Girod P A, Fu H, Zryd J P, Vierstra R D
Laboratoire de Phytogénétique Cellulaire, Université de Lausanne, 1015 Lausanne, Switzerland.
Plant Cell. 1999 Aug;11(8):1457-72. doi: 10.1105/tpc.11.8.1457.
The 26S proteasome, a multisubunit complex, is the primary protease of the ubiquitin-mediated proteolytic system in eukaryotes. We have recently characterized MCB1 (RPN10), a subunit of the 26S complex that has affinity for multiubiquitin chains in vitro and as a result may function as a receptor for ubiquitinated substrates. To define the role of MCB1 further, we analyzed its function in Physcomitrella patens by generating MCB1 gene disruptions using homologous recombination. PpMCB1, which is 50 to 75% similar to orthologs from other eukaryotes, is present in the 26S proteasome complex and has a similar affinity for multiubiquitin chains, using a conserved hydrophobic domain within the C-terminal half of the polypeptide. Unlike yeast Deltamcb1 strains, which grow normally, P. patens Deltamcb1 strains are viable but are under developmental arrest, generating abnormal caulonema that are unable to form buds and gametophores. Treatment with auxin and cytokinin restored bud formation and subsequent partial development of gametophores. Complementation of a Deltamcb1 strain with mutated versions of PpMCB1 revealed that the multiubiquitin chain binding site is not essential for the wild-type phenotype. These results show that MCB1 has an important function in the 26S proteasome of higher order eukaryotes in addition to its ability to bind multiubiquitin chains, and they provide further support for a role of the ubiquitin/26S proteasome proteolytic pathway in plant developmental processes triggered by hormones.
26S蛋白酶体是一种多亚基复合物，是真核生物中泛素介导的蛋白水解系统的主要蛋白酶。我们最近鉴定了MCB1（RPN10），它是26S复合物的一个亚基，在体外对多聚泛素链具有亲和力，因此可能作为泛素化底物的受体发挥作用。为了进一步确定MCB1的作用，我们通过同源重组产生MCB1基因破坏，分析了其在小立碗藓中的功能。PpMCB1与其他真核生物的直系同源物有50%至75%的相似性，存在于26S蛋白酶体复合物中，并且利用多肽C端一半内的保守疏水结构域，对多聚泛素链具有相似的亲和力。与正常生长的酵母Δmcb1菌株不同，小立碗藓Δmcb1菌株是存活的，但处于发育停滞状态，产生异常的茎丝，无法形成芽和配子体。用生长素和细胞分裂素处理可恢复芽的形成以及随后配子体的部分发育。用PpMCB1的突变版本对Δmcb1菌株进行互补分析表明，多聚泛素链结合位点对野生型表型不是必需的。这些结果表明，MCB1除了具有结合多聚泛素链的能力外，在高等真核生物的26S蛋白酶体中还具有重要功能，并且它们为泛素/26S蛋白酶体蛋白水解途径在激素触发的植物发育过程中的作用提供了进一步的支持。
