The UBR Domain of Plant Ubr1 Homolog PRT6 Accommodates Basic and Hydrophobic Amino Termini for Substrate Recognition

• Published in: Journal of molecular biology 2025-02, Vol.437 (4), p.168939, Article 168939
• Main Authors: Rudi, Olga, Hodakova, Zuzana, Farias Saad, Carolina, Winter, Nikola, Grishkovskaya, Irina, Böhm, Jessica, Jarck, Greta, Schleiffer, Alexander, Haselbach, David, Bachmair, Andreas
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Arabidopsis - genetics; Arabidopsis - metabolism; Arabidopsis Proteins - chemistry; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; Hydrophobic and Hydrophilic Interactions; N-degron pathway; Phylogeny; Protein Domains; Proteolysis; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Substrate Specificity; synthetic biology; ubiquitin ligase; Ubiquitin-Protein Ligases - chemistry; Ubiquitin-Protein Ligases - genetics; Ubiquitin-Protein Ligases - metabolism; UBR box
• ISSN: 0022-2836; 1089-8638; 1089-8638
• DOI: 10.1016/j.jmb.2025.168939

[Display omitted] •Using functional expression in yeast, we show that PRT6, a plant ubiquitin ligase of the N-degron pathway, can accept substrates with basic, and with hydrophobic amino terminal amino acid residues.•Whereas yeast and animal homologs Ubr1 have separate domains for each class of termini, PRT6 employs one domain for both classes, its UBR box.•The work shows an unexpected versatility of a UBR box not previously described. N-degrons are amino-terminal degradation signals. Non-acetylated first residues with bulky side chains were the first discovered N-degrons. In yeast, their ability to destabilize a protein depends on ubiquitin ligase Ubr1, which has a binding site for basic first residues, the UBR box, and one for hydrophobic first residues, the N domain. We investigated PRT6, the Arabidopsis homolog of Ubr1, by expression in a yeast strain devoid of Ubr1. Phylogenetic analysis and structure prediction indicate that PRT6 lacks the N domain and thus should not accept hydrophobic N-degrons. However, we show that PRT6 mediates the turnover of proteins with Leu, Phe, Tyr and Trp as the first residue. By functional analysis in the heterologous host, we show that the PRT6 UBR box can accommodate these N-degrons. The data indicate a surprising evolutionary flexibility of the UBR box that may also be found in UBR box proteins of other organisms.