Lysine Residues Are Not Required for Proteasome-Mediated Proteolysis of the Auxin/Indole Acidic Acid Protein IAA1
Lysine Residues Are Not Required for Proteasome-Mediated Proteolysis of the Auxin/Indole Acidic Acid Protein IAA1
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DOI：
10.1104/pp.15.00402
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发表时间：
2015-06-01
期刊：
影响因子：
7.4
通讯作者：
Callis, Judy
中科院分区：
文献类型：
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作者：
Gilkerson, Jonathan;Kelley, Dior R.;Callis, Judy
虽然许多泛素-蛋白酶体底物已在植物中得到表征，但对调节蛋白水解的相应泛素附着知之甚少。目前的教条认为，泛素通常通过泛素羧基末端和底物赖氨酰氨基之间的异肽键共价连接到底物上。然而，在其他真核生物中也观察到了非赖氨酸（non-Lys）泛素连接，包括N末端、半胱氨酸和丝氨酸/苏氨酸修饰。在这里，我们研究的网站（S）的泛素连接的吲哚-3-乙酸（IAA 1），一个短命的拟南芥（拟南芥）生长素/吲哚-3-乙酸（Aux/IAA）家族成员。大多数Aux/IAA蛋白作为生长素反应的负调节剂起作用，并且在泛素连接酶SCFTIR 1/AFB（S-期激酶相关蛋白1，Cullin，F-box [SCF]与转运抑制剂反应1 [TIR 1]/生长素信号F-box [AFB]）通过生长素直接促进的相互作用进行泛素化后被靶向降解。令人惊讶的是，使用在体内表达的组氨酸-血凝素（HIS 6x-HA（3x））表位标记的形式，无赖氨酸的IAA 1被泛素化并在体内快速降解。赖氨酸取代版本的IAA 1定位于细胞核的黄色荧光蛋白融合体和相互作用的TIR 1和IAA 7在酵母（酿酒酵母）双杂交实验，表明这些蛋白质的功能。HIS 6x-HA（3x）-IAA 1和无赖氨酸HIS 6x-HA（3x）-IAA 1蛋白上的泛素化对氢氧化钠处理敏感，表明丝氨酸或苏氨酸残基上的泛素氧酯形成。此外，在HIS 6x-HA（3x）-IAA 1中观察到泛素化IAA 1的碱基抗性形式，表明该蛋白质上存在额外的赖氨酰连接的泛素。对其他Aux/IAA蛋白的表征表明，它们具有不同的降解速率，这增加了生长素信号传导的额外复杂性。总之，这些数据表明，Aux/IAA家族成员具有蛋白质特异性降解速率，并且Aux/IAA的泛素化可以发生在多种类型的氨基残基上，以促进生长素介导的快速降解。
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 SCFTIR1/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 (HIS6x-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 HIS6x-HA(3x)-IAA1 and Lys-less HIS6x-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 HIS6x-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.