Up-regulation of ubiquitin-proteasome activity upon loss of NatA-dependent N-terminal acetylation.

Thomas Ruppert,Ilia Kats,Michael Knop,Laura Armbruster,Marc Kschonsak,Christian Reinbold,Anton Khmelinskii

doi:10.26508/lsa.202000730

Abstract

N-terminal acetylation is a prominent protein modification, and inactivation of N-terminal acetyltransferases (NATs) cause protein homeostasis stress. Using multiplexed protein stability profiling with linear ubiquitin fusions as reporters for the activity of the ubiquitin proteasome system, we observed increased ubiquitin proteasome system activity in NatA, but not NatB or NatC mutants. We find several mechanisms contributing to this behavior. First, NatA-mediated acetylation of the N-terminal ubiquitin-independent degron regulates the abundance of Rpn4, the master regulator of the expression of proteasomal genes. Second, the abundance of several E3 ligases involved in degradation of UFD substrates is increased in cells lacking NatA. Finally, we identify the E3 ligase Tom1 as a novel chain-elongating enzyme (E4) involved in the degradation of linear ubiquitin fusions via the formation of branched K11, K29, and K48 ubiquitin chains, independently of the known E4 ligases involved in UFD, leading to enhanced ubiquitination of the UFD substrates.

Highlights

Selective protein degradation is essential for proteome homeostasis, to remove unnecessary or abnormal proteins as part of quality control pathways or in response to changes in the environment
In the course of that study, we observed that reporters with a proline residue immediately following the ubiquitin moiety (Ubi-PZ-tFT reporters) exhibited increased turnover in strains lacking the N-terminal acetyltransferase NatA (Fig 1A), whereas no destabilization was observed in NatB and NatC mutants
The N-terminal ubiquitin moiety is usually co-translationally cleaved by endogenous deubiquitinating enzymes (DUBs) (Bachmair et al, 1986), which enables the exposure of non-native amino acid residues at the N-terminus of the reporter protein

Summary

Introduction

Selective protein degradation is essential for proteome homeostasis, to remove unnecessary or abnormal proteins as part of quality control pathways or in response to changes in the environment. The bulk of selective protein degradation is handled by the ubiquitin–proteasome system (UPS). Global activity of the UPS is tightly regulated and responds to environmental challenges such as heat stress, DNA damage, or cytotoxic compounds, which can damage or induce misfolding of proteins (Hahn et al, 2006). In the budding yeast Saccharomyces cerevisiae, the transcription factor Rpn is a master regulator of proteasome capacity. It trans-activates promoters of all proteasomal subunits and several other proteins of the UPS (Mannhaupt et al, 1999; Shirozu et al, 2015). Expression of Rpn is in turn regulated by several stress-induced transcription factors such as Hsf and Yap (Hahn et al, 2006)

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