Hannah R Bridges

Ljuban Grgic

Michael E Harbour

Judy Hirst

The respiratory complexes I from the mitochondria of two Pichia species

Keywords: NADH:ubiquinone oxidoreductase Complex I Pichia pastoris Pichia angusta Electron transport chain Mitochondria ACMA, 9-amino-6-chloro-2-methoxyacridine, FeS cluster, iron-sulfur cluster, FMN, flavin mononucleotide, HAR, hexaammineruthenium

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. Here, 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 media, as yeast-like, spheroidal cells. Both Pichia enzymes catalyze 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 proton motive force. Interestingly, the rate of NADH-induced superoxide production by the Pichia enzymes is more than twice as fast 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.

Introduction

In mitochondria, NADH:ubiquinone oxidoreductase (complex I) catalyses the oxidation of NADH and the reduction of ubiquinone, coupled to the translocation of four protons across the mitochondrial inner membrane, contributing to the proton motive force [START_REF] Brandt | Energy converting NADH:quinone oxidoreductase (complex I)[END_REF][START_REF] Hirst | Energy transduction by respiratory complex I -an evaluation of current knowledge[END_REF]. Fourteen 'core subunits' are conserved throughout the complex I family. They comprise seven membrane bound subunits, encoded by the mitochondrial genome in eukaryotes, and seven hydrophilic subunits, encoded in the nucleus, which form a domain extending into the mitochondrial matrix. The hydrophilic subunits bind a flavin mononucleotide (FMN), at the active site for NADH oxidation, and eight iron sulfur (FeS) clusters. The structure of the hydrophilic domain from Thermus thermophilus complex I shows how the FeS clusters enable electron transfer from the flavin toward the quinone binding site [START_REF] Sazanov | Structure of the hydrophilic domain of respiratory complex I from Thermus thermophilus[END_REF]. In addition, eukaryotic complexes I contain a variable number of 'supernumerary' subunits. Complex I from Bos taurus heart mitochondria has been characterized extensively and contains a total of 45 different subunits [START_REF] Hirst | The nuclear encoded subunits of complex I from bovine heart mitochondria[END_REF]. In two fungal model systems, 40 different subunits have been identified in complex I from Yarrowia lipolytica [START_REF] Morgner | Subunit mass fingerprinting of mitochondrial complex I[END_REF], and 39 in the enzyme from Neurospora crassa [START_REF] Marques | Composition of complex I from Neurospora crassa and disruption of two accessory subunits[END_REF]. Gabaldon and coworkers have used genomic data to propose how the subunit composition of eukaryotic complex I has evolved [START_REF] Gabaldón | Tracing the evolution of a large protein complex in the eukaryotes, NADH:ubiquinone oxidoreductase (complex I)[END_REF]; mammalian and fungal complexes I have 21 supernumerary subunits in common, while mammals have ten additional subunits, fungi have three. Mutations in both the nuclearly-and mitochondrially-encoded subunits contribute to the increasing number of complex I deficiencies which have been identified in a wide range of neuromuscular diseases, including Leigh syndrome, leukodystrophy, and Parkinson's disease [START_REF] Dimauro | Mitochondrial respiratory-chain diseases[END_REF][START_REF] Greenamyre | Parkinson's -divergent causes, convergent mechanisms[END_REF].

Studies of eukaryotic complex I rely on model systems to provide sufficient quantities of the enzyme for investigation of its structure and mechanism, and to provide insights into the dysfunction of the human enzyme. The B. taurus enzyme is highly homologous to the human enzyme [START_REF] Hirst | The nuclear encoded subunits of complex I from bovine heart mitochondria[END_REF], and has proved invaluable in the elucidation of its protein composition, as well as for spectroscopic and functional studies. However, lack of control over the source material, and the impracticality of genetic manipulation, are significant limitations. The intensively studied yeast, Saccharomyces cerevisiae, does not encode complex I, but N. crassa [START_REF] Videira | Complex I from the fungus Neurospora crassa[END_REF], and more recently Y. lipolytica [START_REF] Kerscher | Yarrowia lipolytica, a yeast genetic system to study mitochondrial complex I[END_REF], have provided two genetically manipulable fungal enzymes for study.

Figure 1 shows a phylogenetic tree for the dikarya (adapted from [START_REF] Kriventseva | OrthoDB: the hierarchical catalog of eukaryotic orthologs[END_REF]), showing that N. crassa belongs to the pezizomycotina, and Y. lipolytica to the saccharomycetes. Y. lipolytica diverged relatively early in evolution from the saccharomyceteceae, which include Saccharomyces, Kluveromyces, Candida and Pichia. Some of the saccharomycetaceae have lost the complex I genes during evolution, and, in fact, no complex I has been isolated from any of them. Thus, although the Pichia genus shows many promising characteristics for the development of a complex I model system (see below), it has not been clear, until now, whether Pichia species even express complex I. Sequences for some of the subunits of complex I which are encoded in the mitochondrial genome (known as the ND subunits) have been detected in several species, including P. pastoris [START_REF] Nosek | NADH dehydrogenase subunit genes in the mitochondrial DNA of yeasts[END_REF], but attempts to identify an NADH dehydrogenase from P. pastoris which is inhibited by a canonical complex I inhibitor, rotenone, were unsuccessful [START_REF] González-Barroso | Isolation and bioenergetic characterization of mitochondria from Pichia pastoris[END_REF] The two species of yeast described here are Pichia pastoris and Pichia angusta (previously named Hansenula polymorpha). They are both methylotrophic ascomycetes, able to catabolize methanol as their sole carbon source. They are obligate aerobes, and grow on inexpensive, simple media to very high cell densities, particularly in batch-fed conditions. Notably, Pichia species grow almost exclusively as yeast-like, spheroidal cells, whereas N. crassa grows as filaments, and Y. lipolytica switches between yeast and filamentous growth in response to various environmental stresses [START_REF] Ruiz-Herrera | Different effectors of dimorphism in Yarrowia lipolytica[END_REF], a transition known to affect cellular metabolism and protein expression [START_REF] Morín | Proteomic analysis reveals metabolic changes during yeast to hypha transition in Yarrowia lipolytica[END_REF]. Both species can be used for overexpression, commonly controlled by the promoter for alcohol oxidase, and induced with methanol [START_REF] Gellissen | New yeast expression platforms based on methylotrophic Hansenula polymorpha and Pichia pastoris and on dimorphic Arxula adeninivorans and Yarrowia lipolytica -a comparison[END_REF]. In addition, P. angusta can grow at temperatures as high as 50 °C [START_REF] Van Uden | Temperature functions of thermal death in yeasts and their relation to the maximum temperature for growth[END_REF], and therefore may express more stable proteins as part of its heat tolerance. Here, we describe the purification and characterization of the complexes I from P. pastoris and P. angusta, discuss their suitability as eukaryotic model systems, and compare their properties to those of other members of the complex I family.

Experimental Methods

All chemicals were from Sigma Aldrich or BDH (unless otherwise stated), and chromatographic media were from GE Healthcare. Buffer pH values are reported at room temperature.

Yeast strains and growth

Haploid P. pastoris strain X33 (Invitrogen) was grown at 30 °C in 1% yeast extract (ForMedium), 2% peptone (ForMedium) and 2% glycerol (VWR International), initially at pH 5. 60 L of media, in a 70 L Applikon ADI 1075 fermentor stirred at 500 rpm, maintained at 80% pO 2 , was inoculated with 2 L of culture, and cells grown until early stationary phase (approx. 24 hours). P. angusta strain A-16 (NCYC 2310), a haploid leucine auxotroph of CBS4732, was from The National Collection of Yeast Cultures (UK). Cells were grown at 40 °C in 800 mL aliquots in 2 L flasks (shaken at 250 rpm) until mid stationary phase (approx. 48 hours) in 1% yeast extract, 2% peptone, and 4% glucose, initially at pH 5. Y. lipolytica strain GB10 was provided by Prof. U. Brandt (Frankfurt). It was grown at 27 ºC in 2% yeast extract, 4% peptone, and 4% glucose, initially at pH 5.5, in the 70 L fermentor stirred at 500 rpm, maintained at 2% pO 2 (once this value was reached), until late exponential phase (approx. 20 hours). All cells were harvested by centrifugation (5,000 g for 15 min.), then twice resuspended in MilliQ water and recentrifuged. If necessary, cell pellets were frozen at -80 °C. The yields were approximately 41, 27 and 29 g of wet cells per liter of medium for P. pastoris, P. angusta, and Y. lipolytica, respectively. All subsequent steps were at 4 °C.

Isolation of mitochondrial membranes

Methods for cell disruption and membrane isolation were based on those described for Y. lipolytica [START_REF] Kerscher | A single external enzynme confers alternative NADH:ubiqinone oxidoreductase activity in Yarrowia lipolytica[END_REF][START_REF] Kashani-Poor | Efficient large scale purification of his-tagged proton translocating NADH:ubiquinone oxidoreductase (complex I) from the strictly aerobic yeast Yarrowia lipolytica[END_REF]. Cells were resuspended to 500 g L -1 , in 20 mM MOPS pH 7.2, 400 mM sorbitol (NBS Biologicals Ltd), 0.2% BSA, 5 mM EDTA, 1 mM benzamidine, and 1 mM ε-caproic acid. 1 mM phenylmethanesulfonylfluoride (PMSF) was added immediately prior to cell disruption in a DYNO®-MILL bead mill (Willy A. Bachofen UK Ltd.) maintained below 10 °C (two passes at 10 mL min -1 ). Cell debris was removed by centrifugation (5,000 g for 15 min.), then the membranes were collected by centrifugation (one hour at 100,000 g (P. pastoris and Y. lipolytica) or 30,000 g (P. angusta)), rehomogenized in 20 mM MOPS pH 7.2, 5 mM EDTA, 1 mM benzamidine, and 1 mM ε-caproic acid, and recollected by recentrifugation. If necessary, membrane pellets were frozen at -80 C.

Complex I purification

Mitochondrial membranes were homogenized in 20 mM sodium citrate pH 6.5, to 20 mg protein mL -1 , and solubilized for 45 min. by the addition of dodecyl-β-d-maltoside (DDM, Glycon GmbH) to 2.3% (P. pastoris) or 2.9% (P. angusta). Following centrifugation (120,000 g for 45 min.), the supernatant was passed through a MacroCap SP cation exchange column, and dialyzed against 20 mM Tris-Cl pH A c c e p t e d M a n u s c r i p t 7.5 for at least 3 hours. Then, it was loaded onto a Q Sepharose High Performance anion exchange column, pre-equilibrated in 20 mM Tris-Cl (pH 7.5) and 0.15% DDM, and eluted at 2 mL min -1 . First, 0.27 M NaCl (P. pastoris) or 0.22 M NaCl (P. angusta) was applied until the 280 and 420 nm absorbancies became negligible, then complex I was eluted in a sharp gradient to 0.33 NaCl (P. pastoris) or 0.27 M NaCl (P. angusta), and confirmed using its dNADH:hexaammineruthenium (III) (HAR) oxidoreductase activity. Pooled fractions were concentrated (100 kDa cut-off Vivaspin centrifugal concentrators, Sartorius AG), then injected onto a Superose 6 size exclusion column (0.24 mL min -1 , in 20 mM MOPS pH 7.5, 0.05% DDM and 150 mM NaCl). Monomeric complex I eluted from 11 to 14 mL. If necessary, these complex I containing fractions were pooled, concentrated, and reapplied to the column, providing a single, sharp peak. Complex I from Y. lipolytica was isolated as described previously, by affinity purification on a Ni-NTA column using a hexahistidine tag on the Cterminus of the NUGM subunit [START_REF] Kashani-Poor | Efficient large scale purification of his-tagged proton translocating NADH:ubiquinone oxidoreductase (complex I) from the strictly aerobic yeast Yarrowia lipolytica[END_REF][START_REF] Dröse | Proton pumping by complex I from Yarrowia lipolytica reconstituted into proteoliposomes[END_REF], followed by size exclusion chromatography on a Superose 6 column B. taurus complex I was prepared as described previously [START_REF] Sharpley | Interactions between phospholipids and NADH:ubiquinone oxidoreductase (complex I) from bovine mitochondria[END_REF].

Kinetic measurements

Kinetic measurements were carried out at 32 °C, either in 1 mL cuvettes (Ocean Optics diode array spectrometer), or in 200 µL wells (Molecular Devices microtiter plate reader). Deamino-NADH (dNADH) and NADH were added to 100 μM for ferricyanide (hexacyanoferrate (III), FeCN), HAR and decylubiquinone (DQ) measurements, and HAR, FeCN and DQ to 3.5 mM, 2 mM and 100 μM, respectively. For measurements using DQ, soybean asolectin was added to 0.35 mg mL -1 from a 10 mg mL -1 stock solution in 20 mM Tris-HCl pH 7.5 and 1% w/v CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate), Anatrace). Rotenone or piericidin A were added to 5 or 2 µM, respectively, from ethanolic stock solutions. Initial rates of NADH oxidation (ε 340-380 nm = 4.81 mM -1 cm -1 ) were obtained using linear regression. H 2 O 2 and superoxide production were measured at pH 7.5, using 30 μM NADH and either 10 μM Amplex Red and 2 U mL -1 horse radish peroxidase (Invitrogen) or 50 μM acetylated cytochrome c, as described previously [START_REF] Kussmaul | The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria[END_REF].

To produce proteoliposomes containing complex I, 440 L of 10 mg mL -1 soy asolectin (Avanti Polar Lipids Inc.) in 20 mM potassium 3-(N-morpholino) propanesulfonic acid at pH 7.5 (K-MOPS) was homogenised with 60 L of 10% octyl glucoside. 50 L of 2 mg mL -1 complex I was added, then the detergent was removed by three successive additions of 50 mg Amberlite XAD 2 biobeads (Supelpak) at 1 hour intervals. The proteoliposomes were separated from the biobeads using a syringe fitted with a filter, collected by centrifugation (1 hour at 90,000 g in a Beckman-Coulter MLA 130 rotor), then resuspended in 100 L K-MOPS. Respiratory control ratios (RCRs) were calculated from the rate of NADH:DQ oxidoreduction, measured (as described above) in the presence and absence of 7 g mL -1 gramicidin (RCR = uncoupled rate / coupled rate). The quenching of the 9-amino-6-chloro-2methoxyacridine (ACMA) fluorescence at 475 nm was measured using an excitation wavelength of 430 nm, in the presence of 500 nM ACMA and 5 M valinomycin. Typically, 20 L of resuspended proteoliposomes were added to 2 mL of assay buffer. When required carbonylcyanide-ptrifluoromethoxy-phenylhydrazone (8 M, FCCP) or piericidin A (2 M) were added.

EPR spectroscopy

EPR samples (15 mg mL -1 ) were reduced anaerobically with 5 mM NADH, and frozen immediately. Spectra were recorded on a Bruker EMX X-band spectrometer using an ER 4119HS cavity, maintained at low temperature by an ESR900 continuous-flow liquid helium cryostat (Oxford Instruments); the sample temperature was measured with a calibrated Cernox resistor (Lake Shore Cryotronics Inc.).

Analytical methods

Protein concentrations were measured using the Pierce bicinchoninic acid assay. FMN concentrations were analyzed fluorometrically using the method of Burch [START_REF] Burch | Fluorimetric assay of FAD, FMN and riboflavin[END_REF], modified for the plate reader. Iron concentrations were determined using ferene (5,5'(3-(2-pyridyl)-1,2,4-triazine-5,6 diyl)-bis-2furansulfonate), by the method of Pieroni et al. [START_REF] Pieroni | Comparison of bathophenanthroline sulfonate and ferene as chromogens in colorimetric measurement of low hepatic iron concentration[END_REF] (except the protein was denatured in 1% HCl and 1.5% trichloroacetic acid, the reducing agent was 0.5% hydroxylamine, and 5 mM thiosemicarbazide was added to bind any copper present).

Protein analyses SDS-PAGE was carried out on an 18-22% acrylamide gradient, and gels were stained with 0.2% Coomassie Blue R250. Peptide mass fingerprinting and tandem MS were carried out using an Applied Biosystems / MDS SCIEX model 4800 Plus MALDI TOF / TOF mass spectrometer. Database matches to peptide mass fingerprinting and tandem MS data were identified using Mascot (Matrix Science) [START_REF] Perkins | Probability-based protein identification by searching sequence databases using mass spectrometry data[END_REF]. Sequences for proposed subunits of P. angusta complex I were identified by performing tblastn searches to compare known Y. lipolytica and N. crassa sequences with the genome survey sequence information from P. angusta which is available on the NCBI website.

Results

The presence of both complex I and alternative NADH dehydrogenases in Pichia species

Alternative NADH dehydrogenases are single subunit, non-energy transducing NADH:ubiquinone oxidoreductases which are found in many aerobically respiring organisms, either alongside complex I, or, in cases such as S. cerevisiae, as a replacement for it (see Figure 1) [START_REF] Melo | New insights into type II NAD(P)H: quinone oxidoreductases[END_REF][START_REF] Lavín | Comparative genomics of the oxidative phosphorylation system in fungi[END_REF]. Alternative NADH dehydrogenases complicate the identification, analysis, and purification of complex I, because the catalytic properties of the two enzymes overlap. Table 1 presents the catalytic properties of mitochondrial membranes prepared from P. pastoris and P. angusta; in both cases approximately 90% of the NADH binding sites were accessible to the NADH, FeCN and HAR reactants. Three different electron acceptors were used: FeCN and HAR are considered to react at, or close to, the flavin site in complex I, so, unlike DQ, an analogue of the physiological ubiquinone, they do not rely on the functional integrity of the quinone binding site or the membrane domain. First, based on a study of the NADH:ubiquinone oxidoreductases in E. coli, dNADH is assumed to react only with complex I [START_REF] Matsushita | NADH-ubiquinone oxidoreductases of the Escherichia coli aerobic respiratory chain[END_REF], so comparing the rates of NADH and dNADH:DQ oxidoreduction by the P. pastoris membranes suggests that 80% of the NADH:DQ oxidoreductase activity is due to complex I. In contrast, for P. angusta the rate with dNADH is only 10% of that with NADH, indicating that one or more alternative NADH dehydrogenases contribute significantly. There is little difference, in either case, between the rates of dNADH and NADH:HAR oxidoreduction, consistent with previous suggestions that HAR does not react with the alternative enzymes [START_REF] Kerscher | Yarrowia lipolytica, a yeast genetic system to study mitochondrial complex I[END_REF]. dNADH:FeCN oxidoreduction is significantly faster than dNADH:HAR oxidoreduction in both species, suggesting that FeCN reduction by the Pichia complexes I is much faster than HAR reduction. Furthermore, additional FeCN reductases may be present, as suggested by comparison of the dNADH and NADH:FeCN oxidoreductase activities. Note that the concentrations of FeCN and HAR used were chosen because they give close to maximal rates for complex I from B. taurus; an extensive investigation of the kinetics of the two Pichia enzymes has not been undertaken so far. Second, NADH:DQ and dNADH:DQ oxidoreduction by the P. pastoris membranes is 85 and 90% sensitive to the complex I inhibitor piericidin A, consistent with the results described above; the weaker inhibition from rotenone suggests that it is a relatively poor inhibitor in this case. NADH:DQ oxidoreduction by P. angusta is not inhibited strongly by either compound, A c c e p t e d M a n u s c r i p t 7 although, as expected, the dNADH:DQ activity is more strongly inhibited. Taken together, the results in Table 1 indicate that only 10-20% of the NADH:DQ oxidoreductase activity from the P. angusta membranes is due to complex I. Interestingly, dNADH:HAR oxidoreduction by the P. angusta membranes was optimised by harvesting the cells during stationary phase, and by growing them under high aeration. Similar observations were made previously for P. jadinii (also known as Torulopsis utilis or Candida utilis) [START_REF] Katz | Aquisition and loss of rotenone sensitivity in Torulopsis utilis[END_REF][START_REF] Grossman | Reduced nicotinamide adenine dinucleotide dehydrogenase, piericidin sensitivity, and site 1 phosphorylation in different growth phases of Candida utilis[END_REF] and N. crassa [START_REF] Schwitzguébel | Properties of mitochondria as a function of the growth stages of Neurospora crassa[END_REF]. In particular, mitochondria from P. jadinii cells harvested in stationary phase displayed piericidin A sensitive NADH oxidation and the complex I EPR signals N1b and N2, but mitochondria from cells harvested in exponential phase did not.

Purification and confirmation of the complexes I from P. pastoris and P. angusta dNADH:HAR oxidoreductase activities were relied upon to guide the development of purification protocols for the two complexes I (see Figure 2). Methods for isolating the complexes I were based on the established protocol for B. taurus complex I: solubilization with DDM, anion exchange chromatography, and size exclusion chromatography [START_REF] Sharpley | Interactions between phospholipids and NADH:ubiquinone oxidoreductase (complex I) from bovine mitochondria[END_REF]. Passing the solubilized membranes through a MacroCap cation exchange column, prior to application to the anion exchange column, improved the efficiency of the anion exchange step, particularly for P. angusta. For P. pastoris, dNADH:HAR oxidoreduction was observed in only one of the major peaks eluting from the anion exchange column (270 -300 mM NaCl). For P. angusta, dNADH:HAR oxidoreductase activity eluted in two major peaks, at 50 to 120 mM NaCl, and 220 to 270 mM NaCl. Only the latter peak catalyzed dNADH:DQ oxidoreduction, and the results below confirm it was complex I. Typically, 1 g of wet cells yielded 12.7 μg of P. pastoris complex I, or 7.6 μg of P. angusta complex I. In comparison, Y. lipolytica has been reported to yield 30 μg per gram of wet cells by conventional methods [START_REF] Djafarzadeh | Biophysical and structural characterization of proton-translocating NADHdehydrogenase (complex I) from the strictly aerobic yeast Yarrowia lipolytica[END_REF] or 50 -100 μg by affinity chromatography [START_REF] Kashani-Poor | Efficient large scale purification of his-tagged proton translocating NADH:ubiquinone oxidoreductase (complex I) from the strictly aerobic yeast Yarrowia lipolytica[END_REF].

During analytical size exclusion chromatography both Pichia complexes I eluted as single, symmetrical peaks at 11.9 ± 0.2 mL (P. pastoris) or 12.1 ± 0.1 mL (P. angusta). The well characterized complexes from B. taurus and Y. lipolytica eluted at 11.8 and 11.9 mL, respectively. Their protein molecular masses of 980 kDa [START_REF] Carroll | Bovine complex I is a complex of forty-five different subunits[END_REF] and 940 kDa [START_REF] Morgner | Subunit mass fingerprinting of mitochondrial complex I[END_REF], respectively, were included in a molecular mass calibration to estimate masses for the P. pastoris and P. angusta enzymes of 920 and 890 kDa, respectively. The FMN and iron contents of the purified enzymes were 1.0 ± 0.1 FMN and 33.2 ± 3.6 Fe per P. pastoris complex I, and 1.1 ± 0.2 FMN and 33.4 ± 4.5 Fe per P. angusta complex I, using these estimated molecular masses.

The EPR spectra of both the P. pastoris and P. angusta enzymes (see Figure 3), are typical complex I spectra, and confirm, unambiguously, the identity of the purified enzyme [START_REF] Ohnishi | Iron-sulphur clusters/semiquinones in complex I[END_REF]. Comparison with the spectra from B. taurus complex I [START_REF] Ohnishi | Iron-sulphur clusters/semiquinones in complex I[END_REF][START_REF] Reda | Reduction of the iron-sulfur clusters in mitochondrial NADH:ubiquinone oxidoreductase (complex I) by Eu II -DTPA, a very low potential reductant[END_REF] is straightforward, and four signals (N1b, N2, N3 and N4) are immediately identified by their similar g-values. The N5 signal, observed in complex I from B. taurus and Y. lipolytica [START_REF] Djafarzadeh | Biophysical and structural characterization of proton-translocating NADHdehydrogenase (complex I) from the strictly aerobic yeast Yarrowia lipolytica[END_REF], and the N1a signal, observed in complex I from E. coli [START_REF] Uhlmann | EPR signals assigned to Fe/S cluster N1c of the Escherichia coli NADH:ubiquinone oxidoreductase derive from cluster N1a[END_REF], are not observed. The slowly relaxing N2 spectrum, assigned to the [4Fe-4S] cluster in NUKM (PSST) 1 , is evident at around 12 K, with g z -values matching those from the B. taurus and Y. lipolytica enzymes (g z = 2.06 for both P. pastoris and P. angusta). At 40 K the N1b spectrum, from the [2Fe-2S] cluster in NUAM (75 kDa), is observed, again with g z -values matching those from B. taurus and Y. lipolytica complex I (g z = 2.03 for both P. pastoris and P angusta). At lower temperatures (most clearly 7 and 9 K) two further [4Fe-4S] spectra are observed. The g x value for N3, assigned to the [4Fe-4S] cluster in NUBM (51 1 We name the subunits according to their SwissProt codes, and present the names for the B. taurus homologues alongside where appropriate. A c c e p t e d M a n u s c r i p t kDa), is 1.86 for both P. pastoris and P. angusta (the g z signal cannot be distinguished). The origin of N4 is controversial [START_REF] Yakovlev | Reevaluating the relationship between EPR spectra and enzyme structure for the iron-sulfur clusters in NADH:quinone oxidoreductase[END_REF][START_REF] Ohnishi | Were there any misassignments among iron-sulfur clusters N4, N5 and N6b in NADH-quinone oxidoreductase (complex I)[END_REF] but the g z and g x signals of 2.11 and 1.89 (in both cases) match closely to those from Y. lipolytica and B. taurus complex I.

Catalytic activities of the purified enzymes

Table 2 summarises the catalytic properties of the complexes I from P. pastoris and P. angusta, and compares them with those from Y. lipolytica and B. taurus (measured in this study). All the rates of NADH:HAR oxidoreduction are similar, but the rates of NADH:FeCN oxidoreduction are significantly higher for the two Pichia enzymes. Note that our reported values refer to only a single condition, so they are only an indication of the relative catalytic activities of the enzymes, and do not allow the cause of the variation to be identified. Using the structure of the hydrophilic domain of T. thermophilus complex I as a template, sequence comparisons for the protein around the NADH binding site in B. taurus, Y. lipolytica, and P. angusta have not provided any insight. The rates of NADH:DQ oxidoreduction are similar in all cases, except for the P. pastoris enzyme, which displays a markedly higher rate. Note that the highest published values for Y. lipolytica (6-7 μmol min -1 mg -1 [START_REF] Dröse | Full recovery of the NADH:ubiquinone activity of complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica by the addition of phospholipids[END_REF]) exceed those achieved here, but are comparable to our values from P. pastoris. Interestingly, all four enzymes are close to completely inhibited by 2 M piericidin A, but the three yeast enzymes are much less effectively inhibited by rotenone than the B. taurus enzyme (see below).

The markedly more active P. pastoris complex I could be reconstituted into liposomes, using methods based on those for Y. lipolytica [START_REF] Dröse | Proton pumping by complex I from Yarrowia lipolytica reconstituted into proteoliposomes[END_REF] (see Experimental Methods), to provide well coupled proteoliposomes, typically with respiratory control ratios (RCR, the ratio of the rates of NADH oxidation in the presence and absence of an uncoupler) of 2. Proteoliposomes formed from the less active P. angusta enzyme did not show any coupling (RCR = 1). Figure 4 shows typical traces monitoring the fluorescence of ACMA, a measure of ΔpH across the liposomal membrane [START_REF] Dufour | Active proton uptake in lipid vesicles reconstituted with the purified yeast plasma membrane ATPase[END_REF], for reconstituted P. pastoris complex I. In the presence of valinomycin (so that potassium ion transport collapses Δψ and facilitates ΔpH formation) the ACMA fluorescence is rapidly quenched when catalysis is initiated. It is re-established upon the addition of a proton ionophore, FCCP, to dissipate the proton motive force, or an inhibitor (piericidin A) to prevent further proton translocation. The results shown in Figure 4 are strong evidence for proton translocation by complex I from P. pastoris.

Rates of reactive oxygen species (ROS) generation by the complexes I from both Pichia species were measured as H 2 O 2 (detected using Amplex Red) or superoxide (detected using acetylated cytochrome c), and verified by comparison with independent measurements of the rates of NADH oxidation (see Table 2). Within experimental error, the rates of NADH oxidation matched the rates of H 2 O 2 production in a 1:1 ratio, validating both measurements. In both cases, the rates of superoxide formation (one electron) are close to twice the rates of H 2 O 2 formation (two electrons), indicating that the enzymes from P. pastoris and P. angusta, like the enzyme from B. taurus [START_REF] Kussmaul | The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria[END_REF], produce ROS predominantly as superoxide. Table 2 shows that complex I from Y. lipolytica falls into the same class, although the rate of superoxide production measured here (90 nmol min -1 mg -1 , validated using the rate of NADH oxidation) is significantly higher than reported previously (18 nmoles min -1 mg -1 [START_REF] Galkin | Superoxide radical formation by pure complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica[END_REF]). The cause of this discrepancy is not clear. Table 2 shows that the three yeast complexes I produce significantly more ROS than the B. taurus enzyme.

The protein composition of the complexes I from P. pastoris and P. angusta

Figure 5 shows an SDS PAGE analysis of the protein composition of the complexes I from B. taurus, Y. lipolytica, P. pastoris and P. angusta. The overall similarity of the pattern, particularly for the high molecular weight subunits, is striking, though differences in mass and/or migration become apparent lower down the gel. Bands corresponding to the NUAM (75 kDa), NUBM (51 kDa), NUCM (49 kDa), The complexes I from B. taurus and Y. lipolytica have already been characterized extensively, using publically available sequence data, but there is little available for P. pastoris and P. angusta. In order to identify as many subunits as possible in the two Pichia complexes, all the visible protein bands were excised from the gels, digested with trypsin, and subjected to MALDI-TOF and tandem MS analysis. For both P. pastoris and P. angusta the observed peptide masses (from MALDI-TOF MS) were searched (for matches within 70 ppm) against the NCBI non-redundant sequence database, and against an in-house database of expected complex I subunits from P. angusta (derived from the NCBI genome survey sequences by comparison with known sequences from other species). Then, the closest matches to the tandem MS data (within 120 ppm) were re-evaluated using error tolerance searches, allowing for modifications and amino acid substitutions between species. A summary of the data assigned is presented in Supplementary Table S1. All seven hydrophilic core subunits, and two supernumerary subunits, were identified in the enzyme from P. pastoris. Six of the hydrophilic core subunits (the exception being NUKM (PSST)), and nine supernumerary subunits were identified in the enzyme from P. angusta. The subunits of P. pastoris complex I were identified using the similarity of observed peptide masses to predicted peptide masses from other fungal species, including P. stipitis (NUAM (75 kDa), NUBM (51 kDa), NUCM (49 kDa), NUEM (39 kDa), NUGM (30 kDa), NUHM (24 kDa), NUYM (18 kDa), NUIM (TYKY) and NUKM (PSST)), Lodderomyces elongisporus (NUAM), Debaryomyces hansenii (NUBM, NUCM, and NUHM), Y. lipolytica (NUBM, NUCM, and NUIM), P. guilleromondii (NUEM), N. crassa (NUKM), Aspergillus fumigatus (NUKM), and Candida albicans (NUYM). These matches confirm that P. pastoris complex I contains a number of canonical complex I subunits, but the lack of complete sequences for them precludes further discussion about its subunit composition and homology to other species. The genome sequence for P. pastoris is available commercially, and the composition of its complex I will be described in detail in a future publication. In the same way, masses from five subunits in P. angusta complex I were found to be similar to those in other species: P. stipitis (NUAM (75 kDa), and NUCM (49 kDa)), N. crassa (NUBM (51 kDa)), D. hansenii (NUCM) Dekkera bruxellensis (NUHM (24 kDa)), Magnaporthe grisea (NUHM), and A. niger (NB6M (B16.6)). For P. angusta there are several full or partial sequences available publically as genome survey sequences, and they allowed the identification of eleven complex I subunit sequences (NUBM (51 kDa), NUGM (30 kDa), NUIM (TYKY), NUZM, NIAM (ASHI), N7BM (B17.2), NUKM (B14.7), NUFM (B13), NUMM (13 kDa), NIMM (MWFE) and NIDM (PDSW)). A comparison of observed peptide masses from P. pastoris against these P. angusta sequences only provided a match to one complex I subunit (NUIM (TYKY)).

Discussion

The presence of complex I in Pichia species

The data presented here show that both P. pastoris and P. angusta assemble a piericidin A sensitive, multisubunit NADH:ubiquinone oxidoreductase in their mitochondria, with closely similar catalytic and spectroscopic properties to the enzymes from B. taurus and Y. lipolytica. A number of the core subunits of complex I have been identified in both Pichia species, along with several supernumerary subunits, which vary between kingdoms, subkingdoms and between genera and species also [START_REF] Gabaldón | Tracing the evolution of a large protein complex in the eukaryotes, NADH:ubiquinone oxidoreductase (complex I)[END_REF]. Our data and preliminary sequence comparisons suggest that, within the species included in Figure 1, A c c e p t e d M a n u s c r i p t 10 complex I from P. pastoris is most closely related to the enzyme from P. stipitis, whereas P. angusta complex I is most closely related to the enzymes from Y. lipolytica, L. elongisporus, P. stipitis, P. guilleromondii and D. hansenii (but not to any one of them in particular).

The presence of the complex I ND genes in the mitochondrial genomes of many fungi (including Pichia species) [START_REF] Nosek | NADH dehydrogenase subunit genes in the mitochondrial DNA of yeasts[END_REF][START_REF] Lavín | Comparative genomics of the oxidative phosphorylation system in fungi[END_REF], suggests that most (if not all) obligate aerobic fungi are able to express complex I. Those species capable of facultative anaerobic fermentation encode only the single subunit enzyme (see Figure 1). Without a larger set of sequences for comparison, it is difficult to speculate further on the evolutionary development of complex I in some species, and its loss in others. However, intriguing correlations can be identified within the species in Figure 1. Only species that encode complex I encode the alternative, single subunit, non-proton pumping ubiquinol oxidase [START_REF] Lavín | Comparative genomics of the oxidative phosphorylation system in fungi[END_REF]: if complexes III IV are substituted by the alternative oxidase, complex I is retained in order to maintain the proton motive force (or vice versa). Only species in the subphylum pezizomycota encode the complex I supernumerary subunit NURM [START_REF] Lavín | Comparative genomics of the oxidative phosphorylation system in fungi[END_REF], and they are the only species that encode the three subunit, proton pumping transhydrogenase also. In N. crassa NURM is a 17.8 kDa protein containing one transmembrane helix, with no known homology to any other protein.

The catalytic activities of the complexes I from Pichia species

The preparation of a highly pure eukaryotic complex I with native NADH:ubiquinone oxidoreductase activity has proved challenging, particularly for the mammalian complexes. Recently, by including phospholipids in the preparation, highly pure complex I has been prepared from B. taurus mitochondria with NADH:DQ oxidoreductase activity up to 4 mol NADH min -1 mg -1 [START_REF] Sharpley | Interactions between phospholipids and NADH:ubiquinone oxidoreductase (complex I) from bovine mitochondria[END_REF]. For Y. lipolytica, preparations with an activity of ~2.8 mol min -1 mg -1 were obtained by conventional purification [START_REF] Djafarzadeh | Biophysical and structural characterization of proton-translocating NADHdehydrogenase (complex I) from the strictly aerobic yeast Yarrowia lipolytica[END_REF], but a histidine tag on the NUGM (30 kDa) subunit increased the activity to 6 -7 mol min -1 mg -1 [START_REF] Dröse | Full recovery of the NADH:ubiquinone activity of complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica by the addition of phospholipids[END_REF].

Here, conventional methods were used to purify the two Pichia complexes I. Initially, it was hoped that the P. angusta enzyme would be more stable because of the organism's thermotolerance [START_REF] Van Uden | Temperature functions of thermal death in yeasts and their relation to the maximum temperature for growth[END_REF], but the P. pastoris enzyme gave a significantly higher activity than that of P. angusta (6.3 and 4.0 mol NADH min -1 mg -1 , respectively). It is possible that phospholipid content is key to understanding the relative activities. The B. taurus enzyme is readily depleted of phospholipids during preparation [START_REF] Sharpley | Interactions between phospholipids and NADH:ubiquinone oxidoreductase (complex I) from bovine mitochondria[END_REF]. The P. angusta enzyme requires more exogenous phospholipids in activity assays than the P. pastoris enzyme, to attain its highest activity, and it cannot be incorporated properly into liposomes. It is possible that this stems from its lower relative abundance in the membranesit must be washed more extensively on the anion exchange column, a procedure which is known to 'strip' the B. taurus enzyme [START_REF] Sharpley | Interactions between phospholipids and NADH:ubiquinone oxidoreductase (complex I) from bovine mitochondria[END_REF]. Finally, variations in the DQ reductase activity are not reflected in any other activity measurements (see Table 2); in particular, NADH:HAR oxidoreduction is essentially the same in all four cases. Previously, a study of the complexes I from B. taurus and P. denitrificans also found that, while FeCN was reduced relatively slowly by the prokaryotic enzyme, the HAR reduction rates were similar [START_REF] Zickermann | The NADH oxidation domain of complex I: do bacterial and mitochondrial enzymes catalyze ferricyanide reduction similarly ?[END_REF].

'Tests' which distinguish complex I from other NADH dehydrogenases, and inhibitor sensitivity

It is widely assumed that dNADH reacts only with complex I, not with alternative NADH dehydrogenases, although this fact has only been demonstrated in E. coli [START_REF] Matsushita | NADH-ubiquinone oxidoreductases of the Escherichia coli aerobic respiratory chain[END_REF]. All the complexes I tested here (Table 2) react at comparable rates with dNADH and NADH. However, if the dNADH:HAR and dNADH:DQ oxidoreductase activities in the membranes are only from complex I, then the ratio between them should be conserved throughout the preparation. In fact, the ratio (HAR / DQ) increases from 1.1 (P. pastoris) and 5.0 (P. angusta) in the membranes to 9.8 and 21.1, respectively. Because the specific dNADH:HAR oxidoreductase activity is unlikely to vary significantly, the results indicate at least one additional enzyme in the membrane preparations catalyses Consequently, dNADH may differentiate less clearly between complex I and alternative NADH dehydrogenases than commonly supposed. It is widely assumed that only complex I catalyses NADH:HAR oxidoreduction also (see, for example [START_REF] Kerscher | Yarrowia lipolytica, a yeast genetic system to study mitochondrial complex I[END_REF]) but we have been unable to find any published data to support this fact. During the preparation of P. angusta complex I, two fractions with NADH:HAR oxidoreductase activity eluted from the anion exchange column, but the second catalysed dNADH:DQ oxidoreduction. Although the protein composition of the first peak was complex, SDS PAGE, mass spectrometry and Western blotting analyses detected no evidence for complex I or any portion it, suggesting that this second 'test' for complex I is also not definitive.

Table 2 shows that all the NADH:DQ oxidoreductase activities tested are strongly inhibited by piericidin A, but rotenone does not inhibit the three yeast enzymes as strongly as the B. taurus enzyme. This observation may explain why rotenone-sensitive NADH oxidation was not observed previously in P. pastoris mitochondria [START_REF] González-Barroso | Isolation and bioenergetic characterization of mitochondria from Pichia pastoris[END_REF]. Studies of the inhibition of the activity of P. pastoris complex I over a range of rotenone concentrations showed that almost complete inhibition can be observed, but only at much higher concentrations (~96% in 50 μM rotenone). Relatively weak inhibition of Y. lipolytica complex I was observed previously also [START_REF] Galkin | Superoxide radical formation by pure complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica[END_REF]. Thus, rotenone is regarded as an archetypal complex I inhibitor simply because of the predominance of the B. taurus enzyme in early studies.

The production of reactive oxygen species

Previously, we described a mechanism for ROS production by the reduced flavin in complex I from B. taurus, and demonstrated the same mechanism in complex I from E. coli [START_REF] Kussmaul | The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria[END_REF]44]. Here, we compare ROS production by the complexes I from P. pastoris, P. angusta and Y. lipolytica with that by the B. taurus enzyme. Values for all three yeast species were significantly higher than those from B. taurus, and those for the two Pichia species were the highest. This is particularly interesting because B. taurus and E. coli complex I exhibit similar rates [44]. When the two Pichia species are grown on methanol the induction of alcohol oxidases results in significant H 2 O 2 production, which is metabolised by catalase and cytochrome c peroxidase [START_REF] Gellissen | New yeast expression platforms based on methylotrophic Hansenula polymorpha and Pichia pastoris and on dimorphic Arxula adeninivorans and Yarrowia lipolytica -a comparison[END_REF][START_REF] Duff | Metabolism of hydrogen peroxide by Pichia pastoris[END_REF]. The additional contribution from complex I may not be significant in this context. Furthermore, all four eukaryotic enzymes produce predominantly superoxide, not H 2 O 2 . This observation maintains the correlation between superoxide to H 2 O 2 ratio and the reduction of cluster N1a described previously [44]: the N1a EPR signal from the [2Fe-2S] cluster in NUHM (24 kDa) is not observed in any of the eukaryotic enzymes described here (see Figure 3). Therefore, the cluster is predominantly oxidized in the presence of NADH, and is, in principle, available to transiently accept the second electron from the fully reduced flavin, promoting the escape of nascent superoxide from the active site. A c c e p t e d M a n u s c r i p t S1).

Biochemical

  Journal Immediate Publication. Published on 22 May 2009 as manuscript BJ20090492 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090492 A c c e p t e d M a n u s c r i p t 4

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  Biochemical Journal Immediate Publication. Published on 22 May 2009 as manuscript BJ20090492 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090492

Biochemical

  Journal Immediate Publication. Published on 22 May 2009 as manuscript BJ20090492 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090492

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  Journal Immediate Publication. Published on 22 May 2009 as manuscript BJ20090492 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090492 A c c e p t e d M a n u s c r i p t 9 NUDM/NUEM (42/39 kDa) and NUGM (30 kDa) subunits are clearly visible in all cases (NUBM and NUCM comigrate in Y. lipolytica), but one of the bands present in the mammalian enzyme, probably the 42 kDa subunit, does not appear in the lanes from the three yeast species.

Biochemical

  Journal Immediate Publication. Published on 22 May 2009 as manuscript BJ20090492 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090492

Biochemical

  Journal Immediate Publication. Published on 22 May 2009 as manuscript BJ20090492 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090492 A c c e p t e d M a n u s c r i p t dNADH:DQ oxidoreduction also (or a significant portion of the dNADH:DQ activity has been lost).

A c c e

  p t e d M a n u s c r i p t Biochemical Journal Immediate Publication. Published on 22 May 2009 as manuscript BJ20090492 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090492 A c c e p t e d M a n u s c r i p t Biochemical Journal Immediate Publication. Published on 22 May 2009 as manuscript BJ20090492 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090492 A c c e p t e d M a n u s c r i p t Tables

Figure 1 .

 1 Figure1. Phylogenetic tree showing the distribution of complex I within the subkingdom of dikarya. Species in bold have complex I subunits encoded in their nuclear and mitochondrial genomes, and they encode an alternative oxidase and at least one alternative, single subunit NADH:ubiquinone oxidoreductase. Species in grey are able to anaerobically ferment, and they only encode alternative NADH:ubiquinone oxidoreductases. (*) denotes the presence of the genes for the three subunit mitochondrial transhydrogenase, and the NURM gene, which has been observed previously only in N. crassa complex I. The phylogenetic tree was adapted from[START_REF] Kriventseva | OrthoDB: the hierarchical catalog of eukaryotic orthologs[END_REF].

Figure 2 .

 2 Figure 2. Variation of the specific dNADH:HAR oxidoreductase activity during preparation of the complexes I from P. pastoris and P. angusta. Memb., mitochondrial membranes; Sol. Memb., membranes following solubilization in DDM; QS, pooled fractions from the Q-sepharose ion exchange chromatography step; SE2, pooled fractions from the second size exclusion chromatography step.

Figure 3 .

 3 Figure 3. EPR spectra showing signals from the FeS clusters in the complexes I from P. pastoris and P. angusta reduced by NADH. Spectra are presented at different temperatures because each cluster signal is only observed over a limited range. The signals have been identified, and labeled, by matching their g-values and temperature dependence to those from the well characterized complexes I from B. taurus and Y. lipolytica. Conditions: 15 mg mL -1 complex I, 5 mM NADH, 20 mM MOPS pH 7.5, 150 mM NaCl and 0.02% DDM; microwave power 1 mW, conversion time 81.92 ms, time constant 20.48 ms, modulation amplitude 10 G, microwave frequency ~9.38 MHz.

Figure 4 .

 4 Figure 4. Proton pumping by complex I from P. pastoris reconstituted into liposomes. The formation of ΔpH was monitored by the fluorescence of ACMA, which is quenched upon accumulation into the lumen of the liposomes. A small decrease in fluorescence, not related to the formation of ΔpH, is observed upon the addition of DQ. Significant quenching (ΔpH formation) is observed in the presence of both NADH and DQ, and is dissipated by the presence of either the proton ionophore FCCP, or the complex I inhibitor piericidin A. The potassium ionophore, valinomycin, is present to collapse Δ and allow ΔpH formation. The traces all begin at zero fluorescence intensity and have been offset from one another along the y-axis. Conditions: 100 M NADH, 100 M DQ, 500 nM ACMA, 5 M valinomycin, 20 mM MOPS pH 7.5 and 50 mM KCl, excitation 430 nm, emission 475 nm.

Figure 5 .

 5 Figure 5. Analysis of the subunit composition of the complexes I from P. pastoris and P. angusta, and comparison with the enzymes from B. taurus and Y. lipolytica. SDS-PAGE analysis was performed on a 12 -22% gradient gel, and the bands excised and analyzed by peptide mass fingerprinting and tandem mass spectrometry. Peptides were identified using publicly available genome sequences (see Supplementary TableS1).

Biochemical

  Journal Immediate Publication. Published on 22 May 2009 as manuscript BJ20090492 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090492 A c c e p t e d M a n u s c r i p t

Figure 1 Biochemical

 1 Figure 1

Figure 2 Biochemical

 2 Figure 2

Figure 3 Biochemical

 3 Figure 3

Figure 4 Biochemical

 4 Figure 4

Figure 5 Biochemical

 5 Figure 5

Table 1 . Specific activities of the mitochondrial membranes from P. pastoris and P. angusta.

 1

	Catalytic activity
	(μmol NADH min -1 mg -1 )
	P. pastoris	P. angusta

Table 2 . Specific activities of the complexes I purified from P. pastoris and P. angusta, and comparison with the enzymes from Y. lipolytica and B. taurus.

 2 Catalytic activity (μmol NADH min -1 mg -1 )

		P. pastoris	P. angusta	Y. lipolytica	B. taurus
	NADH:FeCN oxidoreduction	302.6 ± 22.9	248.9 ± 22.9	140.8 ± 3.1	111.1 ± 8.3
	NADH:HAR oxidoreduction	70.5 ± 9.4	63.4 ± 3.2	68.8 ± 13	72.5 ± 6.8
	dNADH:HAR oxidoreduction	68.6 ± 7.4	69.0 ± 5.3	-	-
	NADH:DQ oxidoreduction	6.27 ± 0.38	4.00 ± 0.04	3.80 ± 0.55	3.82 ± 0.17
	dNADH:DQ oxidoreduction	7.00 ± 0.86	3.27 ± 0.26	3.55 ± 0.40	3.62 ± 0.50
	dNADH:DQ oxidoreduction +	0.37 ± 0.30	0.06 ± 0.01	0.28 ± 0.02	0.24 ± 0.11
	piericidin A	(95%)	(98%)	(92%)	(93%)
	dNADH:DQ oxidoreduction +	2.02 ± 0.53	0.56 ± 0.11	1.31 ± 0.67	0.14 ± 0.01
	rotenone	(71%)	(83%)	(63%)	(96%)
		H 2 O 2 or superoxide production (nmol min -1 mg -1 )
	H 2 O 2	63.3 ± 4.9	76.3 ± 14.7	42.5 ± 4.4	23.6 ± 5.6
	superoxide	105.0 ± 11.6	127.2 ± 7.5	90.3 ± 2.8	41.3 ± 1.8
	NADH:O 2 oxidoreduction	55.1 ± 5.8	68.6 ± 15.6	41.3 ± 1.8	20.1 ± 1.7
	Conditions: 20 mM Tris-HCl pH 7.5, 100 μM NADH, 100 μM DQ, 3.5 mM HAR, 2 mM FeCN, 5 μM
	rotenone, 2 μM piericidin A, 32 °C. H 2 O 2 measurements: 20 mM Tris-HCl pH 7.5, 30 μM NADH, 10
	μM Amplex Red, 2 U ml

-1 horse radish peroxidase. Superoxide measurements: 20 mM Tris-HCl pH 7.5, 30 μM NADH, 50 μM partially acetylated cytochrome c. Values for Y. lipolytica and B. taurus were measured alongside those for P. pastoris and P. angusta for direct comparison. Biochemical Journal Immediate Publication. Published on 22 May 2009 as manuscript BJ20090492 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090492

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Acknowledgements

We thank Professor Ulrich Brandt (Frankfurt) for providing the strain of Y. lipolytica, Dr. Ian M. Fearnley (MRC) for advice on mass spectrometry, and Martin King (MRC) for providing B. taurus complex I. This work was supported by The Medical Research Council.


Oliver Schieweck

Markus Damme

Bernd Schröder

Andrej Hasilik

Bernhard Schmidt

Torben Lübke

NCU-G1 is a highly glycosylated integral membrane protein of the lysosome

Keywords: NCL, neuronal ceroid lipofuscinosis; MEF, mouse embryonic fibroblasts;

Until recently, a modest number of approximately 40 lysosomal membrane proteins had been identified and even fewer were characterized in their function. In a proteomic study, using lysosomal membranes from human placenta we identified several candidate lysosomal membrane proteins and proved the lysosomal localization of two of them. Here, we demonstrate the lysosomal localization of the mouse ortholog of the human C1orf85 protein that had been termed kidney predominant protein NCU-G1 (Acc.No: AB027141). NCU-G1 encodes a 404 amino acid protein with a calculated molecular mass of 39 kDa. The bioinformatics analysis of its amino acid sequence suggests it is a type I transmembrane protein containing a single tyrosine-based consensus lysosomal sorting motif at position 400 within the 12-residue C-terminal tail. Its lysosomal localization was confirmed using immunofluorescence with a C-terminally His-tagged NCU-G1 and the lysosomal marker LAMP-1 as a reference, and by subcellular fractionation of mouse liver after a tyloxapol-induced density shift of the lysosomal fraction using an anti-NCU-G1 antiserum. In transiently transfected HT1080 and HeLa cells the His-tagged NCU-G1 was detected in two molecular forms with apparent protein sizes of 70 and 80 kDa, and in mouse liver the endogenous wild type NCU-G1 was detected as a 75 kDa protein. The remarkable difference between the apparent and the calculated molecular masses of NCU-G1 was shown by digesting the protein with N-glycosidase F to be due to an extensive glycosylation. The lysosomal localization was impaired by mutational replacement of an alanine for the tyrosine residue within the putative sorting motif.

Introduction

Lysosomes are membrane-limited organelles of eukaryotic cells that degrade intracellular and extracellular materials they obtain by autophagy and endocytosis, respectively. Within the lysosomes, more than 60 acid hydrolases and associated cofactors degrade the imported macromolecules [START_REF] Lubke | Proteomics of the lysosome[END_REF]. The products of this breakdown are exported into the cytoplasm where they are metabolized. The metabolite export through the lysosomal membrane is less understood than the macromolecular degradation within the lysosomal matrix. Although, the lysosomal v-ATPase, which is required for the acidification of the matrix, is well characterized, the number of transporters is less than expected from that of the solutes which must be released from lysosomes [START_REF] Saftig | Lysosomes. Landes Bioscience/Eurekah.com[END_REF][START_REF] Sagne | Molecular physiology and pathophysiology of lysosomal membrane transporters[END_REF]. The importance of these transporters is underscored by the occurrence of lysosomal storage diseases [4]. Thus, a loss of the cystine transporter cystinosin results in cystinosis [5], that of the sialic acid transporter sialin manifests as sialic acid storage diseases [START_REF] Verheijen | A new gene, encoding an anion transporter, is mutated in sialic acid storage diseases[END_REF], of mucolipin as mucolipidosis IV [START_REF] Bargal | Identification of the gene causing mucolipidosis type IV[END_REF][START_REF] Sun | Mucolipidosis type IV is caused by mutations in a gene encoding a novel transient receptor potential channel[END_REF][START_REF] Bassi | Cloning of the gene encoding a novel integral membrane protein, mucolipidin-and identification of the two major founder mutations causing mucolipidosis type IV[END_REF]. Recently, the lysosomal cobalamin transporter has been identified, which is mutated in cblF defect of vitamin B 12 metabolism [START_REF] Rutsch | Identification of a putative lysosomal cobalamin exporter altered in the cblF defect of vitamin B12 metabolism[END_REF]. Inherited defects of the prominent lysosomeassociated membrane protein 2 (LAMP-2) causes Danon disease which is characterized by a (cardio)myopathy due to an abnormal accumulation of phagosomes and autophagosomes [START_REF] Tanaka | Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficient mice[END_REF].

The vast majority of soluble lysosomal proteins are transported from the trans-Golgi network (TGN) to endosomes after receiving a mannose 6-phosphate (M6P) tag at their N-glycans that is recognized by specific mannose 6-phosphate receptors (MPR) [START_REF] Kornfeld | The biogenesis of lysosomes[END_REF]. As an exception, the lysosomal soluble hydrolase -glucocerebrosidase is directed to the lysosomes by a piggy-back transport along with the lysosomal integral membrane protein type 2 (LIMP-2) [START_REF] Reczek | LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase[END_REF]. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

The lysosomal sorting of most integral lysosomal membrane proteins is mediated by short amino acid sequence motifs in their cytosolic loops and tails. Two types of lysosomal sorting motifs are known On the one hand, the tyrosine-based sorting signals NPXY and YXX and, on the other hand, the dileucine-based sorting signals [DE]XXXL [LI] and DXXLL [START_REF] Bonifacino | Signals for sorting of transmembrane proteins to endosomes and lysosomes[END_REF][START_REF] Braulke | Sorting of lysosomal proteins[END_REF]. These motifs resemble endocytic and trans-Golgi network (TGN) sorting signals, however, they differ from the latter by their close proximity (mostly 6-13 amino acids) to the transmembrane segment [START_REF] Geisler | Leucine-based receptor sorting motifs are dependent on the spacing relative to the plasma membrane[END_REF][START_REF] Rohrer | The targeting of Lamp1 to lysosomes is dependent on the spacing of its cytoplasmic tail tyrosine sorting motif relative to the membrane[END_REF]. They are recognized by the heterotetrameric adaptor proteins AP-1 through -4 or by the multidomain Golgi-localized, gamma-ear-containing, ADP ribosylation factor-binding proteins (GGA) (reviewed by [START_REF] Bonifacino | Signals for sorting of transmembrane proteins to endosomes and lysosomes[END_REF][START_REF] Braulke | Sorting of lysosomal proteins[END_REF]).

Recently, several novel lysosomal membrane proteins were identified using different strategies such as subcellular fractionation (p40, [START_REF] Boonen | Intracellular localization of p40, a protein identified in a preparation of lysosomal membranes[END_REF]), genetic approaches (TMEM74 [START_REF] Yu | TMEM74, a lysosome and autophagosome protein, regulates autophagy[END_REF], TMEM76 [START_REF] Hrebicek | Mutations in TMEM76* cause mucopolysaccharidosis IIIC (Sanfilippo C syndrome)[END_REF], MFSD8 [START_REF] Siintola | The novel neuronal ceroid lipofuscinosis gene MFSD8 encodes a putative lysosomal transporter[END_REF]) and proteome studies [START_REF] Fan | Identification of the gene encoding the enzyme deficient in mucopolysaccharidosis IIIC (Sanfilippo disease type C)[END_REF][START_REF] Schroder | Integral and associated lysosomal membrane proteins[END_REF]. Among these, TMEM76 and MFSD8, have been shown to be associated with the lysosomal diseases mucopolysacharidosis IIIC [START_REF] Hrebicek | Mutations in TMEM76* cause mucopolysaccharidosis IIIC (Sanfilippo C syndrome)[END_REF][START_REF] Fan | Identification of the gene encoding the enzyme deficient in mucopolysaccharidosis IIIC (Sanfilippo disease type C)[END_REF] and a late-infantile onset form of neuronal ceroid lipofuscinosis (NCL) [START_REF] Siintola | The novel neuronal ceroid lipofuscinosis gene MFSD8 encodes a putative lysosomal transporter[END_REF]. In a proteomics analysis of lysosomal membranes purified from human placenta we identified 124 lysosomally enriched proteins including 12 novel proteins of unknown function [START_REF] Schroder | Integral and associated lysosomal membrane proteins[END_REF]. Two of these, LOC201931 and LOC51622, were shown to localize mainly to the lysosomal compartment using their YFP-tagged versions and immunofluorescence [START_REF] Schroder | Integral and associated lysosomal membrane proteins[END_REF]. Here, we verify the lysosomal localization of a third candidate called kidney predominant protein NCU-G1 and show that it represents a highly glycosylated integral membrane protein of the lysosomal compartment. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Materials and Methods

Cell lines and cell culture -If not stated otherwise HT1080 cells, HeLa cells and mouse embryonic fibroblasts (MEF) were grown in complete Dulbecco's modified Eagle's medium (DMEM, GIBCO Life Technologies) supplemented with 10% fetal bovine serum (PAN Biotech GmbH), 1% penicillin/streptomycin (GIBCO Life Technologies) and 1% L-glutamine (GIBCO Life Technologies) at 37°C with 5% CO 2 .

Antibodies -Antibodies used for this study include monoclonal anti-RGS-His4-tag and anti-His5-Alexa488 conjugated (Qiagen), anti-porin HL31 (Calbiochem), antiglyceraldehyde 3-phosphate dehydrogenase (Gapdh, Santa Cruz), anti-cathepsin D [START_REF] Pohlmann | The two mannose 6phosphate receptors transport distinct complements of lysosomal proteins[END_REF], anti-human LAMP-2 (H4B4, Developmental Studies Hybridoma Bank, Iowa), anti-mouse LAMP-1 (1D4B; Developmental Studies Hybridoma Bank, Iowa) and antiserine carboxypeptidase, Scpep1 [START_REF] Kollmann | Molecular characterization and gene disruption of mouse lysosomal putative serine carboxypeptidase 1[END_REF], antisera. Horse raddish peroxidase, HRP-and fluorescence-conjugated secondary antibodies were supplied by Dianova and Invitrogen, respectively. A c c e p t e d M a n u s c r i p t

Antibody production

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unintentional mutations. HT1080 cells, HeLa cells and mouse embryonic fibroblasts (MEF) were transfected with Fugene6 reagent (Roche) as recommended by the manufacturer.

Immunofluorescence -MEF or HeLa cells were transiently transfected and cultivated for 24 -36 h. After fixation with methanol, the cells were analysed for immunofluorescence as described before [START_REF] Kollmann | Identification of novel lysosomal matrix proteins by proteome analysis[END_REF] using anti-LAMP-1 (1D4B) and anti-LAMP-2 (H4B4) antibodies. His-tagged NCU-G1 protein was detected with an Alexa488-coupled monoclonal anti-pentaHis antibody (Qiagen). The LAMP antibodies were detected with Alexa546-conjugated goat anti-rat or goat anti-mouse IgG secondary antibodies (Invitrogen). Immunofluorescence images were obtained using a Leica TCS Sp2 AOBS laser scan microscope (Leica Microsystems GmbH, Heidelberg, Germany).

Site-directed mutagenesis of NCU-G1 -We inserted the Y400A mutation into the NCU-G1-RGS-His5 construct using the QuikChange site-directed mutagenesis kit (Stratagene) with the 5'-TATTCTGAGGCCCAGTCCATAAAC-3' forward primer and the 5'-GTTTATGGACTGGGCCTCAGAATA-3' reverse primer according to the manufacturer's instruction.

Northern blot -Northern blot analysis was performed as described before [START_REF] Lubke | Complementation cloning identifies CDG-IIc, a new type of congenital disorders of glycosylation, as a GDP-fucose transporter deficiency[END_REF] with the exception that total mouse RNA from various tissues was separated. NCU-G1 transcripts were detected with the full length NCU-G1 cDNA as a probe. The Northern blot membrane was stripped and re-hybridized with the full length mouse Gapdh cDNA for RNA loading control. were subjected to extraction with either 0.1 M sodium carbonate, pH 11.5, or 1%

Membrane extraction assay

Triton X100 in lysis buffer, for 30 min on ice. Subsequently, the soluble and the solid fractions were separated by centrifugation at 100.000 g and 4 °C for 90 min. The presence of different antigens in the fractions was examined by Western blotting.

Deglycosylation by PNGase F -Cleared lysates from NCU-G1 transiently transfected HT1080 cells were subjected to PNGase F (Roche) treatment as described previously [28].

Lysosome isolation (tritosomes) -Mice were treated by a single injection of 0.75 mg tyloxapol (Triton WR1339; Sigma)/ g body weight 4 d prior to liver withdrawal.

The isolation of tritosomes from tyloxapol-treated and control mice included differential centrifugation and isopycnic centrifugation and resulted in a lysosomeenriched fraction F2 as described before [START_REF] De Duve | Tissue fraction studies. 6. Intracellular distribution patterns of enzymes in rat liver tissue[END_REF]30].

For the continuous sucrose gradient, the "light mitochondria" fraction (L) fraction as described by de Duve et al. [START_REF] De Duve | Tissue fraction studies. 6. Intracellular distribution patterns of enzymes in rat liver tissue[END_REF] was layered onto a pre-formed 15 -30% continuous sucrose gradient and centrifuged at 190.000 g max for 150 min in a Beckman SW 41 Ti rotor. Fractions (1 ml) were collected from the top and analysed by Western blotting. In addition, protein and the activity of -hexosaminidase were determined using photometric assays [START_REF] Koster | Targeted disruption of the M(r) 46,000 mannose 6-phosphate receptor gene in mice results in misrouting of lysosomal proteins[END_REF]. A c c e p t e d M a n u s c r i p t

Bioinformatics analysis

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Results

Bioinformatics analysis of mouse NCU-G1

Human C1orf85 protein, gI 15079485, was identified in a comprehensive proteomics analysis of lysosomal integral and associated membrane proteins from human placenta [START_REF] Schroder | Integral and associated lysosomal membrane proteins[END_REF].

Cloning and expression of NCU-G1

The NCU-G1 cDNA was received in the pCMV-SPORT6 vector from the Deutsche Ressourcenzentrum für Genomforschung GmbH (RZPD) and expressed as a C- polypeptides at molecular weights comparable to the anti-His-tag antibody (Figure 2 A, right panel).

N-glycosylation of NCU-G1

Incubations of HT1080 (Figure 2

NCU-G1 is an integral membrane protein

In order to demonstrate that mouse NCU-G1 is an integral membrane protein, we performed an extraction assay. Homogenates from NCU-G1 transfected and untransfected HeLa cells (50 µg protein) were incubated for 30 min on ice with homogenisation buffer as a control, 0.1 M sodium carbonate (pH 11.5) or 1% Triton X-100. After the incubation, supernatant and pellet fractions were separated by ultracentrifugation at 100.000 g and analysed by immunoblotting for NCU-G1-His with the anti-His antibody and the mitochondrial integral membrane protein porin 31HL using a porin-specific monoclonal antibody (Figure 2 C). In the Western blot analysis, NCU-G1-His was almost completely recovered in the pellet fraction after incubation in the homogenisation buffer. However, NCU-G1-His was partially solubilized in the presence of sodium carbonate and largely solubilized in the presence of Triton X-100 thus resembling the integral membrane protein porin 31HL

(Figure 2 C).

Tissue distribution of NCU-G1

The transcription pattern of NCU-G1 was analysed by using a mouse multi-tissue Northern blot (MTN) containing 10 µg of total RNA derived from various tissues. The MTN was hybridized with the 1215 bp full-length cDNA probe that identified a single NCU-G1 transcript of 1.5 kb widely expressed in most mouse tissues (Figure 3 A). As expected, NCU-G1 (also termed kidney predominant protein) expression was highest in kidney. The signal was significantly less in samples from liver, brain, intestine, testis, spleen, heart and lung. Interestingly, no NCU-G1 transcript could be detected in skeletal muscle.

The Western blot analyses of various tissues with the NCU-G1 antiserum that was raised against the peptide from amino acid 245 to 263 were hampered by a number of probably non-specific signals (data not shown). As shown before, NCU-G1 was detectable in a lysosomally-enriched fraction from mouse liver with an apparent size of 75 kDa (Figure 2 B, lane 5). Furthermore, we loaded the L fraction from tyloxapol-treated and control mice onto a preformed continuous sucrose gradient ranging from 15 -30% and observed a similar tyloxapol-induced shift specific to lysosomes. In the control mouse, NCU-G1, LAMP-1 and the soluble lysosomal protein Scpep1 [START_REF] Kollmann | Molecular characterization and gene disruption of mouse lysosomal putative serine carboxypeptidase 1[END_REF] were collected in the pellet of the sucrose gradient (Figure 5 A). After tyloxapol treatment all three proteins as well as the lysosomal marker -hexosaminidase entered the gradient (Figure 5 B,C). In the control 6% of the total activity of the marker was found in fractions 7 -10, A c c e p t e d M a n u s c r i p t

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whereas, in tyloxapol treated samples this proportion was increased to 47% of total.

These results in combination with the immunofluorescence data suggest clearly that NCU-G1 is a lysosomal or endosomal protein.

Discussion

In a recent proteome analysis of the lysosomal membrane from human placenta, we identified 58 known lysosomal membrane proteins including 17 polypeptides of the v-ATPase and most interestingly twelve novel proteins of so far unknown function that were highly enriched in the lysosomal membrane fraction [START_REF] Schroder | Integral and associated lysosomal membrane proteins[END_REF]. For two of these twelve candidates (LOC201931 and LOC51622), we were able to confirm their lysosomal localization with the help of YFP-tagged variants of the proteins [START_REF] Schroder | Integral and associated lysosomal membrane proteins[END_REF]. Here, we validate the lysosomal localization and provide first insights into the molecular features of the third candidate derived from that particular subproteome analysis called NCU-G1 (C1orf85) in mouse. Using a proteomics approach, the human NCU-G1 orthologue (RGSV2553) was identified as a candidate lysosomal membrane protein. Although, the sequence coverage has reached merely 20% of the 406 amino acid residues of the human precursor form, all the recovered peptides were detected in similar enrichments as the LAMP-1 and -2 proteins [START_REF] Schroder | Integral and associated lysosomal membrane proteins[END_REF]. An inspection of the sequence indicated that most of the remaining peptides of NCU-G1 were not suited for detection either because of size limits or due to glycosylation.

NCU-G1 is a lysosomal protein

Using immunofluorescence and two different density centrifugation techniques, we show that mouse NCU-G1 co-localizes and/or co-fractionates with the lysosomal marker proteins LAMP-1, Scpep1 and -hexosaminidase. This conclusion is supported by the finding that NCU-G1 is subject to the tyloxapol (Triton WR1339)induced density shift with a selective decrease in the buoyant density of lysosomes, which has been described by others [30,[START_REF] Wattiaux | Phagocytosis by rat liver: relationships between phagosomes and lysosomes[END_REF]. Recently, other putative lysosomal proteins were confirmed to be of lysosomal origin by comparable density-shift techniques [START_REF] Boonen | Intracellular localization of p40, a protein identified in a preparation of lysosomal membranes[END_REF][START_REF] Valle | Demonstration of lysosomal localization for the mammalian ependymin-related protein using classical approaches combined with a novel density shift method[END_REF]. Our results in conjunction with the enrichment of human NCU-G1 in a lysosomal membrane fraction derived from human placenta [START_REF] Schroder | Integral and associated lysosomal membrane proteins[END_REF] strongly support the lysosomal localization of NCU-G1.

Our observation that a mutant of the single putative lysosomal sorting motif (Y400QSI) of NCU-G1 only marginally co-localized with the lysosomal marker LAMP-1 indicates that this signal is sufficient to mediate transport towards the lysosomal compartment. Typically, the canonical tyrosine-based sorting signals YXX ) are Nterminally flanked by amino acids with a small side chain, mostly a glycine residue as known in e.g. the LAMP proteins (-1; -2A-C; -3), cystinosin or the acid phosphatase precursor [START_REF] Braulke | Sorting of lysosomal proteins[END_REF]. However, the mucin-like type I membrane protein endolyn exhibits a tyrosine-based sorting signal that is preceded by an asparagine residue instead of glycine [START_REF] Ihrke | Endolyn is a mucin-like type I membrane protein targeted to lysosomes by its cytoplasmic tail[END_REF]. The NCU-G1 sorting signal is a novel variant of the YXX -type since it has a glutamic acid residue in position -1. It should be of interest whether the 12 amino acid NCU-G1 cytoplasmic tail could mediate lysosomal transport of other type I transmembrane proteins such as has been shown for the 10 amino acid tail of endolyn [START_REF] Ihrke | Endolyn is a mucin-like type I membrane protein targeted to lysosomes by its cytoplasmic tail[END_REF].

NCU-G1 is widely expressed and highly glycosylated integral membrane protein

The A c c e p t e d M a n u s c r i p t

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transmembrane segment encompassing residues 370 to 392 and preceding a short cytosolic tail. Initially, the NCU-G1 cDNA was identified in a PCR-based approach to clone a potential prototype of the complement protein C3 from the mouse embryonic carcinoma cell line F9 [START_REF] Kawamura | cDNA of a novel mRNA expressed predominantly in mouse kidney[END_REF]. Although NCU-G1 cDNA was the only PCR product, it showed little homology to mouse C3. Northern blot analyses with a NCU-G1-specific probe revealed a single 1.5-kb transcript [START_REF] Kawamura | cDNA of a novel mRNA expressed predominantly in mouse kidney[END_REF]. The highest expression levels were detected in kidney so that NCU-G1 was also termed synonymously kidney predominant protein 1 [START_REF] Kawamura | cDNA of a novel mRNA expressed predominantly in mouse kidney[END_REF]. Our Northern blot analysis resembles those observations regarding the ubiquitous expression of NCU-G1 with the exception that no transcript was detected in skeletal muscles. On protein level, our NCU-G1 antiserum hardly detected NCU-G1 even in mouse kidney homogenates in which high amounts of NCU-G1 transcripts were shown. However, endogenous NCU-G1 was detectable in a lysosome-enriched fraction from mouse liver, although, at an unexpected molecular range of about 75 kDa. The expression of NCU-G1 in different cell lines came up with comparable apparent molecular sizes of about 70 -80 kDa. Our deglycosylation assays using PNGase F clearly demonstrate that the remarkable difference between calculated and apparent size of NCU-G1 was due to an extensive glycosylation suggesting that most of its nine N-glycosylation sites are used. This observation is not astonishing since many lysosomal membrane proteins including the type I LAMP and the LIMP proteins [38] as well as multipass transmembrane proteins NPC1 [START_REF] Watari | Mutations in the leucine zipper motif and sterol-sensing domain inactivate the Niemann-Pick C1 glycoprotein[END_REF],

cystinosin [START_REF] Cherqui | The targeting of cystinosin to the lysosomal membrane requires a tyrosine-based signal and a novel sorting motif[END_REF] and others show significantly higher apparent molecular weights than calculated from their primary sequence. The function of the extensive glycosylation of many lysosomal proteins and of lysosomal membrane proteins is assigned to a protective function in two respects: It had been estimated that particularly the high amount of LAMP proteins in the lysosomal membrane generate a glycocalyx that A c c e p t e d M a n u s c r i p t

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lines the limiting membrane [START_REF] Neiss | A coat of glycoconjugates on the inner surface of the lysosomal membrane in the rat kidney[END_REF]. In addition, N-glycans are known to protect lysosomal membrane proteins such as LAMP-1 and -2 from proteolysis [START_REF] Kundra | Asparagine-linked oligosaccharides protect Lamp-1 and Lamp-2 from intracellular proteolysis[END_REF].

We also demonstrated by a membrane extraction assay that C-terminally His-tagged NCU-G1 from HeLa cells is most likely an integral membrane protein although a significant amount of NCU-G1 was already solubilized from the lysosomal membrane upon carbonate treatment. It is worth mentioning that NCU-G1 in a lysosomeenriched fraction F2 from mouse liver showed similar extraction behaviour and was also partially solubilized by sodium carbonate (data not shown).

The present results are contrasting a recently published characterization in which human NCU-G1 was reported to function as a transcription factor and as a nuclear receptor activator for the peroxisome proliferators-activated receptors (PPAR)-alpha [START_REF] Steffensen | Human NCU-G1 can function as a transcription factor and as a nuclear receptor co-activator[END_REF]. The authors of that study expressed the human NCU-G1 in Drosophila Schneider S2-cells and detected a protein with an apparent molecular size of around 45 kDa in size using a polyclonal antiserum against a peptide representing the last fifteen amino acids. The authors did not identify the N-terminal signal sequence nor a putative transmembrane segment but allocated two nuclear export signals and several potential Src-homology 3 (SH3) interacting domains to the NCU-G1 protein.

The authors also showed by chloramphenicol acetyl-transferase assays that NCU-G1 might bind to and activate transcription from a cellular retinol-binding protein type I (CRBPI)-promotor [START_REF] Steffensen | Human NCU-G1 can function as a transcription factor and as a nuclear receptor co-activator[END_REF]. However, immunofluorescence studies with a C-terminal GFP-tagged version of NCU-G1 and a RFP-tagged version of PPAR showed negligible co-localization of these proteins. While the nuclear protein PPAR was exclusively sorted to the nucleus, NCU-G1 largely localized to extranuclear compartments and this was attributed to a possible interference by the GFP-moiety [START_REF] Steffensen | Human NCU-G1 can function as a transcription factor and as a nuclear receptor co-activator[END_REF]. A c c e p t e d M a n u s c r i p t

Here, we demonstrate that NCU-G1 is a highly-glycosylated, integral membrane protein of the lysosomal compartment. Although, the function of NCU-G1 is unknown and the protein does not exhibit homology to known proteins, it may be predicted that NCU-G1 fulfils a function similar to other type I lysosomal membrane proteins such as LAMP-1 and -2. Furthermore, NCU-G1 could be involved in the transport of lysosomal hydrolases as described for the LIMP-2 that binds and directs the soluble hydrolase -glucocerebrosidase towards the lysosome in a mannose 6-phosphateindependent manner [START_REF] Reczek | LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase[END_REF] or in forming larger complexes such as described for nicastrin in the -secretase complex [START_REF] Li | Nicastrin is required for assembly of presenilin/gamma-secretase complexes to mediate Notch signaling and for processing and trafficking of beta-amyloid precursor protein in mammals[END_REF]. Unfortunately, our attempts to establish a stably NCU-G1 expressing cell line were not successful, although, we tested the His- (C) Control (-) and NCU-G1-transfected Hela cell extracts were prepared as described in the Material and methods section and subsequently subjected to a membrane extraction assay using either lysis buffer, 0.1 M sodium carbonate or Triton-X100 for 30 min on ice. After a 100000 g centrifugation for 60 min, the obtained supernatants (SN) and pellets (P) were analysed by Western blotting using an anti-His antibody. After stripping with 0.2 N NaOH for 5 min and re-equilibration, the membrane was analysed with an anti-porin 31HL monoclonal antibody. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 02 Jun 2009 as manuscript BJ20090567 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090567

Biochemical

  Journal Immediate Publication. Published on 02 Jun 2009 as manuscript BJ20090567 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090567

  -A rabbit polyclonal antiserum (anti-NCU-G1) . The serum was affinity-purified using the immobilized peptide.Cloning, transfection and expression of NCU-G1-RGS-His5-The NCU-G1 cDNA (AccNo: BC021547; 1563 bp) was received from the Deutsche Ressourcenzentrum für Genomforschung (RZPD; clone IRAVp968F0636D6) and subcloned into the pcDNA3.1/Hygro(+) vector (Invitrogen) by add-on PCR using the following NheIforward primer 5'-gtatgctagcATGTTTCGCTGTTGGGGACCTCAC-3' and the NotI-RGS-His5-reverse primer 5'-gtatgcggccgcTTATCCGTGATGGTGATGGTGCGATCCTCTTCCGTTTATGGACTGG TACTCAGAATACC-3'. Finally, all constructs were full length sequenced to exclude Biochemical Journal Immediate Publication. Published on 02 Jun 2009 as manuscript BJ20090567 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090567

  Its mouse orthologue NCU-G1 is encoded in chromosome 3 and its cDNA is composed of six exons. The mRNA (AccNo: BC021547) is at least 1563 nucleotides (nt) long and consists of a 26 nt 5'-UTR, a 1215 nt coding sequence and a 3'-UTR. The deduced amino acid sequence of the NCU-G1 protein consists of 404 residues with a molecular mass of approximately 40 kDa. The bioinformatics analysis predicts a hydrophobic N-teminal signal peptide (residues 1 -35) followed by the main part of the molecule (residues 36 -369) containing nine putative Nglycosylation sites with the consensus sequence NXS/T, a single transmembrane segment (residues 370 -392) and a C-terminal tail (residues 393 -404) as illustrated in Figure 1 A. The C-terminal residue is preceded by a potential tyrosinebased (YXX ) lysosomal targeting signal with YQSI (aa 400 -403). These data suggest that NCU-G1 is a type I integral membrane protein with a large luminal Nterminal domain, a single transmembrane segment and a short cytoplasmic tail (Figure 1 B). The protein described here does not show any significant sequence similarity to other proteins except in the C-terminal tail, which resembles those in LAMP-1 and -2 proteins. The tails are short, contain several basic residues next to the transmembrane segment and a single tyrosine-based localization motif at or near their C-terminus.

  terminal RGS-His-tagged derivative after subcloning by add-on PCR into the pcDNA3.1/Hygro(+) vector (see Material and Methods section). Expression of the Cterminal His-tagged NCU-G1 in various cell types like HT1080 cells (HT1080-NCU-G1) or HeLa cells and analysis of the cell lysates by Western blotting using an anti-His-antibody resulted in two bands with apparent molecular weights of about 70 and 80 kDa, respectively (Figure2A, left panel). For further characterization of the endogenous NCU-G1 protein, we raised a polyclonal antiserum in rabbit against the peptide CPSVNERNSIDDEYAPAVF (residues 245 -263) that detected two

  B, lane 1 & 2) and HT1080-NCU-G1 cell lysates (Figure 2 B, lane 3 & 4) with glycosidase PNGase F and analysis by Western blotting resulted in a major decrease in the apparent molecular by approximately 40% (Figure 2 B). In the treated sample two band were observed at 37 and 44 kDa (Figure 2 B, lane 4) as compared to the expected value of 43.8 kDa. This result suggests that the existence of two glycosylated molecular forms of NCU-G1 in HT1080 cells is due to differences in the polypeptide rather than in the carbohydrate moieties. Deglycosylation of endogenous NCU-G1 in a lysosome-enriched fraction (F2) that was derived from a tyloxapol-treated mouse liver resulted a similar shift from a 75 kDa glycosylated form of NCU-G1 (Figure 2 B, lane 5) to 40 kDa (Figure 2 B, lane 6).

1 (

 1 Biochemical Journal Immediate Publication. Published on 02 Jun 2009 as manuscript BJ20090567 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090567 A c c e p t e d M a n u s c r i p tSubcellular localization of NCU-G1-His by immunofluorescenceThe anti-NCU-G1 antiserum was not suitable for immunofluorescence since many structures in addition to lysosomes were stained. Therefore, we confirmed the lysosomal localization of NCU-G1 by immunofluorescence with the C-terminal Histagged NCU-G1 derivative in mouse embryonic fibroblasts (MEF) and HeLa cells (not shown). For visualizing we used a monoclonal anti-penta-His antibody that was directly coupled with the chromophore Alexa488. NCU-G1 presented in punctuated vesicular structures throughout the cell. These were shown to represent lysosomes and late endosomes by co-staining with the lysosomal membrane protein LAMP-Figure4 A).Assuming NCU-G1 is a type I integral membrane protein, its topology suggests a single putative tyrosine-based lysosomal sorting motif YQSI in close proximity of the C-terminus and the transmembrane segment. In order to analyse the role of this putative motif we replaced the tyrosyl residue in position 400 by an alanyl residue by site-directed mutagenesis of the His-tagged NCU-G1 construct (NCU-G1-Y400A).The corresponding expression vector was introduced into HeLa cells and the subcellular localization of NCU-G1-Y400A was analysed by immunofluorescence using the Alexa488-coupled anti-His antibody and compared to that of the lysosomal marker LAMP-2. An extensive co-localization was observed with the NCU-G1 wild type construct. However, the NCU-G1-Y400A mutant was found in a diffuse peripheral network resembling the ER (Figure4 B) and rarely in the LAMP-2-positive organelles.Subcellular localization of endogenous NCU-G1 using density centrifugationBiochemical Journal Immediate Publication. Published on 02 Jun 2009 as manuscript BJ20090567 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090567 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited Tyloxapol (Triton WR1339) is a nonionic detergent that upon intravenous injection inhibits the lipoprotein lipase and thus increases serum lipoprotein levels like VLDL. Endocytosis of these lipoproteins and the detergent into hepatocytes causes a decrease in the buoyant density of the lysosomes. Upon fractionation in a density gradient the lysosomes of this cell population are shifted towards the top fraction and the resulting lysosome-enriched fraction (F2) is virtually devoid of other organellesmitochondria in particular. We prepared a "light mitochondria fraction" (L) from either tyloxapol-treated or non-treated mouse liver and applied it to a discontinuous sucrose gradient. After the separation we analysed the resulting fractions F1 -F4 (50 µg protein each) by immunoblotting (Figure 4 C). In the control liver, in fractions of the sucrose gradient very small amounts of endogenous NCU-G1 and LAMP-1 were detected (Figure 4 C, left panel). Both proteins were detected in fraction F4 which corresponds to the load of the gradient with the L fraction, although, their signals are weaker because mitochondrial proteins represent a major part of fraction F4 and hence dilute the lysosomal proteins. . In contrast, in the tyloxapol-treated liver, NCU-G1 (75 kDa) as well as LAMP-1 shifted into fraction F2 due to the selectively altered density of the lysosomes (Figure 4 C, right panel). The tyloxapol-induced shift of both NCU-G1 and LAMP-1 is considered a specific proof for the lysosomal localization of the former.

Biochemical

  Journal Immediate Publication. Published on 02 Jun 2009 as manuscript BJ20090567 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090567

  bioinformatics analysis of mouse NCU-G1 predicted a 40 kDa type I transmembrane protein with a 35 residues N-terminal signal peptide, a large luminal domain with nine putative N-glycosylation sites and a single C-terminal Biochemical Journal Immediate Publication. Published on 02 Jun 2009 as manuscript BJ20090567 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090567

Biochemical

  Journal Immediate Publication. Published on 02 Jun 2009 as manuscript BJ20090567 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090567

Biochemical

  Journal Immediate Publication. Published on 02 Jun 2009 as manuscript BJ20090567 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090567

Figure 1

 1 Figure 1 Predicted structure of NCU-G1

  transmembrane segment and the potential tyrosine-based sorting signal are indicated.

Figure 2

 2 Figure 2 Molecular characterization of NCU-G1

Figure 3

 3 Figure 3 Expression pattern of NCU-G1

LAMP- 1

 1 antibody (1D4B). F2 represents the lysosome-enriched fraction in tyloxapoltreated mice.

Figure 5

 5 Figure 5 Tyloxapol-induced density shift of lysosomes in a continuous sucrose

C 3 Biochemical

 3 Figure 1

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

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Acknowledgements

We thank Ellen Eckermann-Felkl, Nicole Eiselt and Klaus Neifer for excellent technical assistance as well as Karthikeyan Radhakrishnan and Sebastian Krol for their help and discussions. Biochemical Journal Immediate Publication. Published on 02 Jun 2009 as manuscript BJ20090567 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090567

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Funding

This work was supported by the Deutsche Forschungsgemeinschaft grant LU1173/1-4 (T.L.) and the BMBF grant 031U204A.

Northern blot analysis of NCU-G1 in various mouse tissues. Aliquots of total RNA from various tissues, 10 µg, were separated and sequently hybridized with a full length cDNA NCU-G1 probe and a full length Gapdh probe, respectively. (C) Schematic centrifugation procedure to obtain a lysosome-enriched fraction F2 which is finally harvested from the interphase between 1.06 g/ml and 1.14 g/ml density layers. Liver lysosomes from tyloxapol-treated mice are shifted towards this interphase due to their lower density (tritosomes) while lysosomes from non-treated mice are collected at the density of 1.21 g/ml (F4). A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.