Background: The production of high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences that has yet to be addressed in a truly rational manner. Typically eukaryotic protein production experiments have relied on varying expression construct cassettes such as promoters and tags, or culture process parameters such as pH, temperature and aeration to enhance yields. These approaches require repeated rounds of trial-and-error optimization and cannot provide a mechanistic insight into the biology of recombinant protein production. We published an early transcriptome analysis that identified genes implicated in successful membrane protein production experiments in yeast. While there has been a subsequent explosion in such analyses in a range of production organisms, no one has yet exploited the genes identified. The aim of this study was to use the results of our previous comparative transcriptome analysis to engineer improved yeast strains and thereby gain an understanding of the mechanisms involved in highyielding protein production hosts.

Proton pumping respiratory complex I (NADH:ubiquinone oxidoreductase) is a major component of the oxidative phosphorylation system in mitochondria and many bacteria. In mammalian cells it provides 40% of the proton motive force needed to make ATP. Defects in this giant and most complicated membrane-bound enzyme cause numerous human disorders. Yet the mechanism of complex I is still elusive. A group exhibiting redox-linked protonation that is associated with iron-sulfur cluster N2 of complex I has been proposed to act as a central component of the proton pumping machinery. Here we show that a histidine in the 49-kDa subunit that resides near ironsulfur cluster N2 confers this redox-Bohr effect. Mutating this residue to methionine in complex I from Yarrowia lipolytica resulted in a marked shift of the redox midpoint potential of iron-sulfur cluster N2 to the negative and abolished the redoxBohr effect. However, the mutation did not significantly affect the catalytic activity of complex I and protons were pumped with an unchanged stoichiometry of 4 H ؉ /2e ؊ . This finding has significant implications on the discussion about possible proton pumping mechanism for complex I.In mammalian cells mitochondrial complex I (NADH: ubiquinone oxidoreductase) provides 40% of the proton motive force needed to make ATP. Defects in this giant and most complicated membrane-bound enzyme (1) cause numerous human disorders (2). Very recently, the x-ray structure of the peripheral arm of complex I from Thermus thermophilus has been solved (3). However, it is still unclear how complex I couples electron transfer from NADH to ubiquinone with the vectorial translocation of four protons across the bioenergetic membrane (4, 5). Numerous hypothetical mechanisms have been proposed for this energy conversion over the years (6 -9). None of the known redox groups of complex I (FMN and 8 -9 ironsulfur clusters) resides in the membrane domain (3, 10). The Fe 4 S 4 cluster N2 is the last component in a "wire" of seven iron-sulfur clusters connecting FMN, the immediate oxidant for NADH, and substrate ubiquinone (3,11). While all other clusters in this chain have a redox midpoint potential of around Ϫ250 mV, iron-sulfur cluster N2 has a significantly more positive E m7 of typically Ϫ150 mV (12, 13). Moreover, the redox midpoint potential of cluster N2 exhibits pronounced pH dependence between pH 6 and 8 (12). Based on this "redoxBohr" effect (14) and the comparably more positive potential, cluster N2 has been implicated as a key component of the proton pump (6, 15).The structural model for the ubiquinone reducing catalytic core of complex I (1, 16) that had been deduced from distinct homologies between [NiFe] hydrogenase and complex I (17, 18) was now confirmed by the structure of the peripheral arm of complex I (3). Like the proximal iron-sulfur cluster of hydrogenase, iron-sulfur cluster N2 is at the interface between two subunits, the PSST subunit and the 49-kDa subunit (Fig. 1). In water-soluble [NiFe] hydrogenases a conserved histidine of the l...

We have studied the ubiquinone-reducing catalytic core of NADH:ubiquinone oxidoreductase (complex I) from Yarrowia lipolytica by a series of point mutations replacing conserved histidines and arginines in the 49-kDa subunit. Our results show that histidine 226 and arginine 141 probably do not ligate iron-sulfur cluster N2 but that exchanging these residues specifically influences the properties of this redox center. Histidines 91 and 95 were found to be essential for ubiquinone reductase activity of complex I. Mutations at the C-terminal arginine 466 affected ubiquinone affinity and inhibitor sensitivity but also destabilized complex I. These results provide further support for a high degree of structural conservation between the 49-kDa subunit of complex I and its ancestor, the large subunit of water-soluble [NiFe] hydrogenases. In several mutations of histidine 226, arginine 141, and arginine 466 the characteristic EPR signatures of iron-sulfur cluster N2 became undetectable, but specific, inhibitor-sensitive ubiquinone reductase activity was only moderately reduced. As we could not find spectroscopic indications for a modified cluster N2, we concluded that these complex I mutants were lacking most of this redox center but were still capable of catalyzing inhibitor-resistant ubiquinone reduction at near normal rates. We discuss that this at first surprising scenario may be explained by electron transfer theory; after removal of a single redox center in a chain, electron transfer rates are predicted to be still much faster than steady-state turnover of complex I. Our results question some of the central mechanistic functions that have been put forward for iron-sulfur cluster N2.

NADH:ubiquinone oxidoreductase (complex I) is an entry point for electrons into the respiratory chain in many eukaryotes. It couples NADH oxidation and ubiquinone reduction to proton translocation across the mitochondrial inner membrane. Because complex I deficiencies occur in a wide range of neuromuscular diseases, including Parkinson's disease, there is a clear need for model eukaryotic systems to facilitate structural, functional and mutational studies. In the present study, we describe the purification and characterization of the complexes I from two yeast species, Pichia pastoris and Pichia angusta. They are obligate aerobes which grow to very high cell densities on simple medium, as yeast-like, spheroidal cells. Both Pichia enzymes catalyse inhibitor-sensitive NADH:ubiquinone oxidoreduction, display EPR spectra which match closely to those from other eukaryotic complexes I, and show patterns characteristic of complex I in SDS/PAGE analysis. Mass spectrometry was used to identify several canonical complex I subunits. Purified P. pastoris complex I has a particularly high specific activity, and incorporating it into liposomes demonstrates that NADH oxidation is coupled to the generation of a protonmotive force. Interestingly, the rate of NADH-induced superoxide production by the Pichia enzymes is more than twice as high as that of the Bos taurus enzyme. Our results both resolve previous disagreement about whether Pichia species encode complex I, furthering understanding of the evolution of complex I within dikarya, and they provide two new, robust and highly active model systems for study of the structure and catalytic mechanism of eukaryotic complexes I.