| 2021 |
Cryo-EM structures of Ubr1 in complex with Ubc2, ubiquitin, and N-degron peptides revealed the structural basis for both initiation and elongation steps of ubiquitination. Key structural elements including a Ubc2-binding region and an acceptor ubiquitin-binding loop on Ubr1 were identified and characterized by mutagenesis, explaining how Ubr1 mediates linkage-specific polyubiquitin chain formation through a single E2 enzyme. |
Cryo-EM structure determination with chemical mimics of reaction intermediates, plus mutagenesis of key structural elements |
Nature |
High |
34789879
|
| 2008 |
The type-1 and type-2 substrate-binding sites of UBR1 (recognizing basic and bulky hydrophobic N-terminal residues, respectively) reside within the first ~700 residues of the 1,950-residue protein. Selective inactivation of each site by distinct mutations was demonstrated, and binding affinities (Kd ~1 μM) were measured by fluorescence polarization and surface plasmon resonance. A third substrate-binding site targets the internal degron of CUP9, and CUP9 ubiquitylation dependent on dipeptide binding to type-1/2 sites was reconstituted in a fully defined in vitro system. |
Genetic screen for loss-of-function mutations, fluorescence polarization, surface plasmon resonance, in vitro reconstitution with purified components |
The Journal of biological chemistry |
High |
18566452
|
| 2010 |
The RING-type Ubr1 E3 and the HECT-type Ufd4 E3 physically interact and form a complex that is more processive in polyubiquitylation than Ubr1 alone, producing longer substrate-linked polyubiquitin chains. Ubr1 can also recognize the N-terminal ubiquitin moiety of UFD substrates, and can enhance polyubiquitylation of UFD substrates as part of the Ubr1-Ufd4 complex. |
Co-immunoprecipitation, in vitro ubiquitylation assays, genetic epistasis |
Nature cell biology |
High |
21076411
|
| 2009 |
Ubr1 mediates chaperone-dependent ubiquitination of misfolded cytoplasmic proteins for proteasomal degradation in yeast, acting in parallel with nuclear San1. This cytoplasmic quality control function of Ubr1 is distinct from its N-end rule role and depends on chaperone assistance. |
Genetic epistasis (double mutant analysis), biochemical ubiquitination assays, phenotypic analysis of proteotoxic stress |
Proceedings of the National Academy of Sciences of the United States of America |
High |
20080635
|
| 2008 |
Ubr1 is the E3 ligase responsible for targeting misfolded cytoplasmic proteins to proteasomal degradation in yeast, establishing its role as a cytoplasmic protein quality control ubiquitin ligase independent of N-end rule activity. |
Genetic deletion analysis, ubiquitination assay with misfolded reporter substrates |
FEBS letters |
Medium |
19041308
|
| 2010 |
Saccharomyces cerevisiae Ubr1 directly binds denatured (but not native) luciferase and catalyzes its ubiquitinylation in a purified system; Hsp70 stimulates polyubiquitinylation of the denatured substrate. Loss of Ubr1 and Ubr2 suppresses growth arrest caused by chaperone mutation, indicating these E3s compete with chaperones for misfolded substrates. |
In vitro ubiquitylation assay with purified components, genetic epistasis (suppressor analysis of chaperone mutants) |
Molecular biology of the cell |
High |
20462952
|
| 2020 |
Hsp70 ATP hydrolysis is directly required for Ubr1-mediated ubiquitination of misfolded substrates. Using a bead-based in vitro assay with covalently immobilized misfolded protein, only Hsp70 (with its ATPase cycle and cochaperones including nucleotide exchange factor Sse1 and J-proteins) was required for Ubr1-mediated ubiquitination. |
In vitro bead-based ubiquitination assay with immobilized misfolded substrate, ATPase cycle mutant analysis |
Molecular biology of the cell |
High |
32966159
|
| 2013 |
The Type II Hsp40 Sis1 cooperates sequentially with Hsp70 and Ubr1 to target misfolded cytosolic proteins for proteasomal degradation. Sis1 is required for proteasomal degradation of a misfolded reporter (slGFP), acting upstream of Ubr1; in the absence of Ubr1, slGFP accumulates in relatively stable perinuclear and peripheral puncta. |
Genetic deletion/knockdown, fluorescence imaging of misfolded reporter localization, proteasomal degradation assays |
PloS one |
Medium |
23341891
|
| 2013 |
Cytosolic Ubr1 participates in ER-associated protein degradation (ERAD) in yeast. Two polytopic ERAD substrates (Ste6* and CFTR) undergo Ubr1-dependent degradation, with Ubr1 acting together with Hsp70 chaperone Ssa1 and the AAA-ATPase Cdc48 to direct retrotranslocated substrates to proteasomal degradation. |
Genetic epistasis (ubr1 deletion in combination with canonical ERAD ligase deletions), degradation assays, protein-protein interaction |
Proceedings of the National Academy of Sciences of the United States of America |
Medium |
23988329
|
| 2006 |
UBR1 interacts with c-Fos and ubiquitylates it in the cytoplasm, promoting its accelerated degradation. ERK5 inhibits this process by phosphorylating c-Fos at Ser32 (disrupting UBR1–c-Fos interaction) and Thr232 in the NES (blocking nuclear export). UBR1 expression is induced by STAT3 upon IL-6/gp130 stimulation. |
Co-immunoprecipitation, in vivo ubiquitylation assay, siRNA knockdown, phosphorylation site mutagenesis |
Molecular cell |
Medium |
17018293
|
| 2004 |
RECQL4 (mutated in Rothmund-Thomson syndrome) forms a stable complex with UBR1 and UBR2 in HeLa cells. Despite association with these E3 ligases, RECQL4 was not ubiquitylated in vivo and was a long-lived protein. The isolated RECQL4-UBR1/2 complex had DNA-stimulated ATPase activity. |
Co-immunoprecipitation with antibodies to RECQL4, in vivo ubiquitylation analysis, ATPase activity assay |
Human molecular genetics |
Medium |
15317757
|
| 2008 |
Ubr1 is phosphorylated in vivo at multiple sites including Ser300 and Tyr277. Casein kinases Yck1/Yck2 phosphorylate Ubr1 at Ser300, priming subsequent phosphorylations at Ser296, Ser292, Thr288, and Tyr277 by the GSK3-family kinase Mck1. Phosphorylation at Ser300 plays a major role in controlling peptide import regulation by the N-end rule pathway through modulation of Cup9 degradation. |
In vivo phosphorylation mapping, kinase deletion/mutation analysis, in vitro kinase assays, peptide import assays |
Proceedings of the National Academy of Sciences of the United States of America |
High |
19033468
|
| 2010 |
Phosphorylation of Ubc2/Rad6 at Ser120 by CDK1/2 negatively regulates N-end rule-dependent degradation by E3α/UBR1. The S120D phosphomimetic mutant shows 20-fold reduced activity with UBR1 (8-fold increased Km, 2.5-fold decreased Vmax for initial conjugation), while S120A shows reduced Vmax for polyubiquitin chain elongation. |
In vitro E3α/Ubr1-catalyzed ubiquitination kinetics with Ubc2 phosphorylation-site mutants, cell-based N-end rule degradation assay |
The Journal of biological chemistry |
Medium |
21041297
|
| 2001 |
Mouse UBR1, in the presence of a cognate mouse E2 enzyme, can rescue the N-end rule pathway in ubr1Δ S. cerevisiae, confirming its conserved E3 ubiquitin ligase activity. UBR1−/− mice lack the N-end rule pathway in skeletal muscle but not in fibroblasts (due to compensating UBR1-like proteins), and exhibit abnormal regulation of fatty acid synthase in skeletal muscle upon starvation. |
Functional complementation in yeast, knockout mouse construction, biochemical analysis of N-end rule activity in tissues |
Molecular and cellular biology |
High |
11689692
|
| 2006 |
UBR1−/−UBR2−/− double-knockout mouse embryos die at midgestation with defects in neurogenesis (reduced proliferation, precocious migration/differentiation of neural progenitor cells) and cardiovascular development. Altered expression of D-type cyclins and Notch1 was detected, placing UBR1 and UBR2 upstream of these developmental regulators. |
Double-knockout mouse construction, embryological analysis, molecular marker analysis (cyclin D, Notch1) |
Proceedings of the National Academy of Sciences of the United States of America |
Medium |
16606826
|
| 2005 |
Loss-of-function mutations in UBR1 cause Johanson-Blizzard syndrome in humans. UBR1−/− mice exhibit exocrine pancreatic insufficiency with impaired stimulus-secretion coupling and increased susceptibility to pancreatic injury, indicating UBR1 is required for acinar cell function through degradation of N-end rule pathway substrates. |
Human genetics (mutation identification in 12 families), UBR1−/− mouse phenotypic analysis, pancreatic functional assays |
Nature genetics |
High |
16311597
|
| 2018 |
Upon proteotoxic stress, the hydrophilin Roq1 is cleaved by the HtrA-type protease Ynm3, and cleaved Roq1 interacts with Ubr1, reprogramming its substrate specificity. This SHRED (stress-induced homeostatically regulated protein degradation) cascade accelerates proteasomal degradation of both misfolded and some native proteins at the ER membrane and in the cytosol. |
Genetic epistasis, protein interaction assays, substrate degradation assays under stress, protease cleavage assays |
Molecular cell |
Medium |
29861160
|
| 2025 |
Roq1 reprograms Ubr1 via two cooperating multifunctional motifs: an N-terminal arginine that engages the Ubr1 substrate-binding pocket and a short hydrophobic motif. The N-terminal arginine modulates ubiquitination of N-degron substrates and folded proteins, while the hydrophobic motif accelerates ubiquitination of misfolded proteins. This heterobivalent binding mechanism underlies Ubr1 reprogramming. |
Mutational analysis of Roq1 motifs, in vivo and in vitro ubiquitination assays, protein interaction assays |
The EMBO journal |
Medium |
39920309
|
| 2011 |
UBR1 ubiquitin ligase activity, mediated by its RING-H2 domain, promotes degradation of the glucocorticoid receptor and androgen receptor (but not estrogen receptor α) upon Hsp90 inhibition in mammalian cells, demonstrating client-specific quality control activity beyond the N-end rule. |
UBR1 deletion mouse embryonic fibroblasts, geldanamycin treatment, Western blot for client degradation kinetics |
Experimental cell research |
Medium |
21983172
|
| 2013 |
UBR1 deletion results in impaired degradation of the glucocorticoid receptor and androgen receptor, but not estrogen receptor α, upon Hsp90 inhibition, demonstrating specificity in UBR1's recognition of misfolded Hsp90 clients. |
UBR1 deletion cells, Hsp90 inhibitor treatment, receptor degradation assays |
FEBS open bio |
Medium |
24251101
|
| 2010 |
UBR1 and UBR2 bind leucine via their substrate-recognition domains, functioning as leucine-binding proteins identified from leucine-responsive cells using leucine-immobilized affinity beads. Overexpression of UBR1/UBR2 reduces mTOR-dependent S6K1 phosphorylation, while their knockdown increases S6K1 phosphorylation; leucine binding inhibits N-end rule substrate degradation in vitro, suggesting UBR1 is a negative regulator of leucine-mTOR signaling. |
Leucine-affinity bead pulldown, overexpression/knockdown with S6K1 phosphorylation readout, in vitro N-end rule substrate degradation assay |
Genes to cells : devoted to molecular & cellular mechanisms |
Medium |
20298436
|
| 2023 |
Ubr1 targets the lipid droplet protein Plin2 for ubiquitin-proteasome degradation in an amino acid-dependent manner. Ubr1 uses its two canonical substrate-binding pockets (for basic and bulky hydrophobic amino acids) to bind amino acids, which allosterically activates Ubr1 by relieving auto-inhibition. This was directly demonstrated in cell-free reconstituted systems with purified proteins. |
In vitro cell-free reconstitution system with purified proteins, amino acid-binding assays, pulldown assays, in vitro ubiquitination assays, immunoprecipitation in mammalian cells |
Journal of cachexia, sarcopenia and muscle |
High |
37057345
|
| 2019 |
Ubr1 preferentially targets mistranslocated secretory and mitochondrial proteins in the cytosol, recognizing cellular location signals encoded in the P2 (second amino acid) position. This establishes that the N-end rule pathway enforces protein compartmentalization by degrading proteins that fail to reach their intended subcellular locations. |
Systematic P2-position mutant analysis, bioinformatic proteome analysis, genetic deletion assays |
Journal of cell science |
Medium |
30940687
|
| 2022 |
UBR1 functions as a protein quality control E3 ubiquitin ligase at endosomes, ubiquitinating mutant MLC1 via the ESCRT pathway. Under endosomal stress or cytosolic Ca2+ increase, UBR1 and SQSTM1/p62 cooperate to target arginylated and ubiquitinated cargo for selective endosomal autophagy (endophagy). Loss of UBR1 or arginylation causes endosomal compartment stress. |
Co-immunoprecipitation, ubiquitination assays, immunofluorescence of endosomal compartments, autophagy flux assays, siRNA knockdown |
Cellular and molecular life sciences : CMLS |
Medium |
35233680
|
| 2024 |
UBR1 directly interacts with YAP and promotes its monoubiquitylation, which competitively suppresses YAP polyubiquitylation and extends YAP protein half-life, thereby stabilizing YAP and promoting anaplastic thyroid carcinoma cell proliferation and migration. |
siRNA screening, co-immunoprecipitation, ubiquitination assay (mono vs. poly), protein half-life assay, xenograft experiments |
Scientific reports |
Medium |
39174635
|
| 2024 |
UBR1 promotes sex-dependent ubiquitination and degradation of ACE2 in hypertension. UBR1 was identified by proteomics and validated by co-immunoprecipitation as a direct binding partner of ACE2. Testosterone (but not estradiol) upregulates UBR1, and in vivo UBR1 knockdown restores ACE2 levels and transiently reduces blood pressure in hypertensive male mice. UBR1 and Nedd4-2 appear to work synergistically to ubiquitinate ACE2. |
Proteomics, co-immunoprecipitation validation, siRNA knockdown in vivo and in vitro, blood pressure measurement, sex hormone treatment of cells |
Hypertension (Dallas, Tex. : 1979) |
Medium |
39601126
|
| 2018 |
In C. elegans, loss of UBR-1 leads to a specific motor deficit during reversal movements due to synchronized A-class motor neuron activation. This phenotype is rescued by removing GOT-1, a transaminase converting aspartate to glutamate. ubr-1 mutants show elevated glutamate levels and ubr-1 and got-1 are both required in premotor interneurons, placing UBR-1 in a pathway regulating glutamate metabolism for motor circuit function. |
C. elegans genetics (loss-of-function), epistasis with got-1, neuronal calcium imaging, metabolite measurement, cell-specific rescue experiments |
PLoS genetics |
Medium |
29649217
|
| 2011 |
In fission yeast, Ubr1 is concentrated in the nucleus and mediates degradation of activated (nuclear) Pap1 bZIP transcription factor, providing a mechanism to terminate the oxidative stress response. Loss of ubr1 causes increased Pap1 levels and hydrogen peroxide resistance; inactive mutations in the Pap1 bZIP domain stabilize the protein but rescue lethality in ubr1 mutants, showing that DNA-binding-competent Pap1 must be eliminated by Ubr1. |
Genetic deletion, epistasis with nuclear-export-deficient and DNA-binding mutants, Western blot, growth assays |
Molecular microbiology |
Medium |
21410566
|
| 2011 |
Missense mutations in the RING-H2 and UBR domains of human UBR1 (corresponding to JBS alleles) can be modeled in yeast Ubr1 and show graded activity defects: H160R (in UBR box) is completely inactive, Q1224E has weak activity, and V146L has reduced but significant activity, correlating with clinical severity of JBS in corresponding patients. |
Yeast-based functional complementation assay, N-end rule substrate degradation assay, site-directed mutagenesis |
PloS one |
Medium |
21931868
|
| 2025 |
In C. elegans, UBR-1 physically associates with glutamate metabolic enzymes (GLN-3, GOT-2.2, GFAT-1, GDH-1) identified by CRISPR-based proteomics. Genetic interaction studies show UBR-1, GLN-3, and GOT-2.2 form a signaling axis regulating glutamate homeostasis required for locomotion and developmental viability, with UBR-1 functioning via both ubiquitin ligase and scaffolding mechanisms. |
CRISPR-based proteomics, genetic interaction analysis, super-resolution imaging, pharmacological rescue |
bioRxivpreprint |
Medium |
40766417
|