (i.e., urmylation). We show both functions of Urm1 (and its activator Uba4) are conserved between yeast and humans. On analysing whether and how both functions may be interlinked, we found that both tRNA and protein modification outputs are sensitive to elevated cultivation temperatures and importantly, to sulfur stravation. Sulfur requirement directly correlates with the fact that Urm1 is activated in an S-dependent fashion by Uba4 and an upstream S-relay system. As a result, Urm1 itself becomes sulfurated (Urm1-COSH), and this thioform engages in the tRNA thiolation and protein urmylation branches. In the former, Urm1-COSH feeds the sulfur to the enzyme (tRNA thiolase) in action while in the latter, protein urmylation by Urm1-COSH seems to be induced under conditions of oxidative stress by ROS (organic peroxide; H2O2). Using antioxidant enzyme Ahp1 as an Urm1 target, we found that oxidation at a thiol in its redox-active center is necessary for urmylation at nearby lysine residues. Based on genetic and biochemical evidence, we hypothesise that following oxidation and urmylation, Ahp1 is sulfurated at its active center, to protect the antioxidant enzyme from hyperoxidation or alter it otherwise. Thus, active Urm1-COSH not only donates sulfur to the thiolase for S- transfer to tRNA (see above) but also may couple S-transfer to Ahp1 with urmylation. Our project thus elucidates redox requirements critical for urmylation and provides innovative insights into a potential link between Urm1 utilisation and oxidant stress defence of a cell. Still, to advance our knowledge on Urm1 at the mechanistic level, it will be important to have the Ahp1 urmylation reaction reconstituted in vitro upon exposure to ROS. Although it is unknown whether S-donation from Urm1 to the tRNA thiolase involves urmylation, we show in here that thiolation output is crucial for tRNA decoding function during mRNA translation. In line with this, Urm1 dependent anticodon thiolation is found to protect cells against translational errors and proteotoxic stress. Together with its new protein modifier role in oxidant stress response, we consider that the tRNA thiolation and urmylation functions of Urm1 have their share in contributing to redox and protein homeostasis of cells. Based on the unique nature underlying S-dependent urmylation and S-transfer by Urm1, our project output challenges Urm1 indexing among the ubiquitin-like protein family. Instead, it paves the way to tackle whether Urm1 rather represents an evolutionary intermediate or stepping-stone towards the emergence of present-day ubiquitin and ubiquitinlike proteins. In potential support of this notion is the reported capacity of a stand-alone bacterial SCP (ThiS) to conjugate to proteins in a lysine directed fashion like Urm1.
Publications
- (2015). Urmylation and tRNA thiolation functions of ubiquitin-like Uba4•Urm1 systems are conserved from yeast to man. FEBS Lett 589: 904-909
Jüdes A, Ebert F, Bär C, Thüring KL, Harrer A, Klassen R, Helm M, Stark MJR, Schaffrath R
(See online at https://doi.org/10.1016/j.febslet.2015.02.024) - (2016). Sulfur transfer and activation by ubiquitin-like modifier system Uba4•Urm1 link protein urmylation and tRNA thiolation in yeast. Microb Cell 3: 554-564
Jüdes A, Bruch A, Klassen R, Helm M, Schaffrath R
(See online at https://doi.org/10.15698/mic2016.11.539) - (2016). tRNA anticodon loop modifications ensure protein homeostasis and cell morphogenesis in yeast. Nucleic Acids Res 44: 10946-10959
Klassen R, Ciftci A, Johanna Funk J, Bruch A, Butter F, Schaffrath R
(See online at https://doi.org/10.1093/nar/gkw705) - (2017). Independent suppression of ribosomal +1 frameshifts by different tRNA anticodon loop modifications. RNA Biol 14: 1252-1259
Klassen R, Bruch A, Schaffrath R
(See online at https://doi.org/10.1080/15476286.2016.1267098) - (2017). Wobble uridine modifications – a reason to live, a reason to die?! RNA Biol 14: 1209-1222
Schaffrath R, Leidel SA
(See online at https://doi.org/10.1080/15476286.2017.1295204) - (2018) Protein urmylation in yeast - mechanism and functional relevance (DFG SCHA750/15-2). CINSaT Newsletter 1/2018, p. 10f
Brachmann C, Schaffrath R
- (2018). Unfolded protein response suppression in yeast by loss of tRNA modifications. Genes 9: 516
Bruch A, Klassen R, Schaffrath R
(See online at https://doi.org/10.3390/genes9110516) - (2020) Urm1 - the (not so) ubiquitin related modifier. CINSaT Newsletter 1/2020, p. 9f
Kaduhr L, Schaffrath R
- (2020). Redox requirements for ubiquitin-like urmylation of Ahp1, a 2-Cys peroxiredoxin from yeast. Redox Biol 30: 101438
Brachmann C, Jüdes A, Keerthiraju ER, Kaduhr L, West JD, Glatt S, Schaffrath R
(See online at https://doi.org/10.1016/j.redox.2020.101438)
