Functional analysis of Arabidopsis DOA10-like E3 ligases

Etherington, Ross David ORCID: 0000-0002-2909-4771 (2022). Functional analysis of Arabidopsis DOA10-like E3 ligases. University of Birmingham. Ph.D.

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Abstract

The ubiquitin proteasome system (UPS) performs the bulk of targeted protein degradation within eukaryotic cells. As such it is crucial for regulating protein abundance, destroying harmful misfolded proteins and for facilitating responses to stimuli. UPS substrates are identified by E3 ligase enzymes, which catalyse their ubiquitination through recognition of degrons (sequences of amino acids encoding degradation signals).

ScDoa10 is an E3 ligase of the yeast endoplasmic reticulum-associated degradation (ERAD) system that has been reported to recognise acetylated N-termini as degrons, as part of the Ac/N-degron pathway. It is shown here that one Arabidopsis thaliana homolog of ScDoa10 (AtDOA10A) can functionally complement the yeast E3 both in the response to hygromycin and in the degradation of a substrate (AtSQE1), demonstrating conservation of function. A second homolog, AtDOA10B, which is specific to Brassicaceae, was unable to complement ScDoa10 despite sequence and structural similarities.

Despite evidence of functional conservation for AtDOA10A, multiomic analyses, including transcriptomics, quantitative proteomics and N-terminal (Nt-)acetylomics, of seedlings with reduced levels of AtDOA10s revealed no evidence of an accumulation of Nt-acetylated substrates, suggesting that an AtDOA10-mediated Ac/N-degron pathway is not a significant contributor to global protein degradation in Arabidopsis. Similarly, targeting of AtSQE1, the first identified likely substrate of AtDOA10A, was also independent of its Nt-acetylation status, although, interestingly, disruption of Nt-acetylation machinery did indirectly affect AtSQE1’s stability. This supports other recent investigations which suggest that Nt-acetylation does not act as a universal degradation signal but also highlights the significant indirect impacts of this post-translational modification on proteostasis.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):