Kus Bart M, Caldon Catherine E, Andorn-Broza Ronit, Edwards Aled M
Banting and Best Department of Medical Research, University of Toronto, Ontario.
Proteins. 2004 Feb 15;54(3):455-67. doi: 10.1002/prot.10620.
SCF complexes are multi-subunit ubiquitin ligases that, in concert with the E1 and E2 ubiquitination enzymes, catalyze the ubiquination of specific target proteins. Only three yeast SCFs have been reconstituted and characterized to date; each of these ubiquitinates its target protein with the E2 Cdc34. We have reconstituted and purified 1 known and 12 novel yeast SCF complexes, and explored the ability of these complexes to function with 5 different purified E2 enzymes; Ubc1, Cdc34, Ubc4, Ubc8 and Ubc11. We have found that the ubiquitination of Sic1 by the reconstituted SCF(Cdc4) complex was specifically catalyzed by two of the five E2 enzymes tested in vitro; Cdc34 and Ubc4. We also show that at least eight of the purified SCF complexes clearly ubiquitinated their F-box proteins in vitro, lending support for a regulatory mechanism in which F-box proteins catalyze their own destruction. The autoubiquitination of each F-box was in some cases catalyzed only by Cdc34, and in other cases preferentially catalyzed by Ubc4. Ubc4 thus interacts with multiple SCFs in vitro, and the interactions among SCF and E2 components of the ubiquitination machinery may allow further diversification of the roles of SCFs in vivo.
SCF复合物是多亚基泛素连接酶，与E1和E2泛素化酶协同作用，催化特定靶蛋白的泛素化。迄今为止，仅重构并鉴定了三种酵母SCF；它们各自都通过E2 Cdc34使靶蛋白泛素化。我们重构并纯化了1种已知的和12种新型酵母SCF复合物，并探究了这些复合物与5种不同纯化的E2酶（Ubc1、Cdc34、Ubc4、Ubc8和Ubc11）共同发挥作用的能力。我们发现，重构的SCF(Cdc4)复合物对Sic1的泛素化在体外是由所测试的5种E2酶中的两种特异性催化的，即Cdc34和Ubc4。我们还表明，至少8种纯化的SCF复合物在体外能明确地使它们的F-box蛋白泛素化，这为一种调节机制提供了支持，即F-box蛋白催化自身的降解。每种F-box的自泛素化在某些情况下仅由Cdc34催化，而在其他情况下则优先由Ubc4催化。因此，Ubc4在体外与多种SCF相互作用，泛素化机制中SCF与E2组分之间的相互作用可能使SCF在体内的作用进一步多样化。
