Haracska L, Udvardy A
Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged.
FEBS Lett. 1997 Jul 28;412(2):331-6. doi: 10.1016/s0014-5793(97)00808-9.
Short-lived intracellular proteins, after being marked by multiubiquitination, are degraded by the 26S protease. This large ATP-dependent protease is composed of two multiprotein complexes: the regulatory complex and the 20S proteosome. The selective recognition of ubiquitinated proteins is ensured by the regulatory complex. Using an overlay assay a single 54-kDa multiubiquitin-chain-binding subunit was detected in the regulatory complex of the Drosophila 26S protease. Overlay assay with the recombinant p54 subunit confirmed its ubiquitin-binding property. The recombinant protein showed pronounced preference for higher ubiquitin multimers, in agreement with the known preference of the 26S protease for multiubiquitinated proteins as substrates. To map the ubiquitin-binding domain of the p54 subunit different segments of the recombinant protein were expressed in E. coli and tested by the overlay assay. The p54 subunit carries two independent ubiquitin-binding domains. The central domain carries two highly conserved sequence blocks: the FGVDP sequence (at position 207), which is 100% conserved from yeast till human, and the DPELALALRVSMEE sequence (at position 214), which is 100% conserved in higher eukaryotes with two amino acid changes in yeast. In the C-terminal ubiquitin-binding domain the GVDP sequence motif is repeated and 100% conserved in higher eukaryotes. This domain, however, due to the shorter size of the yeast multiubiquitin-binding subunit, is present only in higher eukaryotes.
短寿命的细胞内蛋白质在被多聚泛素化标记后，会被26S蛋白酶降解。这种大型的ATP依赖性蛋白酶由两个多蛋白复合物组成：调节复合物和20S蛋白酶体。调节复合物确保了对泛素化蛋白质的选择性识别。通过覆盖分析，在果蝇26S蛋白酶的调节复合物中检测到一个单一的54 kDa多聚泛素链结合亚基。用重组p54亚基进行覆盖分析证实了其泛素结合特性。重组蛋白对较高的泛素多聚体表现出明显的偏好，这与26S蛋白酶对多聚泛素化蛋白质作为底物的已知偏好一致。为了绘制p54亚基的泛素结合结构域，在大肠杆菌中表达了重组蛋白的不同片段，并通过覆盖分析进行测试。p54亚基携带两个独立的泛素结合结构域。中央结构域有两个高度保守的序列块：FGVDP序列（位于第207位），从酵母到人类100%保守；DPELALALRVSMEE序列（位于第214位），在高等真核生物中100%保守，在酵母中有两个氨基酸变化。在C端泛素结合结构域中，GVDP序列基序重复出现，在高等真核生物中100%保守。然而，由于酵母多聚泛素结合亚基的尺寸较短，该结构域仅存在于高等真核生物中。
