Miriam Vázquez-Acevedo

Unusual location of a mitochondrial gene - Subunit III of cytochrome c oxidase is encoded in the nucleus of chlamydomonad algae

The algae of the family Chlamydomonadaceae lack the gene cox3 that encodes subunit III of cytochromec 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 cerevisiaesubunit 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 fromPolytomella 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 Polytomelladiverged from the photosynthetic Chlamydomonas lineage and that this transfer may have occurred in all chlamydomonad algae.

In Yarrowia lipolytica mitochondria, the alternative NADH dehydrogenase interacts specifically with the cytochrome complexes of the classic respiratory pathway.

In Yarrowia lipolytica, mitochondria contain a branched respiratory chain constituted by the classic complexes I, II, III and IV, plus an alternative external NADH dehydrogenase (NDH2e) and an alternative oxidase (AOX). The alternative enzymes are peripheral, single-subunit oxido-reductases that do not pump protons. Thus, the oxidation of NADH via NDH2e-ubiquinone-AOX would not contribute to the proton-motive force. The futile oxidation of NADH may be prevented if either NDH2e or AOX bind to the classic complexes, channelling electrons. By oxymetry, it was observed that the electrons from complex I reached both cytochrome oxidase and AOX. In contrast, NDH2e-derived electrons were specifically channelled/directed to the cytochrome complexes. In addition, the presence of respiratory supercomplexes plus the interaction of NDH2e with these complexes was evaluated using blue native PAGE, clear native PAGE, in-gel activities, immunoblotting, mass spectrometry, and N-terminal sequencing. NDH2e (but not the redirected matrix NDH2i from a mutant strain, Deltanubm) was detected in association with the cytochromic pathway; this interaction seems to be strong, as it was not disrupted by laurylmaltoside. The association of NDH2e to complex IV was also suggested when both enzymes coeluted from an ion exchange chromatography column. In Y. lipolytica mitochondria the cytochrome complexes probably associate into supercomplexes; those were assigned as follows: I-III(2), I-IV, I-III(2)-IV(4), III(2)-IV, III(2)-IV(2), IV(2) and V(2). The molecular masses of all the complexes and putative supercomplexes detected in Y. lipolytica were estimated by comparison with the bovine mitochondrial complexes. To our knowledge, this is the first evidence of supercomplex formation in Y. lipolytica mitochondria and also, the first description of a specific association between an alternative NADH dehydrogenase and the classic cytochrome pathway.

What limits the allotopic expression of nucleus-encoded mitochondrial genes? The case of the chimeric Cox3 and Atp6 genes.

Allotopic expression is potentially a gene therapy for mtDNA-related diseases. Some OXPHOS proteins like ATP6 (subunit a of complex V) and COX3 (subunit III of complex IV) that are typically mtDNA-encoded, are naturally nucleus-encoded in the alga Chlamydomonas reinhardtii. The mitochondrial proteins whose genes have been relocated to the nucleus exhibit long mitochondrial targeting sequences ranging from 100 to 140 residues and a diminished overall mean hydrophobicity when compared with their mtDNA-encoded counterparts. We explored the allotopic expression of the human gene products COX3 and ATP6 that were re-designed for mitochondrial import by emulating the structural properties of the corresponding algal proteins. In vivo and in vitro data in homoplasmic human mutant cells carrying either a T8993G mutation in the mitochondrial atp6 gene or a 15bp deletion in the mtDNA-encoded cox3 gene suggest that these human mitochondrial proteins re-designed for nuclear expression are targeted to the mitochondria, but fail to functionally integrate into their corresponding OXPHOS complexes.

Subunit-subunit interactions and overall topology of the dimeric mitochondrial ATP synthase of Polytomella sp.

Mitochondrial F1F0-ATP synthase of chlorophycean algae is a dimeric complex of 1600 kDa constituted by 17 different subunits with varying stoichiometries, 8 of them conserved in all eukaryotes and 9 that seem to be unique to the algal lineage (subunits ASA1-9). Two different models proposing the topological assemblage of the nine ASA subunits in the ATP synthase of the colorless alga Polytomella sp. have been put forward. Here, we readdressed the overall topology of the enzyme with different experimental approaches: detection of close vicinities between subunits based on cross-linking experiments and dissociation of the enzyme into subcomplexes, inference of subunit stoichiometry based on cysteine residue labelling, and general three-dimensional structural features of the complex as obtained from small-angle X-ray scattering and electron microscopy image reconstruction. Based on the available data, we refine the topological arrangement of the subunits that constitute the mitochondrial ATP synthase of Polytomella sp.

Swi/SNF‐GCN5‐dependent chromatin remodelling determines induced expression of GDH3, one of the paralogous genes responsible for ammonium assimilation and glutamate biosynthesis in Saccharomyces cerevisiae

It is accepted that Saccharomyces cerevisiae genome arose from complete duplication of eight ancestral chromosomes; functionally normal ploidy was recovered because of the massive loss of 90% of duplicated genes. There is evidence that indicates that part of this selective conservation of gene pairs is compelling to yeast facultative metabolism. As an example, the duplicated NADP‐glutamate dehydrogenase pathway has been maintained because of the differential expression of the paralogous GDH1 and GDH3 genes, and the biochemical specialization of the enzymes they encode. The present work has been aimed to the understanding of the regulatory mechanisms that modulate GDH3 transcriptional activation. Our results show that GDH3 expression is repressed in glucose‐grown cultures, as opposed to what has been observed for GDH1, and induced under respiratory conditions, or under stationary phase. Although GDH3 pertains to the nitrogen metabolic network, and its expression is Gln3p‐regulated, complete derepression is ultimately determined by the carbon source through the action of the SAGA and SWI/SNF chromatin remodelling complexes. GDH3 carbon‐mediated regulation is over‐imposed to that exerted by the nitrogen source, highlighting the fact that operation of facultative metabolism requires strict control of enzymes, like Gdh3p, involved in biosynthetic pathways that use tricarboxylic acid cycle intermediates.

Subunit structures of purified beef mitochondrial cytochrome bc1 complex from liver and heart.

The existence of tissue-specific isozymes of cytochromec oxidase has been widely documented. We have now studied if there are differences between subunits of mitochondrialbc1 complexes isolated from liver and heart. For this purpose, we have developed a method for the purification of an active ubiquinol-cytochromec oxidoreductase from adult bovine liver that includes solubilization of submitochondrial particles with deoxycholate, ammonium acetate fractionation, resolubilization with dodecyl maltoside, and ion exchange chromatography. The electrophoretic pattern of the liver preparation showed the presence of 11 subunits, with apparent molecular weights identical to the ones reported for the heart complex. Western blot analysis and isoelectric focusing followed by two-dimensional gels ofbc1 complexes from liver and heart were compared, and no qualitative differences were observed. In addition, the high-molecular-weight subunits of the purified complexes from both tissues, subunits I, II, V, and VI, were isolated by PAGE in the presence of Coomasie Blue and subjected to limited proteolysis and to chemical digestion with cyanogen bromide and BNPS-skatol, and the peptide patterns were compared. Finally, two of the small-molecular-weight subunits from the liver complex were isolated (subunits VII and X), partially analyzed by amino terminal sequencing, and found to be identical with the reported sequence of their heart counterparts. The data suggest that, in contrast to the case of cytochromec oxidase,bc1 complexes from liver and heart do not exhibit tissue-specific differences.

Interactions of subunits Asa2, Asa4 and Asa7 in the peripheral stalk of the mitochondrial ATP synthase of the chlorophycean alga Polytomella sp.

Mitochondrial F1FO-ATP synthase of chlorophycean algae is a complex partially embedded in the inner mitochondrial membrane that is isolated as a highly stable dimer of 1600kDa. It comprises 17 polypeptides, nine of which (subunits Asa1 to 9) are not present in classical mitochondrial ATP synthases and appear to be exclusive of the chlorophycean lineage. In particular, subunits Asa2, Asa4 and Asa7 seem to constitute a section of the peripheral stalk of the enzyme. Here, we over-expressed and purified subunits Asa2, Asa4 and Asa7 and the corresponding amino-terminal and carboxy-terminal halves of Asa4 and Asa7 in order to explore their interactions in vitro, using immunochemical techniques, blue native electrophoresis and affinity chromatography. Asa4 and Asa7 interact strongly, mainly through their carboxy-terminal halves. Asa2 interacts with both Asa7 and Asa4, and also with subunit α in the F1 sector. The three Asa proteins form an Asa2/Asa4/Asa7 subcomplex. The entire Asa7 and the carboxy-terminal half of Asa4 seem to be instrumental in the interaction with Asa2. Based on these results and on computer-generated structural models of the three subunits, we propose a model for the Asa2/Asa4/Asa7 subcomplex and for its disposition in the peripheral stalk of the algal ATP synthase.

During the stationary growth phase, Yarrowia lipolytica prevents the overproduction of reactive oxygen species by activating an uncoupled mitochondrial respiratory pathway

In the branched mitochondrial respiratory chain from Yarrowia lipolytica there are two alternative oxido-reductases that do not pump protons, namely an external type II NADH dehydrogenase (NDH2e) and the alternative oxidase (AOX). Direct electron transfer between these proteins is not coupled to ATP synthesis and should be avoided in most physiological conditions. However, under low energy-requiring conditions an uncoupled high rate of oxygen consumption would be beneficial, as it would prevent overproduction of reactive oxygen species (ROS). In mitochondria from high energy-requiring, logarithmic-growth phase cells, most NDH2e was associated to cytochrome c oxidase and electrons from NADH were channeled to the cytochromic pathway. In contrast, in the low energy requiring, late stationary-growth phase, complex IV concentration decreased, the cells overexpressed NDH2e and thus a large fraction of this enzyme was found in a non-associated form. Also, the NDH2e-AOX uncoupled pathway was activated and the state IV external NADH-dependent production of ROS decreased. Association/dissociation of NDH2e to/from complex IV is proposed to be the switch that channels electrons from external NADH to the coupled cytochrome pathway or allows them to reach an uncoupled, alternative, ΔΨ-independent pathway.

In Polytomella sp. mitochondria, biogenesis of the heterodimeric COX2 subunit of cytochrome c oxidase requires two different import pathways

In the vast majority of eukaryotic organisms, the mitochondrial cox2 gene encodes subunit II of cytochrome c oxidase (COX2). However, in some lineages including legumes and chlorophycean algae, the cox2 gene migrated to the nucleus. Furthermore, in chlorophycean algae, this gene was split in two different units. Thereby the COX2 subunit is encoded by two independent nuclear genes, cox2a and cox2b, and mitochondria have to import the cytosol-synthesized COX2A and COX2B subunits and assemble them into the cytochrome c oxidase complex. In the chlorophycean algae Chlamydomonas reinhardtii and Polytomella sp., the COX2A precursor exhibits a long (130-140 residues), cleavable mitochondrial targeting sequence (MTS). In contrast, COX2B lacks an MTS, suggesting that mitochondria use different mechanisms to import each subunit. Here, we explored the in vitro import processes of both, the Polytomella sp. COX2A precursor and the COX2B protein. We used isolated, import-competent mitochondria from this colorless alga. Our results suggest that COX2B is imported directly into the intermembrane space, while COX2A seems to follow an energy-dependent import pathway, through which it finally integrates into the inner mitochondrial membrane. In addition, the MTS of the COX2A precursor is eliminated. This is the first time that the in vitro import of split COX2 subunits into mitochondria has been achieved.

Subunit Asa1 spans all the peripheral stalk of the mitochondrial ATP synthase of the chlorophycean alga Polytomella sp.

Mitochondrial F1FO-ATP synthase of chlorophycean algae is dimeric. It contains eight orthodox subunits (alpha, beta, gamma, delta, epsilon, OSCP, a and c) and nine atypical subunits (Asa1 to 9). These subunits build the peripheral stalk of the enzyme and stabilize its dimeric structure. The location of the 66.1kDa subunit Asa1 has been debated. On one hand, it was found in a transient subcomplex that contained membrane-bound subunits Asa1/Asa3/Asa5/Asa8/a (Atp6)/c (Atp9). On the other hand, Asa1 was proposed to form the bulky structure of the peripheral stalk that contacts the OSCP subunit in the F1 sector. Here, we overexpressed and purified the recombinant proteins Asa1 and OSCP and explored their interactions in vitro, using immunochemical techniques and affinity chromatography. Asa1 and OSCP interact strongly, and the carboxy-terminal half of OSCP seems to be instrumental for this association. In addition, the algal ATP synthase was partially dissociated at relatively high detergent concentrations, and an Asa1/Asa3/Asa5/Asa8/a/c10 subcomplex was identified. Furthermore, Far-Western analysis suggests an Asa1-Asa8 interaction. Based on these results, a model is proposed in which Asa1 spans the whole peripheral arm of the enzyme, from a region close to the matrix-exposed side of the mitochondrial inner membrane to the F1 region where OSCP is located. 3D models show elongated, helix-rich structures for chlorophycean Asa1 subunits. Asa1 subunit probably plays a scaffolding role in the peripheral stalk analogous to the one of subunit b in orthodox mitochondrial enzymes.