Modular assembly of cytochrome oxidase

描述（由申请方提供）：细胞色素氧化酶（考克斯）是呼吸链的末端复合物，由11种不同的亚基多肽组成。Cox 1 p、Cox 2 p和Cox 3 p亚基是由核基因编码的催化核心，而其他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，考克斯的生物发生是一个高度整合的过程，是由许多辅助蛋白和调节因子协调平衡输出的区室分离的基因。用分离的线粒体进行的脉冲追踪研究表明，在它们被同化为考克斯之前，Cox 1 p和Cox 3 p各自通过一系列中间体转变，这些中间体由它们的辅助因子和结构亚基的组成区分。这一发现 使我们提出了一个生物起源模型，涉及三个独立的途径，模块化的产品，最终联合收割机形成全酶。 该提案有四个目标，每个目标都与考克斯生物发生的一个重要方面有关。1)我们希望巩固的模块化组装模型与直接的实验证据表明，Cox 2 p也预装作为一个独立的模块。这将通过识别和表征脉冲追踪标记的线粒体中的Cox 2 p中间体来解决。2)我们有间接的证据表明，线粒体基因产物的考克斯和bc 1复合物，这两者都存在于一个超复合物，插入在同一个或相邻的亚隔室的膜在其生物合成。这表明，组装的考克斯/bc 1超复合物可能是协调的物理和时间。我们将通过遗传学和生物化学手段来检验这一假设。3)最近的一些研究结果表明，考克斯和ATP合酶的适当的化学计量可以通过转子亚基Atp 9 p和考克斯外周亚基Cox 6 p的复合物来实现。我们将通过研究cox 6无效突变体中ATP合酶的组装和atp 9突变体中考克斯组装的相反组装来进一步测试这种有趣的可能性。4)我们目前对考克斯生物发生的认识大多来自于对Cox 1 p-Cox 3 p组装途径不同阶段的辅助因子的功能研究。许多但不是所有的这些因素是通过研究酵母突变体显示特定缺陷的考克斯。本建议的第四个也是最后一个目标是继续挖掘一系列核呼吸缺陷酵母pet突变体，以寻找尚未发现的考克斯生物合成所必需的基因。 这些研究将扩大我们的理解的机制和因素，管理装配的一个重要的线粒体呼吸复合体来自遗传信息居住在两个空间上不同的车厢的细胞。

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.