Cytochrome c oxidase (complex IV) of the respiratory chain is assembled from nuclear and mitochondrially‐encoded subunits. Defects in the assembly process lead to severe human disorders such as Leigh syndrome. Shy1 is an assembly factor for complex IV in Saccharomyces cerevisiae and mutations of its human homolog, SURF1, are the most frequent cause for Leigh syndrome. We report that Shy1 promotes complex IV biogenesis through association with different protein modules; Shy1 interacts with Mss51 and Cox14, translational regulators of Cox1. Additionally, Shy1 associates with the subcomplexes of complex IV that are potential assembly intermediates. Formation of these subcomplexes depends on Coa1 (YIL157c), a novel assembly factor that cooperates with Shy1. Moreover, partially assembled forms of complex IV bound to Shy1 and Cox14 can associate with the bc1 complex to form transitional supercomplexes. We suggest that Shy1 links Cox1 translational regulation to complex IV assembly and supercomplex formation.

Mitochondria are crucial for numerous cellular processes, yet the regulation of mitochondrial functions is only understood in part. Recent studies indicated that the number of mitochondrial phosphoproteins is higher than expected; however, the effect of reversible phosphorylation on mitochondrial structure and function has only been defined in a few cases. It is thus crucial to determine authentic protein phosphorylation sites from highly purified mitochondria in a genetically tractable organism. The yeast Saccharomyces cerevisiae is a major model organism for the analysis of mitochondrial functions. We isolated highly pure yeast mitochondria and performed a systematic analysis of phosphorylation sites by a combination of different enrichment strategies and mass spectrometry. We identified 80 phosphorylation sites in 48 different proteins. These mitochondrial phosphoproteins are involved in critical mitochondrial functions, including energy metabolism, protein biogenesis, fatty acid metabolism, metabolite transport, and redox regulation. By combining yeast genetics and in vitro biochemical analysis, we found that phosphorylation of a serine residue in subunit g (Atp20) regulates dimerization of the mitochondrial ATP synthase. The authentic phosphoproteome of yeast mitochondria will represent a rich source to uncover novel roles of reversible protein phosphorylation. Molecular & Cellular Proteomics 6:1896 -1906, 2007.Mitochondria are the central organelle for the energy metabolism of eukaryotic cells and are critical for numerous metabolic pathways, including that for amino acids, lipids, heme, and iron-sulfur clusters, and play key roles in the regulation of programmed cell death (1-5). It is evident that these processes have to be tightly regulated to permit a mitochondrial response to changes in energy demand, cellular metabolism, or environmental conditions (6, 7). Until recently the most common regulatory mechanism of eukaryotic cells, reversible phosphorylation (8 -10), was considered to represent an exception in the case of mitochondria, including the E1 subunit of pyruvate dehydrogenase and the branched-chain ␣-ketoacid dehydrogenase (11)(12)(13)(14).A number of recent studies have provided evidence that phosphorylation of mitochondrial proteins is much more frequent than expected (15-23). (i) Incubation of isolated mitochondria with radiolabeled ATP or staining of mitochondrial proteins with phosphospecific dyes suggested that a substantial fraction of mitochondrial proteins are phosphorylated (24 -27). A limitation of these approaches is the inability to identify the specific phosphorylated amino acid residues in addition to the possibility of nonspecific labeling of proteins.(ii) Proteomics analysis of isolated mitochondria by mass spectrometry revealed the presence of numerous protein kinases and phosphatases (28 -34), implying that reversible protein phosphorylation may be a widespread mechanism of regulating mitochondrial function. The most comprehensive proteomics analysis of mitochondria, the PROM...

Members of the Pbx family are involved in a diverse range of developmental processes including axial patterning and organogenesis. Pbx functions are in part mediated by the interaction of Pbx proteins with members of the Hox and Meis/Prep families. We have identified a fourth mammalian Pbx family member. Pbx4 in the mouse and PBX4 in humans are located on chromosome 8 and chromosome 19, respectively. Pbx4 expression is confined to the testis, especially to spermatocytes in the pachytene stage of the first meiotic prophase.

The mitochondrial inner membrane contains preprotein translocases that mediate insertion of hydrophobic proteins. Little is known about how the individual components of these inner membrane preprotein translocases combine to form multisubunit complexes. We have analyzed the assembly pathway of the three membrane-integral subunits Tim18, Tim22, and Tim54 of the twin-pore carrier translocase. Tim54 displayed the most complex pathway involving four preprotein translocases. The precursor is translocated across the intermembrane space in a supercomplex of outer and inner membrane translocases. The TIM10 complex, which translocates the precursor of Tim22 through the intermembrane space, functions in a new posttranslocational manner: in case of Tim54, it is required for the integration of Tim54 into the carrier translocase. Tim18, the function of which has been unknown so far, stimulates integration of Tim54 into the carrier translocase. We show that the carrier translocase is built via a modular process and that each subunit follows a different assembly route. Membrane insertion and assembly into the oligomeric complex are uncoupled for each precursor protein. We propose that the mitochondrial assembly machinery has adapted to the needs of each membrane-integral subunit and that the uncoupling of translocation and oligomerization is an important principle to ensure continuous import and assembly of protein complexes in a highly active membrane.The majority of mitochondrial proteins are nucleus encoded and imported into mitochondria through protein translocase complexes (6,7,17,25,29,33,42). The translocase of the outer membrane (TOM complex) is the general entry gate for mitochondrial precursor proteins. Two translocases of the inner membrane (TIM), the presequence translocase (TIM23 complex) and the twin-pore carrier translocase (TIM22 complex), mediate signal-selective transport of precursor proteins. While the TIM23 complex translocates the majority of substrates into the matrix and inserts only a limited number of substrates into the inner membrane (7,10,13,17,25,26,40), the TIM22 complex is dedicated to the insertion of multispanning hydrophobic proteins into the inner membrane, including a large number of metabolite carriers (7,17,25,27,29,46). The TIM22 complex is a voltage-dependent 300-kDa complex with three membrane-integral subunits, Tim18, Tim22, and Tim54. Tim22 forms the voltage-sensitive channels of the twin-pore translocase (21, 30). Tim54 was shown to play a role in the assembly of a protease complex (Yme1) of the inner membrane, yet the molecular mechanism of its action has not been elucidated (12). Thus, the molecular functions of Tim54 and Tim18 in the TIM22 complex are unknown (6,7,17,25,29).The precursors of metabolite carriers are not directly transferred from the TOM complex to the TIM22 complex, but the TIM10 translocase complex of the intermembrane space binds to the precursors and functions in a chaperone-like manner to guide them through the aqueous space between outer and inner membranes. ...