Paul G Crichton

Charles Affourtit

Anthony L Moore
email: a.l.moore@sussex.ac.uk

Identification of a mitochondrial alcohol dehydrogenase in Schizosaccharomyces pombenew insights into energy metabolism

Keywords: ADH, alcohol dehydrogenase; CCCP, carbonyl cyanide Schizosaccharomyces pombe mitochondria, alcohol dehydrogenase isozymes, respiration, modular kinetics, NADH dehydrogenase, ubiquinone pool

In this paper we show that mitochondria isolated from Schizosaccharomyces pombe exhibit antimycin A-sensitive oxygen uptake activity that is exclusively dependent on ethanol and is inhibited by trifluoroethanol, a potent inhibitor of alcohol dehydrogenase (ADH). Ethanoldependent respiratory activity has, to our knowledge, not been reported in S. pombe mitochondria to date, and is indeed surprising as it has previously been concluded that only one ADH gene, encoding a cytosolic enzyme, occurs in this yeast. Spectrophotometric enzyme assays reveal that ADH activity in isolated mitochondria is increased ~ 16-fold by Triton X-100, which demonstrates the enzyme is located in the matrix. Using genetic knock-outs, we show conclusively that the novel mitochondrial ADH is encoded by adh4 and, as such, is unrelated to ADH isozymes found in mitochondria of other yeasts. By performing a modular-kinetic analysis of mitochondrial electron transfer, we furthermore show how ethanol-dependent respiratory activity (which involves oxidation of matrix-located NADH) compares to that observed when succinate or externally added NADH are used as substrates. This analysis reveals distinct kinetic differences between substrates which fully explain the lack of respiratory control generally observed during ethanol oxidation in yeast mitochondria.

INTRODUCTION

Over the last two decades, S. pombe has proved an invaluable model organism to study biological processes that are fundamental to higher eukaryotes. This has greatly improved the understanding of, most notably, mechanisms by which the cell cycle is controlled and damaged DNA is repaired (reviewed in ref. [START_REF] Carr | DNA structure dependent checkpoints as regulators of DNA repair[END_REF]). In our laboratory, S. pombe has been used for a different purpose, namely to functionally express the plant alternative oxidase [START_REF] Albury | Targeting the plant alternative oxidase protein to Schizosaccharomyces pombe mitochondria confers cyanideinsensitive respiration[END_REF]. This cyanideinsensitive mitochondrial respiratory enzyme is wasteful in terms of energy conservation, which is why considerable attention has been paid to the clarification of its physiological function [START_REF] Moore | Function of the alternative oxidase: is it still a scavenger?[END_REF].

Functional expression of the protein in S. pombe has provided valuable information on structurefunction relationships of the oxidase [START_REF] Albury | Structure of the plant alternative oxidase: site-directed mutagenesis provides new information on the active site and membrane topology[END_REF][START_REF] Albury | A highly conserved glutamate residue (Glu-270) is essential for alternative oxidase activity[END_REF][START_REF] Crichton | Constitutive activity of Sauromatum guttatum alternative oxidase in Schizosaccharomyces pombe implicates residues other than conserved cysteines in α-keto acid activation[END_REF] and, moreover, has resulted in this yeast becoming a useful model of plant mitochondrial electron transfer [START_REF] Affourtit | Functional expression of the plant alternative oxidase affects growth of the yeast Schizosaccharomyces pombe[END_REF].

Although S. pombe is widely used as a model system, relatively little fundamental information is available on the mitochondrial energy metabolism of this yeast [START_REF] Jault | Preparation of highly phosphorylating mitochondria from the yeast Schizosaccharomyces pombe[END_REF][START_REF] Moore | Schizosaccharomyces pombe mitochondria -morphological, respiratory and protein import characteristics[END_REF]. From the few studies published to date it is clear that the respiratory chain of S. pombe contains at least two substrate dehydrogenases (oxidising succinate and external NADH, respectively) that both reduce ubiquinone, which is subsequently oxidised by the cytochrome pathway [START_REF] Jault | Preparation of highly phosphorylating mitochondria from the yeast Schizosaccharomyces pombe[END_REF][START_REF] Moore | Schizosaccharomyces pombe mitochondria -morphological, respiratory and protein import characteristics[END_REF].

In addition to NADH and succinate, mitochondria from other yeasts (Saccharomyces carlsbergensis and Saccharomyces cerevisiae for example) are able to use ethanol as a respiratory substrate [10][START_REF] Luttik | The Saccharomyces cerevisiae NDE1 and NDE2 genes encode separate mitochondrial NADH dehydrogenases catalyzing the oxidation of cytosolic NADH[END_REF][START_REF] Overkamp | In vivo analysis of the mechanisms for oxidation of cytosolic NADH by Saccharomyces cerevisiae mitochondria[END_REF][START_REF] Von Jagow | Pathways of hydrogen in mitochondria of Saccharomyces carlsbergensis[END_REF]. Ethanol-dependent respiratory activity is generally attributed to the presence of mitochondrial alcohol dehydrogenase (ADH) isozymes [START_REF] Jault | Preparation of highly phosphorylating mitochondria from the yeast Schizosaccharomyces pombe[END_REF][START_REF] Moore | Schizosaccharomyces pombe mitochondria -morphological, respiratory and protein import characteristics[END_REF][10][START_REF] Luttik | The Saccharomyces cerevisiae NDE1 and NDE2 genes encode separate mitochondrial NADH dehydrogenases catalyzing the oxidation of cytosolic NADH[END_REF][START_REF] Overkamp | In vivo analysis of the mechanisms for oxidation of cytosolic NADH by Saccharomyces cerevisiae mitochondria[END_REF][START_REF] Von Jagow | Pathways of hydrogen in mitochondria of Saccharomyces carlsbergensis[END_REF]. In S. pombe only one ADH gene has been reported that relates to the mitochondrial ADH genes identified in other yeasts [START_REF] Russell | The primary structure of the alcohol dehydrogenase gene from the fission yeast Schizosaccharomyces pombe[END_REF]. As this gene is believed to encode a cytosolic enzyme (e.g. [START_REF] Reid | Molecular characterization of microbial alcohol dehydrogenases[END_REF]) it has been widely assumed that S. pombe does not contain a mitochondrial ADH isozyme and, as a result, differs from other yeast in how it metabolises ethanol [16].

In this paper we show unequivocally that S. pombe mitochondria exhibit ADH activity.

Using genetic knock-out strains we confirm that the responsible enzyme is ADH4, the first protein of its type to be shown to play a role in mitochondrial function.

EXPERIMENTAL

Sample preparation

S. pombe cells were batch-cultured in glucose minimal medium [17] supplemented with 0.4 mM adenine, 1.1 mM leucine and 0.7 mM uracil. Mitochondria were isolated as described previously [START_REF] Crichton | Constitutive activity of Sauromatum guttatum alternative oxidase in Schizosaccharomyces pombe implicates residues other than conserved cysteines in α-keto acid activation[END_REF] from the following two strains: Sp.011 (h -, ade6-704, leu1-32, ura4-D18), and JY743 carrying gene disruptions (see [START_REF] Sakurai | A distinct type of alcohol dehydrogenase, adh4 + , complements ethanol fermentation in an adh1-deficient strain of Schizosaccharomyces pombe[END_REF] for details) at either adh1 (h -, leu1-32, ura4-D18, adh1∆::ura4 + ) or adh4 (h -, leu1-32, ura4-D18, adh4∆::leu1 + ). Mitochondrial protein amounts were estimated using bicinchoninic acid [19] with bovine serum albumin as a standard.

Respiratory analysis

Mitochondrial oxygen consumption was measured at room temperature using a Clark-type electrode (Rank Brothers, Cambridge, U.K.). Mitochondria (0.4-1.2 mg) were suspended in 2.4 mL reaction medium containing 0.65 M mannitol, 20 mM MOPS (pH adjusted to 6.8 with NaOH), 1 mM MgCl 2 , 5 mM Na 2 HPO 4 and 10 mM NaCl (medium A). For modular-kinetic experiments, the reduction level of the Q-pool was measured simultaneously with respiratory activity using a vessel adapted to accommodate glassy carbon, platinum and Ag/AgCl reference electrodes as described in [20]. For these experiments, 1 µM Q 1 was included in the reaction medium. Where necessary, Q-reduction measurements were corrected for NADH-or ethanolinduced artefacts by subtracting electrode responses observed in the absence of Q 1 .

Alcohol dehydrogenase activity

ADH activity was followed spectrophotometrically as the production of NADH (ε NADH, 340 nm = 6.22 mM -1 cm -1 ) using a lambda 16-Perkin Elmer spectrophotometer. Assays were performed with 17-24 µg mitochondrial protein at 24-25˚C in medium A (pH 6.8) that included 1.7 µM antimycin A and 2 mM NAD + . Reactions were initiated by adding 100 mM ethanol.

RESULTS

Identification of a mitochondrial ADH

S. pombe exhibits mitochondrial ADH activity

Mitochondria isolated from S. pombe contain an active external NADH dehydrogenase [START_REF] Jault | Preparation of highly phosphorylating mitochondria from the yeast Schizosaccharomyces pombe[END_REF][START_REF] Moore | Schizosaccharomyces pombe mitochondria -morphological, respiratory and protein import characteristics[END_REF] that rapidly oxidises exogenous NADH (Table 1A). This respiratory activity (~ 67 nmol min -1 mg -1 ) is stimulated 5-fold by the addition of the protonophore CCCP, indicating that electron transfer is tightly coupled to proton translocation and, hence, that the mitochondrial inner-membranes are relatively intact. Interestingly, when ethanol is added to S. pombe mitochondria, electron transfer rates are achieved that are comparable to those observed with NADH (Table 1A). This ethanoldependent oxygen uptake activity is marginally increased by the addition of NAD + (Table 1A) and appears to depend on the integrity of the mitochondrial inner-membrane, since it is significantly reduced in osmotically shocked mitochondria (data not shown). NAD + -stimulated ethanol-dependent respiration rates, however, are similar in intact and disrupted mitochondria, implying that ethanol-dependent respiratory activity requires matrix NAD + . NAD + -stimulated, ethanol-dependent oxygen uptake is stimulated ~ 1.4 fold by CCCP (Table 1A).

Ethanol-dependent respiratory activity is sensitive to antimycin A (~ 95% inhibition) and 2,2,2-trifluoroethanol (~ 74% inhibition), specific inhibitors of mitochondrial complex III [21] and ADH [22], respectively (Table 1A). These observations strongly suggest that ethanoldependent oxygen consumption in S. pombe mitochondria, similar to other yeasts (e.g. refs [10][START_REF] Luttik | The Saccharomyces cerevisiae NDE1 and NDE2 genes encode separate mitochondrial NADH dehydrogenases catalyzing the oxidation of cytosolic NADH[END_REF][START_REF] Overkamp | In vivo analysis of the mechanisms for oxidation of cytosolic NADH by Saccharomyces cerevisiae mitochondria[END_REF][START_REF] Von Jagow | Pathways of hydrogen in mitochondria of Saccharomyces carlsbergensis[END_REF]), is accounted for by a matrix-located ADH that oxidises ethanol resulting in reduction of endogenous NAD + , which is subsequently re-oxidised by the respiratory chain. In S. cerevisiae, the acetaldehyde produced during this reaction can also be oxidised, which results in additional formation of NADH [START_REF] Boubekeur | Participation of acetaldehyde dehydrogenases in ethanol and pyruvate metabolism of the yeast Saccharomyces cerevisiae[END_REF]. This appears not to occur in S. pombe mitochondria as acetaldehyde inhibits ethanol-dependent oxygen consumption (~ 98% at 8 mM), and does not sustain a respiratory activity when added separately (data not shown).

The presence of a matrix-located ADH in S. pombe is confirmed unambiguously by the spectrophotometric data presented in Table 1B, which show that oxidation of ethanol can be increased approximately 16 times by the addition of the detergent Triton X-100. This result clearly demonstrates that the major part of ADH activity in our mitochondrial sample is latent and only becomes apparent when membranes are disrupted by detergent. Such latency has been observed in other yeasts (e.g. [10,[START_REF] Young | Evolution of a glucose-regulated ADH gene in the genus Saccharomyces[END_REF]) and implies that nearly all NADH formed in the assay lacking Triton X-100 is shielded from detection by a membrane. This membrane is undoubtedly mitochondrial, since ADH activity is linked to antimycin A-sensitive oxygen consumption (Table 1A). Given that only the mitochondrial inner-and not the outer-membrane is impermeable to NADH [START_REF] Von Jagow | Pathways of hydrogen in mitochondria of Saccharomyces carlsbergensis[END_REF], it can be concluded that the observed ethanol-dependent respiratory activity in S. pombe mitochondria involves a matrix-located ADH.

The molecular nature of mitochondrial ADH in S. pombe

The presence of a mitochondrial ADH in S. pombe is somewhat surprising given that one ADH gene only (adh1), encoding a cytoplasmic protein, has been reported to date [START_REF] Russell | The primary structure of the alcohol dehydrogenase gene from the fission yeast Schizosaccharomyces pombe[END_REF]. Using adh1 as a probe, Southern analysis did not reveal any homologous sequences in S. pombe [START_REF] Russell | The primary structure of the alcohol dehydrogenase gene from the fission yeast Schizosaccharomyces pombe[END_REF], which is in contrast to other yeasts where multiple adh1-like genes are usually present [START_REF] Reid | Molecular characterization of microbial alcohol dehydrogenases[END_REF]. Recently, however, a protein has been identified in S. pombe [START_REF] Sakurai | A distinct type of alcohol dehydrogenase, adh4 + , complements ethanol fermentation in an adh1-deficient strain of Schizosaccharomyces pombe[END_REF] that is unrelated to ADH1 but similar to ADH isozymes from the fermentative bacterium Zymomonas mobilis (ADH2) and S. cerevisiae (ADH4) [25,[START_REF] Williamson | Homology of Saccharomyces cerevisiae ADH4 to an iron-activated alcohol dehydrogenase from Zymomonas mobilis[END_REF]. Using algorithms described in [START_REF] Bannai | Extensive feature detection of N-terminal protein sorting signals[END_REF][28][29] we predict that, unlike ADH1, this novel ADH protein (now termed ADH4) is targetted to mitochondria, and may therefore account for the mitochondrial ADH activity reported in this paper. To test this notion, we measured respiratory activity in mitochondria isolated from S. pombe cell lines in which either ADH1 or ADH4 was ablated (∆adh1 and ∆adh4, respectively).

From the data presented in Table 2 it is clear that mitochondria isolated from ∆adh1 cells exhibit ethanol oxidation rates (60 nmol min -1 mg -1 ) that are comparable to wild-type activities (cf. Table 1). In striking contrast, no ethanol-dependent activity is detectable in mitochondria isolated from the ∆adh4 strain. This clear difference between ∆adh1 and ∆adh4 mitochondria confirms that the observed mitochondrial ADH activity in S. pombe can be attributed entirely to ADH4. Although quantitatively somewhat different from Sp.011, it is clear that mitochondria from JY743 cell lines oxidise NADH at appreciable rates that are stimulated significantly by CCCP (Table 2) confirming the respiratory competence of both the ∆adh1 and ∆adh4 strains.

Modular-kinetic analysis of S. pombe respiration

Respiratory control

The data shown in Table 1A reveal that mitochondrial ethanol-dependent oxygen consumption in S. pombe is not increased significantly by CCCP. This relative lack of respiratory control during ethanol oxidation has previously been observed in other yeast mitochondria, and has been attributed to a relatively high degree of proton leak in the presence of this respiratory substrate [START_REF] Bakker | Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae[END_REF]. However, the coupled rates of ethanol oxidation are comparable to the equivalent rates observed with NADH, which would suggest that the proton conductivity of the mitochondrial inner-membrane in S. pombe is not affected significantly by 1% (v/v) ethanol.

From studies with plant and yeast mitochondria, it has become clear that respiratory control ratios are substrate-dependent, since they reflect the kinetic interaction between several components of the respiratory system, including substrate dehydrogenases [31,32]. For example, oxygen consumption by S. pombe mitochondria is stimulated by CCCP more strongly when NADH is used as a substrate than when succinate is employed [START_REF] Affourtit | Functional expression of the plant alternative oxidase affects growth of the yeast Schizosaccharomyces pombe[END_REF]. To assess if the lack of respiratory control during ethanol oxidation could be explained in a similar manner, we performed a modular-kinetic analysis to gain more detailed insights in the kinetics of the respiratory system in S. pombe mitochondria oxidising succinate, ethanol, or external NADH. In this analysis we simplified the respiratory system conceptually to comprise modules that either reduce or oxidise a common Q-pool (see reference [31] for details).

Oxidation of succinate

From the simultaneous oxygen uptake and Q-reduction measurements shown in Fig. 1, it can be seen that succinate is oxidised in S. pombe mitochondria at ~ 28 nmol min -1 mg -1 , with a concomitant Q-reduction level of ~ 34% (Fig. 1A). Subsequent sequential addition of ATP and glutamate leads to increases in both respiratory activity and Q-reduction since both compounds activate succinate dehydrogenase [START_REF] Affourtit | Functional expression of the plant alternative oxidase affects growth of the yeast Schizosaccharomyces pombe[END_REF][START_REF] Moore | Schizosaccharomyces pombe mitochondria -morphological, respiratory and protein import characteristics[END_REF]. Valuable information may be gained when the respective steady state respiratory rates are plotted as a function of Q-reduction (Fig. 1B). When interpreting such a Q-kinetic plot, it is important to realise that respiratory steady states result from the interplay between Q-reducing (in this case succinate dehydrogenase with the dicarboxylate carrier) and QH 2 -oxidising (cytochrome pathway) modules. Since ATP and glutamate specifically affect Q-reduction, a single model representing coupled QH 2 -oxidation kinetics should suffice to describe the shown steady states. Indeed, the data in Fig. 1B are fitted readily to a single hyperbolic equation (cf. [START_REF] Van Den Bergen | The relationship between electron flux and the redox poise of the quinone pool in plant mitochondria -interplay between quinol-oxidizing and quinone-reducing pathways[END_REF]) that accounts for the theoretically inevitable fact that cytochrome pathway activity is zero when the Q-pool is fully oxidised.

Oxidation of ethanol

Ethanol-dependent respiratory rates are plotted as a function of Q-reduction in Fig. 2A, both in the absence and the cumulative presence of ADP and CCCP. Under coupled conditions, oxygen is consumed at ~ 52 nmol min -1 mg -1 at a Q-redox poise of ~ 78%. Importantly, this steady state is modelled well by the curve that describes cytochrome pathway activity (Fig. 2A-curve 1). This implies that ethanol does not alter the kinetics of QH 2 -oxidation under coupled conditions, indicating that it neither affects the components of the cytochrome pathway nor the integrity of the mitochondrial inner-membrane. The rate of ethanol oxidation is increased only marginally by ADP and CCCP (to ~58 and 61 nmol O 2 min -1 mg -1 , respectively), whereas sequential addition of these compounds decreases the Q-redox poise to 71 and 43%. Obviously, the resulting respiratory steady states do not fall on the curve describing coupled QH 2 -oxidation kinetics (Fig. 2A), since control by the protonmotive force is relieved by ADP and CCCP. During ethanoldependent respiration, the Q-reducing module comprises mitochondrial ADH and a substrate dehydrogenase that oxidises matrix NADH. Since S. pombe does not contain a protonmotive complex I [34], it is unlikely that activity of this internal-NADH dehydrogenase is affected by ADP or CCCP. It is therefore reasonable to expect that all steady states shown in Fig. 2A are describable by a single Q-reduction model, which indeed appears the case (Fig. 2A-curve ethanol). Note that this model accounts for the theoretical inevitability that Q-reducing activity is zero when the Q-pool is fully reduced.

Oxidation of external NADH

In Fig. 2B Q-reduction kinetics during ethanol-oxidation are compared directly with those observed during oxidation of externally added NADH. In the latter case, a Q-reduction level of ~ 95% is achieved allowing a respiratory activity of ~ 65 nmol O 2 min -1 mg -1 (see also Table 1A).

This activity is stimulated significantly by ADP (1.5-fold) and CCCP (5-fold), whilst Qreduction is hardly affected by either compound (Fig. 2B). This shows that the external-NADH dehydrogenase exhibits a rather high elasticity towards the reduction level of the Q-pool. In contrast, Q-reduction kinetics during ethanol oxidation are such that activity (~ 50 to 60 nmol O 2 min -1 mg -1 ) is almost fully independent of the Q-redox poise.

Apparent lack of respiratory control during ethanol oxidation is due to kinetic characteristics of the Q-reduction module

Differences in the behaviour of Q-reducing modules cause clear system-kinetic differences between S. pombe mitochondria oxidising either NADH or ethanol. In Fig. 2C modelled data are shown that reflect both Q-reduction kinetics and cytochrome pathway behaviour under various energetic conditions. It is evident that lowering the protonmotive force with ADP or CCCP predominantly affects the oxygen consumption rate during NADH oxidation, whereas it has an exclusive effect on the Q-redox poise during ethanol oxidation (Fig. 2C, upper and lower case letters, respectively). These results explain clearly why NADH-dependent respiratory activity is stimulated considerably by CCCP, whereas ethanol-dependent activity is not (Table 1A).

DISCUSSION

In this paper we have demonstrated that the fission yeast S. pombe expresses a functional ADH isozyme that is specifically located in the mitochondrial matrix (Table 1), and using genetic knock-out strains we have identified the responsible gene (Table 2). This gene (adh4) encodes for a novel ADH protein that is not homologous to any of the mitochondrial (ADH1like) isozymes found in other yeasts, which would explain why it has not been discovered before.

In this respect, our results are of significant interest as they convincingly challenge the apparent general acceptance in the literature that S. pombe does not contain a mitochondrial ADH. An exception to this general notion is a study by Tsai et al. [35], who reported gel-electrophoretic data that would indeed suggest the occurrence of a mitochondrial ADH in S. pombe. Although the results of these authors are often overlooked, probably because confirmation of the subcellular location of the various isozymes is lacking [35], they are clearly in agreement with our observations.

As is the case in other yeasts [10-13], mitochondrial ADH activity sustains considerable oxygen consumption when S. pombe mitochondria are incubated in the exclusive presence of ethanol (Table 1). This indicates that ADH reduces matrix NAD + , which is subsequently reoxidised by the respiratory chain. Although perhaps not surprising, our data therefore confirm that the mitochondrial electron transfer chain of S. pombe contains an active internal NADH dehydrogenase that couples oxidation of matrix NADH to reduction of ubiquinone. Importantly, however, it turns out that electron transfer is controlled differently during NADH-and ethanoldependent respiration, such that control is exerted predominantly by QH 2 -oxidation when NADH is used as substrate, and by Q-reduction when ethanol is employed (Fig. 2C). The apparent lack of respiratory control (Tables 1 and2), often attributed to detrimental effects of ethanol on the integrity of mitochondrial membranes [START_REF] Bakker | Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae[END_REF], can be explained fully by the particular distribution of control during ethanol oxidation.

In general, the physiological function of yeast mitochondrial ADH enzymes is unclear. It has been proposed, however, that they form an ethanol-acetaldehyde shuttle with their respective cytosolic counterparts, which facilitates equilibration of cytoplasmic and mitochondrial NAD + /NADH ratios [START_REF] Von Jagow | Pathways of hydrogen in mitochondria of Saccharomyces carlsbergensis[END_REF]. In S. cerevisiae, for example, this is thought to be of particular relevance during anaerobic growth, when the NADH produced in the matrix by assimilatory reactions cannot be oxidised by the respiratory chain. S. cerevisiae strains lacking mitochondrial ADH3 display impaired anaerobic growth [10], which would agree with this notion. As S. pombe cells lacking ADH4 also display reduced rates of anaerobic growth [START_REF] Sakurai | A distinct type of alcohol dehydrogenase, adh4 + , complements ethanol fermentation in an adh1-deficient strain of Schizosaccharomyces pombe[END_REF], it is conceivable that ADH4 fulfils a function in this system similar to that of ADH3 in S. cerevisiae. S. pombe ADH4 does not show significant amino acid sequence homology with ADH1 or any other member of the 'group I-type', NAD(P) + -dependent ADH enzyme family (as classified in [START_REF] Reid | Molecular characterization of microbial alcohol dehydrogenases[END_REF]). Instead, it shares considerable sequence identity with ADH4 from S. cerevisiae (56%) and ADH2 from Zymomonas mobilis (52%), both of which belong to the 'group III-type', ironactivated, NAD(P) + -dependent ADH enzyme family [25,[START_REF] Williamson | Homology of Saccharomyces cerevisiae ADH4 to an iron-activated alcohol dehydrogenase from Zymomonas mobilis[END_REF]. To our knowledge, S. pombe ADH4 is the first group III-type protein that has been shown to play a role in mitochondrial metabolism. This raises the possibility that these enzymes also function in mitochondria of other species. Interestingly in this respect, a S. pombe ADH4 homologue has recently also been discovered in human, where it is expressed during foetal development as well as in adult liver [36]. Inspection of the amino acid sequence of this enzyme reveals the presence of an ~ 40 amino acid N-terminal extension that we predict -using the algorithms published in [START_REF] Bannai | Extensive feature detection of N-terminal protein sorting signals[END_REF][28][29] -to target the protein to mitochondria.

To summarise, we have identified a novel mitochondrial ADH enzyme in S. pombe and have demonstrated in detail how the activity of this enzyme is linked to in vitro respiration. With this information we believe that we have provided valuable new insights into the energy metabolism of this widely used model organism.

Figure 1 -

 1 Figure 1 -The kinetic dependency of succinate-dependent oxygen consumption activity on the reduction level of the Q-pool. (A) Typical oxygen consumption (solid line) and Q-reduction (dotted line) traces are shown illustrating the stimulatory effect of ATP (0.2 mM) and glutamate (9 mM) on the oxidation of succinate (9 mM). The numbers adjacent to the traces represent specific O -uptake rates (nmol min -1 mg -1 ) and Q-reduction levels (fraction QH 2 in total Q-pool).(B) Steady state respiratory rates obtained with succinate alone () or in the cumulative presence of ATP () and glutamate (∆) are shown as a function of the Q-redox poise. Data are averaged values from three independent mitochondrial preparations and were modelled with hyperbolic equations as described in[START_REF] Van Den Bergen | The relationship between electron flux and the redox poise of the quinone pool in plant mitochondria -interplay between quinol-oxidizing and quinone-reducing pathways[END_REF].

Figure 2 - 1 .

 21 Figure 2 -Kinetic interplay between QH 2 -oxidising and Q-reducing pathways in S. pombe mitochondria. Respiratory steady states were achieved upon the oxidation of ethanol (0.15 M, closed symbols) or NADH (1.8 mM, open symbols) in the absence (diamonds) or the cumulative presence of ADP (0.1 mM, circles) and CCCP (1 µM, triangles). Data are averaged values from two to three separate mitochondrial preparations and were modelled with hyperbolic equations as described in [33]. Curves representing QH 2 -oxidising kinetics under coupled conditions (labelled 1. in panels A and C) are identical to the one presented in Figure 1B. Descriptions of these kinetics in the presence of ADP and CCCP (labelled 2. and 3. in panel C, respectively) are based on the data shown in panel B (circles and triangles, respectively) and include the origin, since cytochrome pathway kinetics with respect to the Q-redox poise should be substrate-independent and zero when the Q-pool is fully oxidised. The intersects of curves reflecting the kinetics of Qreducing (labelled NADH and ethanol) and QH 2 -oxidising modules (panel C) indicate steady states attained during the oxidation of ethanol (lower case letters) or NADH (upper case letters) in the absence (a) or the cumulative presence of ADP (b) and CCCP (c).

  34 Wood, V. et al. (2002) The genome sequence of Schizosaccharomyces pombe. Nature 415, 871-880 35 Tsai, C. S., Shi, J. L., Beehler, B. W. and Beck, B. (1992) Enzyme-activities of D-glucose metabolism in the fission yeast Schizosaccharomyces pombe. Can. J. Microbiol. 38,

		1313-1319
	36	Deng, Y., Wang, Z., Gu, S., Ji, C., Ying, K., Xie, Y. and Mao, Y. (2002) Cloning and
		characterization of a novel human alcohol dehydrogenase gene (ADHFe1). DNA Seq. 13,
		301-306

Biochemical Journal Immediate Publication. Published on 25 Sep 2006 as manuscript BJ20061181

ACKNOWLEDGEMENTS

We would like to express our gratitude to Prof. Yuko Giga-hama (Asahi Glass Co., Ltd., 1-12-1 Yurakucho, Chiyoda-ku, Tokyo, Japan) for providing the ∆adh1 and ∆adh4 strains of S. pombe. P.G.C. would like to thank the School of Life Sciences, University of Sussex for postgraduate funding.

3.

2.

Figure 2


Anaïs G Jensen

Magali Chemali

Agnès Chapel

Sylvie Kieffer-Jaquinod

Michel Jadot

Jéröme Garin

Agnès Journet
email: agnes.journet@cea.fr

Jérôme Garin

Biochemical characterization and lysosomal localization of the mannose-6-phosphate protein p76 (hypothetical protein LOC196463)

Keywords: mannose-6-phosphate, lysosome, proteomics, sub-cellular fractionation, immunofluorescence, post-translational modifications 2DE, 2-dimensional electrophoresis, HRP: horseradish peroxidase, LAMP, lysosomal-associated membrane protein, LC-MS/MS, liquid chromatography-tandem mass spectrometry, MALDI-TOF, matrix assisted laser desorption ionisation time of flight, Man6P, mannose-6-phosphate, MPR, mannose-6-phosphate receptor, PNGase

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Lysosomes are membrane delimited intracellular organelles with an intraluminal acidic pH, which, for a long time, were considered simply as the main digestive compartment of the cell.

The degradation of macromolecules inside lysosomes is performed through the action of more than 50 hydrolases and associated accessory proteins. About 40 human disorders are related to mutations in genes encoding lysosomal proteins, and named lysosomal storage diseases, according to their cellular effects [START_REF] Futerman | The cell biology of lysosomal storage disorders[END_REF]. Although the precise mechanisms are not yet completely elucidated, lysosomes have been shown to be involved in many other cellular processes, as antigen processing and presentation [START_REF] Li | Compartmentalization of class II antigen presentation: contribution of cytoplasmic and endosomal processing[END_REF]. Moreover, lysosomes could be involved in programmed cell death [START_REF] Kroemer | Lysosomes and autophagy in cell death control[END_REF].

During their biosynthesis, soluble lysosomal proteins acquire on N-linked oligosaccharides a mannose-6-phosphate (Man6P) marker, which is recognised by two Man6P receptors (MPRs), the small cation-dependent MPR and the large cation-independent MPR [START_REF] Hille-Rehfeld | Mannose 6-phosphate receptors in sorting and transport of lysosomal enzymes[END_REF]. The MPRs sort soluble lysosomal proteins towards the endosomes, where they release their cargo, due to the low pH of the organelles. Finally, the soluble lysosomal proteins reach lysosomes where their Man6P tag is dephosphorylated [START_REF] Einstein | Serum factors alter the extent of dephosphorylation of ligands endocytosed via the mannose 6-phosphate/insulin-like growth factor II receptor[END_REF].

Over the last decade, proteomic analyses aiming at identifying soluble lysosomal proteins have been performed on Man6P proteins, purified by affinity on immobilised MPRs. Several of these studies [START_REF] Journet | Towards a human repertoire of monocytic lysosomal proteins[END_REF][START_REF] Journet | Proteomic analysis of human lysosomes: Application to monocytic and breast cancer cells[END_REF][START_REF] Kollmann | Identification of novel lysosomal matrix proteins by proteome analysis[END_REF][START_REF] Czupalla | Proteomic analysis of lysosomal acid hydrolases secreted by osteoclasts: implications for lytic enzyme transport and bone metabolism[END_REF] were carried out on proteins purified from cell secretions. Body fluids such as urine [10] or plasma [11] were also used as a source of extracellular Man6P proteins.

Studies dealing with intracellular Man6P proteins were performed on a few transformed cell lines [START_REF] Sleat | Increased levels of glycoproteins containing mannose 6-phosphate in human breast carcinomas[END_REF], and mainly on brain homogenates [START_REF] Sleat | Rat brain contains high levels of mannose-6-phosphorylated glycoproteins including lysosomal enzymes and palmitoyl-protein thioesterase, an enzyme implicated in infantile neuronal lipofuscinosis[END_REF][START_REF] Sleat | The human brain mannose 6-phosphate glycoproteome: A complex mixture composed of multiple isoforms of many soluble lysosomal proteins[END_REF][START_REF] Sleat | Identification of sites of mannose 6-phosphorylation on lysosomal proteins[END_REF]. Indeed, as lysosomal Man6P protein dephosphorylation is less efficient in the brain than in other organs [START_REF] Sleat | Rat brain contains high levels of mannose-6-phosphorylated glycoproteins including lysosomal enzymes and palmitoyl-protein thioesterase, an enzyme implicated in infantile neuronal lipofuscinosis[END_REF]16], brain is the most appropriate organ for purification of mature lysosomal proteins through their Man6P sugar.

Our own proteomic study of Man6P proteins purified from human cell secretions [START_REF] Journet | Towards a human repertoire of monocytic lysosomal proteins[END_REF][START_REF] Journet | Proteomic analysis of human lysosomes: Application to monocytic and breast cancer cells[END_REF] led us to the discovery of a novel protein that we named "p76" because of its apparent molecular mass. In the present report, we describe its identification as the hypothetical protein LOC196463 and its biochemical characterisation and finally, we effectively demonstrate its lysosomal localisation.

EXPERIMENTALS Mass spectrometry analysis and protein N-terminal sequence determination

Proteins were identified by MALDI-TOF peptide fingerprinting or LC-MS/MS analysis as previously described [START_REF] Journet | Proteomic analysis of human lysosomes: Application to monocytic and breast cancer cells[END_REF][START_REF] Saksouk | Histone-modifying complexes regulate gene expression pertinent to the differentiation of the protozoan parasite Toxoplasma gondii[END_REF]. For N-terminal sequence analysis, Edman degradation of electroblotted proteins [START_REF] Journet | Towards a human repertoire of monocytic lysosomal proteins[END_REF] was performed by the Laboratoire d'Enzymologie Moléculaire (IBS, Grenoble, France) using an Applied Biosystems gas-phase sequencer (model 492) and an Applied Biosystems Model 140C HPLC system with the model 610A data analysis software package (version 2.1).

Bioinformatics

Tools found on the Expasy server (www.expasy.org/) were used for primary sequence analyses (Blast, Compute pI/Mw, SignalP 3.0 and NetNGlyc 1.0). Genomic, transcriptomic and protein data were obtained from the Ensembl (www.ensembl.org/index.html), Symatlas (www.symatlas.gnf.org) and NCBI (www.ncbi.nlm.nih.gov) web sites.

Molecular cloning and northern blot analysis

All inserts were generated by PCR using the Expand High Fidelity System (Roche).

Sequences of the constructs were verified by DNA sequencing. hp76 cDNA was initially obtained by RT-PCR on U937 mRNA with the F1/R1 primers (see Supplemental data, Table1). The vector pFLAG-hp76 was generated by inserting the sequence encoding hp76 from which both signal sequence and stop codon had been removed (F2/R2 primers), within the pFLAG-ATS bacterial expression vector (Sigma-Aldrich). For eukaryotic expression hp76 cDNA was cloned into the pcDNA3.1/Myc-His(+)A vector (Invitrogen), generating pcDNA3.1/hp76-Myc-His. The DNA sequence encoding the hp76-myc-His 6 fusion protein (hp76-myc) was then PCR-amplified from pcDNA3.1/hp76-Myc-His (F3/R3 primer pair), and inserted into the pCEP4 expression vector (Invitrogen), generating pCEP4/hp76-Myc-His. An expression vector encoding hp76 without tag was built, by ligation into the pcDNA3.1 vector (Invitrogen) of the 5' KpnI/BamHI hp76 fragment purified from pCEP4/hp76-Myc-His, and of the 3' BamHI/XhoI hp76 fragment obtained by digestion of a longer PCR-amplified hp76 fragment (F4/R4 primer pair).

The plasmid pCMV-SPORT6/mp76 (clone IRAVp968E0290D6), encoding mouse p76, was obtained from RZPD (Deutsches Ressourcenzentrum für Genomforschung). Copyright 2006 Biochemical Society Northern blot analysis was performed on a human Multiple Tissue Northern blot (Clontech) by hybridisation with a specific hp76 cDNA fragment and with the control human β-actin cDNA, as described [START_REF] Sambrook | Molecular Cloning. A Laboratory Manual[END_REF].

Cell lines and transfections

HeLa, U937 and Rat2 cells were from the American Type Culture Collection (ATCC), and 293-EBNA cells were from Invitrogen. Murine JR11 fibroblasts [19] are a kind gift from Dr B. Hoflack (Technical University of Dresden, Germany). They constitutively secrete Man6P proteins because of their deficiency in both MPRs.

HeLa, 293-EBNA, Rat2 and JR11 cells were grown in DMEM-GlutamaxI supplemented with 10% FBS, while U937 cells were maintained in RPMI-GlutamaxI medium supplemented with 10% FBS. Media and serum were from Invitrogen. HeLa cells were transiently transfected using the Fugene 6 reagent (Roche) and processed 3 or 4 days later for analysis. 293-EBNA cells were transfected with pCEP4 or pCEP4/hp76-Myc-His by calcium phosphate precipitation [START_REF] Chen | High-efficiency transformation of mammalian cells by plasmid DNA[END_REF], and stable transfectants (293/mock or 293/hp76-myc) were selected using hygromycin B (0.3 mg/ml, Invitrogen).

Expression and purification of recombinant proteins

The FLAG-p76 recombinant protein was expressed and purified as inclusion bodies from transformed Escherichia coli BL21 bacteria as described [START_REF] Harlow | Antibodies: a laboratory manual[END_REF]. For analysis of hp76-myc, supernatants from confluent 293/mock or 293/hp76-myc cells cultivated in DMEM-GlutaMax I without FBS nor hygromycin were collected 3 times a week for a duration of 1 month, and stored at -20°C.

Production of anti-p76 antibodies

Purified FLAG-hp76 inclusion bodies were injected into rabbits to raise hp76Ab polyclonal antibodies (Charles River Laboratories, Chatillon-sur-Chalaronne, France), as described [START_REF] Harlow | Antibodies: a laboratory manual[END_REF].

Two rat peptides (TRNPRAKIFQRDQS and SQPDLWMFSPVKVPWD, residues 76-89 and 196-211, respectively, according to the incomplete NCBI entry AAM23313), and the human N-terminal peptide (IPAPGGRWARDGQVPPASR, residues 42-60, according to LOC196463) were synthesised with an additional cysteine residue (NeoMPS, Strasbourg, France) and coupled to activated maleimide ovalbumin (Pierce). The lyophilised antigens were injected into rabbits (Charles River Laboratories or NeoMPS; [START_REF] Harlow | Antibodies: a laboratory manual[END_REF]) to raise respectively the Irp76Ab, Crp76Ab and Nhp76Ab antisera. Copyright 2006 Biochemical Society hp76Ab and Nhp76Ab react against human p76 only. Irp76Ab reacts against mouse and rat p76. Crp76Ab reacts against the three species. As purification did not improve the reactivity nor the specificity of the antisera, these were used without purification. Non-immune sera did not reveal any significant protein species.

Electrophoresis, Western blot and MPR overlay SDS-PAGE separation of the reduced proteins and 2DE were performed as described [START_REF] Journet | Proteomic analysis of human lysosomes: Application to monocytic and breast cancer cells[END_REF]22].

Proteins were stained by silver nitrate or colloidal blue (Bio-Safe, Bio-Rad).

Western blot analyses [START_REF] Harlow | Antibodies: a laboratory manual[END_REF] were performed with the following primary antibodies: i) rabbit polyclonal antibodies: hp76Ab, 1/1000; Nhp76Ab, 1/500; Crp76Ab, 1/1000; Irp76Ab, 1/400; anti-mitochondrial ATPase (anti-F 0 subunit), 1/3000 (generous gift of Drs Dupuis and Lunardi); ii) mouse monoclonal anti-LAMP1, 1/500 (1D4B, Developmental Studies Hybridoma Bank); iii) goat polyclonal anti-mouse CD98, 1/200 (M-20, Santa Cruz).

Membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies: goat anti-rabbit (1/50000, Pierce), goat anti-mouse (1/3000, Bio-Rad), or mouse anti-goat (1/10000, Sigma-Aldrich). Signal detection was performed using the SuperSignal West Pico chemiluminescent substrate (Pierce) and photographic revelation. MPR overlay assays were adapted from Valenzano et al. [START_REF] Valenzano | An assay to detect glycoproteins that contain mannose 6-phosphate[END_REF]. Soluble CI-MPR (sCI-MPR, purified as described in [START_REF] Journet | Towards a human repertoire of monocytic lysosomal proteins[END_REF]) was biotinylated using the Biotin Protein Labeling Kit (Roche), according to the manufacturer protocol. 2DE-resolved Man6P proteins were electrotransferred onto nitrocellulose membranes (Bio-Rad), that were blocked in TBS, 0.1% Tween-20, 3% (w/v) BSA, incubated with biotinylated sCI-MPR in TBS, 0.1% Tween-20, 1% BSA with or without Man6P (Sigma-Aldrich), and then with neutravidin-HRP (1/75000; Pierce) in TBS, 0.1% Tween-20, 1% BSA. Bound biotinylated sCI-MPR was revealed using the SuperSignal West Pico chemiluminescent substrate (Pierce) and photographic revelation.

Production and purification of Man6P proteins from cell lines or mouse brains

Man6P proteins were produced and purified from U937, Rat2, JR11 and 293/hp76-myc cells as described [START_REF] Journet | Towards a human repertoire of monocytic lysosomal proteins[END_REF]. Purification was performed either on an Affigel-10-sCI-MPR column or on sCI-MPR immobilised on Epoxy membranes (Sartorius).

Mouse brains were homogenised and Man6P proteins were affinity purified as described [START_REF] Sleat | The human brain mannose 6-phosphate glycoproteome: A complex mixture composed of multiple isoforms of many soluble lysosomal proteins[END_REF], on sCI-MPR immobilised on an Epoxy membrane (Sartorius).

Immunofluorescence studies

Transiently transfected HeLa cells were processed for immunofluorescence [START_REF] Harlow | Antibodies: a laboratory manual[END_REF] 4 days after transfection. Cells were fixed at room temperature, in methanol:acetone (50:50) previously chilled at -20°C. Antibodies were used at the following dilutions: rabbit polyclonal Crp76Ab, 1/40; mouse monoclonal anti-human LAMP1, 1/1000 (CD107a, BD PharMingen); goat polyclonal anti-human cathepsin D, 1/25 (R-20, Santa Cruz); Cy3-conjugated goat anti-rabbit, 1/1500 (Jackson); Alexa 488-conjugated goat anti-mouse, 1/1000 (Molecular Probes); FITCconjugated donkey anti-goat, 1/200 (Serotec). Fluorescence was examined using a TSC-SP2 confocal laser-scanning microscope (Leica).

Sub-cellular fractionation and osmotic release experiments

Livers were obtained from female adult NMRI mice (20g) that received, when specified, an intravenous injection of 17 mg Triton WR-1339 (tyloxapol, Sigma-Aldrich) 4 days prior to sacrifice. Fractionation of sub-cellular organelles by differential centrifugation was performed according to [24]. Isopycnic centrifugation was carried out on the total mitochondrial "ML" fraction as described [START_REF] Beaufay | Tissue fractionation studies. 18. Resolution of mitochondrial fractions from rat liver into three distinct populations of cytoplasmic particles by means of density equilibration in various gradients[END_REF].

For osmotic release experiments, an ML fraction from NMRI mouse liver was diluted in sucrose solutions of decreasing concentrations, and analysed as described [START_REF] Appelmans | Tissue fractionation studies. 3. Further observations on the binding of acid phosphatase by rat-liver particles[END_REF].

Enzymatic assays

The activities of β-galactosidase, β-hexosaminidase and β-glucuronidase were measured as described [START_REF] Peters | Lysosomes of the arterial wall. I. Isolation and subcellular fractionation of cells from normal rabbit aorta[END_REF].

RESULTS

Identification of a novel human protein among Man6P proteins: p76 (hypothetical protein LOC196463)

Our previous proteomic analysis of purified Man6P proteins from U937 and MCF7 human cells revealed a few novel proteins such as cystatin F and CREG [START_REF] Journet | Towards a human repertoire of monocytic lysosomal proteins[END_REF][START_REF] Journet | Proteomic analysis of human lysosomes: Application to monocytic and breast cancer cells[END_REF]. One of these novel proteins, which was not mentioned in these papers, was initially identified on a 2DE gel of a U937 sample, in a series of spots of neutral to alkaline pI (approximately pH 7-8) and at a molecular mass of 70 kDa by a unique MS/MS peptide sequence (LASDGATWADIFK, residues 355-367 of LOC196463). These spots were hardly detected by colloidal blue staining (Figure 1A) and were not visible on MCF7 2DE gels. The same protein was also identified from a series of spots of more acidic pI (approximately pH 5) and migrating at a slightly higher molecular mass of 76 kDa, in both U937 and MCF7 samples (Figure 1A and Supplemental data, Table 2). We named it "hp76", due to its apparent molecular mass. In the U937 sample, 2 peptides were identified by LC-MS/MS. In the MCF7 sample, 11 peptides of hp76 were identified by MALDI-TOF mass fingerprinting, leading to a sequence coverage of 20%. Complementary analyses performed by LC-MS/MS on both U937 and MCF7 samples separated by SDS-PAGE led to the identification of 7 tryptic peptides of hp76 in the U937 sample, and 9 peptides in the MCF7 one (respective coverages, 13 and 17%; Supplemental data, Table 2).

This novel protein, reported in the NCBI protein database as the "hypothetical protein LOC196463", is 589 amino acids long with a predicted signal peptide of 41 amino acids (Supplemental data, Figure 1). Since 6 potential N-glycosylation sites are predicted, carbohydrate modifications are likely to explain the difference between the apparent and predicted molecular masses of the secreted protein, i.e. 76 and 61 kDa respectively. hp76 appears to be evolutionarily conserved as proteins exhibiting high amino acid sequence analogy are found in several mammals and other species, namely Caenorhabditis elegans, Dictyostelium discoideum and Trypanosoma brucei (Supplemental data, Table 3). However, no similar protein is found in yeast. All these proteins are members of a family, based on the presence of a domain named "Laminin A domain". This name likely originates from the organ where the first p76 encoding gene was identified, namely in the Drosophila "lamina" glia [START_REF] Sleat | The human brain mannose 6-phosphate glycoproteome: A complex mixture composed of multiple isoforms of many soluble lysosomal proteins[END_REF][START_REF] Morgan | Identification of phospholipase B from Dictyostelium discoideum reveals a new lipase family present in mammals, flies and nematodes, but not yeast[END_REF]. The human and rodent p76 proteins are highly similar (Supplemental data, Figure 1).

The mouse hypothetical protein LOC71772 is 594 amino acids long and shares 82% identity and 91% similarity with hp76 over a 558 amino acids alignment, whereas the rat hypothetical Copyright 2006 Biochemical Society protein LOC246120 (Ensembl peptide ID ENSRNOP00000001872), is 585 amino acids long and shares 82% identity and 92% similarity with hp76 over a 547 amino acid alignment. The similar protein expressed in the amoeba Dictyostelium discoideum is of particular interest, because it is the only one for which a function (phospholipase B) has been demonstrated in vitro so far [START_REF] Morgan | Identification of phospholipase B from Dictyostelium discoideum reveals a new lipase family present in mammals, flies and nematodes, but not yeast[END_REF].

Expression of hp76 in human tissues

The loc196463 gene is located on the long arm of chromosome 12 (12q24.13) and is predicted to contain 12 exons generating a transcript of 2584 bp. The sequence of the corresponding cDNA (BC030618) is composed of a 28 bp 5'-UTR, a 1767 bp ORF and a 789 bp 3'-UTR containing a polyadenylation signal and a short poly(A) stretch.

A Northern blot experiment (Supplemental data, Figure 2) showed that a transcript migrating at 2.4 kb, consistent with hp76 cDNA length, is found in all tissues studied, with a significantly higher expression in heart, brain and liver. The ubiquitous expression correlates well with the transcriptomic data provided by the Symatlas website. Two additional transcripts were observed: a longer one of 4.4 kb, whose expression roughly follows that of the 2.4 kb transcript, and a shorter one of 0.24 kb only detected in the liver. The origin of these 2 additional transcripts is unknown.

Characterisation of human and mouse p76 maturation fragments

Human p76

As recombinant hp76-myc is secreted in large amounts by 293-EBNA cells transfected with pCEP4/hp76-Myc-His (293/hp76-myc cells), we used this model for initial analysis of hp76 maturation. Comparison by SDS-PAGE of protein profiles of culture supernatants from either mock-transfected 293-EBNA cells (not shown) or 293/hp76-myc cells showed that 3 major hp76-myc-related polypeptides were secreted (Figure 2A, lane 1). N-terminal sequencing of the largest (80 kDa) form indicated that it corresponded to the hp76-myc precursor, after cleavage of its signal peptide between positions 41 and 42 as predicted (Supplemental data, Table 4). Edman analysis of the 32 kDa species identified position 42 at its N-terminus, indicating that this fragment is N-terminal (Figure 2B, and Supplemental data, Table 4). As for the 50 kDa fragment, it starts at position 244 of the preprotein (Figure 2B, and Supplemental data, Table 4). Western blot analysis of the 293/hp76-myc supernatant with hp76Ab, an antibody raised against the recombinant FLAG-hp76 protein, revealed the 80 kDa and the 32 kDa bands, but not the 50 kDa one (Figure 2A, lane 2). Thus, hp76Ab targets hp76 Copyright 2006 Biochemical Society N-terminal epitopes only. The same sample was then analysed with Crp76Ab, an antibody that specifically recognises the C-terminus of rodent p76 and cross reacts with human p76.

Crp76Ab revealed the 80 kDa precursor and the 50 kDa fragment, but not the 32 kDa Nterminal one (Figure 2A, lane 5), indicating that the 50 kDa fragment is C-terminal. Other polypeptides at 40 kDa and 35 kDa were also revealed and these might be additional Cterminal fragments generated through limited proteolysis. These fragments could also be seen in the silver-stained gel (Figure 2A, lane 1). Among U937 Man6P proteins, hp76Ab revealed a strong band of 76 kDa as well as a faint 32 kDa one (Figure 2A, lane 4), while Crp76Ab detected the same 76 kDa band and a 45 kDa doublet (Figure 2A, lane 7).

Therefore, in both our models, U937 Man6P proteins and 293/hp76-myc secreted proteins, the hp76 precursor seems to be matured by a cleavage event occurring after signal peptide cleavage (Figure 2B). An N-terminal 32 kDa fragment is identically produced from hp76 or hp76-myc, whereas the C-terminal fragment appears as a doublet of 45 kDa in U937 Man6P proteins or as a unique 50 kDa polypeptide in 293/hp76-myc secretions. The presence of the myc tag on hp76-myc explains the difference in migration observed between the hp76 precursors (76 kDa versus 80 kDa) and between the C-terminal polypeptides (45 kDa versus 50 kDa) from U937 or 293/hp76-myc samples, respectively.

Mouse p76

Rat and mouse p76 proteins were analysed by immunoblotting and detected with Crp76Ab and Irp76Ab, two antibodies that recognise both rat and mouse p76. They were raised against different peptides of the rat p76 sequence, targeting the last 16 C-terminal residues and an internal peptide located in the C-terminal part of the protein, respectively. Western blot analysis of lysates of HeLa cells overexpressing mouse p76 (mp76) allowed us to visualise 3 major bands migrating at 76 kDa, 40 kDa and 27 kDa (Figure 2C, lanes 1 and 2). These same polypeptides were detected in Man6P proteins purified from rat (not shown) or mouse brain (Figure 2C, lane 3), although the 76 kDa precursor was present as a minor species. In Man6P proteins purified from secretions of a rat cell line, Rat2 (not shown), or of a mouse cell line, Copyright 2006 Biochemical Society major intracellular 27 kDa fragment. This 27 kDa fragment will now be referred to as "mp27" (Figure 2D).

Human p76 precursor glycosylation

All potential glycosylation sites are glycosylated

To determine the actual number of occupied N-glycosylations sites, endogenous Man6P proteins purified from U937 cells were subjected to a time-course deglycosylation by PNGase F and subsequently analysed by western blot, using 3 anti-p76 antibodies: the previously mentioned hp76Ab and Crp76Ab antibodies, and Nhp76Ab, an antibody raised against the Nterminal peptide of hp76. Several bands of decreasing molecular masses ranging from 76 kDa to approximately 60 kDa were progressively produced (Figure 3A). Progressive deglycosylation of the 45 kDa C-terminal doublet gave rise to 2 additional bands of lower molecular masses, with the fully deglycosylated polypeptide migrating at 38 kDa (Figure 3B). Therefore, the initial doublet likely represents di-glycosylated and tri-glycosylated forms of the 38 kDa fragment, present in approximately equivalent amounts among U937 Man6P proteins. Similarly, deglycosylation of the 32 kDa N-terminal fragment produced 3 additional species of decreasing molecular masses, indicating the presence of 3 glycosylations on the 22 kDa polypeptide backbone (Figure 3C). Taken together, these data indicate that all 6 hp76 predicted N-glycosylation sites are actually glycosylated.

p76 is a Man6P protein

Although the specificity of the purification procedure was previously demonstrated [START_REF] Journet | Towards a human repertoire of monocytic lysosomal proteins[END_REF], some proteins might be indirectly trapped on the affinity column through their association with a Man6P protein, rather than being Man6P-labelled themselves. To address this question for hp76, we performed a biotinylated sCI-MPR overlay assay on 2DE-resolved Man6P proteins, in the presence or absence of free Man6P competition. As the hp76 spot positions had been established both by mass spectrometry (Figure 1A and Supplemental data, Table 2) and western blot (Figure 1B), we could identify hp76 among the proteins recognised by the biotinylated sCI-MPR (Figure 1C). Addition of free Man6P effectively competed this labelling (Figure 1D), thus confirming the specificity of the MPR-hp76 interaction. Therefore, hp76 is a Man6P protein, which interacts directly with the sCI-MPR on the affinity column.

Human and mouse p76 co-localise with lysosomal markers

As the presence of Man6P sugars on p76 suggested that p76 might be a lysosomal protein, its localisation was first studied by immunofluorescence. In HeLa cells overexpressing hp76, hp76 was observed in vesicles concentrated in a perinuclear area, as well as dispersed throughout the cytoplasm (Figure 4). Double-staining with a lysosomal membrane marker, LAMP1, or a luminal lysosomal marker, Cathepsin D, showed that hp76 co-localised with both of them to a large extent (Figure 4).

A similar immunofluorescence study was carried out on HeLa cells overexpressing mouse p76, leading to the same results. The intracellular distribution of mp76 also consisted in a vesicular staining mainly co-localising with lysosomal markers, as determined by cathepsin D or LAMP1 co-labelling (not shown).

Sub-cellular fractionation shows a lysosomal distribution for mp27, the 27 kDa Cterminal polypeptide fragment of maturated mouse p76

The sub-cellular localisation of endogenous p76 was established by determining the distribution of mp27, the 27 kDa C-terminal polypeptide fragment of mouse p76 (Figure 2D), in fractions obtained by sub-cellular fractionation of liver homogenates [START_REF] Beaufay | Lysosomes[END_REF]. In the following study, experiments were performed on both rat and mouse samples. As we obtained similar results for both species, experiments carried out on mouse samples only are presented here.

Differential centrifugation

Differential centrifugation of mouse liver homogenates was performed to separate nuclear (N), heavy mitochondrial (M), light mitochondrial (L), microsomal (P) and soluble (S) fractions [24]. As shown in Figure 5A, the distribution of mp27, LAMP1, mitochondrial ATPase F 0 subunit, and CD98 were determined by western blot analysis, or by enzymatic assays for β-galactosidase and β-hexosaminidase. For western blot analysis, the same quantity of proteins was loaded onto each lane of the gel, and thus the intensity of the signal revealed after immunoblotting corresponds to the relative enrichment of the protein in each fraction. with Crp76Ab in all fractions (Figure 5A). Although this signal is not detected by the nonimmune serum (not shown), we assume that it is not related to mp76 because i) Irp76Ab does not reveal this 29 kDa band in those fractions (not shown) and ii) Crp76Ab and Irp76Ab detect this band neither in HeLa cells overexpressing mp76 nor in mouse brain purified Man6P proteins (Figure 2C). The distribution of the mitochondrial ATPase F 0 subunit (strongest signal in the M fraction) and the plasma membrane protein CD98 (in N and P fractions) agreed with the previously reported distribution of these sub-cellular compartments from liver samples [24]. These patterns were clearly distinct from that of lysosomal markers.

Density gradient fractionation

An ML fraction was prepared and further analysed by isopycnic centrifugation on a linear sucrose density gradient. Lysosomes, represented by β-galactosidase and β-hexosaminidase, equilibrated in the heavy fractions of the gradient (Figure 5B). mp27 was detected in the same fractions as these lysosomal markers. However, to assess the significance of this codistribution, lysosomes were submitted to a density shift experiment. We injected mice with a non-haemolytic detergent, Triton WR 1339, that specifically accumulates in lysosomes and strongly reduces their density [START_REF] Wattiaux | Influence of the injection of "Triton WR-1339" on lysosomes of a rat transplantable hepatoma[END_REF]. As shown in Figure 5C, mp27 accompanied the shift of the lysosomes toward the low densities of the gradient after this treatment, providing strong evidence for mp27 residence in lysosomes.

Osmotic release of mp27

We studied the sensitivity of mp27-containing vesicles to an hypo-osmotic treatment [START_REF] Appelmans | Tissue fractionation studies. 3. Further observations on the binding of acid phosphatase by rat-liver particles[END_REF] and compared the release of mp27 to that of known lysosomal markers. Aliquots of an ML fraction were incubated in decreasing sucrose concentration. As shown in Figure 6, the osmotic release curve of the luminal lysosomal hydrolases β-galactosidase, β-hexosaminidase and β-glucuronidase was similar to that of mp27. This result is therefore consistent with mp27 being a soluble polypeptide present in the lysosome. Conversely, the non-specific 29 kDa signal mentioned above (see "differential centrifugation" section) was never released from the membrane fraction.

DISCUSSION

During the course of our proteomic analyses of Man6P proteins [START_REF] Journet | Towards a human repertoire of monocytic lysosomal proteins[END_REF][START_REF] Journet | Proteomic analysis of human lysosomes: Application to monocytic and breast cancer cells[END_REF], a novel protein was identified in two human cell lines, U937 and MCF7. This protein migrated mainly at an apparent molecular mass of 76 kDa, which led us to name it "p76". While we were carrying out the present study, human p76 appeared in the NCBI protein database as the hypothetical protein LOC196463, and the human and murine p76 proteins were identified in proteomic analyses of similarly affinity-purified Man6P proteins from human brain extracts and from mouse cell culture supernatants, respectively [START_REF] Kollmann | Identification of novel lysosomal matrix proteins by proteome analysis[END_REF][START_REF] Sleat | The human brain mannose 6-phosphate glycoproteome: A complex mixture composed of multiple isoforms of many soluble lysosomal proteins[END_REF]. In the latter study, murine p76 was named "66.3 kDa protein". p76 was also identified in a proteomic analysis of purified neuromelanin granules from human brain [START_REF] Tribl | Subcellular proteomics of neuromelanin granules isolated from the human brain[END_REF]. Human p76 is a 589 residue protein with a putative 41 amino acid signal peptide and 6 potential glycosylation sites.

All potential glycosylations sites of human p76 are glycosylated, and some of them are effectively mannose-6-phosphorylated

The extent of human p76 glycosylations was determined on U937 Man6P proteins, and we unambiguously showed that all 6 potential hp76 glycosylation sites were occupied. Some glycosylation heterogeneity was observed, residing on either one of the 3 positions 436, 465, or 515. Such heterogeneous glycosylations are common among Man6P proteins [START_REF] Von Figura | Lysosomal enzymes and their receptors[END_REF].

However, in a recent global proteomic study which aimed at listing the mannose-6phosphorylated glycopeptides of purified brain Man6P proteins from both human and mouse origin [START_REF] Sleat | Identification of sites of mannose 6-phosphorylation on lysosomal proteins[END_REF], Sleat et al. did not observe heterogeneous glycosylation at any of these 3 positions. This discrepancy might be due to the respective p76 sources, i.e. brain Man6P proteins versus Man6P proteins secreted from a monocytic cell line. Since hp76 could have been be purified on the MPR affinity column through association with a true Man6P-bearing protein, without being mannose-6-phosphorylated itself, we checked hp76 Man6P status. We showed unambiguously a direct and Man6P-dependent interaction between hp76 and sCI-MPR, indicating that hp76 is a true Man6P protein. Our overlay results confirm the proteomic analysis published by Lobel's group, which detects the presence of Man6P on 5 glycopeptides of brain hp76 [START_REF] Sleat | Identification of sites of mannose 6-phosphorylation on lysosomal proteins[END_REF]. This high number of Man6P glycopeptides in hp76 is interesting with regard to other known lysosomal Man6P proteins. Indeed, for the 43 known lysosomal proteins that were identified through their Man6P-glycopeptides, an average Copyright 2006 Biochemical Society sulfatase (9 peptides) [START_REF] Sleat | Identification of sites of mannose 6-phosphorylation on lysosomal proteins[END_REF]. The high number of Man6P in hp76 might have implications for the specificity of its recognition by the MPR and for its potential lysosomal targeting efficiency. Besides, this result might suggest that although the 6 human p76 glycosylation sites are occupied (our study), only 5 of them are mannose-6-phosphorylated. The Nglycosylation in position 110 of hp76 could be of the complex type and therefore absent from the Man6P glycopeptides identified by Sleat et al.. Differences in the sugar types borne by lysosomal proteins do exist, such as for human β-glucuronidase and human β-hexosaminidase B, which both have only 2 mannose-6-phosphorylated N-glycosylations out of their 4 Nglycosylated sites [START_REF] Sonderfeld-Fresko | Analysis of the glycosylation and phosphorylation of the lysosomal enzyme, beta-hexosaminidase B, by site-directed mutagenesis[END_REF][START_REF] Shipley | The role of glycosylation and phosphorylation in the expression of active human beta-glucuronidase[END_REF].

Human and mouse p76 are subject to maturation

In human secreted Man6P proteins, we observed an N-terminal fragment of 32 kDa as well as a 45 kDa C-terminal doublet, both specific for hp76. The corresponding fragments were also observed on the secreted recombinant hp76-myc protein. Their N-terminal sequencing identified the signal peptide cleavage site, between positions 41 and 42, in addition to an internal cleavage site between position 243 and 244. In human brain Man6P proteins [START_REF] Sleat | The human brain mannose 6-phosphate glycoproteome: A complex mixture composed of multiple isoforms of many soluble lysosomal proteins[END_REF], hp76 was found in numerous spots in the 42-30 kDa molecular mass range. An N-terminal sequence starting at position 291 of hp76 was determined, indicating the presence of at least a C-terminal fragment in brain. Nonetheless, the 32 kDa N-terminal chain might also be present in these spots. The 2 different N-terminal sequences obtained for the C-terminal fragment might be due to different proteolytic events occurring for the respective proteins, i.e. the recombinant protein secreted from cells in culture (this study) versus the intracellular protein from brain [START_REF] Sleat | The human brain mannose 6-phosphate glycoproteome: A complex mixture composed of multiple isoforms of many soluble lysosomal proteins[END_REF].

In Man6P proteins purified from mouse cell secretions, our antibodies allowed us to detect a 40 kDa mp76 C-terminal fragment. Intracellularly, this 40 kDa fragment appeared to be further processed, since an extra 27 kDa C-terminal fragment (mp27) was detected in mp76 cDNA transfected cell lysates, in the enriched ML fraction from mouse liver, and in mouse brain Man6P proteins. As the 40 kDa mp76 fragment was detected in cell secretions, the first proteolytic mp76 cleavage is likely to occur before segregation of lysosomal enzymes from the secretory pathway. The cleavage event responsible for mp27 formation would happen once the 40 kDa fragment is engaged in the endocytic pathway. In a proteomic study of secreted mouse Man6P proteins [START_REF] Kollmann | Identification of novel lysosomal matrix proteins by proteome analysis[END_REF], murine p76 was found on a 2DE gel in 3 series of spots, migrating at molecular masses of 66 kDa, 40 kDa and 30 kDa. These species might correspond to the precursor, the C-terminal and the N-terminal fragments, respectively. The Copyright 2006 Biochemical Society difference in the apparent molecular masses of the p76 precursor (76 kDa versus 66 kDa) in our study and in Kollmann's one might be due to lower mp76 glycosylation or differences in the respective electrophoresis gels and standards. Maturation events of this type are not uncommon for lysosomal proteins. The human 53 kDa Cathepsin D or the 85 kDa β-galactosidase precursors are proteolytically processed so that the mature forms are hetero-dimers composed of a heavy and a light chain in lysosomes [START_REF] Gieselmann | Biosynthesis and transport of cathepsin D in cultured human fibroblasts[END_REF][START_REF] Van Der Spoel | Processing of lysosomal betagalactosidase. The C-terminal precursor fragment is an essential domain of the mature enzyme[END_REF].

Conversely, human β-glucuronidase loses a small C-terminal peptide upon maturation [START_REF] Erickson | Carboxyl-terminal proteolytic processing during biosynthesis of the lysosomal enzymes beta-glucuronidase and cathepsin D[END_REF]. In conclusion, we propose that the 76 kDa p76 precursor is cleaved into a 32/30 kDa N-terminal polypeptide and a 45/40 kDa C-terminal polypeptide (human versus mouse; this study and [START_REF] Kollmann | Identification of novel lysosomal matrix proteins by proteome analysis[END_REF]). Intracellularly, a further cleavage would occur in the C-terminal chain, at least for the rat and mouse proteins, generating mp27.

p76 is a lysosomal protein

Proteins bearing Man6P glycosylations are mainly soluble lysosomal proteins, despite the known existence of a few non-lysosomal Man6P proteins [START_REF] Faust | Renin, a secretory glycoprotein, acquires phosphomannosyl residues[END_REF][START_REF] Herzog | Thyroglobulin, the major and obligatory exportable protein of thyroid follicle cells, carries the lysosomal recognition marker mannose-6-phosphate[END_REF][START_REF] Lee | Proliferin secreted by cultured cells binds to mannose 6-phosphate receptors[END_REF][START_REF] Purchio | Identification of mannose 6phosphate in two asparagine-linked sugar chains of recombinant transforming growth factor-β1 precursor[END_REF][START_REF] Todderud | Presence of mannose phosphate on the epidermal growth factor receptor in A-431 cells[END_REF][START_REF] Cacia | Human DNase I contains mannose 6-phosphate and binds the cation-independent mannose 6-phosphate receptor[END_REF]. Nonetheless, the recent identification of numerous non-lysosomal Man6P proteins in plasma weakened this idea [11].

This finding reinforces the necessity to experimentally assess the sub-cellular localisation of any newly identified Man6P protein. Accordingly, Sleat and collaborators defined 3 criteria to select new candidate lysosomal proteins: i) enrichment of proteins by MPR purification cycles, ii) low abundance in plasma, and iii) predicted or known function compatible with a lysosomal localisation. Hence, among the 44 Man6P proteins that were identified in plasma and were not known to be lysosomal, 9 candidates were selected, among which hp76 [11].

Besides, the identification of hp76 in a proteomic analysis of neuromelanin granules from human brain also brought support to the hypothesis that p76 could be a lysosomal protein [START_REF] Tribl | Subcellular proteomics of neuromelanin granules isolated from the human brain[END_REF]. Indeed, the authors demonstrated that neuromelanin granules belonged to the lysosomelike organelles family. Moreover, Kollmann et al. have shown that purified recombinant mp76 was internalized via MPR-mediated endocytosis [START_REF] Kollmann | Identification of novel lysosomal matrix proteins by proteome analysis[END_REF], giving another argument in favor of a lysosomal localisation of p76.

To determine p76 sub-cellular localisation, we firstly used immunofluorescence on cells overexpressing either human or mouse p76, and showed a clear co-localisation of p76 with the lysosomal markers LAMP1 and Cathepsin D. To analyse the sub-cellular localisation of endogenous p76, we performed biochemical studies on rat and mouse liver samples, in which p76 was detected as the mp27 maturated polypeptide. The similar behaviour of mp27 and lysosomal marker proteins in sub-cellular fractionation experiments (differential Copyright 2006 Biochemical Society centrifugation, isopycnic centrifugation of ML fractions on linear sucrose density gradients, with or without a specific reduction of the lysosome density), and in osmotic release experiments strongly indicates residence of p76 in lysosomes. Taken together, these results give very strong evidence for p76 being a lysosomal protein.

Concluding remarks

Now that the lysosomal localisation of p76 has been clearly assessed, the next step will be to determine its function. As the only protein displaying significant sequence similarity with p76 and for which a function has been experimentally demonstrated is a Dictyostelium discoideum phospholipase B [START_REF] Morgan | Identification of phospholipase B from Dictyostelium discoideum reveals a new lipase family present in mammals, flies and nematodes, but not yeast[END_REF], we are setting up functional assays for the analysis of the putative phospholipid degradation by p76. As little is known about phospholipid catabolism, the identification of p76 as a novel lysosomal phospholipase and the analysis of the possible effects of a deficiency in this protein would be an important contribution to lysosome comprehension. Note: While the present report was under revision, a study has been published on the mouse p76 (66.3 kDa protein; [START_REF] Deuschl | Molecular characterization of the hypothetical 66.3-kDa protein in mouse: Lysosomal targeting, glycosylation, processing and tissue distribution[END_REF]). The experimental data presented in this publication complement our own work and strongly support our conclusions.

Acknowledgements

  JR11 [19], only the 76 kDa and 40 kDa polypeptides were revealed (Figure 2C, lane 4). The presence of an additional 27 kDa p76 polypeptide fragment in the HeLa cell lysate and the brain samples is probably due to an additional maturation event occurring inside the cell only. As the antibodies target C-terminal peptides, the 40 kDa and 27 kDa polypeptides are Cterminal fragments of the 76 kDa precursor. The 40 kDa polypeptide, which is probably the equivalent of the 45 kDa doublet observed for human p76, must be cleaved to generate a Biochemical Journal Immediate Publication. Published on 15 Nov 2006 as manuscript BJ20061205

For

  enzymatic assays, β-galactosidase and β-hexosaminidase activities are represented as proposed by de Duve [24]: bar height (specific activity) is indicative of the lysosome purification in the N, M, L, P and S fractions. As shown in Figure 5A, a strong mp27 signal was detected in the total mitochondrial fraction (M and L) with the strongest signal in the L fraction. This pattern is highly indicative of liver lysosomes, as shown by the distributions of LAMP1 and lysosomal enzymes. It should be noted that a 29 kDa fragment was also detected Biochemical Journal Immediate Publication. Published on 15 Nov 2006 as manuscript BJ20061205 Copyright 2006 Biochemical Society

of 2

 2 Man6P-glycopeptides was identified per protein. Only 2 hydrolases displayed 5 or more Man6P-glycopeptides: the acid ceramidase (5 peptides) and the N-acetylglucosamine-6-Biochemical Journal Immediate Publication. Published on 15 Nov 2006 as manuscript BJ20061205

Biochemical

  Journal Immediate Publication. Published on 15 Nov 2006 as manuscript BJ20061205

:

  The authors are grateful to D. Grünwald for help and guidance with confocal microscopy, B. Hoflack for the kind gift of JR11 cells, A. Dupuis for helpful discussions and M. Gallagher-Gambarelli for reading of the manuscript. This work was supported by the INSERM, the Life Sciences Division of the CEA (Commissariat à l'Energie Atomique), the Joseph Fourier University and the Fonds de la Recherche Fondamentale Collective (Contract no. 2.4505.01). A.G.J. and M.C. are recipients of fellowships from the CEA.

Figure 1 :

 1 Figure 1: Identification of hp76 as a Man6P protein(A) Man6P proteins purified from 2.5×10 9 U937 cells were resolved by 2DE over a 4-8 linear pH range, 10% SDS-PAGE gel (20×20 cm) before colloidal blue staining (modified from[START_REF] Journet | Proteomic analysis of human lysosomes: Application to monocytic and breast cancer cells[END_REF] with permission). (B to D) Man6P proteins purified from 200×10 6 U937 cells were resolved by 2DE over a 4-8 linear pH range, 10% SDS-PAGE minigels (9×9 cm), transferred onto nitrocellulose membranes and probed with hp76Ab (B) or with 3.3 nM of biotinylated sCI-MPR without (C) or with (D) 5 mM Man6P. White oval: main series of hp76 spots; black oval: position of the minor series of hp76-containing spots which could only be detected with high protein amounts.

Figure 2 :

 2 Figure 2: Human and mouse p76 maturation (A) Characterisation of human p76-derived polypeptides. Protein samples were loaded onto a 12% SDS-PAGE gel and either silver-stained (lane 1) or transferred for western blot analysis with hp76Ab (lanes 2-4) or with Crp76Ab (lanes 5-7). Lane 1: Man6P proteins purified from 293/hp76-myc supernatant; lanes 2 and 5: 293/hp76-myc supernatant; lanes 3 and 6: 293/mock supernatant; lanes 4 and 7: Man6P proteins purified from U937. Lane 4 is overexposed compared to the other lanes. (B) Hypothetical maturation scheme for human p76. hp76Ab targets epitopes in the Nterminal part of hp76 (light grey). Crp76Ab targets the C-terminal peptide of hp76 (black). (C) Characterisation of mouse p76-derived polypeptides. Protein samples were loaded onto a 12% SDS-PAGE gel and transferred for western blot analysis with Crp76Ab. Lane 1: untransfected HeLa cell lysate; lane 2: lysate of HeLa cells transfected with wild-type mp76 cDNA; lane 3: purified mouse brain Man6P proteins; lane 4: purified JR11 Man6P proteins.(D) Hypothetical maturation scheme for mouse p76. Crp76Ab targets the C-terminal peptide of rat and mouse p76 (black). Irp76Ab targets an internal peptide of rat and mouse p76 (dark grey).

Figure 3 :

 3 Figure 3: hp76 N-glycosylations U937 Man6P proteins were subjected to deglycosylation by PNGase F for the indicated times, loaded onto 7.5% (A; equivalent of 6 × 10 6 cells/lane) or 9% (B, C; equivalent of 30 × 10 6 cells/lane) SDS-PAGE gels and transferred for western blot analysis. Additional PNGase F

Figure 4 :

 4 Figure 4: Co-localisation of hp76 with lysosomal markers HeLa cells were transfected with wild type hp76 cDNA. At 72 h post-transfection, cells were fixed and double-immunolabelled for hp76 (with Crp76Ab, in red) and LAMP1 (A) or cathepsin D (B) (in green). Arrowheads denote some vesicles that are positive for both stainings. Scale bars, 10 µm.

Figure 5 :

 5 Figure 5: Distribution of mp27 in mouse liver sub-cellular fractions.(A) Differential centrifugation of mouse liver. The distribution of various markers and mp27 was analysed either by western blotting (mp27, LAMP1, mitochondrial ATPase and CD98) and quantified using the NIH (National Institutes of Health) Image software or by enzymatic activity measurement (β-galactosidase, β-hexosaminidase). Fractions correspond to nuclear (N), heavy mitochondrial (M), light mitochondrial (L), microsomal (P) and soluble (S) fractions. Equal amounts of proteins (40 µg) were loaded for each fraction onto a 10% SDS-PAGE gel and transferred for western blot analysis. mp27 was detected by Crp76Ab. For all graphs, the ordinate is the relative specific activity or intensity and the abscissa is the relative protein content plotted cumulatively from left to right. Asterisk: non-specific band.

FIGURES Figure 1

 1 FIGURES

Biochemical Journal Immediate Publication. Published on 15 Nov 2006 as manuscript BJ20061205 Copyright 2006 Biochemical Society

Biochemical Journal Immediate Publication. Published on 15 Nov 2006 as manuscript BJ20061205

Biochemical Journal Immediate Publication. Published on 15 Nov 2006 as manuscript BJ20061205

Biochemical Journal Immediate Publication. Published on 15 Nov 2006 as manuscript BJ20061205 Copyright 2006 Biochemical Society

	Carbohydrate modification analysis
	Purified U937 Man6P proteins were deglycosylated by PNGase F (E.C. 3.5.1.52, Roche),
	according to the manufacturer's instructions. The reaction was stopped by addition of
	Laemmli sample buffer [22], or by boiling. In the latter case, proteins were concentrated on
	Strataclean beads (Stratagene), and eluted by boiling in Laemmli sample buffer prior to
	Western blot analysis.

Biochemical Journal Immediate Publication. Published on 15 Nov 2006 as manuscript BJ20061205 Copyright 2006 Biochemical Society

Biochemical Journal Immediate Publication. Published on 15 Nov 2006 as manuscript BJ20061205

Biochemical Journal Immediate Publication. Published on 15 Nov 2006 as manuscript BJ20061205

Biochemical Journal Immediate Publication. Published on 15 Nov 2006 as manuscript BJ20061205 Copyright 2006 Biochemical Society

Biochemical Journal Immediate Publication. Published on 15 Nov 2006 as manuscript BJ20061205

Biochemical Journal Immediate Publication. Published on 15 Nov 2006 as manuscript BJ20061205