Tzagoloff A, Crivellone M D, Gampel A, Muroff I, Nishikimi M, Wu M
Department of Biological Sciences, Columbia University, New York, New York 10027.
Philos Trans R Soc Lond B Biol Sci. 1988 May 31;319(1193):107-20. doi: 10.1098/rstb.1988.0035.
The synthesis of cytochrome b in yeast depends on the expression of both mitochondrial and nuclear gene products that act at the level of processing of the pre-mRNA, translation of the mRNA, and maturation of the apoprotein during its assembly with the nuclear-encoded subunits of coenzyme QH2-cytochrome c reductase. Previous studies indicated one of the nuclear genes (CBP2) to code for a protein that is needed for the excision of the terminal intervening sequence from the pre-mRNA. We show here that the intervening sequence can promote its own excision in the presence of high concentrations of magnesium ion (50 mM), but that at physiological concentrations of the divalent cation (5 mM), the splicing reaction requires the presence of the CBP2-encoded product. These results provide strong evidence for a direct participation of the protein in splicing, most likely in stabilizing a splicing competent structure in the RNA. The conversion of apocytochrome b to the functional cytochrome has been examined in mutants lacking one or multiple structural subunits of the coenzyme QH2-cytochrome c reductase complex. Based on the phenotypes of the different mutants studied, the following have been concluded. (i) The assembly of catalytically active enzyme requires the synthesis of all except the 17 kDa subunit. (ii) Membrane insertion of the individual subunits is not contingent on protein-protein interactions. (iii) Assembly of the subunits occurs in the lipid bilayer following their insertion. (iv) The attachment of haem to apocytochrome b is a late event in assembly after an intermediate complex of the structural subunits has been formed. This complex minimally is composed of apocytochrome b, the non haem iron protein and all the non-catalytic subunits except for the 17 kDa core 3 subunit.
酵母中细胞色素b的合成取决于线粒体和核基因产物的表达，这些产物作用于前体mRNA的加工、mRNA的翻译以及脱辅基蛋白与辅酶QH2 - 细胞色素c还原酶的核编码亚基组装过程中的成熟。先前的研究表明，其中一个核基因（CBP2）编码一种蛋白质，该蛋白质是从前体mRNA中切除末端间隔序列所必需的。我们在此表明，间隔序列在高浓度镁离子（50 mM）存在下可促进自身切除，但在二价阳离子的生理浓度（5 mM）下，剪接反应需要CBP2编码产物的存在。这些结果为该蛋白质直接参与剪接提供了有力证据，很可能是在稳定RNA中的剪接活性结构。在缺乏辅酶QH2 - 细胞色素c还原酶复合物的一个或多个结构亚基的突变体中，已对脱辅基细胞色素b向功能性细胞色素的转化进行了研究。基于所研究的不同突变体的表型，得出了以下结论。（i）催化活性酶的组装需要合成除17 kDa亚基之外的所有亚基。（ii）各个亚基的膜插入不依赖于蛋白质 - 蛋白质相互作用。（iii）亚基在插入脂质双层后发生组装。（iv）血红素与脱辅基细胞色素b的结合是在结构亚基的中间复合物形成后的组装后期事件。该复合物至少由脱辅基细胞色素b、非血红素铁蛋白和除17 kDa核心3亚基之外的所有非催化亚基组成。
