Structural analyses of the plant PRT6-UBR box in the Cys-Arg/N-degron pathway and insights into the plant submergence response

Kim, Leehyeon; Lin, Chih-Cheng; Lin, Ting-Jhen; Cao, Yan-Cih; Chen, Minchun; Chou, Mei-Yi; Lin, Wen-Hsuan W.; Kim, Minki; Wu, Jianli; Shih, Ming-Che; Song, Hyun Kyu; Ho, Meng-Chiao

doi:10.1101/2022.08.19.504472

Cited by 4 publications

References 53 publications

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Ubiquitin E3 ligases in the plant Arg/N-degron pathway

Oldham,

Mabbitt

2024

Biochemical Journal

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Regulation of protein longevity via the ubiquitin (Ub) — proteasome pathway is fundamental to eukaryotic biology. Ubiquitin E3 ligases (E3s) interact with substrate proteins and provide specificity to the pathway. A small subset of E3s bind to specific exposed N-termini (N-degrons) and promote the ubiquitination of the bound protein. Collectively these E3s, and other N-degron binding proteins, are known as N-recognins. There is considerable functional divergence between fungi, animal, and plant N-recognins. In plants, at least three proteins (PRT1, PRT6, and BIG) participate in the Arg/N-degron pathway. PRT1 has demonstrated E3 ligase activity, whereas PRT6 and BIG are candidate E3s. The Arg/N-degron pathway plays a central role in plant development, germination, and submersion tolerance. The pathway has been manipulated both to improve crop performance and for conditional protein degradation. A more detailed structural and biochemical understanding of the Arg/N-recognins and their substrates is required to fully realise the biotechnological potential of the pathway. This perspective focuses on the structural and molecular details of substrate recognition and ubiquitination in the plant Arg/N-degron pathway. While PRT1 appears to be plant specific, the PRT6 and BIG proteins are similar to UBR1 and UBR4, respectively. Analysis of the cryo-EM structures of Saccharomyces UBR1 suggests that the mode of ubiquitin conjugating enzyme (E2) and substrate recruitment is conserved in PRT6, but regulation of the two N-recognins may be significantly different. The structurally characterised domains from human UBR4 are also likely to be conserved in BIG, however, there are sizeable gaps in our understanding of both proteins.

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Ubiquitin E3 ligases in the plant Arg/N-degron pathway

Oldham,

Mabbitt

2024

Biochemical Journal

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BIG enhances Arg/N-degron pathway-mediated protein degradation to regulate Arabidopsis hypoxia responses and suberin deposition

Zhang,

Rundle,

Winter

et al. 2024

The Plant Cell

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BIG/DARK OVEREXPRESSION OF CAB1/TRANSPORT INHIBITOR RESPONSE3 is a 0.5-MDa protein associated with multiple functions in Arabidopsis (Arabidopsis thaliana) signalling and development. However, the biochemical functions of BIG are unknown. We investigated a role for BIG in the Arg/N-degron pathways, in which substrate protein fate is influenced by the N-terminal (Nt) residue. We crossed a big loss-of-function allele to two N-degron pathway E3 ligase mutants, proteolysis6 (prt6) and prt1, and examined the stability of protein substrates. Stability of model substrates was enhanced in prt6-1 big-2 and prt1-1 big-2 relative to the respective single mutants and the abundance of the PRT6 physiological substrates, HYPOXIA-RESPONSIVE ERF2 (HRE2) and VERNALIZATION2 (VRN2) was similarly increased in prt6 big double mutants. Hypoxia marker expression was enhanced in prt6 big double mutants; this constitutive response required arginyltransferase activity and RAP-type ERFVII transcription factors. Transcriptomic analysis of roots not only demonstrated increased expression of multiple hypoxia-responsive genes in the double mutant relative to prt6, but also revealed other roles for PRT6 and BIG, including regulation of suberin deposition through both ERFVII-dependent and independent mechanisms, respectively. Our results show that BIG acts together with PRT6 to regulate the hypoxia response and broader processes in Arabidopsis.

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BIG participates in the Arg/N-degron pathways and the hypoxia response inArabidopsis thaliana

Zhang

Rundle

Winter

et al. 2023

Preprint

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BIG (also known as DOC1 and TIR3) is an 0.5 MDa protein that has been associated with multiple important functions in signalling and development through forward genetic screens in Arabidopsis thaliana. However, the biochemical function(s) of BIG are unknown. Here, we investigated whether BIG plays a role in the Arg/N-degron pathways, protein regulatory mechanisms in which substrate protein fate is influenced by the N-terminal (Nt) residue. In Arabidopsis, PROTEOLYSIS1 (PRT1) is an E3 ligase with specificity for aromatic amino acids, whereas PROTEOLYSIS6 (PRT6) targets basic N-terminal residues. We crossed a big loss-of-function allele toprt6andprt1mutants and examined the stability of protein substrates. Stability of model N-degron pathway substrates was enhanced inprt6-1 big-2andprt1-1 big-2relative to the respective single mutants. Abundance of the PRT6 physiological substrates, HYPOXIA RESPONSIVE ERF (HRE)2 and VERNALIZATION (VRN)2 was similarly increased in prt6 big double mutants, without increase in transcripts. Accordingly, hypoxia marker expression was enhanced inprt6 bigdouble mutants, in a manner requiring arginyltransferase activity and RAP-type ERFVII transcription factors. Transcriptomic analysis of roots not only demonstrated synergistically increased expression of a plethora of hypoxia responsive genes in the double mutant relative to prt6 but also revealed other roles for PRT6 and BIG, including regulation of suberin deposition through both ERFVII-dependent and independent mechanisms, respectively. Our results show that BIG acts together with PRT6 to regulate the hypoxia response and wider processes.

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Structural analyses of the plant PRT6-UBR box in the Cys-Arg/N-degron pathway and insights into the plant submergence response

Cited by 4 publications

References 53 publications

Ubiquitin E3 ligases in the plant Arg/N-degron pathway

Ubiquitin E3 ligases in the plant Arg/N-degron pathway

BIG enhances Arg/N-degron pathway-mediated protein degradation to regulate Arabidopsis hypoxia responses and suberin deposition

BIG participates in the Arg/N-degron pathways and the hypoxia response inArabidopsis thaliana

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