Modular assembly of cytochrome oxidase

描述(申请人提供)：细胞色素氧化酶(COX)，呼吸链的末端复合体，由11个不同的亚基多肽组成。线粒体编码的Cox1p、Cox2p和Cox3p亚基构成催化核心，其他8个结构亚基是核基因的产物。由于其遗传和组成的复杂性，COX的生物发生是一个高度整合的过程，它受到大量辅助蛋白和调节因子的辅助，以协调分隔分离的基因的平衡输出。对分离的线粒体的脉冲追逐研究表明，在它们被同化为COX、Cox1p和Cox3p之前，每个线粒体都通过一系列中间产物过渡，这些中间产物由辅助因子和结构亚基的组成来区分。这一发现已经 导致我们提出了一个生物发生模型，涉及三个独立的途径，具有模块化的产物，最终结合形成全酶。这一提议有四个目标，每个目标都与COX生物发生的一个重要方面有关。1)我们希望用直接的实验证据巩固模块化组装模型，证明Cox2p也作为一个独立的模块进行预组装。这将通过鉴定和表征脉冲追逐标记线粒体中的Cox2p中间体来解决。2)我们有间接证据表明，在生物发生过程中，COX和BC1复合体的线粒体基因产物都以超复合体的形式存在，它们被插入到膜的相同或相邻的亚室中。这表明COX/BC1超复合体的组装可能在物理上和时间上都是协调的。我们将通过遗传和生化手段来检验这一假说。3)最近的一些结果表明，COX和ATP合成酶的适当化学计量比可能是通过转子亚基Atp9p和COX外周亚基Cox6p的复合体实现的。我们将通过研究ATP合成酶在cox6缺失突变体中的组装，以及反过来在atp9突变体中的cox组装来进一步测试这一有趣的可能性。4)我们目前对COX生物发生的大部分知识来自对辅助因子的功能研究，这些辅助因子参与了Cox1p-Cox3p组装途径的不同阶段。许多但不是所有这些因素都是通过对酵母突变株的研究确定的，这些突变株显示了COX的一个特定缺陷。这项建议的第四个也是最后一个目标是继续挖掘核呼吸缺陷酵母宠物突变体的集合，以寻找对COX生物发生至关重要的仍未发现的基因。这些研究将扩大我们对控制一个重要的线粒体呼吸复合体组装的机制和因素的理解，该复合体来自位于细胞两个空间不同隔室的遗传信息。

DESCRIPTION (provided by applicant): Cytochrome oxidase (COX), the terminal complex of the respiratory chain, consists of 11 different subunit polypeptides. The mitochondrially encoded Cox1p, Cox2p and Cox3p subunits constitute the catalytic core while eight other structural subunits are products of nuclear genes. Because of its genetic and compositional complexity, biogenesis of COX is a highly integrated process that is assisted by numerous accessory proteins and regulatory factors for coordinating a balanced output of compartmentally separated genes. Pulse-chase studies with isolated mitochondria have shown that prior to their assimilation into COX, Cox1p and Cox3p, each, transitions through a series of intermediates differentiated by their compositions of accessory factors and structural subunits. This finding has led us to propose a biogenesis model involving three separate pathways with modular products that ultimately combine to form the holoenzyme. This proposal has four objectives, each related to an important facet of COX biogenesis. 1) We wish to consolidate the modular assembly model with direct experimental evidence that Cox2p also preassembles as an independent module. This will be addressed by identifying and characterizing Cox2p intermediates in pulse-chase labeled mitochondria. 2) We have indirect evidence that mitochondrial gene products of COX and the bc1 complex, both of which exist in a supercomplex, are inserted in the same or neighboring subcompartments of the membrane during their biogenesis. This suggests that assembly of the COX/bc1 supercomplex may be coordinated both physically and temporally. We will test this hypothesis by genetic and biochemical means. 3) Some recent results suggest that the proper stoichiometry of COX and ATP synthase may be achieved via a complex of the rotor subunit Atp9p and the COX peripheral subunit Cox6p. We will further test this interesting possibility by studying assembly of the ATP synthase in cox6 null mutants and conversely of COX assembly in atp9 mutants. 4) Much of our current knowledge of COX biogenesis has come from functional studies of accessory factors that intervene at different stages of the Cox1p-Cox3p assembly pathways. Many but not all of these factors were identified through studies of yeast mutants displaying a specific deficiency in COX. The fourth and last objective of this proposal is to continue mining a collection of nuclear respiratory defective yeast pet mutants for still undiscovered genes essential for COX biogenesis. These studies will enlarge our understanding of the mechanisms and the factors that govern assembly of an important mitochondrial respiratory complex derived from genetic information residing in two spatially distinct compartments of the cell.