Genetic code differences prevent expression of nuclear genes within Saccharomyces cerevisiae mitochondria. To bridge this gap a synthetic gene, ARG8m, designed to specify an arginine biosynthetic enzyme when expressed inside mitochondria, has been inserted into yeast mtDNA in place of the COX3 structural gene. This mitochondrial cox3::ARG8m gene fully complements a nuclear arg8 deletion at the level of cell growth, and it is dependent for expression upon nuclear genes that encode subunits of the COX3 mRNAspecific translational activator. Thus, cox3::ARG8m serves as a mitochondrial reporter gene. Measurement of cox3::ARG8m expression at the levels of steady-state protein and enzymatic activity reveals that glucose repression operates within mitochondria. The levels of this reporter vary among strains whose nuclear genotypes lead to under-and overexpression of translational activator subunits, in particular Pet494p, indicating that mRNA-specific translational activation is a rate-limiting step in this organellar system. Whereas the steady-state level of cox3::ARG8m mRNA was also glucose repressed in an otherwise wild-type strain, absence of translational activation led to essentially repressed mRNA levels even under derepressing growth conditions. Thus, the mRNA is stabilized by translational activation, and variation in its level may be largely due to modulation of translation.

The core of the cytochrome c oxidase complex is composed of its three largest subunits, Cox1p, Cox2p, and Cox3p, which are encoded in mitochondrial DNA of Saccharomyces cerevisiae and inserted into the inner membrane from the inside. Mitochondrial translation of the COX1,COX2, and COX3 mRNAs is activated mRNA specifically by the nuclearly coded proteins Pet309p, Pet111p, and the concerted action of Pet54p, Pet122p, and Pet494p, respectively. Because the translational activators recognize sites in the 5′-untranslated leaders of these mRNAs and because untranslated mRNA sequences contain information for targeting their protein products, the activators are likely to play a role in localizing translation. Herein, we report physical associations among the mRNA-specific translational activator proteins, located on the matrix side of the inner membrane. These interactions, detected by coimmune precipitation and by two-hybrid experiments, suggest that the translational activator proteins could be organized on the surface of the inner membrane such that synthesis of Cox1p, Cox2p, and Cox3p would be colocalized in a way that facilitates assembly of the core of the cytochrome c oxidase complex. In addition, we found interactions between Nam1p/Mtf2p and the translational activators, suggesting an organized delivery of mitochondrial mRNAs to the translation system.

Functional interactions of the translational activator Mss51 with both the mitochondrially encoded COX1 mRNA 5-untranslated region and with newly synthesized unassembled Cox1 protein suggest that it has a key role in coupling Cox1 synthesis with assembly of cytochrome c oxidase. Mss51 is present at levels that are near rate limiting for expression of a reporter gene inserted at COX1 in mitochondrial DNA, and a substantial fraction of Mss51 is associated with Cox1 protein in assembly intermediates. Thus, sequestration of Mss51 in assembly intermediates could limit Cox1 synthesis in wild type, and account for the reduced Cox1 synthesis caused by most yeast mutations that block assembly. Mss51 does not stably interact with newly synthesized Cox1 in a mutant lacking Cox14, suggesting that the failure of nuclear cox14 mutants to decrease Cox1 synthesis, despite their inability to assemble cytochrome c oxidase, is due to a failure to sequester Mss51. The physical interaction between Mss51 and Cox14 is dependent upon Cox1 synthesis, indicating dynamic assembly of early cytochrome c oxidase intermediates nucleated by Cox1. Regulation of COX1 mRNA translation by Mss51 seems to be an example of a homeostatic mechanism in which a positive effector of gene expression interacts with the product it regulates in a posttranslational assembly process. INTRODUCTIONThe largest subunit of mitochondrial cytochrome c oxidase, Cox1, is encoded in the mitochondrial DNA (mtDNA) of all eukaryotic species that have been examined (Gray et al., 2004), and it is synthesized by their organellar genetic systems. Cox1 is highly hydrophobic, spanning the inner mitochondrial membrane 12 times, and it is complexed with several metal ions and two heme A moieties that participate directly in electron transport (Tsukihara et al., 1996). It is assembled into the core of cytochrome c oxidase, largely surrounded by subunits encoded by nuclear genes. The processes by which Cox1 is assembled with the other subunits and cofactors into an active enzyme are highly complex, requiring at least 30 genes in Saccharomyces cerevisiae (Herrmann and Funes, 2005;Khalimonchuk and Rodel, 2005;Cobine et al., 2006;Fontanesi et al., 2006;Barrientos et al., 2009). The assembly pathway is not understood in detail. In mammals, analysis of mutant and drug-treated cell lines indicates that Cox1 is a component of the earliest assembly intermediates (Nijtmans et al., 1998;Williams et al., 2004), and similar analysis in yeast is consistent with this idea (Horan et al., 2005).An important function of this assembly process may be to prevent incompletely assembled components of cytochrome c oxidase from generating damaging reactive oxygen species, before they are contained by the holoenzyme. Indeed, mutations in several yeast genes required for cytochrome c oxidase assembly cause hypersensitivity to hydrogen peroxide (Pungartnik et al., 1999;Williams et al., 2005;Banting and Glerum, 2006), and a key component of the reactive prooxidant species is Cox1 . One feature of the assembly p...

A number of mechanisms have been proposed in the literature as contributors to the strength development at the polymer-polymer interface during fusion bonding of thermoplastic composites. Of these, healing and intimate contact emerge as fundamental mechanisms governing bonding. Intimate contact refers to the development of the amount of surface area that is physically contacted at the interface at any time, and healing describes the migration of polymer chains across the interface in intimate contact. This work provides a new theoretical development of a coupled bonding model that accounts for variability in initiation time for healing due to growth in the area in intimate contact. The generalized coupled bonding model is valid for any set of processing conditions and reduces to the proper controlling mechanism as dictated by the process. Analysis revealed a key dimensionless group, Q, that captures the coupled nature of the mechanisms governing fusion bonding. By evaluating Q, which is a function of material and process parameters, one can determine the relative contributions of each mechanism. Experimental validation of the coupled model using two different processes, tow placement and resistance welding, is also presented. An evaluation of Q for the tow-placement process indicates that both mechanisms are controlling. For this case, the coupled model demonstrates better strength predictions than the conventional healing model alone. In contrast, the resistance welding process is shown to be intimate-contact controlled, in which case the coupled model reduces to a more simplified model. The ability to rigorously determine the controlling mechanisms is of critical importance to accurately model the strength development during fusion bonding processes.

The protein specified by the Saccharomyces cerevisiae nuclear gene PET111 specifically activates translation of the mitochondrially coded mRNA for cytochrome c oxidase subunit II (Cox2p). We found Pet111p specifically in mitochondria of both wild-type cells and cells expressing a chromosomal gene for a functional epitopetagged form of Pet111p. Pet111p was associated with mitochondrial membranes and was highly resistant to extraction with alkaline carbonate. Pet111p was protected from proteolytic digestion by the mitochondrial inner membrane. Thus, it is exposed only on the matrix side, where it could participate directly in organellar translation and localize Cox2p synthesis by virtue of its functional interaction with the COX2 mRNA 5-untranslated leader. We also found that Pet111p is present at levels limiting the synthesis of Cox2p by examining the effect of altered PET111 gene dosage in the nucleus on expression of a reporter gene, cox2::ARG8 m , that was inserted into mitochondrial DNA. The level of the reporter protein, Arg8p, was one-half that of wild type in a diploid strain heterozygous for a pet111 deletion mutation, whereas it was increased 2.8-fold in a strain bearing extra copies of PET111 on a high-copy plasmid. Thus, Pet111p could play dual roles in both membrane localization and regulation of Cox2p synthesis within mitochondria.

Synthesis of the largest cytochrome c oxidase (CcO) subunit, Cox1, on yeast mitochondrial ribosomes is coupled to assembly of CcO. The translational activator Mss51 is sequestered in early assembly intermediate complexes by an interaction with Cox14 that depends on the presence of newly synthesized Cox1. If CcO assembly is prevented, the level of Mss51 available for translational activation is reduced. We deleted the C-terminal 11 or 15 residues of Cox1 by site-directed mutagenesis of mtDNA. Although these deletions did not prevent respiratory growth of yeast, they eliminated the assembly-feedback control of Cox1 synthesis. Furthermore, these deletions reduced the strength of the Mss51-Cox14 interaction as detected by co-immunoprecipitation, confirming the importance of the Cox1 C-terminal residues for Mss51 sequestration. We surveyed a panel of mutations that block CcO assembly for the strength of their effect on Cox1 synthesis, both by pulse labeling and expression of the ARG8 m reporter fused to COX1. Deletion of the nuclear gene encoding Cox6, one of the first subunits to be added to assembling CcO, caused the most severe reduction in Cox1 synthesis. Deletion of the C-terminal 15 amino acids of Cox1 increased Cox1 synthesis in the presence of each of these mutations, except pet54. Our data suggest a novel activity of Pet54 required for normal synthesis of Cox1 that is independent of the Cox1 C-terminal end.

In the nuclear genome of Saccharomyces cerevisiae, simple, repetitive DNA sequences (microsatellites) mutate at rates much higher than nonrepetitive sequences. Most of these mutations are deletions or additions of repeat units. The yeast mitochondrial genome also contains many microsatellites. To examine the stability of these sequences, we constructed a reporter gene (arg8 m ) containing out-of-frame insertions of either poly(AT) or poly(GT) tracts within the coding sequence. Yeast strains with this reporter gene inserted within the mitochondrial genome were constructed. Using these strains, we showed that poly(GT) tracts were considerably less stable than poly(AT) tracts and that alterations usually involved deletions rather than additions of repeat units. In contrast, in the nuclear genome, poly(GT) and poly(AT) tracts had similar stabilities, and alterations usually involved additions rather than deletions. Poly(GT) tracts were more stable in the mitochondria of diploid cells than in haploids. In addition, an msh1 mutation destabilized poly(GT) tracts in the mitochondrial genome. Microsatellites are regions of DNA (usually 20-60 bp in length) with repetitions of small numbers of base pairs (1, 2). Microsatellite alterations occur at a rate much higher than the mutation rate in nonrepetitive DNA (3). These alterations are likely to reflect DNA polymerase slippage (1). During DNA replication, a transient dissociation of the DNA strands, followed by incorrect reassociation, results in one or more unpaired repeat units on either the template or the nascent strand (4). If these unpaired loops are not repaired, another round of replication will result in a tract that is shorter (if the unpaired repeats are on the template strand) or longer (if the unpaired repeats are on the nascent strand) than the original tract.The instability of microsatellites observed in wild-type yeast strains is elevated by several different types of mutations, most of which affect DNA synthesis or DNA repair (1, 5). Mutations affecting DNA polymerase or polymerase cofactors presumably destabilize microsatellites by increasing the rate of DNA polymerase slippage. The simplest interpretation of the destabilizing effect of mutations of the DNA mismatch repair system is that most of the DNA loops created by DNA polymerase slippage are repaired in wild-type strains.The mtDNA of the yeast Saccharomyces cerevisiae is approximately 75-85 kb in size and extremely AT-rich (82%) (6). Consequently, most of the microsatellites found in the mtDNA are AT-rich [for example, poly(AT) repeats]. There are about 50 copies of mtDNA per haploid yeast cell and about 100 copies per diploid cell (7). The mtDNA polymerase (DNA polymerase ␥) is encoded by the nuclear MIP1 gene (8, 9). DNA polymerase ␥, which has an associated proofreading exonuclease activity, replicates DNA in vitro with high fidelity compared with a number of other DNA polymerases (10).In the nuclear genome, most misincorporated bases that escape correction by the proofreading exonuclease are cor...

Rsm28p is a dispensable component of the mitochondrial ribosomal small subunit in Saccharomyces cerevisiae that is not related to known proteins found in bacteria. It was identified as a dominant suppressor of certain mitochondrial mutations that reduced translation of the COX2 mRNA. To explore further the function of Rsm28p, we isolated mutations in other genes that caused a synthetic respiratory defective phenotype together with rsm28D. These mutations identified three nuclear genes: IFM1, which encodes the mitochondrial translation initiation factor 2 (IF2); FMT1, which encodes the methionyl-tRNA-formyltransferase; and RMD9, a gene of unknown function. The observed genetic interactions strongly suggest that the ribosomal protein Rsm28p and Ifm1p (IF2) have similar and partially overlapping functions in yeast mitochondrial translation initiation. Rmd9p, bearing a TAP-tag, was localized to mitochondria and exhibited roughly equal distribution in soluble and membrane-bound fractions. A small fraction of the Rmd9-TAP sedimented together with presumed monosomes, but not with either individual ribosomal subunit. Thus, Rmd9 is not a ribosomal protein, but may be a novel factor associated with initiating monosomes. The poorly respiring rsm28D, rmd9-V363I double mutant did not have a strong translationdefective phenotype, suggesting that Rmd9p may function upstream of translation initiation, perhaps at the level of localization of mitochondrially coded mRNAs.