title: Lysine Residues Are Not Required for Proteasome-Mediated Proteolysis of the Auxin/Indole Acidic Acid Protein IAA1.
abstract: Although many ubiquitin-proteasome substrates have been characterized in plants, very little is known about the corresponding ubiquitin attachment(s) underlying regulated proteolysis. Current dogma asserts that ubiquitin is typically covalently attached to a substrate through an isopeptide bond between the ubiquitin carboxy terminus and a substrate lysyl amino group. However, nonlysine (non-Lys) ubiquitin attachment has been observed in other eukaryotes, including the N terminus, cysteine, and serine/threonine modification. Here, we investigate site(s) of ubiquitin attachment on indole-3-acetic acid1 (IAA1), a short-lived Arabidopsis (Arabidopsis thaliana) Auxin/indole-3-acetic acid (Aux/IAA) family member. Most Aux/IAA proteins function as negative regulators of auxin responses and are targeted for degradation after ubiquitination by the ubiquitin ligase SCF(TIR1/AFB) (for S-Phase Kinase-Associated Protein1, Cullin, F-box [SCF] with Transport Inhibitor Response1 [TIR1]/Auxin Signaling F-box [AFB]) by an interaction directly facilitated by auxin. Surprisingly, using a Histidine-Hemaglutinin (HIS(6x)-HA(3x)) epitope-tagged version expressed in vivo, Lys-less IAA1 was ubiquitinated and rapidly degraded in vivo. Lys-substituted versions of IAA1 localized to the nucleus as Yellow Fluorescent Protein fusions and interacted with both TIR1 and IAA7 in yeast (Saccharomyces cerevisiae) two-hybrid experiments, indicating that these proteins were functional. Ubiquitination on both HIS(6x)-HA(3x)-IAA1 and Lys-less HIS(6x)-HA(3x)-IAA1 proteins was sensitive to sodium hydroxide treatment, indicative of ubiquitin oxyester formation on serine or threonine residues. Additionally, base-resistant forms of ubiquitinated IAA1 were observed for HIS(6x)-HA(3x)-IAA1, suggesting additional lysyl-linked ubiquitin on this protein. Characterization of other Aux/IAA proteins showed that they have diverse degradation rates, adding additional complexity to auxin signaling. Altogether, these data indicate that Aux/IAA family members have protein-specific degradation rates and that ubiquitination of Aux/IAAs can occur on multiple types of amino residues to promote rapid auxin-mediated degradation.
authors.affiliation: Department of Molecular and Cellular Biology and Plant Biology Graduate Group, University of California, Davis, California 95616 (J.G., R.T., J.C.); andDivision of Biological Sciences and Howard Hughes Medical Institute, University of California, San Diego, La Jolla, California 92093-0116 (D.R.K., M.E.).
authors.forename: Jonathan
authors.initials: J
authors.lastname: Gilkerson
authors.affiliation: Department of Molecular and Cellular Biology and Plant Biology Graduate Group, University of California, Davis, California 95616 (J.G., R.T., J.C.); andDivision of Biological Sciences and Howard Hughes Medical Institute, University of California, San Diego, La Jolla, California 92093-0116 (D.R.K., M.E.).
authors.forename: Dior R
authors.identifier: http://orcid.org/0000-0001-9186-4186
authors.initials: DR
authors.lastname: Kelley
authors.affiliation: Department of Molecular and Cellular Biology and Plant Biology Graduate Group, University of California, Davis, California 95616 (J.G., R.T., J.C.); andDivision of Biological Sciences and Howard Hughes Medical Institute, University of California, San Diego, La Jolla, California 92093-0116 (D.R.K., M.E.).
authors.forename: Raymond
authors.identifier: http://orcid.org/0000-0002-7292-0709
authors.initials: R
authors.lastname: Tam
authors.affiliation: Department of Molecular and Cellular Biology and Plant Biology Graduate Group, University of California, Davis, California 95616 (J.G., R.T., J.C.); andDivision of Biological Sciences and Howard Hughes Medical Institute, University of California, San Diego, La Jolla, California 92093-0116 (D.R.K., M.E.).
authors.forename: Mark
authors.initials: M
authors.lastname: Estelle
authors.affiliation: Department of Molecular and Cellular Biology and Plant Biology Graduate Group, University of California, Davis, California 95616 (J.G., R.T., J.C.); andDivision of Biological Sciences and Howard Hughes Medical Institute, University of California, San Diego, La Jolla, California 92093-0116 (D.R.K., M.E.) jcallis@ucdavis.edu.
authors.forename: Judy
authors.identifier: http://orcid.org/0000-0002-0622-078X
authors.initials: J
authors.lastname: Callis
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D007210:Indoleacetic Acids / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding; D059748:Proteolysis; D025801:Ubiquitin / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D029681:Arabidopsis Proteins; D004268:DNA-Binding Proteins; C491592:IAA1 protein, Arabidopsis; D007210:Indoleacetic Acids; D009687:Nuclear Proteins; D025801:Ubiquitin; C030737:indoleacetic acid; D008156:Luciferases; D046988:Proteasome Endopeptidase Complex; D008239:Lysine