Mapping Degradation Signals and Pathways in a Eukaryotic N-terminome

Abstract

Most eukaryotic proteins are N-terminally acetylated. This modification can be recognized as a signal for selective protein degradation (degron) by the N-end rule pathways. However, the prevalence and specificity of such degrons in the proteome are unclear. Here, by systematically examining how protein turnover is affected by N-terminal sequences, we perform a comprehensive survey of degrons in the yeast N-terminome. We find that approximately 26% of nascent protein N termini encode cryptic degrons. These degrons exhibit high hydrophobicity and are frequently recognized by the E3 ubiquitin ligase Doa10, suggesting a role in protein quality control. In contrast, N-terminal acetylation rarely functions as a degron. Surprisingly, we identify two pathways where N-terminal acetylation has the opposite function and blocks protein degradation through the E3 ubiquitin ligase Ubr1. Our analysis highlights the complexity of N-terminal degrons and argues that hydrophobicity, not N-terminal acetylation, is the predominant feature of N-terminal degrons in nascent proteins.

N-terminal acetylation and the N-end rule pathway control degradation of the lipid droplet protein PLIN2

“…Another plausible explanation for the stabilization of PLIN2 on LD most likely stems from the spatial segregation between LD-anchored PLIN2 and ER-embedded TEB4, which would prevent the interaction of the Ac/N-degron of PLIN2 with TEB4 E3 Ub ligase. Nonetheless, we cannot exclude the possibility that TEB4 is still in proximity to the PLIN2 protein, and targets it for degradation while LD is budding from ER membrane, as Doa10 (a counterpart of TEB4 in S. cerevisiae) regulates the abundance of the LD-associated proteins during LD biogenesis from the ER (39).…”

Mechanisms of up-regulation of Ubiquitin-Proteasome activity in the absence of NatA dependent N-terminal acetylation

“…The tFT reports on protein stability independently of expression through the intensity ratio of the slow maturing mCherry and the fast maturing sfGFP fluorescent proteins, which increases as a function of protein half-life in steady state (Khmelinskii et al, 2012;Khmelinskii et al, 2016). 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 (see Supplementary Figure S3 in (Kats et al, 2018)). 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.…”

“…The protein stability index (PSI) is a measure of protein turnover resulting from high-throughput analysis of tFT-tagged constructs and increases as a function of the mCherry/sfGFP ratio and is therefore anticorrelated with degradation rate. Data from Kats et al (Kats et al, 2018). Boxplots show median, 1 st and 3 rd quartile, whiskers extend to ± 1.5x interquartile range (IQR) from the box.…”

“…methionine aminopeptidases 1 and 2 (MAP1/2), which, in the absence of Nt-acetylation, can remove the initiator methionine (iMet) form the nascent chain. This leads to the exposure of the second residue, which in the case of NatB and NatC N-termini will lead to the exposure of an Arg/N-degron that can targets the protein for Ubr1 dependent degradation (Kats et al, 2018;Nguyen et al, 2018).…”

“…We have developed multiplexed protein stability (MPS) profiling, a quantitative and highthroughput compatible method that enables the degradation profiling of large peptide libraries using fluorescence activated cell sorting (FACS) and analysis of enriched fractions by deep sequencing (Kats et al, 2018). We used MPS profiling to explore the degron propensity of native and non-native N-termini and a large fraction of the yeast N-termini (N-terminome) (Kats et al, 2018). In this work we explore the influence of NatA on protein degradation in the budding yeast S. cerevisiae starting with the observation that artificial UFD substrates are degraded faster in NatA-deficient cells.…”