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Mitochondrial proton-translocating NADH:ubiquinone oxidoreductase (complex I) couples the transfer of two electrons from NADH to ubiquinone to the translocation of four protons across the mitochondrial inner membrane. Subunit PSST is the most likely carrier of iron-sulfur cluster N2, which has been proposed to play a crucial role in ubiquinone reduction and proton pumping. To explore the function of this subunit we have generated site-directed mutants of all eight highly conserved acidic residues in the Yarrowia lipolytica homologue, the NUKM protein. Mutants D99N and D115N had only 5 and 8% of the wild type catalytic activity, respectively. In both cases complex I was stably assembled but electron paramagnetic resonance spectra of the purified enzyme showed a reduced N2 signal (about 50%). In terms of complex I catalytic activity, almost identical results were obtained when the aspartates were individually changed to glutamates or to glycines. Mutations of other conserved acidic residues had less dramatic effects on catalytic activity and did not prevent assembly of iron-sulfur cluster N2. This excludes all conserved acidic residues in the PSST subunit as fourth ligands of this redox center. The results are discussed in the light of the structural similarities to the homologous small subunit of water-soluble [NiFe] hydrogenases.

The modular evolutionary origin of NADH:ubiquinone oxidoreductase (complex I) provides useful insights into its functional organization. Iron–sulfur cluster N2 and the PSST and 49 kDa subunits were identified as key players in ubiquinone reduction and proton pumping. Structural studies indicate that this ‘catalytic core’ region of complex I is clearly separated from the membrane. Complex I from Escherichia coli and Klebsiella pneumoniae was shown to pump sodium ions rather than protons. These new insights into structure and function of complex I strongly suggest that proton or sodium pumping in complex I is achieved by conformational energy transfer rather than by a directly linked redox pump.

Respiratory chain complex I deficiencies represent a genetically heterogeneous group of diseases resulting from mutations in either mitochondrial or nuclear DNA. Combination of denaturing high performance liquid chromatography and sequence analysis allowed us to show that a 4-bp deletion in intron 2 (IVS2+5_+8delGTAA) of the NDUFV2 gene (encoding NADH dehydrogenase ubiquinone flavoprotein 2) causes complex I deficiency and early onset hypertrophic cardiomyopathy with trunk hypotonia in three affected sibs of a consanguineous family. The homozygous mutation altering the consensus splice-donor site of exon 2 resulted in 70% decreased NDUFV2 protein and complex I deficiency. While mutation in a number of genes encoding complex I subunits essentially result in neurological symptoms, this first mutation in NDUFV2 is strikingly associated with cardiomyopathy, as previously observed in the unique case of NDFUS2 mutations.

The yeast Yarrowia lipolytica has been used for industrial bioconversions since the late 1960’s, mainly by the food and chemical industries. The most important process is presently the production of citric acid from rapeseed oil, but Y. lipolytica has also been considered for various other applications, ranging from single cell protein to heterologous proteins production. Several of these procedures have received a GRAS status, which facilitates further applications of this yeast in the food and pharmaceutical industries. This review focuses on the acquisition of basic knowledge on Y. lipolytica, which is physiologically and taxonomically very distant from Saccharomyces cerevisiae. Thanks to the development of advanced genetic tools for both classical approaches and recombinant DNA technology, significant progresses have been achieved in several aspects of its unusual biology. Particular attention has been paid to the study of early steps of protein secretion, peroxisome biogenesis, carbon metabolism and utilisation of hydrophobic substrates, mitochondrial biology, morphogenetic control, and proteae regulation. An international effort relayed by the Genoscope will permit access to its full genome sequence by the end of 2003, thus facilitating further developments of its functional genetics.

Complex I is the first and largest enzyme in the respiratory chain located in the inner mitochondrial membrane and in the membrane of many bacteria [1]. In the process of oxidizing NADH, Complex I translocates 4 protons across the membrane. This is followed by additional proton translocation by complexes III and IV. The resulting membrane potential powers ATP production by the F-ATPase. The minimal bacterial complex I contains 14 different subunits that are conserved throughout species. Eukaryotic complex I contains more than 26 additional (accessory) subunits. Complex I is L-shaped with a membrane arm and a peripheral arm that contains the NADH binding site and reaches into the mitochondrial matrix. The membrane arm contains 7 conserved subunits, ND1–ND4, ND4L-ND6. ND2, ND4 and ND5 having sequence homology to certain antiporters [2] and are the putative subunits for active proton translocation [3]. Here we have analyzed the structure of a subcomplex of Y. lipolytica complex I, created by the deletion of the accessory subunit NB8M located in the membrane arm with a molecular mass of 11kDa. This resulted in the assembly of a subcomplex of molecular mass of approximately 680 kDa lacking the central subunits ND4, and ND5 [4]. This complex showed reduced (30%) electron transfer activity and the stoichiometry of proton pumping was reduced by half. The 3D structure was determined by single particle reconstruction from electron micrographs using the Random Conical reconstruction Technique (RCT)[5]. The sample was prepared on carbon coated grids, deep stain embedded [6,7] in NanoW (Nanoprobes, Yaphank, NY). Tilt pairs were recorded at a nominal magnification of 67 KX with a 55° tilt angle. 68 tilt pairs were digitized with a final calibrated pixel size of 3.14. The microscope transfer function was corrected for defocus and astigmatism in both 0°- and tilt-images as described in [8]. The 0° images were first centered and then aligned by iterations of multireference alignments and classifications, using correspondence analysis and classification as the last step. All rotation/translation alignments were carried out using the simultaneous translation/rotation alignments based on Radon transforms [9]. 3D reconstructions were calculated from the 10 final classes. (Fig. 2). The differences between the 10 reconstructions were mainly differences in preferred orientation, which showed that the sample was homogeneous and allowed us to merge all 10 classes into a single reconstruction. The final reconstruction from 10586 images has a resolution of 23 A determined by Fourier Shell Correlation with a cutoff of 0.3 [10]. Figure 2 Ten volumes reconstructed after classification of the projections. The main difference is the particle orientation. Scale bar 10 nm. A comparison with our earlier 3D structure of the complete complex I [11] and recent Xray-crystallographic results [12,13] show that the subcomplex is missing the distal portion of the membrane arm. This study confirms the location of ND4 and ND5 as being located in the distal region of the membrane, and further shows that a third proton pumping subunit, probably ND2, located proximal to the matrix arm is still active.

We have used the obligate aerobic yeast Yarrowia lipolytica to reconstruct and analyse three missense mutations in the nuclear coded subunits homologous to bovine TYKY and PSST of mitochondrial complex I (proton translocating NADH:ubiquinone oxidoreductase) that have been shown to cause Leigh syndrome (MIM 25600), a severe progressive neurodegenerative disorder. While homozygosity for a V122M substitution in NDUFS7 (PSST) has been found in two siblings with neuropathologically proven Leigh syndrome (R. Triepels et al., Ann. Neurol. 45 (1999) 787), heterozygosity for a P79L and a R102H substitution in NDUFS8 (TYKY) has been found in another patient (J. Loeffen et al., Am. J. Hum. Genet. 63 (1998) 1598). Mitochondrial membranes from Y. lipolytica strains carrying any of the three point mutations exhibited similar complex I defects, with Vmax being reduced by about 50%. This suggests that complex I mutations that clinically present as Leigh syndrome may share common characteristics. In addition changes in the Km for n-decyl-ubiquinone and I50 for hydrophobic complex I inhibitors were observed, which provides further evidence that not only the hydrophobic, mitochondrially coded subunits, but also some of the nuclear coded subunits of complex I are involved in its reaction with ubiquinone.

Vacuolar proton-translocating ATPase (holoATPase and free membrane sector) was isolated from bovine chromaffin granules by blue native polyacrylamide gel electrophoresis. A 5-fold excess of membrane sector over holoenzyme was determined in isolated chromaffin granule membranes. M9.2, a novel extremely hydrophobic 9.2-kDa protein comprising 80 amino acids, was detected in the membrane sector. It shows sequence and structural similarity to Vma21p, a yeast protein required for assembly of vacuolar ATPase. A second membrane sector-associated protein (M8-9) was identified and characterized by amino-terminal protein sequencing.

Glycosylation is an important mechanism for modulating the physicochemical and biological properties of proteins in a stage- and tissue-specific manner. The enzymology of this process is just beginning to be understood. Here we present the molecular analysis of mas-1 (mannosidase-1), a Drosophila gene with significant homologies to mammalian and Saccharomyces cerevisiae glycoprotein processing α1,2-mannosidases. An enhancer-trap P-element inserted upstream of mas-1 leads to highly specific lacZ expression in the lobula plate giant neurons, cells that mediate the large-field optomotor response. This staining, however, seems to reflect only a small part of the complex expression pattern of the mas-1 gene: Two promoters produce alternative transcripts that show individual spatial distributions during embryonic development, including a maternal contribution. Both transcripts code for type II transmembrane proteins which differ in their N-terminal parts. Null mutants in mas-1 display defects in the embryonic PNS, in the wing, and in the adult eye. These findings illustrate that the processing of N-linked glycans plays a functional role in Drosophila development. There is, however, ample evidence for genetic and biochemical redundancy in the mannose-trimming steps of this pathway.

The X-chromosomal complementation unit lethal(1)optomotor-blind [l(1)omb] is defined by lack of complementation among over a dozen recessive lethal mutations that map to the omb gene locus. Mutations in l(1)omb also fail to complement viable mutations of three seemingly unrelated functions in this region: bifid (bi), manifesting defective wings, Quadroon (Qd), a semi-dominant mutation expressing abnormal tergite pigmentation, and In(1)ombH31, giving rise to a normal external morphology but with discrete defects in the optic lobes and behavior. The locus encodes a 70-kilobase primary transcript that is spliced into a 6-kilobase mature RNA. cDNAs for this transcript were isolated and sequenced and the derived amino acid sequence was analyzed. Certain features of this sequence suggest that the l(1)omb gene product is a nuclear regulatory protein. The lethal phase of various apparent null mutants was determined and found to occur mainly in the pupal stage. A large proportion of all hemizygous mutant males develop to pharate adults that eclose only rarely but can be rescued from the pupal case. These animals show a severe maldevelopment of the optic lobes. In addition they have only rudimentary wings as well as a Quadroon-like abdominal pigmentation. Thus, in the lethal mutants those parts of the body are affected for which independent viable mutations have been previously described in the omb locus, such as optomotor-blind, bifid, and Quadroon.

Mitochondria from various organisms, especially plants, fungi and many bacteria contain so-called alternative NADH:ubiquinone oxidoreductases that catalyse the same redox reaction as respiratory chain complex I, but do not contribute to the generation of transmembrane proton gradients. In eucaryotes, these enzymes are associated with the mitochondrial inner membrane, with their NADH reaction site facing either the mitochondrial matrix (internal alternative NADH:ubiquinone oxidoreductases) or the cytoplasm (external alternative NADH:ubiquinone oxidoreductases). Some of these enzymes also accept NADPH as substrate, some require calcium for activity. In the past few years, the characterisation of several alternative NADH:ubiquinone oxidoreductases on the DNA and on the protein level, of substrate specificities, mitochondrial import and targeting to the mitochondrial inner membrane has greatly improved our understanding of these enzymes. The present review will, with an emphasis on yeast model systems, illuminate various aspects of the biochemistry of alternative NADH:ubiquinone oxidoreductases, address recent developments and discuss some of the questions still open in the field.

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.

While diagnosis and genetic analysis of mitochondrial disorders has made remarkable progress, we still do not understand how given molecular defects are correlated to specific patterns of symptoms and their severity. Towards resolving this dilemma for the largest and therefore most affected respiratory chain enzyme, we have established the yeast Yarrowia lipolytica as a eucaryotic model system to analyse respiratory chain complex I. For in vivo analysis, eYFP protein was attached to the 30-kDa subunit to visualize complex I and mitochondria. Deletions strains for nuclear coded subunits allow the reconstruction of patient alleles by site-directed mutagenesis and plasmid complementation. In most of the pathogenic mutations analysed so far, decreased catalytic activities, elevated KM values, and/or elevated I50 values for quinone-analogous inhibitors were observed, providing plausible clues on the pathogenic process at the molecular level. Leigh mutations in the 49-kDa and PSST homologous subunits are found in regions that are at the boundaries of the ubiquinone-reducing catalytic core. This supports the proposed structural model and at the same time identifies novel domains critical for catalysis. Thus, Y. lipolytica is a useful lower eucaryotic model that will help to understand how pathogenic mutations in complex I interfere with enzyme function.

We have employed laser induced liquid bead ion desorption (LILBID) mass spectrometry to determine the total mass and to study the subunit composition of respiratory chain complex I from Yarrowia lipolytica . Using 5–10 pmol of purified complex I, we could assign all 40 known subunits of this membrane bound multiprotein complex to peaks in LILBID subunit fingerprint spectra by comparing predicted protein masses to observed ion masses. Notably, even the highly hydrophobic subunits encoded by the mitochondrial genome were easily detectable. Moreover, the LILBID approach allowed us to spot and correct several errors in the genome-derived protein sequences of complex I subunits. Typically, the masses of the individual subunits as determined by LILBID mass spectrometry were within 100 Da of the predicted values. For the first time, we demonstrate that LILBID spectrometry can be successfully applied to a complex I band eluted from a blue-native polyacrylamide gel, making small amounts of large multiprotein complexes accessible for subunit mass fingerprint analysis even if they are membrane bound. Thus, the LILBID subunit mass fingerprint method will be of great value for efficient proteomic analysis of complex I and its assembly intermediates, as well as of other water soluble and membrane bound multiprotein complexes.

Mitochondrial NADH:ubiquinone oxidoreductase is the largest and most complicated proton pump of the respiratory chain. Here we report the preparation and characterization of a subcomplex of complex I selectively lacking the flavoprotein part of the N-module. Removing the 51-kDa and the 24-kDa subunit resulted in loss of catalytic activity. The redox centers of the subcomplex could be reduced neither by NADH nor NADPH demonstrating that physiological electron input into complex I occurred exclusively via the N-module and that the NADPH binding site in the 39-kDa subunit and further potential nucleotide binding sites are isolated from the electron transfer pathway within the enzyme. Taking advantage of the selective removal of two of the eight iron–sulfur clusters of complex I and providing additional evidence by redox titration and site-directed mutagenesis, we could for the first time unambiguously assign cluster N1 of fungal complex I to mammalian cluster N1b.