24 results on '"Pérez-Martínez X"'
Search Results
2. Effect of metal salts on freshly lysed chloroplasts.
- Author
-
Castillo-Blum, S.E., primary, Mendoza-Arizmendi, J.L., additional, King, B., additional, Pérez-Martínez, X., additional, Barba-Behrens, N., additional, and Lotina-Hennsen, B., additional
- Published
- 1993
- Full Text
- View/download PDF
3. Modulation of the autophagy-lysosomal pathway and endoplasmic reticulum stress by ketone bodies in experimental models of stroke.
- Author
-
Montiel T, Gómora-García JC, Gerónimo-Olvera C, Heras-Romero Y, Bernal-Vicente BN, Pérez-Martínez X, Tovar-Y-Romo LB, and Massieu L
- Subjects
- Rats, Animals, Ketone Bodies pharmacology, Ketone Bodies metabolism, Endoribonucleases pharmacology, Protein Serine-Threonine Kinases, Endoplasmic Reticulum Stress, 3-Hydroxybutyric Acid metabolism, 3-Hydroxybutyric Acid pharmacology, Glucose metabolism, Autophagy, Infarction, Middle Cerebral Artery, Models, Theoretical, Brain Injuries, Stroke drug therapy
- Abstract
Ischemic stroke is a leading cause of disability worldwide. There is no simple treatment to alleviate ischemic brain injury, as thrombolytic therapy is applicable within a narrow time window. During the last years, the ketogenic diet (KD) and the exogenous administration of the ketone body β-hydroxybutyrate (BHB) have been proposed as therapeutic tools for acute neurological disorders and both can reduce ischemic brain injury. However, the mechanisms involved are not completely clear. We have previously shown that the D enantiomer of BHB stimulates the autophagic flux in cultured neurons exposed to glucose deprivation (GD) and in the brain of hypoglycemic rats. Here, we have investigated the effect of the systemic administration of D-BHB, followed by its continuous infusion after middle cerebral artery occlusion (MCAO), on the autophagy-lysosomal pathway and the activation of the unfolded protein response (UPR). Results show for the first time that the protective effect of BHB against MCAO injury is enantiomer selective as only D-BHB, the physiologic enantiomer of BHB, significantly reduced brain injury. D-BHB treatment prevented the cleavage of the lysosomal membrane protein LAMP2 and stimulated the autophagic flux in the ischemic core and the penumbra. In addition, D-BHB notably reduced the activation of the PERK/eIF2α/ATF4 pathway of the UPR and inhibited IRE1α phosphorylation. L-BHB showed no significant effect relative to ischemic animals. In cortical cultures under GD, D-BHB prevented LAMP2 cleavage and decreased lysosomal number. It also abated the activation of the PERK/eIF2α/ATF4 pathway, partially sustained protein synthesis, and reduced pIRE1α. In contrast, L-BHB showed no significant effects. Results suggest that protection elicited by D-BHB treatment post-ischemia prevents lysosomal rupture allowing functional autophagy, preventing the loss of proteostasis and UPR activation., (© 2023 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)
- Published
- 2023
- Full Text
- View/download PDF
4. The cytochrome b carboxyl terminal region is necessary for mitochondrial complex III assembly.
- Author
-
Flores-Mireles D, Camacho-Villasana Y, Lutikurti M, García-Guerrero AE, Lozano-Rosas G, Chagoya V, Gutiérrez-Cirlos EB, Brandt U, Cabrera-Orefice A, and Pérez-Martínez X
- Subjects
- Electron Transport Complex III, Saccharomyces cerevisiae metabolism, Mitochondria metabolism, Carrier Proteins, Membrane Proteins metabolism, Molecular Chaperones metabolism, Mitochondrial Proteins genetics, Cytochromes b genetics, Cytochromes b metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Mitochondrial bc
1 complex from yeast has 10 subunits, but only cytochrome b (Cyt b ) subunit is encoded in the mitochondrial genome. Cyt b has eight transmembrane helices containing two hemes b for electron transfer. Cbp3 and Cbp6 assist Cyt b synthesis, and together with Cbp4 induce Cyt b hemylation. Subunits Qcr7/Qcr8 participate in the first steps of assembly, and lack of Qcr7 reduces Cyt b synthesis through an assembly-feedback mechanism involving Cbp3/Cbp6. Because Qcr7 resides near the Cyt b carboxyl region, we wondered whether this region is important for Cyt b synthesis/assembly. Although deletion of the Cyt b C-region did not abrogate Cyt b synthesis, the assembly-feedback regulation was lost, so Cyt b synthesis was normal even if Qcr7 was missing. Mutants lacking the Cyt b C-terminus were non-respiratory because of the absence of fully assembled bc1 complex. By performing complexome profiling, we showed the existence of aberrant early-stage subassemblies in the mutant. In this work, we demonstrate that the C-terminal region of Cyt b is critical for regulation of Cyt b synthesis and bc1 complex assembly., (© 2023 Flores-Mireles et al.)- Published
- 2023
- Full Text
- View/download PDF
5. The ARG8 m Reporter for the Study of Yeast Mitochondrial Translation.
- Author
-
Flores-Mireles D, Camacho-Villasana Y, and Pérez-Martínez X
- Subjects
- Protein Biosynthesis, DNA, Mitochondrial genetics, Mitochondria metabolism, Mitochondrial Proteins genetics, Mitochondrial Proteins metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Mitochondrial translation is an intricate process involving both general and mRNA-specific factors. In addition, in the yeast Saccharomyces cerevisiae, translation of mitochondrial mRNAs is coupled to assembly of nascent polypeptides into the membrane. ARG8
m is a reporter gene widely used to study the mechanisms of yeast mitochondrial translation. This reporter is a recodified gene that uses the mitochondrial genetic code and is inserted at the desired locus in the mitochondrial genome. After deletion of the endogenous nuclear gene, this reporter produces Arg8, an enzyme necessary for arginine biosynthesis. Since Arg8 is a soluble protein with no relation to oxidative phosphorylation, it is a reliable reporter to study mitochondrial mRNAs translation and dissect translation form assembly processes. In this chapter, we explain how to insert the ARG8m reporter in the desired spot in the mitochondrial DNA, how to analyze Arg8 synthesis inside mitochondria, and how to follow steady-state levels of the protein. We also explain how to use it to find spontaneous suppressors of translation defects., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)- Published
- 2023
- Full Text
- View/download PDF
6. IRE1α RIDD activity induced under ER stress drives neuronal death by the degradation of 14-3-3 θ mRNA in cortical neurons during glucose deprivation.
- Author
-
Gómora-García JC, Gerónimo-Olvera C, Pérez-Martínez X, and Massieu L
- Abstract
Altered protein homeostasis is associated with neurodegenerative diseases and acute brain injury induced under energy depletion conditions such as ischemia. The accumulation of damaged or unfolded proteins triggers the unfolded protein response (UPR), which can act as a homeostatic response or lead to cell death. However, the factors involved in turning and adaptive response into a cell death mechanism are still not well understood. Several mechanisms leading to brain injury induced by severe hypoglycemia have been described but the contribution of the UPR has been poorly studied. Cell responses triggered during both the hypoglycemia and the glucose reinfusion periods can contribute to neuronal death. Therefore, we have investigated the activation dynamics of the PERK and the IRE1α branches of the UPR and their contribution to neuronal death in a model of glucose deprivation (GD) and glucose reintroduction (GR) in cortical neurons. Results show a rapid activation of the PERK/p-eIF2α/ATF4 pathway leading to protein synthesis inhibition during GD, which contributes to neuronal adaptation, however, sustained blockade of protein synthesis during GR promotes neuronal death. On the other hand, IRE1α activation occurs early during GD due to its interaction with BAK/BAX, while ASK1 is recruited to IRE1α activation complex during GR promoting the nuclear translocation of JNK and the upregulation of Chop. Most importantly, results show that IRE1α RNase activity towards its splicing target Xbp1 mRNA occurs late after GR, precluding a homeostatic role. Instead, IRE1α activity during GR drives neuronal death by positively regulating ASK1/JNK activity through the degradation of 14-3-3 θ mRNA, a negative regulator of ASK and an adaptor protein highly expressed in brain, implicated in neuroprotection. Collectively, results describe a novel regulatory mechanism of cell death in neurons, triggered by the downregulation of 14-3-3 θ mRNA induced by the IRE1α branch of the UPR.
- Published
- 2021
- Full Text
- View/download PDF
7. Conservation and Variability of the AUG Initiation Codon Context in Eukaryotes.
- Author
-
Hernández G, Osnaya VG, and Pérez-Martínez X
- Subjects
- Codon, Initiator physiology, Eukaryota physiology, Nucleotide Motifs, Peptide Chain Initiation, Translational physiology, RNA, Messenger metabolism
- Abstract
Selection of the translation initiation site (TIS) is a crucial step during translation. In the 1980s Marylin Kozak performed key studies on vertebrate mRNAs to characterize the optimal TIS consensus sequence, the Kozak motif. Within this motif, conservation of nucleotides in crucial positions, namely a purine at -3 and a G at +4 (where the A of the AUG is numbered +1), is essential for TIS recognition. Ever since its characterization the Kozak motif has been regarded as the optimal sequence to initiate translation in all eukaryotes. We revisit here published in silico data on TIS consensus sequences, as well as experimental studies from diverse eukaryotic lineages, and propose that, while the -3A/G position is universally conserved, the remaining variability of the consensus sequences enables their classification as optimal, strong, and moderate TIS sequences., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
8. Cbp3 and Cbp6 are dispensable for synthesis regulation of cytochrome b in yeast mitochondria.
- Author
-
García-Guerrero AE, Camacho-Villasana Y, Zamudio-Ochoa A, Winge DR, and Pérez-Martínez X
- Subjects
- Cytochromes b genetics, Cytochromes c1 genetics, Cytochromes c1 metabolism, Membrane Proteins genetics, Methyltransferases genetics, Methyltransferases metabolism, Mitochondria genetics, Mitochondrial Proteins genetics, Mitochondrial Proteins metabolism, Molecular Chaperones genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Cytochromes b biosynthesis, Membrane Proteins metabolism, Mitochondria metabolism, Mitochondrial Proteins biosynthesis, Molecular Chaperones metabolism, Protein Biosynthesis, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Cytochrome b (Cyt b ) is the only mitochondrial encoded subunit from the bc
1 complex. Cbp3 and Cbp6 are chaperones necessary for translation of the COB mRNA and Cyt b hemylation. Here we demonstrate that their role in translation is dispensable in some laboratory strains, whereas their role in Cyt b hemylation seems to be universally conserved. BY4742 yeast requires Cbp3 and Cbp6 for efficient COB mRNA translation, whereas the D273-10b strain synthesizes Cyt b at wildtype levels in the absence of Cbp3 and Cbp6. Steady-state levels of Cyt b are close to wildtype in mutant D273-10b cells, and Cyt b forms non-functional, supercomplex-like species with cytochrome c oxidase, in which at least core 1, cytochrome c1 , and Rieske iron-sulfur subunits are present. We demonstrated that Cbp3 interacts with the mitochondrial ribosome and with the COB mRNA in both BY4742 and D273-10b strains. The polymorphism(s) causing the differential function of Cbp3, Cbp6, and the assembly feedback regulation of Cyt b synthesis is of nuclear origin rather than mitochondrial, and Smt1, a COB mRNA-binding protein, does not seem to be involved in the observed differential phenotype. Our results indicate that the essential role of Cbp3 and Cbp6 is to assist Cyt b hemylation and demonstrate that in the absence of heme b , Cyt b can form non-functional supercomplexes with cytochrome c oxidase. Our observations support that an additional protein or proteins are involved in Cyt b synthesis in some yeast strains., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)- Published
- 2018
- Full Text
- View/download PDF
9. Mitochondrial versus nuclear gene expression and membrane protein assembly: the case of subunit 2 of yeast cytochrome c oxidase.
- Author
-
Rubalcava-Gracia D, Vázquez-Acevedo M, Funes S, Pérez-Martínez X, and González-Halphen D
- Abstract
Deletion of the yeast mitochondrial gene COX2 , encoding subunit 2 (mtCox2) of cytochrome c oxidase (C c O), results in a respiratory-incompetent Δcox2 strain. For a cytosol-synthesized Cox2 to restore respiratory growth, it must carry the W56R mutation (cCox2
W56R ). Nevertheless, only a fraction of cCox2W56R is matured in mitochondria, allowing ∼60% steady-state accumulation of C c O. This can be attributed either to the point mutation or to an inefficient biogenesis of cCox2W56R . We generated a strain expressing the mutant protein mtCox2W56R inside mitochondria which should follow the canonical biogenesis of mitochondria-encoded Cox2. This strain exhibited growth rates, C c O steady-state levels, and C c O activity similar to those of the wild type; therefore, the efficiency of Cox2 biogenesis is the limiting step for successful allotopic expression. Upon coexpression of cCox2W56R and mtCox2, each protein assembled into C c O independently from its genetic origin, resulting in a mixed population of C c O with most complexes containing the mtCox2 version. Notably, the presence of the mtCox2 enhances cCox2W56R incorporation. We provide proof of principle that an allotopically expressed Cox2 may complement a phenotype due to a mutant mitochondrial COX2 gene. These results are relevant to developing a rational design of genes for allotopic expression intended to treat human mitochondrial diseases.- Published
- 2018
- Full Text
- View/download PDF
10. The Cox1 C-terminal domain is a central regulator of cytochrome c oxidase biogenesis in yeast mitochondria.
- Author
-
García-Villegas R, Camacho-Villasana Y, Shingú-Vázquez MÁ, Cabrera-Orefice A, Uribe-Carvajal S, Fox TD, and Pérez-Martínez X
- Subjects
- Amino Acid Substitution, Electron Transport Complex IV genetics, Membrane Proteins genetics, Membrane Proteins metabolism, Mitochondria genetics, Mitochondrial Proteins genetics, Mitochondrial Proteins metabolism, Mutation, Missense, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Transcription Factors genetics, Transcription Factors metabolism, Electron Transport Complex IV metabolism, Mitochondria enzymology, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Cytochrome c oxidase (C c O) is the last electron acceptor in the respiratory chain. The C c O core is formed by mitochondrial DNA-encoded Cox1, Cox2, and Cox3 subunits. Cox1 synthesis is highly regulated; for example, if C c O assembly is blocked, Cox1 synthesis decreases. Mss51 activates translation of COX1 mRNA and interacts with Cox1 protein in high-molecular-weight 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 (Cox1ΔC15), reduced binding of Mss51 and Cox14 to COA complexes. Mss51 was enriched in a translationally active form that maintains full Cox1 synthesis even if C c O assembly is blocked in these mutants. Moreover, Cox1ΔC15, but not Cox1-P521A/P522A and Cox1-V524E, promoted formation of aberrant supercomplexes in C c O 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 C c O is inactive and not fully assembled. Our results indicate that the Cox1 C-terminal end is a key regulator of C c O biogenesis and that it is important for supercomplex formation/stability., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2017
- Full Text
- View/download PDF
11. A Novel Function of Pet54 in Regulation of Cox1 Synthesis in Saccharomyces cerevisiae Mitochondria.
- Author
-
Mayorga JP, Camacho-Villasana Y, Shingú-Vázquez M, García-Villegas R, Zamudio-Ochoa A, García-Guerrero AE, Hernández G, and Pérez-Martínez X
- Subjects
- 5' Untranslated Regions physiology, Electron Transport Complex IV genetics, Mitochondrial Proteins genetics, RNA, Fungal genetics, RNA, Fungal metabolism, RNA-Binding Proteins genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Transcription Factors genetics, Transcription Factors metabolism, Electron Transport Complex IV biosynthesis, Mitochondrial Proteins biosynthesis, Protein Biosynthesis physiology, RNA-Binding Proteins metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins biosynthesis, Saccharomyces cerevisiae Proteins metabolism
- Abstract
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 aI5β 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., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2016
- Full Text
- View/download PDF
12. The Pet309 pentatricopeptide repeat motifs mediate efficient binding to the mitochondrial COX1 transcript in yeast.
- Author
-
Zamudio-Ochoa A, Camacho-Villasana Y, García-Guerrero AE, and Pérez-Martínez X
- Subjects
- Binding Sites, Electron Transport Complex IV metabolism, Gene Expression Regulation, Fungal, Membrane Proteins genetics, Mitochondrial Proteins genetics, Mutation, Peptide Initiation Factors genetics, RNA, Fungal metabolism, Ribosomes metabolism, Saccharomyces cerevisiae genetics, Transcription Factors genetics, Electron Transport Complex IV genetics, Membrane Proteins metabolism, Mitochondrial Proteins metabolism, Peptide Initiation Factors metabolism, RNA, Messenger metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Mitochondrial synthesis of Cox1, the largest subunit of the cytochrome c oxidase complex, is controlled by Mss51 and Pet309, two mRNA-specific translational activators that act via the COX1 mRNA 5'-UTR through an unknown mechanism. Pet309 belongs to the pentatricopeptide repeat (PPR) protein family, which is involved in RNA metabolism in mitochondria and chloroplasts, and its sequence predicts at least 12 PPR motifs in the central portion of the protein. Deletion of these motifs selectively disrupted translation but not accumulation of the COX1 mRNA. We used RNA coimmunoprecipitation assays to show that Pet309 interacts with the COX1 mRNA in vivo and that this association is present before processing of the COX1 mRNA from the ATP8/6 polycistronic mRNA. This association was not affected by deletion of 8 of the PPR motifs but was undetectable after deletion of the entire 12-PPR region. However, interaction of the Pet309 protein lacking 12 PPR motifs with the COX1 mRNA was detected after overexpression of the mutated form of the protein, suggesting that deletion of this region decreased the binding affinity for the COX1 mRNA without abolishing it entirely. Moreover, binding of Pet309 to the COX1 mRNA was affected by deletion of Mss51. This work demonstrates an in vivo physical interaction between a yeast mitochondrial translational activator and its target mRNA and shows the cooperativity of the PPR domains of Pet309 in interaction with the COX1 mRNA.
- Published
- 2014
- Full Text
- View/download PDF
13. The cytosol-synthesized subunit II (Cox2) precursor with the point mutation W56R is correctly processed in yeast mitochondria to rescue cytochrome oxidase.
- Author
-
Cruz-Torres V, Vázquez-Acevedo M, García-Villegas R, Pérez-Martínez X, Mendoza-Hernández G, and González-Halphen D
- Subjects
- Amino Acid Sequence, Cell Respiration physiology, Electron Transport Complex IV chemistry, Electron Transport Complex IV genetics, Immunoassay, Mitochondria genetics, Mitochondria metabolism, Mitochondrial Proteins chemistry, Mitochondrial Proteins metabolism, Molecular Sequence Data, Native Polyacrylamide Gel Electrophoresis, Protein Conformation, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins metabolism, Tandem Mass Spectrometry, Cytoplasm enzymology, Electron Transport Complex IV metabolism, Oxygen metabolism, Point Mutation genetics, Saccharomyces cerevisiae enzymology
- Abstract
Deletion of the yeast mitochondrial gene COX2 encoding subunit 2 (Cox2) of cytochrome c oxidase (CcO) results in loss of respiration (Δcox2 strain). Supekova et al. (2010) [1] transformed a Δcox2 strain with a vector expressing Cox2 with a mitochondrial targeting sequence (MTS) and the point mutation W56R (Cox2(W56R)), restoring respiratory growth. Here, the CcO carrying the allotopically-expressed Cox2(W56R) was characterized. Yeast mitochondria from the wild-type (WT) and the Δcox2+Cox2(W56R) strains were subjected to Blue Native electrophoresis. In-gel activity of CcO and spectroscopic quantitation of cytochromes revealed that only 60% of CcO is present in the complemented strain, and that less CcO is found associated in supercomplexes as compared to WT. CcOs from the WT and the mutant exhibited similar subunit composition, although activity was 20-25% lower in the enzyme containing Cox2(W56R) than in the one with Cox2(WT). Tandem mass spectrometry confirmed that W(56) was substituted by R(56) in Cox2(W56R). In addition, Cox2(W56R) exhibited the same N-terminus than Cox2(WT), indicating that the MTS of Oxa1 and the leader sequence of 15 residues were removed from Cox2(W56R) during maturation. Thus, Cox2(W56R) is identical to Cox2(WT) except for the point mutation W56R. Mitochondrial Cox1 synthesis is strongly reduced in Δcox2 mutants, but the Cox2(W56R) complemented strain led to full restoration of Cox1 synthesis. We conclude that the cytosol-synthesized Cox2(W56R) follows a rate-limiting process of import, maturation or assembly that yields lower steady-state levels of CcO. Still, the allotopically-expressed Cox2(W56R) restores CcO activity and allows mitochondrial Cox1 synthesis to advance at WT levels., (Copyright © 2012 Elsevier B.V. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
14. The carboxyl-terminal end of Cox1 is required for feedback assembly regulation of Cox1 synthesis in Saccharomyces cerevisiae mitochondria.
- Author
-
Shingú-Vázquez M, Camacho-Villasana Y, Sandoval-Romero L, Butler CA, Fox TD, and Pérez-Martínez X
- Subjects
- Amino Acid Sequence, Electron Transport Complex IV genetics, Membrane Proteins genetics, Membrane Proteins metabolism, Mitochondria genetics, Mitochondrial Proteins genetics, Mitochondrial Proteins metabolism, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae growth & development, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Sequence Deletion, Transcription Factors genetics, Transcription Factors metabolism, Transcription, Genetic physiology, Electron Transport Complex IV biosynthesis, Mitochondria enzymology, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae Proteins biosynthesis
- Abstract
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.
- Published
- 2010
- Full Text
- View/download PDF
15. In Saccharomyces cerevisiae, the phosphate carrier is a component of the mitochondrial unselective channel.
- Author
-
Gutiérrez-Aguilar M, Pérez-Martínez X, Chávez E, and Uribe-Carvajal S
- Subjects
- Animals, Mersalyl pharmacology, Mitochondria drug effects, Mitochondria metabolism, Mitochondrial Swelling drug effects, Permeability drug effects, Phosphate Transport Proteins antagonists & inhibitors, Phosphates metabolism, Polyethylene Glycols pharmacology, Potassium Channels chemistry, Potassium Channels deficiency, Potassium Channels genetics, Saccharomyces cerevisiae cytology, Saccharomyces cerevisiae drug effects, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae Proteins genetics, Sequence Deletion, Vanadates pharmacology, Voltage-Dependent Anion Channels metabolism, Phosphate Transport Proteins metabolism, Potassium Channels metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
The mitochondrial permeability transition (PT) involves the opening of a mitochondrial unselective channel (MUC) resulting in membrane depolarization and increased permeability to ions. PT has been observed in many, but not all eukaryotic species. In some species, PT has been linked to cell death, although other functions, such as matrix ion detoxification or regulation of the rate of oxygen consumption have been considered. The identification of the proteins constituting MUC would help understand the biochemistry and physiology of this channel. It has been suggested that the mitochondrial phosphate carrier is a structural component of MUC and we decided to test this in yeast mitochondria. Mersalyl inhibits the phosphate carrier and it has been reported that it also triggers PT. Mersalyl induced opening of the decavanadate-sensitive Yeast Mitochondrial Unselective Channel (YMUC). In isolated yeast mitochondria from a phosphate carrier-null strain the sensitivity to both phosphate and mersalyl was lost, although the permeability transition was still evoked by ATP in a decavanadate-sensitive fashion. Polyethylene glycol (PEG)-induced mitochondrial contraction results indicated that in mitochondria lacking the phosphate carrier the YMUC is smaller: complete contraction for mitochondria from the wild type and the mutant strains was achieved with 1.45 and 1.1 kDa PEGs, respectively. Also, as expected for a smaller channel titration with 1.1 kDa PEG evidenced a higher sensitivity in mitochondria from the mutant strain. The above data suggest that the phosphate carrier is the phosphate sensor in YMUC and contributes to the structure of this channel., (2009 Elsevier Inc. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
16. The pentatricopeptide repeats present in Pet309 are necessary for translation but not for stability of the mitochondrial COX1 mRNA in yeast.
- Author
-
Tavares-Carreón F, Camacho-Villasana Y, Zamudio-Ochoa A, Shingú-Vázquez M, Torres-Larios A, and Pérez-Martínez X
- Subjects
- Amino Acid Motifs, Amino Acids, Electron Transport Complex IV biosynthesis, Gene Expression Regulation, Fungal, Mitochondria metabolism, Mitochondrial Proteins, Models, Molecular, Mutagenesis, Peptide Initiation Factors, Protein Structure, Tertiary, Protein Transport, RNA, Fungal metabolism, RNA, Mitochondrial, Repetitive Sequences, Amino Acid, Saccharomyces cerevisiae cytology, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae growth & development, Saccharomyces cerevisiae Proteins biosynthesis, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae Proteins metabolism, Structure-Activity Relationship, Electron Transport Complex IV genetics, Membrane Proteins chemistry, Membrane Proteins metabolism, Protein Biosynthesis, RNA Stability, RNA, Messenger metabolism, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae Proteins genetics
- Abstract
Pet309 is a protein essential for respiratory growth. It is involved in translation of the yeast mitochondrial COX1 gene, which encodes subunit I of the cytochrome c oxidase. Pet309 is also involved in stabilization of the COX1 mRNA. Mutations in a similar human protein, Lrp130, are associated with Leigh syndrome, where cytochrome c oxidase activity is affected. The sequence of Pet309 reveals the presence of at least seven pentatricopeptide repeats (PPRs) located in tandem in the central portion of the protein. Proteins containing PPR motifs are present in mitochondria and chloroplasts and are in general involved in RNA metabolism. Despite the increasing number of proteins from this family found to play essential roles in mitochondria and chloroplasts, little is understood about the mechanism of action of the PPR domains present in these proteins. In a series of in vivo analyses we constructed a pet309 mutant lacking the PPR motifs. Although the stability of the COX1 mRNA was not affected, synthesis of Cox1 was abolished. The deletion of one PPR motif at a time showed that all the PPR motifs are required for COX1 mRNA translation and respiratory growth. Mutations of basic residues in PPR3 caused reduced respiratory growth. According to a molecular model, these residues are facing a central cavity that could be involved in mRNA-binding activity, forming a possible path for this molecule on Pet309. Our results show that the RNA metabolism function of Pet309 is found in at least two separate domains of the protein.
- Published
- 2008
- Full Text
- View/download PDF
17. Protein synthesis and assembly in mitochondrial disorders.
- Author
-
Pérez-Martínez X, Funes S, Camacho-Villasana Y, Marjavaara S, Tavares-Carreón F, and Shingú-Vázquez M
- Subjects
- Humans, Mitochondrial Diseases genetics, Mitochondrial Diseases pathology, Oxidative Phosphorylation, Peptide Chain Initiation, Translational genetics, Ribosomes genetics, Ribosomes metabolism, Mitochondrial Diseases metabolism, Protein Biosynthesis genetics
- Abstract
Human mitochondrial DNA (mtDNA) codes for 13 polypeptides which constitute the central core of the oxidative phosphorylation (OXPHOS) complexes. The machinery for mitochondrial protein synthesis has a dual origin: a full set of tRNAs, as well as the 12S and 16S rRNAs are encoded in the mitochondrial genome, while most factors necessary for translation are encoded by nuclear genes. The mitochondrial translation apparatus is highly specialized in expressing membrane proteins, and couples the synthesis of proteins to the insertion into the mitochondrial inner membrane. In recent years it has become clear that defects of mitochondrial translation and protein assembly cause several mitochondrial disorders. Since direct studies on protein synthesis in human mitochondria are still a relatively difficult task, we owe our current knowledge of this field to the large amount of genetic and biochemical studies performed in the yeast Saccharomyces cerevisiae. These studies have allowed the identification of several genes involved in mitochondrial protein synthesis and assembly, and have provided insights into the conserved mechanisms of mitochondrial gene expression. In the present review we will discuss the most recent advances in the understanding of the mechanisms and factors that govern mammalian mitochondrial translation/protein insertion, as well as known pathologies associated with them.
- Published
- 2008
- Full Text
- View/download PDF
18. Genetic correction of mitochondrial diseases: using the natural migration of mitochondrial genes to the nucleus in chlorophyte algae as a model system.
- Author
-
González-Halphen D, Funes S, Pérez-Martínez X, Reyes-Prieto A, Claros MG, Davidson E, and King MP
- Subjects
- Animals, Chlamydomonas reinhardtii metabolism, Genetic Therapy, Humans, Mitochondria metabolism, Models, Biological, Mutation, Peptides chemistry, Protein Structure, Secondary, Protein Structure, Tertiary, Cell Nucleus metabolism, DNA, Mitochondrial metabolism, Eukaryota genetics, Eukaryota metabolism, Genome
- Abstract
Mitochondrial diseases display great diversity in clinical symptoms and biochemical characteristics. Although mtDNA mutations have been identified in many patients, there are currently no effective treatments. A number of human diseases result from mutations in mtDNA-encoded proteins, a group of proteins that are hydrophobic and have multiple membrane-spanning regions. One method that has great potential for overcoming the pathogenic consequences of these mutations is to place a wild-type copy of the affected gene in the nucleus, and target the expressed protein to the mitochondrion to function in place of the defective protein. Several respiratory chain subunit genes, which are typically mtDNA encoded, are nucleus encoded in the chlorophyte algae Chlamydomonas reinhardtii and Polytomella sp. Analysis of these genes has revealed adaptations that facilitated their expression from the nucleus. The nucleus-encoded proteins exhibited diminished physical constraints for import as compared to their mtDNA-encoded homologues. The hydrophobicity of the nucleus-encoded proteins is diminished in those regions that are not involved in subunit-subunit interactions or that contain amino acids critical for enzymatic reactions of the proteins. In addition, these proteins have unusually large mitochondrial targeting sequences. Information derived from these studies should be applicable toward the development of genetic therapies for human diseases resulting from mutations in mtDNA-encoded polypeptides.
- Published
- 2004
- Full Text
- View/download PDF
19. On the evolutionary origins of apicoplasts: revisiting the rhodophyte vs. chlorophyte controversy.
- Author
-
Funes S, Reyes-Prieto A, Pérez-Martínez X, and González-Halphen D
- Subjects
- Animals, Chlorophyta genetics, Phylogeny, Rhodophyta genetics, Apicomplexa genetics, Biological Evolution, Plastids genetics, Symbiosis
- Abstract
Apicomplexans are parasites of great medical and veterinary importance. They contain a vestigial plastid, the apicoplast, that originated through the secondary endosymbiosis of the photosynthetic unicellular alga. The nature of this alga remains controversial. Here, we revisit the available evidence and critically summarize the "green vs. red" debate.
- Published
- 2004
- Full Text
- View/download PDF
20. Structure of nuclear-localized cox3 genes in Chlamydomonas reinhardtii and in its colorless close relative Polytomella sp.
- Author
-
Pérez-Martínez X, Funes S, Tolkunova E, Davidson E, King MP, and González-Halphen D
- Subjects
- Amino Acid Sequence, Animals, Conserved Sequence, Electron Transport Complex IV chemistry, Evolution, Molecular, Membrane Proteins chemistry, Molecular Sequence Data, RNA Splice Sites genetics, Saccharomyces cerevisiae Proteins, Cell Nucleus genetics, Chlamydomonas reinhardtii genetics, Chlorophyta genetics, Electron Transport Complex IV genetics, Introns genetics, Membrane Proteins genetics
- Abstract
Several chlorophyte algae do not have the cox3 gene, encoding subunit III of cytochrome c oxidase, in their mitochondrial genomes. The cox3 gene is nuclear-encoded in the photosynthetic alga Chlamydomonas reinhardtii and in the colorless alga Polytomella sp. In this work, the genomic sequences of the cox3 genes of these two closely related algae are reported. The cox3 genes of both C. reinhardtii and Polytomella sp. contain four introns in the region encoding the putative mitochondrial-targeting sequences. These four introns show low sequence identities, but their locations are conserved between these species. The cox3 gene of C. reinhardtii has five additional introns in the region encoding the mature subunit III of cytochrome c oxidase. Sequence analysis of intron 6 of the cox3 gene of C. reinhardtii revealed similarity with two sequence elements present in introns of several other nuclear genes from this green alga. In the majority of the genes, these conserved sequences are located either near the 3' end or near the 5' end of the introns. Based on these data, we propose that the colorless genus Polytomella separated from C. reinhardtii after the cox3 gene was transferred to the nucleus. The data also support the evolutionary hypothesis of a recent acquisition of introns in C. reinhardtii.
- Published
- 2002
- Full Text
- View/download PDF
21. The typically mitochondrial DNA-encoded ATP6 subunit of the F1F0-ATPase is encoded by a nuclear gene in Chlamydomonas reinhardtii.
- Author
-
Funes S, Davidson E, Claros MG, van Lis R, Pérez-Martínez X, Vázquez-Acevedo M, King MP, and González-Halphen D
- Subjects
- Amino Acid Sequence, Animals, Cell Membrane enzymology, Chlamydomonas reinhardtii enzymology, Cloning, Molecular, Expressed Sequence Tags, Mitochondrial Proton-Translocating ATPases, Models, Molecular, Molecular Sequence Data, Protein Conformation, Sequence Alignment, Sequence Homology, Amino Acid, Adenosine Triphosphatases genetics, Cell Nucleus genetics, Chlamydomonas reinhardtii genetics, DNA, Mitochondrial genetics, Proton-Translocating ATPases genetics
- Abstract
The atp6 gene, encoding the ATP6 subunit of F(1)F(0)-ATP synthase, has thus far been found only as an mtDNA-encoded gene. However, atp6 is absent from mtDNAs of some species, including that of Chlamydomonas reinhardtii. Analysis of C. reinhardtii expressed sequence tags revealed three overlapping sequences that encoded a protein with similarity to ATP6 proteins. PCR and 5'- and 3'-RACE were used to obtain the complete cDNA and genomic sequences of C. reinhardtii atp6. The atp6 gene exhibited characteristics of a nucleus-encoded gene: Southern hybridization signals consistent with nuclear localization, the presence of introns, and a codon usage and a polyadenylation signal typical of nuclear genes. The corresponding ATP6 protein was confirmed as a subunit of the mitochondrial F(1)F(0)-ATP synthase from C. reinhardtii by N-terminal sequencing. The predicted ATP6 polypeptide has a 107-amino acid cleavable mitochondrial targeting sequence. The mean hydrophobicity of the protein is decreased in those transmembrane regions that are predicted not to participate directly in proton translocation or in intersubunit contacts with the multimeric ring of c subunits. This is the first example of a mitochondrial protein with more than two transmembrane stretches, directly involved in proton translocation, that is nucleus-encoded.
- Published
- 2002
- Full Text
- View/download PDF
22. Subunit II of cytochrome c oxidase in Chlamydomonad algae is a heterodimer encoded by two independent nuclear genes.
- Author
-
Pérez-Martínez X, Antaramian A, Vazquez-Acevedo M, Funes S, Tolkunova E, d'Alayer J, Claros MG, Davidson E, King MP, and González-Halphen D
- Subjects
- Amino Acid Sequence, Animals, Cell Nucleus, Gene Expression Regulation, Enzymologic, Genes, Plant, Genes, Protozoan, Molecular Sequence Data, RNA, Messenger analysis, RNA, Messenger genetics, Sequence Alignment, Chlamydomonas enzymology, Chlamydomonas genetics, Electron Transport Complex IV analysis, Electron Transport Complex IV genetics
- Abstract
The mitochondrial genomes of Chlamydomonad algae lack the cox2 gene that encodes the essential subunit COX II of cytochrome c oxidase. COX II is normally a single polypeptide encoded by a single mitochondrial gene. In this work we cloned two nuclear genes encoding COX II from both Chlamydomonas reinhardtii and Polytomella sp. The cox2a gene encodes a protein, COX IIA, corresponding to the N-terminal portion of subunit II of cytochrome c oxidase, and the cox2b gene encodes COX IIB, corresponding to the C-terminal region. The cox2a and cox2b genes are located in the nucleus and are independently transcribed into mRNAs that are translated into separate polypeptides. These two proteins assemble with other cytochrome c oxidase subunits in the inner mitochondrial membrane to form the mature multi-subunit complex. We propose that during the evolution of the Chlorophyte algae, the cox2 gene was divided into two mitochondrial genes that were subsequently transferred to the nucleus. This event was evolutionarily distinct from the transfer of an intact cox2 gene to the nucleus in some members the Leguminosae plant family.
- Published
- 2001
- Full Text
- View/download PDF
23. Unusual location of a mitochondrial gene. Subunit III of cytochrome C oxidase is encoded in the nucleus of Chlamydomonad algae.
- Author
-
Pérez-Martínez X, Vazquez-Acevedo M, Tolkunova E, Funes S, Claros MG, Davidson E, King MP, and González-Halphen D
- Subjects
- Amino Acid Sequence, Animals, Chlorophyta enzymology, Electron Transport Complex IV classification, Electron Transport Complex IV isolation & purification, Eukaryota enzymology, Eukaryota genetics, Magnoliopsida enzymology, Magnoliopsida genetics, Membrane Proteins classification, Molecular Sequence Data, Phylogeny, Polymerase Chain Reaction, Protein Sorting Signals, Protein Structure, Quaternary, Saccharomyces cerevisiae Proteins, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Cell Nucleus genetics, Chlorophyta genetics, Electron Transport Complex IV genetics, Membrane Proteins genetics, Mitochondria enzymology
- Abstract
The algae of the family Chlamydomonadaceae lack the gene cox3 that encodes subunit III of cytochrome c oxidase in their mitochondrial genomes. This observation has raised the question of whether this subunit is present in cytochrome c oxidase or whether the corresponding gene is located in the nucleus. Cytochrome c oxidase was isolated from the colorless chlamydomonad Polytomella spp., and the existence of subunit III was established by immunoblotting analysis with an antibody directed against Saccharomyces cerevisiae subunit III. Based partly upon the N-terminal sequence of this subunit, oligodeoxynucleotides were designed and used for polymerase chain reaction amplification, and the resulting product was used to screen a cDNA library of Chlamydomonas reinhardtii. The complete sequences of the cox3 cDNAs from Polytomella spp. and C. reinhardtii are reported. Evidence is provided that the genes for cox3 are encoded by nuclear DNA, and the predicted polypeptides exhibit diminished physical constraints for import as compared with mitochondrial-DNA encoded homologs. This indicates that transfer of this gene to the nucleus occurred before Polytomella diverged from the photosynthetic Chlamydomonas lineage and that this transfer may have occurred in all chlamydomonad algae.
- Published
- 2000
- Full Text
- View/download PDF
24. An atypical cytochrome b in the colorless alga Polytomella spp.: the high potential bH heme exhibits a double transition in the alpha-peak of its absorption spectrum.
- Author
-
Gutiérrez-Cirlos EB, Gómez-Lojero C, Vázquez-Acevedo M, Pérez-Martínez X, and González-Halphen D
- Subjects
- Animals, Chlamydomonas reinhardtii enzymology, Chromatography, High Pressure Liquid, Spectrophotometry, Atomic, Chlorophyta enzymology, Cytochrome b Group chemistry, Electron Transport Complex III chemistry, Heme chemistry
- Abstract
Polytomella spp. is a colorless alga of the family Chlamydomonadaceae that lacks chloroplasts and cell wall. A highly active ubiquinol-cytochrome c oxidoreductase (bc1 complex), sensitive to antimycin and myxothiazol, has been purified and characterized from this alga (Gutiérrez-Cirlos et al., 1994, J. Biol. Chem. 269, 9147-9154). Both in mitochondrial membranes and in the isolated complex, the visible spectrum of cytochrome b from Polytomella spp. exhibits an atypical alpha-band with a maximum at 567 nm. This maximum is shifted 3-4 nm to the red when compared with b-type cytochromes from other organisms. Analysis of the b hemes of the bc1 complex by high performance liquid chromatography revealed no differences in the retention time and in the absorption spectra of the b-type hemes from Polytomella spp. and hemin, indicating that the prosthetic group in this alga is protoheme and thus ruling out the possibility that the red-shift could be due to different chemical substitutions in the porphyrin rings of the bL or bH hemes. The two b hemes were characterized by electrochemical redox titration; at pH 7.8-8.0, the midpoint potential for bL was-143 mV and for bH +25 mV. The spectra of the two b-type hemes were recorded in the presence of different reductants, at selected electrochemical potentials, and in the presence of antimycin A, to distinguish between the contribution of bL and bH to the visible spectrum. Both hemes bL and bH of the algal cytochrome b contribute to the observed bathochromic absorption maximum in the alpha-band of the spectrum. The data also show that the low potential bL heme from Polytomella spp. is spectroscopically similar to that of other organisms, with two transitions in the alpha-peak at 558.7 and 568.4 nm. The high-potential heme bH also exhibits a spectrum with two transitions at 557.2 and 568.9 nm, which surprisingly differs from the spectra of cytochrome bH of mammals, plants, yeasts, and bacteria, which all exhibit a single transition centered around 560 nm.
- Published
- 1998
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.