Novel multiubiquitin chain linkages catalyzed by the conjugating enzymes E2(EPF) and RAD6 are recognized by 26 S proteasome subunit 5
Novel multiubiquitin chain linkages catalyzed by the conjugating enzymes E2(EPF) and RAD6 are recognized by 26 S proteasome subunit 5
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DOI：
10.1074/jbc.271.5.2823
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发表时间：
1996-02-02
影响因子：
4.8
通讯作者：
Haas, AL
中科院分区：
文献类型：
--
作者：
Baboshina, OV;Haas, AL
靶向ATP降解底物，泛素依赖性途径需要形成多泛素链，其中8.6-kDa多肽通过羧基末端和连续单体的Lys-48残基之间的异肽键连接。26 S蛋白酶体调节复合物的亚基5与Lys-48连接链的结合使所连接的靶蛋白降解，伴随着链解体后游离泛素单体的释放。泛素的点突变体（Lys-> Arg）被用于绘制先前证明形成不通过Lys-48连接的新型多泛素链的泛素缀合酶的连接特异性。重组人E2（EPF）仅通过泛素的Lys-11催化多泛素链的形成，而重组酵母RAD 6仅通过Lys-6形成多泛素链，通过Lys-6、Lys-11或Lys-48连接的多泛素链各自以相当的亲和力结合部分纯化的人26 S蛋白酶体的亚基5。由于带有不同键的链预期包装成不同的结构，所以Lys-11和Lys-48链之间结合亚基5的竞争表明，后者具有识别交替连接的链的决定因素，并且排除了识别不同结构的亚基5同种型的存在。竞争研究提供了对于连接数n> 4的Lys-48连接链的内在结合的K-d小于或等于18 nM的估计。这一结果表明，多遍在蛋白链形成的主要机制优势是相对于未缀合的靶蛋白，增强相关底物对26 S复合物的亲和力。用E1/E2耗尽的兔网织红细胞提取物进行的互补研究表明，RAD6仅通过Lys-48连接的链支持异肽连接酶依赖性降解，而E2（EPF）保留了通过Lys-11连接的链靶向模型放射性标记底物的能力。因此，这些E2同工酶所表现出的连接特异性取决于它们对异肽连接酶的催化作用。
Targeting of substrates for degradation by the ATP, ubiquitin-dependent pathway requires formation of multiubiquitin chains in which the 8.6-kDa polypeptide is linked by isopeptide bonds between carboxyl termini and Lys-48 residues of successive monomers. Binding of Lys-48-linked chains by subunit 5 of the 26 S proteasome regulatory complex commits the attached target protein to degradation with concomitant release of free ubiquitin monomers following disassembly of the chains. Point mutants of ubiquitin (Lys --> Arg) were used to map the linkage specificity for ubiquitin-conjugating enzymes previously demonstrated to form novel multiubiquitin chains not attached through Lys-48. Recombinant human E2(EPF) catalyzed multiubiquitin chain formation exclusively through Lys-11 of ubiquitin while recombinant yeast RAD6 formed chains linked only through Lys-6, Multiubiquitin chains linked through Lys-6, Lys-11, or Lys-48 each bound to subunit 5 off partially purified human 26 S proteasome with comparable affinities. Since chains bearing different linkages are expected to pack into distinct structures, competition between Lys-11 and Lys-48 chains for binding to subunit 5 demonstrates that the latter possesses determinants for recognizing alternatively linked chains and precludes the existence of subunit 5 isoforms recognizing distinct structures, In addition, competition studies provided an estimate of K-d less than or equal to 18 nM for the intrinsic binding of Lys-48-linked chains of linkage number n > 4. This result suggests that the principal mechanistic advantage of multiubiquitin chain formation is to enhance the affinity of the associated substrate for the 26 S complex relative to that of unconjugated target protein. Complementation studies with E1/E2-depleted rabbit reticulocyte extract demonstrated RAD6 supported isopeptide ligase-dependent degradation only through Lys-48-linked chains, while E2(EPF) retained the ability to target a model radiolabeled substrate through Lys-11-linked chains. Therefore, the linkage specificity exhibited by these E2 isozymes depends on their catalytic context with respect to isopeptide ligase.