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

André Jüdes,Andre Juedes,Mark Helm,Raffael Schaffrath,Alexander Bruch,Roland Klassen

doi:10.15698/mic2016.11.539

André Jüdes, Andre Juedes + Show 4 more

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https://doi.org/10.15698/mic2016.11.539

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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.

Highlights

In eukaryotes, the activity of many diverse proteins can be modulated by conjugation to ubiquitin and ubiquitin-like modifiers [1, 2]
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 contribute to proper urmylation
One was kept at 30°C, the other shifted to 39°C and both cultivated for three hours prior to protein urmylation analysis

Summary

Introduction

The activity of many diverse proteins can be modulated by conjugation to ubiquitin and ubiquitin-like modifiers [1, 2] Among the latter, Urm from Saccharomyces cerevisiae [3] is unique since it can act as a lysine-directed protein modifier in ubiquitin-like urmylation and as a sulfur donor for tRNA anticodon thiolation [4,5,6,7,8,9]. In contrast to ATP-dependent ubiquitin adenylation and conjugation by E1-E3 enzymes, E2/E3 activities for Urm are unknown, and Urm activation by its E1-like enzyme Uba results in C-terminal thiocarboxylation (Urm1COSH) [18, 19] This is similar to E1-like (MoeB or ThiF) activation of bacterial S-carrier proteins (MoaD or ThiS) that donate sulfur for molybdopterin or thiamine synthesis rather than being involved in protein conjugation [20, 21]. Upon reductive cleavage of this bond, Urm1-COSH gets released [18, 23] to donate the activated sulfur species for S-insertion into tRNAs (Fig. 1) by thiolase (Ncs2Ncs6) [7,8,9]

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