Translational Regulation of Mitochondrial Gene Expression by Nuclear Genes of Saccharomyces cerevisiae (original) (raw)
Related papers
Genetics, 1987
Mutations in the nuclear gene PET111 are recessive and specifically block accumulation of cytochrome c oxidase subunit II (coxII), the product of a mitochondrial gene. However, the coxII mRNA is present in pet111 mutants at a level approximately one-third that of wild type. The simplest explanation for this phenotype is that PET111 is required for translation of the coxII mRNA. The reduced steady-state level of this mRNA is probably a secondary effect, caused by increased degradation of the untranslated transcript. Mitochondrial suppressors of pet111, carried on rho- mtDNAs, bypass the requirement for PET111 in coxII translation. Three suppressors are fusions between the coxII structural gene and other mitochondrial genes, that encode chimeric proteins consisting of the N-terminal portions of other mitochondrially coded proteins fused to the coxII precursor protein. When present together with rho + mtDNA in a heteroplasmic state, these suppressors allow coxII synthesis in pet111 mu...
2003
Mutations in the nuclear gene PETl I I are recessive and specifically block accumulation of cytochrome c oxidase subunit I1 (coxII), the product of a mitochondrial gene. However, the cox11 mRNA is present in p e t l I I mutants at a level approximately one-third that of wild type. The simplest explanation for this phenotype is that PETl I I is required for translation of the cox11 mRNA. The reduced steady-state level of this mRNA is probably a secondary effect, caused by increased degradation of the untranslated transcript. Mitochondrial suppressors of petl I I, carried on rhomtDNAs, bypass the requirement for PETl 1 I in cox11 translation. Three suppressors are fusions between the cox11 structural gene and other mitochondrial genes, that encode chimeric proteins consisting of the N-terminal portions of other mitochondrially coded proteins fused to the cox11 precursor protein. When present together with rho+ mtDNA in a heteroplasmic state, these suppressors allow cox11 synthesis in ...
Proceedings of the National Academy of Sciences, 1996
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 mRNA-specific 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-li...
Molecular and cellular biology, 1991
Expression of the yeast mitochondrial genes COX1 and COX3, which encode subunits I and III of cytochrome oxidase, respectively, is controlled by a common nuclear-encoded trans-acting factor. This protein, encoded by the PET54 gene, controls expression of COX1 at the level of RNA splicing and COX3 at the level of mRNA translation. While the steps of COX1 and COX3 gene expression affected by the PET54 gene product are different, it is possible that the PET54 protein is monofunctional and affects expression of each gene by a single mechanism, such as modulation of RNA secondary structure. The goal of this study was to address whether the PET54 protein is monofunctional or multifunctional with respect to its role in COX1 and COX3 gene expression. Ten insertion mutations, which each resulted in the in-frame addition of four amino acids within the PET54 polypeptide, were generated, and the resulting mutants were characterized for respiration phenotype and mitochondrial gene expression. Fi...
Nucleic Acids …, 2011
Pentatricopeptide repeat (PPR) proteins are particularly numerous in plant mitochondria and chloroplasts, where they are involved in different steps of RNA metabolism, probably due to the repeated 35 amino acid PPR motifs that are thought to mediate interactions with RNA. In non-photosynthetic eukaryotes only a handful of PPR proteins exist, for example the human LRPPRC, which is involved in a mitochondrial disease. We have conducted a systematic study of the PPR proteins in the fission yeast Schizosaccharomyces pombe and identified, in addition to the mitochondrial RNA polymerase, eight proteins all of which localized to the mitochondria, and showed some association with the membrane. The absence of all but one of these PPR proteins leads to a respiratory deficiency and modified patterns of steady state mt-mRNAs or newly synthesized mitochondrial proteins. Some cause a general defect, whereas others affect specific mitochondrial RNAs, either coding or non-coding: cox1, cox2, cox3, 15S rRNA, atp9 or atp6, sometimes leading to secondary defects. Interestingly, the two possible homologs of LRPPRC, ppr4 and ppr5, play opposite roles in the expression of the cox1 mt-mRNA, ppr4 being the first mRNA-specific translational activator identified in S. pombe, whereas ppr5 appears to be a general negative regulator of mitochondrial translation.
Journal of Biological Chemistry, 2007
Downloaded from FIGURE 7. Basic residues facing the PPR groove are important for the function of Pet309. A, 10-fold serial dilutions of strains bearing the wild-type PET309-HA, the point mutations on Pet309 or empty vector (pet309⌬), in low copy or high copy plasmids were spotted on synthetic complete medium lacking uracil with either glucose or ethanol/glycerol, and incubated for 4 days at 30°C. B, the same plasmids were transformed in a strain bearing the cox1⌬::ARG8 m gene. 10-fold serial dilutions were spotted on synthetic complete medium lacking uracil (ϩArg) or arginine (ϪArg) and incubated for 4 days at 30°C.
The EMBO Journal, 1989
Communicated by L.Grivell The nuclear PETS4 gene of Saccharomyces cerevisiae was cloned and a pet54::LEU2 gene disruption strain was constructed. Analysis of the phenotype of this strain revealed a defect in expression of two mitochondrial genes: COX], which encodes cytochrome c oxidase subunit I, and COX3, which encodes cytochrome c oxidase subunit HI. The defect in COX] gene expression in the petS4 mutant was shown to be the result of inefficient excision of COX] intron a15f. Two lines of evidence indicate that inefficient excision of intron a15,B is the sole defect in COX] gene expression. First, a petS4::LEU2 cytoductant bearing the 'short' mitochondrial genome that lacks both COX] introns aI5a and a15f3 is defective only in COX3 gene expression and not in COX] mRNA splicing or mRNA translation. Second, Northern analysis of COX] transcipts from the pet54 mutant showed that a 3.8 kb COX] transcript containing unexcised intron aISfl and lacking intron aI5a is accumulated while the amount of 2.2 kb mature COXI mRNA is diminished. In an effort to relate the role of the PET54 gene product in splicing of COX] pre-mRNA to the previously characterized role for PETS4 in translation of mitochondrial COX3 mRNA, the sequence of the PET54-responsive portion of the COX3 5' untranslated leader region was compared to the COX] intron aI5S sequence. Two blocks of RNA sequence present in COX3 have similar counterparts within intron aI5f of COX]. The possibility that the PETS4 protein binds to one or the other of these blocks of RNA sequence and the potential consequences of this interaction are discussed.
1991
Expression oftheyeast mitochondrial genesCOX)andCOX3,which encode subunits IandIIIofcytochrome coxidase, respectively, iscontrolled bya common nuclear-encoded trans-acting factor. Thisprotein, encoded bythePET54gene,controls expression ofCOXIatthelevel ofRNA splicing andCOX3atthelevel ofmRNA translation. Whilethesteps ofCOX)andCOX3geneexpression affected bythePET54geneproduct are different, itispossible that thePET54protein ismonofunctional andaffects expression ofeach genebyasingle mechanism, such asmodulation ofRNAsecondary structure. Thegoal ofthis study was toaddress whether the PET54protein ismonofunctional ormultifunctional withrespect toits role inCOX)andCOX3geneexpression. Teninsertion mutations, whicheachresulted inthein-frame addition offouraminoacids within thePET54 polypeptide, were generated, andtheresulting mutants were characterized forrespiration phenotype and mitochondrial geneexpression. Fiveofthetenmutants were respiration deficient. Twoofthese five mutants werede...
A Novel Function of Pet54 in Regulation of Cox1 Synthesis in Saccharomyces cerevisiae Mitochondria
Journal of Biological Chemistry, 2016
Cytochrome c oxidase assembly requires the synthesis of the mitochondria-encoded core subunits, Cox1, Cox2, and Cox3. In yeast, Pet54 protein is required to activate translation of the COX3 mRNA and to process the aI5 intron on the COX1 transcript. Here we report a third, novel function of Pet54 on Cox1 synthesis. We observed that Pet54 is necessary to achieve an efficient Cox1 synthesis. Translation of the COX1 mRNA is coupled to the assembly of cytochrome c oxidase by a mechanism that involves Mss51. This protein activates translation of the COX1 mRNA by acting on the COX1 5-UTR, and, in addition, it interacts with the newly synthesized Cox1 protein in high molecular weight complexes that include the factors Coa3 and Cox14. Deletion of Pet54 decreased Cox1 synthesis, and, in contrast to what is commonly observed for other assembly mutants, double deletion of cox14 or coa3 did not recover Cox1 synthesis. Our results show that Pet54 is a positive regulator of Cox1 synthesis that renders Mss51 competent as a translational activator of the COX1 mRNA and that this role is independent of the assembly feedback regulatory loop of Cox1 synthesis. Pet54 may play a role in Mss51 hemylation/conformational change necessary for translational activity. Moreover, Pet54 physically interacts with the COX1 mRNA, and this binding was independent of the presence of Mss51. Cytochrome c oxidase (CcO) 3 is the last electron acceptor of the mitochondrial respiratory chain. In the yeast Saccharomyces cerevisiae, this enzyme contains 12 subunits, three of which (Cox1, Cox2, and Cox3) are encoded by the mitochondrial DNA. Assembly of CcO is a complex process regulated by more than 25 factors and chaperones (for reviews, see Ref. 1). The first steps of CcO biogenesis involve the translational activation of the mitochondria-encoded mRNAs COX1, COX2, and COX3 by mRNA-specific proteins. Translational activation of the COX1 mRNA depends on Pet309 and Mss51 (2, 3), whereas COX2 translation depends on Pet111 (4, 5), and COX3 mRNA translation depends on Pet54, Pet122, and Pet494 (6-9). These proteins act on the target mRNA 5Ј-UTRs to allow translation by the mitochondrial ribosomes. They interact with each other and with the mitochondrial inner membrane and are thought to tether translation initiation close to the assembly sites of CcO in the membrane (for a review, see Ref. 10) (11). The mitochondria-encoded Cox1, Cox2, and Cox3 subunits are proposed to assemble from three different modules, each containing a specific subset of nucleus-encoded subunits (12-14). Cox1, the largest subunit of the CcO, carries the heme aa 3-Cu B center to reduce oxygen. Synthesis of Cox1 inside mitochondria is highly regulated. If CcO assembly is blocked by mutations on either integral subunits or accessory chaperones, Cox1 synthesis is down-regulated (15, 16). It is proposed that by this mechanism, mitochondria avoids accumulation of pro-oxidant Cox1 intermediates (17). In addition to its role as translational activator of COX1 mRNA, Mss51 also physically interacts with Cox1 protein to form the first high molecular weight intermediaries (COA complexes) that include the chaperones Cox14 and Coa3 (15, 18-20). The current model (reviewed in Refs. 1 and 21) states that if CcO assembly is defective, then Mss51 is sequestered on COA intermediaries to reduce the effective concentration of Mss51 as a translational activator of the COX1 mRNA, resulting in a decrease of Cox1 synthesis. In this context, Cox14 and Coa3 are negative regulators, because their deletion restores Cox1 synthesis when assembly of CcO is deficient (15, 18, 19). The C-terminal end of Cox1 is also a negative regulator of Cox1 synthesis. Deletion of the last 11-15 residues of the Cox1 C-terminal end results in normal Cox1 synthesis even if CcO is not assembled (16). In addition to the first intermediates formed by Cox1, Mss51, Cox14, and Coa3, subunit 1 forms subsequent intermediates (12, 22) that include proteins like Coa1 (23, 24), Shy1 (23, 25, 26), subunits Cox5/ Cox6 (19), Coa2 (25), and Cox15 (26). Moreover, the Hsp70 chaperone Ssc1 is associated with both Mss51 and Mss51-containing complexes and regulates Cox1 synthesis as well (27). Mss51 contains two heme-regulating motifs. These motifs are * This work was supported by Consejo Nacional de Ciencia y Tecnología Grant 47514 (to X. P.-M.
Journal of Biological Chemistry, 2017
Cytochrome c oxidase (CcO) is the last electron acceptor in the respiratory chain. The CcO core is formed by mitochondrial DNA-encoded Cox1, Cox2, and Cox3 subunits. Cox1 synthesis is highly regulated; for example, if CcO assembly is blocked, Cox1 synthesis decreases. Mss51 activates translation of COX1 mRNA and interacts with Cox1 protein in high-molecularweight complexes (COA complexes) to form the Cox1 intermediary assembly module. Thus, Mss51 coordinates both Cox1 synthesis and assembly. We previously reported that the last 15 residues of the Cox1 C terminus regulate Cox1 synthesis by modulating an interaction of Mss51 with Cox14, another component of the COA complexes. Here, using site-directed mutagenesis of the mitochondrial COX1 gene from Saccharomyces cerevisiae, we demonstrate that mutations P521A/P522A and V524E disrupt the regulatory role of the Cox1 C terminus. These mutations, as well as C terminus deletion (Cox1⌬C15), reduced binding of Mss51 and Cox14 to COA complexes. Mss51 was enriched in a translationally active form that maintains full Cox1 synthesis even if CcO assembly is blocked in these mutants. Moreover, Cox1⌬C15, but not Cox1-P521A/P522A and Cox1-V524E, promoted formation of aberrant supercomplexes in CcO assembly mutants lacking Cox2 or Cox4 subunits. The aberrant supercomplex formation depended on the presence of cytochrome b and Cox3, supporting the idea that supercomplex assembly factors associate with Cox3 and demonstrating that supercomplexes can be formed even if CcO is inactive and not fully assembled. Our results indicate that the Cox1 C-terminal end is a key regulator of CcO biogenesis and that it is important for supercomplex formation/stability. Cytochrome c oxidase (CcO) 2 is the last redox multisubunit complex of the mitochondrial respiratory chain. This enzyme couples the transference of electrons from cytochrome c to oxygen with the translocation of protons from the matrix to the intermembrane space. In Saccharomyces cerevisiae, CcO contains at least 12 subunits, where Cox1, Cox2, and Cox3 are encoded by mitochondrial genes and constitute the catalytic core. The corresponding mRNAs are translated by the organelle's ribosomes, and the proteins are subsequently integrated into the CcO. The remaining subunits are encoded by nuclear DNA and imported from the cytosol (1, 2). CcO assembly requires at least 30 factors to correctly coordinate the incorporation of subunits and to add prosthetic groups (1, 2). Yeast CcO is assembled after formation of three subassembly modules, each containing a mtDNA-encoded subunit and a subset of cytosolic subunits (3-6). The Cox1 module stabilizes the Cox3 module and possibly the Cox2 module (6), suggesting that assembly of Cox1 orchestrates CcO assembly. One or two monomers of CcO interact with the dimeric bc 1 complex to form III 2 /IV and III 2 /IV 2 supercomplexes (7, 8). Formation and stabilization of these supercomplexes is dependent on the Rcf1, Rcf2, Aac2, and Cox13 proteins (6, 9-11). Cardiolipin (12), and the proteins Rcf3 and Cox26 are also components of supercomplexes (13-15). Cox1 is the largest CcO subunit, with 12 transmembrane stretches comprising the bulk of the protein. Its only significant hydrophilic domain is formed by ϳ59 residues exposed on the inner side of the inner membrane in the assembled enzyme. Cox1 contains hemes a and a 3 plus Cu B cofactors for oxygen reduction as well as the channels for proton translocation (1, 2). Partially assembled Cox1 may form pro-oxidant intermediaries containing unassembled heme a 3-Cu B (16). Indeed, yeast Cox1 synthesis is down-regulated when CcO assembly is defective (for reviews, see Refs. 1 and 17). Translation of the COX1 mRNA in mitochondria is specifically activated by Pet309 and Mss51 (18-22). According to the current model, newly synthesized Cox1 enters a progressive series of intermediaries named D1-D5 (or COA complexes) in