PRT6/At5g02310 encodes an Arabidopsis ubiquitin ligase of the N‐end rule pathway with arginine specificity and is not the CER3 locus

Garzon, Marcus; Eifler, Karolin; Faust, Andréa; Scheel, Hartmut; Hofmann, Kay; Koncz, Csaba; Yephremov, Alexander; Bachmair, Andreas

doi:10.1016/j.febslet.2007.06.005

Cited by 105 publications

(144 citation statements)

References 45 publications

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“…In the S. cerevisiae UBR1 E3, in its mammalian sequelogs UBR1/UBR2, and in some other N-recognins of mammals and plants (31,32,35) the UBR domain contains at least the type-1 substrate-binding site. A UBR domain is also present in UBR1 sequelogs that do not function as N-recognins.…”

Section: Discussionmentioning

confidence: 99%

“…We also employed a similarly produced, labeled, and purified py CUP9his 6 with a different N-terminal tag, called polyoma (py) that was bound by a monoclonal antibody (61) (AK6967; a gift from Dr. S. Stevens and Dr. J. Abelson (Caltech)). The in vitro ubiquitylation reactions contained the following components: 7 M Ub, 50 nM UBA1, 50 nM RAD6, 50 nM UBR1, 550 nM 35 S-labeled f CUP9his 6 , 25 mM KCl, 5 mM MgCl 2 , 2 mM ATP, 0.1 mM dithiothreitol, 25 mM HEPES, pH 7.5, and ovalbumin (as a carrier protein) at 0.5 mg/ml. Pairs of dipeptides were added to final concentrations specified in the legend to Fig.…”

Section: Ubr1-dependent In Vitro Ubiquitylationmentioning

confidence: 99%

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Substrate-binding Sites of UBR1, the Ubiquitin Ligase of the N-end Rule Pathway

Xia

Webster

et al. 2008

Journal of Biological Chemistry

128

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Substrates of a ubiquitin-dependent proteolytic system called the N-end rule pathway include proteins with destabilizing N-terminal residues. N-recognins, the pathway's ubiquitin ligases, contain three substrate-binding sites. The type-1 site is specific for basic N-terminal residues (Arg, Lys, and His). The type-2 site is specific for bulky hydrophobic N-terminal residues (Trp, Phe, Tyr, Leu, and Ile). We show here that the type-1/2 sites of UBR1, the sole N-recognin of the yeast Saccharomyces cerevisiae, are located in the first ϳ700 residues of the 1,950-residue UBR1. These sites are distinct in that they can be selectively inactivated by mutations, identified through a genetic screen. Mutations inactivating the type-1 site are in the previously delineated ϳ70-residue UBR motif characteristic of N-recognins. Fluorescence polarization and surface plasmon resonance were used to determine that UBR1 binds, with a K d of ϳ1 M, to either type-1 or type-2 destabilizing N-terminal residues of reporter peptides but does not bind to a stabilizing N-terminal residue such as Gly. A third substrate-binding site of UBR1 targets an internal degron of CUP9, a transcriptional repressor of peptide import. We show that the previously demonstrated in vivo dependence of CUP9 ubiquitylation on the binding of cognate dipeptides to the type-1/2 sites of UBR1 can be reconstituted in a completely defined in vitro system. We also found that purified UBR1 and CUP9 interact nonspecifically and that specific binding (which involves, in particular, the binding by cognate dipeptides to the UBR1 type-1/2 sites) can be restored either by a chaperone such as EF1A or through macromolecular crowding.

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Section: Discussionmentioning

confidence: 99%

Section: Ubr1-dependent In Vitro Ubiquitylationmentioning

confidence: 99%

Substrate-binding Sites of UBR1, the Ubiquitin Ligase of the N-end Rule Pathway

Xia

Webster

et al. 2008

Journal of Biological Chemistry

128

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“…Positional cloning and complementation analysis with previously published alleles identified the PROTEOLYSIS6 (PRT6) gene as one of these loci (allele prt6-4, At5g02310; Fig. S1) (3). After-ripening of prt6 seeds was substantially delayed, although moist chilling or exogenous gibberellic acid (GA) removed this block to germination (Fig.…”

Section: Influence Of Components Of the N-end Rule Pathway On Key Aspmentioning

confidence: 99%

“…In contrast, the Arabidopsis UBR box-containing E3 ligase PROTEOLYSIS6 (PRT6) recognizes only basic amino acids, and PRT1, which does not contain a UBR box, recognizes aromatic amino acids (3,4,6). Other Nrecognins must also exist in Arabidopsis, because other Ndegrons are unstable in prt6 prt1 double mutants (3). N-degrons containing primary destabilizing amino-terminal amino acids can also be created through the conversion of pre-N-degron tertiary or secondary destabilizing residues (Fig.…”

mentioning

confidence: 99%

“…Yeast UBR1 was the first characterized E3 ligase with N-degron activity, and in mammals there are at least 7 proteins that contain the characteristic UBR box (2). In contrast, the Arabidopsis UBR box-containing E3 ligase PROTEOLYSIS6 (PRT6) recognizes only basic amino acids, and PRT1, which does not contain a UBR box, recognizes aromatic amino acids (3,4,6). Other Nrecognins must also exist in Arabidopsis, because other Ndegrons are unstable in prt6 prt1 double mutants (3).…”

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confidence: 99%

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The N-end rule pathway promotes seed germination and establishment through removal of ABA sensitivity in Arabidopsis

Holman

Jones²,

Russell

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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173

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The N-end rule pathway targets protein degradation through the identity of the amino-terminal residue of specific protein substrates. Two components of this pathway in Arabidopsis thaliana, PROTEOLYSIS6 (PRT6) and arginyl-tRNA:protein arginyltransferase (ATE), were shown to regulate seed after-ripening, seedling sugar sensitivity, seedling lipid breakdown, and abscisic acid (ABA) sensitivity of germination. Sensitivity of prt6 mutant seeds to ABA inhibition of endosperm rupture reduced with after-ripening time, suggesting that seeds display a previously undescribed window of sensitivity to ABA. Reduced root growth of prt6 alleles and the ate1 ate2 double mutant was rescued by exogenous sucrose, and the breakdown of lipid bodies and seed-derived triacylglycerol was impaired in mutant seedlings, implicating the N-end rule pathway in control of seed oil mobilization. Epistasis analysis indicated that PRT6 control of germination and establishment, as exemplified by ABA and sugar sensitivity, as well as storage oil mobilization, occurs at least in part via transcription factors ABI3 and ABI5. The N-end rule pathway of protein turnover is therefore postulated to inactivate as-yet unidentified key component(s) of ABA signaling to influence the seed-to-seedling transition.abscisic acid ͉ aminoacyl tRNA protein transferase ͉ lipid bodies ͉ targeted protein degradation

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The Arabidopsis thaliana N‐recognin E3 ligase PROTEOLYSIS1 influences the immune response

et al. 2019

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N‐degron pathways of ubiquitin‐mediated proteolysis (formerly known as the N‐end rule pathway) control the stability of substrate proteins dependent on the amino‐terminal (Nt) residue. Unlike yeast or mammalian N‐recognin E3 ligases, which each recognize several different classes of Nt residues, in Arabidopsis thaliana, N‐recognin functions of different N‐degron pathways are carried out independently by PROTEOLYSIS (PRT)1, PRT6, and other unknown proteins. PRT1 recognizes type 2 aromatic Nt‐destabilizing residues and PRT6 recognizes type 1 basic residues. These two N‐recognin functions diverged as separate proteins early in the evolution of plants, before the conquest of the land. We demonstrate that loss of PRT1 function promotes the plant immune system, as mutant prt1‐1 plants showed greater apoplastic resistance than WT to infection by the bacterial hemi‐biotroph Pseudomonas syringae pv tomato (Pst) DC3000. Quantitative proteomics revealed increased accumulation of proteins associated with specific components of plant defense in the prt1‐1 mutant, concomitant with increased accumulation of salicylic acid. The effects of the prt1 mutation were additional to known effects of prt6 in influencing the immune system, in particular, an observed over‐accumulation of pipecolic acid (Pip) in the double‐mutant prt1‐1 prt6‐1. These results demonstrate a potential role for PRT1 in controlling aspects of the plant immune system and suggest that PRT1 limits the onset of the defense response via degradation of substrates with type 2 Nt‐destabilizing residues.

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PRT6/At5g02310 encodes an Arabidopsis ubiquitin ligase of the N‐end rule pathway with arginine specificity and is not the CER3 locus

Cited by 105 publications

References 45 publications

Substrate-binding Sites of UBR1, the Ubiquitin Ligase of the N-end Rule Pathway

Substrate-binding Sites of UBR1, the Ubiquitin Ligase of the N-end Rule Pathway

The N-end rule pathway promotes seed germination and establishment through removal of ABA sensitivity in Arabidopsis

The Arabidopsis thaliana N‐recognin E3 ligase PROTEOLYSIS1 influences the immune response

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