Tracing the tail of ubiquinone in mitochondrial complex I

Mitochondrial complex I (proton pumping NADH:ubiquinone oxidoreductase) is the largest and most complicated component of the respiratory electron transfer chain. Despite its central role in biological energy conversion the structure and function of this membrane integral multiprotein complex is still poorly understood. Recent insights into the structure of complex I by X-ray crystallography have shown that iron–sulfur cluster N2, the immediate electron donor for ubiquinone, resides about 30 Å above the membrane domain and mutagenesis studies suggested that the active site for the hydrophobic substrate is located next to this redox-center. To trace the path for the hydrophobic tail of ubiquinone when it enters the peripheral arm of complex I, we performed an extensive structure/function analysis of complex I from Yarrowia lipolytica monitoring the interaction of site-directed mutants with five ubiquinone derivatives carrying different tails. The catalytic activity of a subset of mutants was strictly dependent on the presence of intact isoprenoid moieties in the tail. Overall a consistent picture emerged suggesting that the tail of ubiquinone enters through a narrow path at the interface between the 49-kDa and PSST subunits. Most notably we identified a set of methionines that seems to form a hydrophobic gate to the active site reminiscent to the M-domains involved in the interaction with hydrophobic targeting sequences with the signal recognition particle of the endoplasmic reticulum. Interestingly, two of the amino acids critical for the interaction with the ubiquinone tail are different in bovine complex I and we could show that one of these exchanges is responsible for the lower sensitivity of Y. lipolytica complex I towards the inhibitor rotenone. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

A ferredoxin bridge connects the two arms of plant mitochondrial complex I

Mitochondrial complex I is the main site for electron transfer to the respiratory chain and generates much of the proton gradient across the inner mitochondrial membrane. Complex I is composed of two arms, which form a conserved L-shape. We report the structures of the intact, 47-subunit mitochondrial complex I from Arabidopsis thaliana and the 51-subunit complex I from the green alga Polytomella sp., both at around 2.9 Å resolution. In both complexes, a heterotrimeric γ-carbonic anhydrase domain is attached to the membrane arm on the matrix side. Two states are resolved in A. thaliana complex I, with different angles between the two arms and different conformations of the ND1 (NADH dehydrogenase subunit 1) loop near the quinol binding site. The angle appears to depend on a bridge domain, which links the peripheral arm to the membrane arm and includes an unusual ferredoxin. We propose that the bridge domain participates in regulating the activity of plant complex I.

An unusual ferredoxin completes a protein bridge that links the two arms of plant mitochondrial complex I and adjusts their angle in an open or closed conformation.     

Cloning of the isocitrate lyase gene (ICL1) from Yarrowia lipolytica and characterization of the deduced protein

The ICL1 gene encoding isocitrate lyase was cloned from the dimorphic fungus Yarrowia lipolytica by complementation of a mutation (acuA3) in the structural gene of isocitrate lyase of Escherichia coli. The open reading frame of ICL1 is 1668 by long and contains no introns in contrast to currently sequenced genes from other filamentous fungi. The ICL1 gene encodes a deduced protein of 555 amino acids with a molecular weight of 62 kDa, which fits the observed size of the purified monomer of isocitrate lyase from Y. lipolytica. Comparison of the protein sequence with those of known pro- and eukaryotic isocitrate lyases revealed a high degree of homology among these enzymes. The isocitrate lyase of Y. lipolytica is more similar to those from Candida tropicalis and filamentous fungi than to Sacharomyces cerevisiae. This enzyme of Y. lipolytica has the putative glyoxysomal targeting signal S-K-L at the carboxy-terminus. It contains a partial repeat which is typical for eukaryotic isocitrate lyases but which is absent from the E. coli enzyme. Surprisingly, deletion of the ICL1 gene from the genome not only inhibits the utilization of acetate, ethanol, and fatty acids, but also reduces the growth rate on glucose.     

Three-dimensional structure of respiratory complex I from Escherichia coli in ice in the presence of nucleotides

Complex I (NADH:ubiquinone oxidoreductase) is the largest protein complex of bacterial and mitochondrial respiratory chains. The first three-dimensional structure of bacterial complex I in vitrified ice was determined by electron cryo-microscopy and single particle analysis. The structure of the Escherichia coli enzyme incubated with either NAD(+) (as a reference) or NADH was calculated to 35 and 39 A resolution, respectively. The X-ray structure of the peripheral arm of Thermus thermophilus complex I was docked into the reference EM structure. The model obtained indicates that Fe-S cluster N2 is close to the membrane domain interface, allowing for effective electron transfer to membrane-embedded quinone. At the current resolution, the structures in the presence of NAD(+) or NADH are similar. Additionally, side-view class averages were calculated for the negatively stained bovine enzyme. The structures of bovine complex I in the presence of either NAD(+) or NADH also appeared to be similar. These observations indicate that conformational changes upon reduction with NADH, suggested to occur by a range of studies, are smaller than had been thought previously. The model of the entire bacterial complex I could be built from the crystal structures of subcomplexes using the EM envelope described here.     

A structural investigation of complex I and I + III2 supercomplex from Zea mays at 11-13 Å resolution: Assignment of the carbonic anhydrase domain and evidence for structural heterogeneity within complex I

The projection structures of complex I and the I + III₂ supercomplex from the C₄ plant Zea mays were determined by electron microscopy and single particle image analysis to a resolution of up to 11 Å. Maize complex I has a typical L-shape. Additionally, it has a large hydrophilic extra-domain attached to the centre of the membrane arm on its matrix-exposed side, which previously was described for Arabidopsis and which was reported to include carbonic anhydrase subunits. A comparison with the X-ray structure of homotrimeric γ-carbonic anhydrase from the archaebacterium Methanosarcina thermophila indicates that this domain is also composed of a trimer. Mass spectrometry analyses allowed to identify two different carbonic anhydrase isoforms, suggesting that the γ-carbonic anhydrase domain of maize complex I most likely is a heterotrimer. Statistical analysis indicates that the maize complex I structure is heterogeneous: a less-abundant “type II” particle has a 15 Å shorter membrane arm and an additional small protrusion on the intermembrane-side of the membrane arm if compared to the more abundant “type I” particle. The I + III₂ supercomplex was found to be a rigid structure which did not break down into subcomplexes at the interface between the hydrophilic and the hydrophobic arms of complex I. The complex I moiety of the supercomplex appears to be only of “type I”. This would mean that the “type II” particles are not involved in the supercomplex formation and, hence, could have a different physiological role.     

Crystal structure of a new benzoic acid inhibitor of influenza neuraminidase bound with a new tilt induced by overpacking subsite C6

Background

The quaternary structure of eukaryotic NADH:ubiquinone oxidoreductase (complex I), the largest complex of the oxidative phosphorylation, is still mostly unresolved. Furthermore, it is unknown where transiently bound assembly factors interact with complex I. We therefore asked whether the evolution of complex I contains information about its 3D topology and the binding positions of its assembly factors. We approached these questions by correlating the evolutionary rates of eukaryotic complex I subunits using the mirror-tree method and mapping the results into a 3D representation by multidimensional scaling.

Results

More than 60% of the evolutionary correlation among the conserved seven subunits of the complex I matrix arm can be explained by the physical distance between the subunits. The three-dimensional evolutionary model of the eukaryotic conserved matrix arm has a striking similarity to the matrix arm quaternary structure in the bacterium Thermus thermophilus (rmsd=19 ?) and supports the previous finding that in eukaryotes the N-module is turned relative to the Q-module when compared to bacteria. By contrast, the evolutionary rates contained little information about the structure of the membrane arm. A large evolutionary model of 45 subunits and assembly factors allows to predict subunit positions and interactions (rmsd = 52.6 ?). The model supports an interaction of NDUFAF3, C8orf38 and C2orf56 during the assembly of the proximal matrix arm and the membrane arm. The model further suggests a tight relationship between the assembly factor NUBPL and NDUFA2, which both have been linked to iron-sulfur cluster assembly, as well as between NDUFA12 and its paralog, the assembly factor NDUFAF2.

Conclusions

The physical distance between subunits of complex I is a major correlate of the rate of protein evolution in the complex I matrix arm and is sufficient to infer parts of the complex??s structure with high accuracy. The resulting evolutionary model predicts the positions of a number of subunits and assembly factors.     

The three-dimensional structure of complex I from Yarrowia lipolytica: a highly dynamic enzyme

The structure of complex I from Yarrowia lipolytica was determined by three-dimensional electron microscopy. A random conical data set was collected from deep stain embedded particles. More than 14000 image pairs were analyzed. Through extensive classification combined with three-dimensional reconstruction, it was possible for the first time to show a much more detailed substructure of the complex. The peripheral arm is subdivided in at least six domains. The membrane arm shows two major protrusions on its matrix facing side and exhibits a channel like feature on the side facing the cytoplasm. Structures resembling a tether connecting the subunits near the catalytic center with the protrusions of the membrane arm provide a second connection between matrix and membrane domain.     

Tight binding of NADPH to the 39-kDa subunit of complex I is not required for catalytic activity but stabilizes the multiprotein complex

In addition to the 14 central subunits, respiratory chain complex I from the aerobic yeast Yarrowia lipolytica contains at least 24 accessory subunits, most of which are poorly characterized. Here we investigated the role of the accessory 39-kDa subunit which belongs to the heterogeneous short-chain dehydrogenase/reductase (SDR) enzyme family and contains non-covalently bound NADPH. Deleting the chromosomal copy of the gene that codes for the 39-kDa subunit drastically impaired complex I assembly in Y. lipolytica. We introduced several site-directed mutations into the nucleotide binding motif that severely reduced NADPH binding. This effect was most pronounced when the arginine at the end of the second β-strand of the NADPH binding Rossman fold was replaced by leucine or aspartate. Mutations affecting nucleotide binding had only minor or moderate effects on specific catalytic activity in mitochondrial membranes but clearly destabilized complex I. One mutant exhibited a temperature sensitive phenotype and significant amounts of three different subcomplexes were observed even at more permissive temperature. We concluded that the 39-kDa subunit of Y. lipolytica plays a critical role in complex I assembly and stability and that the bound NADPH serves to stabilize the subunit and complex I as a whole rather than serving a catalytic function.     

Cryo-EM Structure of the Yeast ATP Synthase

We have used electron cryomicroscopy of single particles to determine the structure of the ATP synthase from Saccharomyces cerevisiae. The resulting map at 24 Å resolution can accommodate atomic models of the F₁-c₁₀ subcomplex, the peripheral stalk subcomplex, and the N-terminal domain of the oligomycin sensitivity conferral protein. The map is similar to an earlier electron cryomicroscopy structure of bovine mitochondrial ATP synthase but with important differences. It resolves the internal structure of the membrane region of the complex, especially the membrane embedded subunits b, c, and a. Comparison of the yeast ATP synthase map, which lacks density from the dimer-specific subunits e and g, with a map of the bovine enzyme that included e and g indicates where these subunits are located in the intact complex. This new map has allowed construction of a model of subunit arrangement in the F_O motor of ATP synthase that dictates how dimerization of the complex via subunits e and g might occur.     

In Chlamydomonas, the loss of ND5 subunit prevents the assembly of whole mitochondrial complex I and leads to the formation of a low abundant 700 kDa subcomplex

In the green alga Chlamydomonas reinhardtii, a mutant deprived of complex I enzyme activity presents a 1T deletion in the mitochondrial nd5 gene. The loss of the ND5 subunit prevents the assembly of the 950 kDa whole complex I. Instead, a low abundant 700 kDa subcomplex, loosely associated to the inner mitochondrial membrane, is assembled. The resolution of the subcomplex by SDS-PAGE gave rise to 19 individual spots, sixteen having been identified by mass spectrometry analysis. Eleven, mainly associated to the hydrophilic part of the complex, are homologs to subunits of the bovine enzyme whereas five (including gamma-type carbonic anhydrase subunits) are specific to green plants or to plants and fungi. None of the subunits typical of the β membrane domain of complex I enzyme has been identified in the mutant. This allows us to propose that the truncated enzyme misses the membrane distal domain of complex I but retains the proximal domain associated to the matrix arm of the enzyme. A complex I topology model is presented in the light of our results. Finally, a supercomplex most probably corresponding to complex I-complex III association, was identified in mutant mitochondria, indicating that the missing part of the enzyme is not required for the formation of the supercomplex.     

Single particle analysis confirms distal location of subunits NuoL and NuoM in Escherichia coli complex I

Respiratory complex I catalyses the transfer of electrons from NADH to quinone coupled to the translocation of protons across the membrane. The mechanism of coupling and the structure of the complete enzyme are not known. The membrane domain of the complex contains three similar antiporter-like subunits NuoL/M/N, probably involved in proton pumping. We have previously shown that subunits NuoL/M can be removed from the rest of the complex, suggesting their location at the distal end of the membrane domain. Here, using electron microscopy and single particle analysis, we show that subunits NuoL and M jointly occupy a distal half of the membrane domain, separated by about 10nm from the interface with the peripheral arm. This indicates that coupling mechanism of complex I is likely to involve long range conformational changes.     

Direct localization of the 51 and 24 kDa subunits of mitochondrial complex I by three-dimensional difference imaging

Complex I is the largest complex in the respiratory chain, and the least understood. We have determined the 3D structure of complex I from Yarrowia lipolytica lacking the flavoprotein part of the N-module, which consists of the 51 kDa (NUBM) and the 24 kDa (NUHM) subunits. The reconstruction was determined by 3D electron microscopy of single particles. A comparison to our earlier reconstruction of the complete Y. lipolytica complex I clearly assigns the two flavoprotein subunits to an outer lobe of the peripheral arm of complex I. Localizing the two subunits allowed us to fit the X-ray structure of the hydrophilic fragment of complex I from Thermus thermophilus. The fit that is most consistent with previous immuno-electron microscopic data predicts that the ubiquinone reducing catalytic center resides in the second peripheral lobe, while the 75 kDa subunit is placed near the previously seen connection between the peripheral arm and the membrane arm protrusions.     

ErpA is important but not essential for the Fe/S cluster biogenesis of Escherichia coli NADH:ubiquinone oxidoreductase (complex I)

Energy converting NADH:ubiquinone oxidoreductase, complex I, is the first enzyme of respiratory chains in most eukaryotes and many bacteria. The complex comprises a peripheral arm catalyzing electron transfer and a membrane arm involved in proton-translocation. In Escherichia coli, the peripheral arm features a non-covalently bound flavin mononucleotide and nine iron-sulfur (Fe/S)-clusters. Very little is known about the incorporation of the Fe/S-clusters into the E. coli complex I. ErpA, an A-type carrier protein is discussed to act as a Fe/S-cluster carrier protein. To contribute to the understanding of ErpA for the assembly of E. coli complex I, we analyzed an erpA knock-out strain. Deletion of erpA decreased the complex I content in cytoplasmic membranes to approximately one third and the NADH oxidase activity to one fifth. EPR spectroscopy showed the presence of all Fe/S-clusters of the complex in the membrane but only in minor quantities. Sucrose gradient centrifugation and native PAGE revealed the presence of a marginal amount of a stable and fully assembled complex extractable from the membrane. Thus, ErpA is not essential for the assembly of complex I but its absence leads to a strong decrease of a functional complex in the cytoplasmic membrane due to a major lack of all EPR-detectable Fe/S-clusters.     

Production of Lycopene in the Non-Carotenoid-Producing Yeast Yarrowia lipolytica

The codon-optimized genes crtB and crtI of Pantoea ananatis were expressed in Yarrowia lipolytica under the control of the TEF1 promoter of Y. lipolytica. Additionally, the rate-limiting genes for isoprenoid biosynthesis in Y. lipolytica, GGS1 and HMG1, were overexpressed to increase the production of lycopene. All of the genes were also expressed in a Y. lipolytica strain with POX1 to POX6 and GUT2 deleted, which led to an increase in the size of lipid bodies and a further increase in lycopene production. Lycopene is located mainly within lipid bodies, and increased lipid body formation leads to an increase in the lycopene storage capacity of Y. lipolytica. Growth-limiting conditions increase the specific lycopene content. Finally, a yield of 16 mg g⁻¹ (dry cell weight) was reached in fed-batch cultures, which is the highest value reported so far for a eukaryotic host.     

Functional characterization of extracellular chitinase encoded by the YlCTS1 gene in a dimorphic yeast Yarrowia lipolytica

The hemiascomycetes yeast Yarrowia lipolytica is a dimorphic yeast with alternating yeast and mycelia forms. Bioinformatic analysis revealed the presence of three putative chitinase genes, YlCTS1, YlCTS2, and YlCTS3, in the Y. lipolytica genome. Here, we demonstrated that the protein of YlCTS1 (YlCts1p), which contains an N-terminal secretion signal peptide, a long C-terminal Ser/Thr-rich domain, and a chitin-binding domain, is a homologue to Saccharomyces cerevisiae chitinase 1 (ScCts1p). Deletion of YlCTS1 remarkably reduced extracellular endochitinase activity in the culture supernatant of Y. lipolytica and enhanced cell aggregation, suggesting a role of YlCts1p in cell separation as ScCts1p does in S. cerevisiae. However, loss of YlCts1p function did not affect hyphal formation induced by fetal bovine serum addition. The mass of YlCts1p was dramatically decreased by jack bean α-mannosidase digestion but not by PNGase F treatment, indicating that YlCts1p is modified only by O-mannosylation without N-glycosylation. Moreover, the O-glycan profile of YlCts1p was identical to that of total cell wall mannoproteins, supporting the notion that YlCts1p can be used as a good model for studying O-glycosylation in this dimorphic yeast.     

Study of the Accumulation of Rec A from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> in the Mitochondria of a Recombinant Strain of the Yeast <Emphasis Type="Italic">Yarovia lipolytica</Emphasis>

No eukaryotic species has a system for homologous DNA recombination of the mitochondrial genome. We report on an integrative genetic system based on the pQ-SRUS construct that allows the expression of the RecA recombinase from Bacillus subtilis and its transportation to mitochondria of Yarrowia lipolytica. The targeting of recombinant RecA to mitochondria is provided by leader sequences (5'-UTR and 3'-UTR) derived from the SOD2 gene mRNA, which exhibit affinity to the outer mitochondrial membrane and provides cotranslational import of RecA to the inner space of mitochondria. The accumulation of RecA in mitochondria of the Y. lipolytica recombinant strain bearing the pQ-SRUS construct has been shown by immunoblotting of purified mitochondrial preparations.