Oxidative phosphorylation (OXPHOS) is fundamental for life. OXPHOS complexes pose a unique challenge for the cell, because their subunits are encoded on two different genomes, the nuclear genome and the mitochondrial genome. Genomic approaches designed to study nuclear/cytosolic and bacterial gene expression have not been broadly applied to the mitochondrial system; thus the co-regulation of OXPHOS genes remains largely unexplored. Here we globally monitored mitochondrial and nuclear gene expression processes in Saccharomyces cerevisiae during mitochondrial biogenesis, when OXPHOS complexes are synthesized. Nuclear- and mitochondrial-encoded OXPHOS transcript levels do not increase concordantly. Instead, we observe that mitochondrial and cytosolic translation are rapidly and dynamically regulated in a strikingly synchronous fashion. Furthermore, the coordinated translation programs are controlled unidirectionally through the intricate and dynamic control of cytosolic translation. Thus the nuclear genome carefully directs the coordination of mitochondrial and cytosolic translation to orchestrate the timely synthesis of each OXPHOS complex, representing an unappreciated regulatory layer shaping the mitochondrial proteome. Our whole-cell genomic profiling approach establishes a foundation for global gene regulatory studies of mitochondrial biology.

Eukaryotic cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial respiratory chain. COX is a multimeric enzyme formed by subunits of dual genetic origin which assembly is intricate and highly regulated. The COX catalytic core is formed by three mitochondrial DNA encoded subunits, Cox1, Cox2 and Cox3, conserved in the bacterial enzyme. Their biogenesis requires the action of messenger-specific and subunit-specific factors which facilitate the synthesis, membrane insertion, maturation or assembly of the core subunits. The study of yeast strains and human cell lines from patients carrying mutations in structural subunits and COX assembly factors has been invaluable to identify these ancillary factors. Here we review the current state of knowledge of the biogenesis and assembly of the eukaryotic COX catalytic core and discuss the degree of conservation of the players and mechanisms operating from yeast to human.

Cytochrome c-oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, plays a key role in the regulation of aerobic production of energy. Biogenesis of eukaryotic COX involves the coordinated action of two genomes. Three mitochondrial DNA-encoded subunits form the catalytic core of the enzyme, which contains metal prosthetic groups. Another 10 subunits encoded in the nuclear DNA act as a protective shield surrounding the core. COX biogenesis requires the assistance of >20 additional nuclear-encoded factors acting at all levels of the process. Expression of the mitochondrial-encoded subunits, expression and import of the nuclear-encoded subunits, insertion of the structural subunits into the mitochondrial inner membrane, addition of prosthetic groups, assembly of the holoenzyme, further maturation to form a dimer, and additional assembly into supercomplexes are all tightly regulated processes in a nuclear-mitochondrial-coordinated fashion. Such regulation ensures the building of a highly efficient machine able to catalyze the safe transfer of electrons from cytochrome c to molecular oxygen and ultimately facilitate the aerobic production of ATP. In this review, we will focus on describing and analyzing the present knowledge about the different regulatory checkpoints in COX assembly and the dynamic relationships between the different factors involved in the process. We have used information mostly obtained from the suitable yeast model, but also from bacterial and animal systems, by means of large-scale genetic, molecular biology, and physiological approaches and by integrating information concerning individual elements into a cellular system network.

The intricate biogenesis of multimeric organellar enzymes of dual genetic origin entails several levels of regulation. In Saccharomyces cerevisiae, mitochondrial cytochrome c oxidase (COX) assembly is regulated translationally. Synthesis of subunit 1 (Cox1) is contingent on the availability of its assembly partners, thereby acting as a negative feedback loop that coordinates COX1 mRNA translation with Cox1 utilization during COX assembly. The COX1 mRNA-specific translational activator Mss51 plays a fundamental role in this process. Here, we report that Mss51 successively interacts with the COX1 mRNA translational apparatus, newly synthesized Cox1, and other COX assembly factors during Cox1 maturation/assembly. Notably, the mitochondrial Hsp70 chaperone Ssc1 is shown to be an Mss51 partner throughout its metabolic cycle. We conclude that Ssc1, by interacting with Mss51 and Mss51-containing complexes, plays a critical role in Cox1 biogenesis, COX assembly, and the translational regulation of these processes.

SummaryEukaryotic cytochrome c oxidase (COX), the last enzyme of the mitochondrial respiratory chain, is a multimeric enzyme of dual genetic origin, whose assembly is a complicated and highly regulated process. COX displays a concerted accumulation of its constitutive subunits. Data obtained from studies performed with yeast mutants indicate that most catalytic core unassembled subunits are posttranslationally degraded. Recent data obtained in the yeast Saccharomyces cerevisiae have revealed another contribution to the stoichiometric accumulation of subunits during COX biogenesis targeting subunit 1 or Cox1p. Cox1p is a mitochondrially encoded catalytic subunit of COX which acts as a seed around which the full complex is assembled. A regulatory mechanism exists by which Cox1p synthesis is controlled by the availability of its assembly partners. The unique properties of this regulatory mechanism offer a means to catalyze multiple-subunit assembly. New levels of COX biogenesis regulation have been recently proposed. For example, COX assembly and stability of the fully assembled enzyme depend on the presence in the mitochondrial compartments of two partners of the oxidative phosphorylation system, the mobile electron carrier cytochrome c and the mitochondrial ATPase. The different mechanisms of regulation of COX assembly are reviewed and discussed.2008 IUBMB IUBMB Life, 60(9): [557][558][559][560][561][562][563][564][565][566][567][568] 2008

Heme plays fundamental roles as cofactor and signaling molecule in multiple pathways devoted to oxygen sensing and utilization in aerobic organisms. For cellular respiration, heme serves as a prosthetic group in electron transfer proteins and redox enzymes. Here we report that in the yeast Saccharomyces cerevisiae a heme-sensing mechanism translationally controls the biogenesis of cytochrome c oxidase (COX), the terminal mitochondrial respiratory chain enzyme. We show that Mss51, a COX1 mRNA-specific translational activator and Cox1 chaperone, which coordinates Cox1 synthesis in mitoribosomes with its assembly in COX, is a heme-binding protein. Mss51 contains two heme regulatory motifs or Cys-Pro-X domains located in its N-terminus. Using a combination of in vitro and in vivo approaches, we have demonstrated that these motifs are important for heme binding and efficient performance of Mss51 functions. We conclude that heme sensing by Mss51 regulates COX biogenesis and aerobic energy production.

Eukaryotic cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial respiratory chain. COX is a multimeric enzyme formed by subunits of dual genetic origin whose assembly is intricate and highly regulated. In addition to the structural subunits, a large number of accessory factors are required to build the holoenzyme. The function of these factors is required in all stages of the assembly process. They are relevant to human health because devastating human disorders have been associated with mutations in nuclear genes encoding conserved COX assembly factors. The study of yeast strains and human cell lines from patients carrying mutations in structural subunits and COX assembly factors has been invaluable to attain the current state of knowledge, even if still fragmentary, of the COX assembly process. After the identification of the genes involved, the isolation and characterization of genetic and metabolic suppressors of COX assembly defects, reviewed here, have become a profitable strategy to gain insight into their functions and the pathways in which they operate. Additionally, they have the potential to provide useful information for devising therapeutic approaches to combat human disorders associated with COX deficiency.

Signal regulatory proteins (SIRPs) comprise a family of cell surface signaling receptors differentially expressed in leukocytes and the central nervous system. Although the extracellular domains of SIRPs are highly similar, classical motifs in the cytoplasmic or transmembrane domains distinguish them as either activating (␤) or inhibitory (␣) isoforms. We reported previously that human neutrophils (polymorphonuclear leukocytes (PMN)) express multiple SIRP isoforms and that SIRP␣ binding to its ligand CD47 regulates PMN transmigration. Here we further characterized the expression of PMN SIRPs, and we reported that the major SIRP␣ and SIRP␤ isoforms expressed in PMN include Bit/PTPNS-1 and SIRP␤1, respectively. Furthermore, although SIRP␣ (Bit/PTPNS-1) is expressed as a monomer, we showed that SIRP␤1 is expressed on the cell surface as a disulfide-linked homodimer with bond formation mediated by Cys-320 in the membrane-proximal Ig loop. Subcellular fractionation studies revealed a major pool of SIRP␤1 within the plasma membrane fractions of PMN. In contrast, the majority of SIRP␣ (Bit/PTPNS-1) is present in fractions enriched in secondary granules and is translocated to the cell surface after chemoattractant (formylmethionylleucylphenylalanine) stimulation. Functional studies revealed that antibody-mediated ligation of SIRP␤1 enhanced formylmethionylleucylphenylalanine-driven PMN transepithelial migration. Co-immunoprecipitation experiments to identify associated adaptor proteins revealed a 10 -12-kDa protein associated with SIRP␤1 that was tyrosine-phosphorylated after PMN stimulation and is not DAP10/12 or Fc receptor ␥ chain. These results provide new insights into the structure and function of SIRPs in leukocytes and their potential role(s) in fine-tuning responses to inflammatory stimuli. Signal regulatory proteins (SIRPs)3 are a family of transmembrane receptor-like signaling proteins that are abundantly expressed in hematopoietic cells, including granulocytes, monocytes, dendritic cells, and lymphocytes (1-3). In addition, SIRPs are expressed in neuronal cells (4 -6) and certain types of cancer cells (7-10). SIRPs can be divided into two subfamilies, SIRP␣ and SIRP␤, based on the putative structures of their C-terminal intracellular domains (11). SIRPs share typical immunoglobulin superfamily structures with an N-terminal extracellular domain containing three cysteine-bound Ig-like loops, a single membrane-spanning transmembrane domain, and a C-terminal intracellular domain (11). The C-terminal intracellular domains of the SIRP␣ subfamily contain a relatively long amino acid sequence (110 amino acids for SIRP␣1) that includes four tyrosine residues to form two immunoreceptor tyrosine-based inhibition motifs (ITIM). Conversely, SIRP␤ subfamily members have a short intracellular domain containing only a few amino acids (4 amino acids for SIRP␤1). Despite a short cytoplasmic tail, SIRP␤1 contains a positively charged lysine in the transmembrane domain that can mediate interactions with an immunoreceptor tyrosi...