Fu H, Sadis S, Rubin D M, Glickman M, van Nocker S, Finley D, Vierstra R D
Cellular and Molecular Biology Program, University of Wisconsin-Madison 53706, USA.
J Biol Chem. 1998 Jan 23;273(4):1970-81. doi: 10.1074/jbc.273.4.1970.
The 26 S proteasome is a multisubunit proteolytic complex responsible for degrading eukaryotic proteins targeted by ubiquitin modification. Substrate recognition by the complex is presumed to be mediated by one or more common receptor(s) with affinity for multiubiquitin chains, especially those internally linked through lysine 48. We have identified previously a candidate for one such receptor from diverse species, designated here as Mcb1 for Multiubiquitin chain-binding protein, based on its ability to bind Lys48-linked multiubiquitin chains and its location within the 26 S proteasome complex. Even though Mcb1 is likely not the only receptor in yeast, it is necessary for conferring resistance to amino acid analogs and for degrading a subset of ubiquitin pathway substrates such as ubiquitin-Pro-beta-galactosidase (Ub-Pro-beta-gal) (van Nocker, S., Sadis, S., Rubin, D.M., Glickman, M., Fu, H., Coux, O., Wefes, I., Finley, D., and Vierstra, R. D. (1996) Mol. Cell. Biol. 16, 6020-28). To further define the role of Mcb1 in substrate recognition by the 26 S proteasome, a structure/function analysis of various deletion and site-directed mutants of yeast and Arabidopsis Mcb1 was performed. From these studies, we identified a single stretch of conserved hydrophobic amino acids (LAM/LALRL/V (ScMcb1 228-234 and At-Mcb1 226-232)) within the C-terminal half of each polypeptide that is necessary for interaction with Lys48-linked multiubiquitin chains. Unexpectedly, this domain was not essential for either Ub-Pro-beta-gal degradation or conferring resistance to amino acid analogs. The domain responsible for these two activities was mapped to a conserved region near the N terminus. Yeast and Arabidopsis Mcb1 derivatives containing an intact multiubiquitin-binding site but missing the N-terminal region failed to promote Ub-Pro-beta-gal degradation and even accentuated the sensitivity of the yeast delta mcb1 strain to amino acid analogs. This hypersensitivity was not caused by a gross defect in 26 S proteasome assembly as mutants missing either the N-terminal domain or the multiubiquitin chain-binding site could still associate with 26 S proteasome and generate a complex indistinguishable in size from that present in wild-type yeast. Together, these data indicate that residues near the N terminus, and not the multiubiquitin chain-binding site, are most critical for Mcb1 function in vivo.
26S蛋白酶体是一种多亚基蛋白水解复合物，负责降解经泛素修饰靶向的真核生物蛋白质。推测该复合物对底物的识别是由一种或多种对多聚泛素链具有亲和力的共同受体介导的，尤其是那些通过赖氨酸48内部连接的多聚泛素链。我们之前从不同物种中鉴定出了一种此类受体的候选物，基于其结合赖氨酸48连接的多聚泛素链的能力及其在26S蛋白酶体复合物中的位置，在此将其命名为Mcb1（多聚泛素链结合蛋白）。尽管Mcb1可能不是酵母中唯一的受体，但它对于赋予对氨基酸类似物的抗性以及降解泛素途径底物的一个子集（如泛素 - 脯氨酸 - β - 半乳糖苷酶（Ub - Pro - β - gal））是必需的（范·诺克，S.，萨迪斯，S.，鲁宾，D.M.，格利克曼，M.，傅，H.，库克斯，O.，韦费斯，I.，芬利，D.，和维斯特拉，R.D.（1996年）《分子与细胞生物学》16，6020 - 28）。为了进一步确定Mcb1在26S蛋白酶体底物识别中的作用，我们对酵母和拟南芥Mcb1的各种缺失突变体和定点突变体进行了结构/功能分析。通过这些研究，我们在每个多肽的C端一半区域内鉴定出了一段单一的保守疏水氨基酸序列（LAM/LALRL/V（酿酒酵母Mcb1 228 - 234和拟南芥Mcb1 226 - 232）），这是与赖氨酸48连接的多聚泛素链相互作用所必需的。出乎意料的是，该结构域对于Ub - Pro - β - gal的降解或赋予对氨基酸类似物的抗性都不是必需的。负责这两种活性的结构域被定位到N端附近的一个保守区域。含有完整多聚泛素结合位点但缺失N端区域的酵母和拟南芥Mcb1衍生物无法促进Ub - Pro - β - gal的降解，甚至加剧了酵母Δmcb1菌株对氨基酸类似物的敏感性。这种超敏感性不是由26S蛋白酶体组装的严重缺陷引起的，因为缺失N端结构域或多聚泛素链结合位点的突变体仍然可以与26S蛋白酶体结合，并产生一个大小与野生型酵母中存在的复合物无法区分的复合物。总之，这些数据表明N端附近的残基而非多聚泛素链结合位点对于Mcb1在体内的功能最为关键。
