Sulfur transfer and activation by ubiquitin-like modifier system Uba4•Urm1 link protein urmylation and tRNA thiolation in yeast

André Jüdes; Alexander Bruch; Roland Klassen; Mark Helm; Raffael Schaffrath

doi:10.15698/mic2016.11.539

Sulfur transfer and activation by ubiquitin-like modifier system Uba4•Urm1 link protein urmylation and tRNA thiolation in yeast

Microb Cell. 2016 Oct 24;3(11):554-564. doi: 10.15698/mic2016.11.539.

Authors

André Jüdes¹, Alexander Bruch¹, Roland Klassen¹, Mark Helm², Raffael Schaffrath¹

Affiliations

¹ Universität Kassel, Institut für Biologie, FG Mikrobiologie, Heinrich-Plett-Str. 40, 34132 Kassel, Germany.
² Johannes Gutenberg Universität Mainz, Institut für Pharmazie und Biochemie, Staudinger Weg 5, 55128 Mainz, Germany.

Abstract

Urm1 is a unique dual-function member of the ubiquitin protein family and conserved from yeast to man. It acts both as a protein modifier in ubiquitin-like urmylation and as a sulfur donor for tRNA thiolation, which in concert with the Elongator pathway forms 5-methoxy-carbonyl-methyl-2-thio (mcm⁵s²) modified wobble uridines (U34) in anticodons. Using Saccharomyces cerevisiae as a model to study a relationship between these two functions, we examined whether cultivation temperature and sulfur supply previously implicated in the tRNA thiolation branch of the URM1 pathway also contribute to proper urmylation. Monitoring Urm1 conjugation, we found urmylation of the peroxiredoxin Ahp1 is suppressed either at elevated cultivation temperatures or under sulfur starvation. In line with this, mutants with sulfur transfer defects that are linked to enzymes (Tum1, Uba4) required for Urm1 activation by thiocarboxylation (Urm1-COSH) were found to maintain drastically reduced levels of Ahp1 urmylation and mcm⁵s²U34 modification. Moreover, as revealed by site specific mutagenesis, the S-transfer rhodanese domain (RHD) in the E1-like activator (Uba4) crucial for Urm1-COSH formation is critical but not essential for protein urmylation and tRNA thiolation. In sum, sulfur supply, transfer and activation chemically link protein urmylation and tRNA thiolation. These are features that distinguish the ubiquitin-like modifier system Uba4•Urm1 from canonical ubiquitin family members and will help elucidate whether, in addition to their mechanistic links, the protein and tRNA modification branches of the URM1 pathway may also relate in function to one another.

Keywords: E1-like enzyme Uba4; Saccharomyces cerevisiae; protein urmylation; sulfur transferase Tum1; tRNA thiolation; tRNase zymocin; ubiquitin-like modifier Urm1.

Grants and funding

We thank Katrin L. Thüring (Universität Mainz, Germany) for technical assistance with the LC-MS/MS profiles and Dr Christian Bär (Medizinische Hochschule Hannover, Germany) for Uba4•Urm1 pioneering work. Drs Kuge (Tohoku Pharmaceutical University, Japan), Thorner (University of California, USA) and Ohsumi (Tokyo Institute of Technology, Japan) kindly provided anti-Ahp1 & anti-Cdc19 antibodies and the HA-Urm1 expression plasmid. We thank Drs Leimkühler (University of Potsdam, Germany) and Stark (University of Dundee, Scotland, UK) for reading the manuscript. Project support by DFG and funds through their Priority Program 1784 Chemical Biology of Native Nucleic Acid Modifications to RK (KL 2937/1), MH (HE 3397/13) and RS (SCHA 750/15 & 750/20) are gratefully acknowledged.