Ana R Correia

Salvatore Adinolfi

Annalisa Pastore

Cláudio M Gomes
email: gomes@itqb.unl.pt

Conformational stability of human frataxin and effect of Friedreich's ataxia-related mutations on protein folding

Keywords: FRDA, Friedreich ataxia, GdmCl, guanidinium chlroride, Protein folding, clinical mutants, iron-binding protein, trinucleotide expansion Biochemical

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

Introduction

The possibility of identifying genetic mutations causing human diseases is a revolutionary achievement acquired only within the last 3-4 decades, which has opened new avenues towards our understanding of pathologies. We now know that even a single amino acid mismatch from the wild-type protein sequence may result in major metabolic consequences in an organism thus often leading to severe pathologies.

Understanding which effect point mutations have is therefore an important topic essential not only for our basic knowledge of how proteins fold but with direct applications to molecular medicine.

We have recently become interested in single point mutations observed in Friedreich ataxia (FRDA), an autosomal recessive neurodegenerative disease linked to oxidative stress and caused by reduced levels of a small mitochondrial protein, frataxin, encoded by a gene mapped in chromosome 9q13 [START_REF] Pandolfo | Molecular pathogenesis of Friedreich ataxia[END_REF][START_REF] Chamberlain | Mapping of mutation causing Friedreich's ataxia to human chromosome 9[END_REF]. Although the exact function of frataxin is still unknown, increasing evidence suggests a role of the protein in iron-sulphur cluster formation [START_REF] Ramazzotti | Mitochondrial functional interactions between frataxin and Isu1p, the iron-sulfur cluster scaffold protein, in Saccharomyces cerevisiae[END_REF][START_REF] Duby | A nonessential function for yeast frataxin in iron-sulfur cluster assembly[END_REF][START_REF] Gerber | An interaction between frataxin and Isu1/Nfs1 that is crucial for Fe/S cluster synthesis on Isu1[END_REF][START_REF] Yoon | Frataxin-mediated iron delivery to ferrochelatase in the final step of heme biosynthesis[END_REF][START_REF] Yoon | Iron-sulfur cluster biosynthesis. Characterization of frataxin as an iron donor for assembly of [2Fe-2S] clusters in ISU-type proteins[END_REF][START_REF] Bulteau | Frataxin acts as an iron chaperone protein to modulate mitochondrial aconitase activity[END_REF][START_REF] Park | Yeast frataxin sequentially chaperones and stores iron by coupling protein assembly with iron oxidation[END_REF]. The majority of FRDA patients are homozygous for an intronic expansion of a GAA trinucleotide repeat within the frataxin gene [START_REF] Cossee | Friedreich's ataxia: point mutations and clinical presentation of compound heterozygotes[END_REF]. The abnormal triplet expansion does not completely abolish frataxin expression, and a shorter magnitude of the expansion correlates with the late-onset of the disease. However, a few FRDA patients (ca. 4%) are heterozygous, containing an expanded repeat in one allele and a deleterious point mutation in the other [START_REF] Cossee | Friedreich's ataxia: point mutations and clinical presentation of compound heterozygotes[END_REF]. These DNA alterations can either lead to protein truncation or to missense modifications in the mature region of the protein.

The latter are particularly interesting as they have different impacts on disease expression: whereas some mutations lead to the normal phenotype of the disease, others result in atypical, milder clinical presentations of the pathology. At the present stage, at least 15 missense frataxin point-mutations have been described in FRDA patients [START_REF] Cossee | Friedreich's ataxia: point mutations and clinical presentation of compound heterozygotes[END_REF].

Reports on these mutations [START_REF] Cossee | Friedreich's ataxia: point mutations and clinical presentation of compound heterozygotes[END_REF] indicate that they do not interfere with splicing and the fact that some compound heterozygous individuals have atypical phenotypic expressions of the disease is suggestive of the cellular presence of frataxin with a partly reduced function [START_REF] Cossee | Friedreich's ataxia: point mutations and clinical presentation of compound heterozygotes[END_REF][START_REF] Campuzano | Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion[END_REF]. Although mapping these mutations onto the available threedimensional structures of frataxin [START_REF] Adinolfi | A structural approach to understanding the iron-binding properties of phylogenetically different frataxins[END_REF][START_REF] Musco | Towards a structural understanding of Friedreich's ataxia: the solution structure of frataxin[END_REF][START_REF] Dhe-Paganon | Crystal structure of human frataxin[END_REF][START_REF] Musco | Assignment of the 1H, 15N, and 13C resonances of the C-terminal domain of frataxin, the protein responsible for Friedreich ataxia[END_REF] can provide a framework for predicting its effect on the protein structure, the exact impact of these clinically relevant missense mutations on the folding pathway of frataxin remains to be experimentally and quantitatively addressed.

Here, we report our studies on the structural stability and folding properties of human frataxin, and on two mutant variants comprising clinically relevant point mutations, I154F and W155R. These modifications involve highly conserved amino acids and are located in structurally distinct regions of the protein [START_REF] Dhe-Paganon | Crystal structure of human frataxin[END_REF]. Previous studies have suggested that the two mutations represent two extreme possibilities [START_REF] Cossee | Friedreich's ataxia: point mutations and clinical presentation of compound heterozygotes[END_REF][START_REF] Musco | Towards a structural understanding of Friedreich's ataxia: the solution structure of frataxin[END_REF]. The first one, I154F, constitutes the most common clinically occurring mutation and affects a residue which is part of the protein core. This mutant is therefore expected to directly affect the stability of the protein fold and should be structurally important. The second mutation involves instead the conserved W155, which is part of an invariant surface in the exposed region of a β-sheet. The aromatic ring of W155 is fully solvent exposed, suggesting a role of this residue in interaction with a partner. The mutation is expected to be functionally important since conservation of surface residues usually implies that they are directly involved in the protein functions. Both mutations lead to aggressive phenotypic development of the ataxia [START_REF] Cossee | Friedreich's ataxia: point mutations and clinical presentation of compound heterozygotes[END_REF][START_REF] Musco | Towards a structural understanding of Friedreich's ataxia: the solution structure of frataxin[END_REF]. In a preliminary report we showed that both these mutants are folded [START_REF] Musco | Towards a structural understanding of Friedreich's ataxia: the solution structure of frataxin[END_REF]. While this result constitutes little surprise for W155R, the fact that the clinical mutation I154F, which in vivo leads to a severe expression of the pathology, results in a protein which is essentially folded under physiological conditions may seem paradoxical in respect to disease expression.

To understand further this apparent paradox, we have extended and complemented previous preliminary data by carrying out an extensive study of the thermodynamic stability of frataxin and its variants using a number of complementary biochemical and biophysical techniques. By exploring different chemical denaturants and experimental conditions, we find that both mutations affect the thermodynamic stability of the proteins but to a very different extent so that the destabilization does not affect the fold of the proteins at physiological temperatures or pH. We show that the structural dynamics of mutant frataxin variants is altered in respect to the wild type protein, resulting in a partly destabilised conformation, more prone to proteolytic processing. Furthermore, both proteins retain partly their iron binding properties. This suggests, in particular, that W155 and the surface around it are implicated in a function distinct from iron binding. On the basis of our results, we suggest a mechanism to explain the strong pathology associated with I145F. Our study may therefore contribute to a molecular understanding of the frataxin functions and ultimately of its role in FRDA pathology.

Experimental

Chemicals

All reagents were of the highest purity grade commercially available. The chemical denaturant guanidine thiocyanate (GuSCN) was purchased from Promega. Stock solutions were prepared in different buffers and the concentration determined by its density. Guanidinium chloride (GdmCl) was obtained from Sigma and the accurate concentration of the stock solutions in different buffers was confirmed by refractive index measurements.

Gene Expression and Protein purification

The constructs (from the truncated frataxin (91-210), hereon simply Hfra(91-210), and mutant forms) were expressed in E. coli (competent cells BLC21 DE3 from Novagen) as fusion proteins with a His and a GST tag with cleavage site for tobacco etch virus (TEV) or PreScission protease as previously described [START_REF] Musco | Towards a structural understanding of Friedreich's ataxia: the solution structure of frataxin[END_REF][START_REF] Adinolfi | The factors governing the thermal stability of frataxin orthologues: how to increase a protein's stability[END_REF]. Briefly the wild type and the two frataxin variants were purified to homogeneity, as follows the cells were grown in LB medium with 30μg/ml of kanamycin. The soluble cell extract was applied on a glutathione Sepharose column (10ml) previously equilibrated with PBS buffer, and eluted with 30ml of PBS buffer, after which TEV was added (2ml of 25μM TEV per litter of cell culture). The column was closed and left overnight at 4ºC. The purified protein was eluted by washing the column with PBS buffer. The tags as well as TEV remained bound and were subsequently eluted with reduced glutathione. Protein concentration was determined using the extinction coefficient

ε 280nm = 21030 M -1 .cm -1 .
Both mutants were found to be stable in solution although susceptible to precipitate upon slow freezing; however, thawed proteins that had been fast frozen retained their spectroscopic properties and melting temperatures.

Spectroscopic methods

UV/Vis Spectra were recorded at room temperature in a Shimadzu UVPC-1601 spectrometer equipped with cell stirring. Fluorescence spectroscopy was performed on a Cary Varian Eclipse instrument (λ ex =280nm, λ em =340nm, slit ex : 5 nm, slit em : 10 nm unless otherwise noted) equipped with cell stirring and Peltier temperature control. Far-UV CD spectra were recorded typically at 0.2 nm resolution on a Jasco J-715 spectropolarimeter fitted with a cell holder thermostated with a peltier.

Chemical denaturation

The denaturation curves were measured using the dilutions method, and two solutions with the same protein concentration were prepared, one with no denaturation agent and the other with a high concentration of denaturation agent. These were combined in different proportions yelling different denaturant concentrations. After mixing, the solutions were left equilibrating for two hours. Transitions curves were determined plotting the average emission wavelength versus denaturant concentrations, correcting for the pre and pos transitions [START_REF] Shirley | Methods in Molecular Biology -Proteins Stability and Folding Theory and Practice[END_REF][18][19]. A non-linear least-square analysis was used to fit the data to a two state model described by equation ( 1

) [19]: [ ] ( ) [ ] ( ) [ ] [ ] ( ) [ ] [ ] ( ) ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ⎥ ⎥ ⎦ ⎤ ⎢ ⎢ ⎣ ⎡ - ⋅ + ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ⎥ ⎥ ⎦ ⎤ ⎢ ⎢ ⎣ ⎡ - ⋅ ⋅ ⋅ + + ⋅ + = RT D D m RT D D m D m y D m y Y u u f f 2 1 2 1 exp 1 exp (1)
Where y represents the average emission wavelength observed for a given concentration [ ]

Denaturant m G G O H - Δ = Δ 2 (2)

Thermal denaturation

Thermal unfolding was followed by monitoring the intrinsic tryptophan fluorescence (λ exc =280 nm, λ em =340 nm, slit ex =5 nm and slit em =10 nm) and the ellipticity ( Δε mrw at 222nm) variations. It has been previously demonstrated [START_REF] Bolis | Protein stability in nanocages: a novel approach for influencing protein stability by molecular confinement[END_REF] that identical melting temperatures are obtained for frataxin, by both CD and fluorescence spectroscopies, thus ruling out artefacts resulting from changes of fluorescence quantum yield with temperature. Further, DSC experiments confirmed melting temperatures (data not shown). In all experiments a heating rate of 1ºC/min was used, and the temperature was changed from 20 to 90ºC. Data were analysed according to a two-state model described by equation 3,

[ ] ( ) [ ] ( ) ( ) ( ) ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ⎥ ⎦ ⎤ ⎢ ⎣ ⎡ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ - ⋅ ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ Δ + ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ⎥ ⎦ ⎤ ⎢ ⎣ ⎡ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ - ⋅ ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ Δ ⋅ ⋅ + + ⋅ + = m m m m m m u u f f T T T RT H exp 1 T T T RT H exp T m y T m y Y ( 3
)
were Y is the spectroscopic signal observed, m f and m u the slopes of the pre-and posttransition baselines; y f and y u correspond to the value of y for folded and unfolded forms, T m is the midpoint of the thermal unfolding curve, and ΔH m is the enthalpy change for unfolding at T m [19]. The fits to the unfolding transitions were made using Origin (MicroCal). For pH variations, the buffers used were 30mM acetate (pH 4.9), 40mM phosphate (pH 6.0, pH 7.9 and pH 11.6), 10mM HEPES (pH 7.0) and 40mM glycine (pH 8.9 and 9.7). The reversibility of the reaction was investigated by downward temperature ramps, as well as repetition of the upward ramps after cooling down of the sample to 25°C.

Fluorescence quenching

Quenching experiments were preformed using a neutral quencher -acrylamide (Bio-Rad) and an ionic quencher KI (Fluka). The samples were excited at 295nm in order to ensure that the light was absorbed almost entirely by tryptophanyl groups and the fluorescence intensity decrease at 340nm was followed. Results were analysed according to the Stern-Volmer (equation 4).

[ ]

Q K F F SV + = 1 0 (4)
where F 0 and F are the fluorescence intensities in the absence and presence of quencher, respectively, K SV is the collisional quenching constant and [Q] is the quencher concentration [START_REF] Eftink | Fluorescence quenching studies with proteins[END_REF].

Trypsin limited proteolysis

Frataxins were incubated with trypsin (bovine pancreas trypsin, sequencing grade, Sigma) at 37°C in 0.1 M Tris-HCl pH 8.5, in a 100 fold excess over the protease. Aliquots (~0.5 nmol protein) were sampled at different incubation periods and the reaction stopped by the addition of 0.2% (v/v) of trifluoroacetic acid (TFA). The products of the proteolysis reaction were analysed by reverse-phase HPLC (Waters Alliance System 2695), using a C18 Column 150 x 3.9 mm (DeltaPak, Waters) run at a 0.5 ml.min -1 flow rate, monitoring the absorbance at 214nm. The peptidic profiles were obtained running a three-step a gradient from (A) 0.1% (v/v) TFA to (B) 80% (v/v) Acetonitrile + 0.1% (v/v) TFA: 0-37% (in 65 min), 37-75% (in 30 min) and 75-100% (in 20 min). The column was regenerated with 0.1% (v/v) TFA.

Results

Thermodynamics of frataxin chemical and thermal unfolding

To evaluate the effect of the introduced clinical mutations on the human frataxin fold, the chemical and thermal unfolding reactions of human frataxin were investigated in detail. Frataxin unfolding can be monitored either by fluorescence or by far UV circular dichroism spectroscopies (Fig. 1); in the present work, intrinsic tryptophan fluorescence changes were routinely used to monitor unfolding transitions (see Experimental). In most denaturation conditions tested, frataxin unfolding was found to be highly reversible. Hfra(91-210) exhibits a considerable stability over the physiological pH range: its thermal unfolding transition is reversible and essentially invariant between pH 6 and 9 (Table 1). However, below pH 5, which corresponds to the isoelectric point of Hfra(91-210) (pI=5), as well as at higher pH values, an increasingly extent of irreversibility was observed as a result of protein aggregation. At lower pH values this is expected considering the decreased solubility of the unfolded form of a protein near its isoelectric point [19,[START_REF] Creighton | Proteins Structure and molecular properties[END_REF].

Chemical denaturation experiments were performed at pH 7.9 using urea, guanidinium hydrochloride (GdmCl) and guanidinium thiocyanate (GuSCN) as denaturants. The obtained curves were analysed assuming a two-state mechanism, and the unfolded fraction was plotted as a function of denaturant concentration; from the data in the transition region, and using the linear extrapolation model, the thermodynamic parameters ΔG(H 2 O) and m were determined (Fig. 2, Table 2). Frataxin conformational stability ranges from 23.8 to 35.6 kJ.mol -1 (5.7 to 8.5 kcal.mol -1 ).

Conformational dynamics of mutant frataxins

The impact of the clinical mutations on the frataxin fold and dynamics was evaluated from fluorescence quenching studies. Fluorescence quenching experiments were performed at 25°C, using acrylamide and iodine (Fig. 3, Table 3). Acrylamide is a neutral quencher molecule, more prone to access to the protein interior, and quench buried residues, whereas iodide is negatively charged. The Stern-Volmer constant for the I154F Hfra(91-210) is comparable to that of the wild type for both quenchers, which shows that this structural core mutation does not result in protein misfolding. In respect to the W155R Hfra(91-210) variant, a lower Stern-Volmer constant is determined, in agreement with the fact that since the two remaining tryptophans are not superficial, the protein matrix will strongly slow down the penetration of the quencher molecules. For all frataxins studied, the accessibility of the tryptophans is increased in the presence of GdmCl, corresponding to a disrupted tertiary structure. Thus, quenching experiments show that under physiological conditions, the mutant frataxins have a globally stable, wild type like fold and breathing dynamics.

Limited proteolysis experiments evidence destabilization of mutant frataxin forms

The conformational differences between wild type and the mutant frataxin forms were accessed by monitoring the progression of trypsin mediated proteolysis. Trypsin was selected for this comparative mapping as it cleaves with high specificity at the COOHterminal side of lysine and arginine, unless the following position is occupied by a proline [23]. Inspection of the protein sequence identifies 11 possible digestion sites for the wild type and I154F proteins and an additional one for the W155R mutant, as a result of the inserted arginine. A comparison of these limited proteolysis experiments allows evaluating the degree of destabilization of the mutant forms in respect to the native protein, since we expect that a destabilised fold will have more accessible cleavage sites and undergo a faster proteolysis. The obtained peptide maps clearly show that both mutant frataxins are destabilised in respect to the wild type protein, as evidenced by the appearance of a substantially higher number of peptides during the same incubation period (Fig. 4). This observation indicates that the clinical mutant frataxins under study comprise partly destabilised regions which make them more prone to degradation.

Iron binding properties of frataxin clinical variants are impaired by protein destabilisation

Iron binding was also investigated as this aspect may provide useful insights into the biological impact of the mutated variants, since frataxin has been proposed to act as a cellular iron chaperon. Frataxin is known to be able to bind 6-7 irons per molecule, albeit with low affinity [START_REF] Yoon | Iron-sulfur cluster biosynthesis. Characterization of frataxin as an iron donor for assembly of [2Fe-2S] clusters in ISU-type proteins[END_REF][START_REF] Nair | Solution structure of the bacterial frataxin ortholog, CyaY: mapping the iron binding sites[END_REF]. The FRDA associated mutant frataxins were investigated in respect to their ability to bind iron, as well as the wild type protein, which was used as a positive control. Iron binding monitored by fluorescence spectroscopy according to a previously reported procedure [START_REF] Yoon | Iron-sulfur cluster biosynthesis. Characterization of frataxin as an iron donor for assembly of [2Fe-2S] clusters in ISU-type proteins[END_REF] showed that progressive iron binding destabilises the mutants, resulting in protein precipitation. Although this occurs both with Fe(II) and Fe(III), and under pH controlled conditions, the ferric ion seems to have a more destabilizing effect and precipitation is observed above an iron:frataxin ratio of 2. Concerning ferrous ion binding, precipitation was only observed at iron:frataxin ratios ranging from 5 to 7. Thus, although none of these FRDA associated mutations is directly involved in proposed iron binding region [START_REF] Nair | Solution structure of the bacterial frataxin ortholog, CyaY: mapping the iron binding sites[END_REF][START_REF] He | Yeast frataxin solution structure, iron binding, and ferrochelatase interaction[END_REF], its binding may have a negative functional impact as it likely induces structural modifications which destabilise the frataxin fold leading to the formation of precipitates. In any case, iron-mediated colloidal precipitation of the mutant frataxins cannot be ruled out at this stage.

Frataxin mutants I154F and W155R have reduced thermodynamic stability

The conformational properties of the two Hfra(91-210) mutants were spectroscopically investigated using fluorescence and far-UV CD. The differences in conformational stability between the wild type Hfra(91-210) and the two mutants were determined from analysis of their chemical and thermal denaturation transitions, at pH 7 (Fig. 5 and6). In both cases, cooperative unfolding transitions were observed and, under the experimental conditions used, the unfolding reactions were found to be reversible. Concerning chemical unfolding, the order of the midpoint unfolding urea concentrations was determined to be Hfra wild type > W155R > I154F (Table 4). The slightly higher m values determined for the two mutants, also shows that a larger fraction of buried residues is exposed upon unfolding. The mutants midpoint denaturation concentrations was about one unit below the observed for the wild type and this corresponded to a destabilization of 5.9-7.1 kJ (1.4 -1.7kcal) as a result of the mutations. Thermal unfolding experiments also revealed that the mutant I154F is more destabilized than the W155R having a T m ~16ºC lower than the wild type protein, whereas the mutant W155R shows a decrease of only about 5ºC. Altogether these data reveal that both mutations under study reduce the thermodynamic stability of frataxin and that the mutation I154F is more aggressive.

Discussion

We have reported here a detailed comparison of the thermodynamic properties of wild type frataxin and of two clinically relevant mutations (I154F and W155R) in FRDA.

We show that under chemically or thermally destabilizing conditions, the two mutants show distinct behaviours, but that they both are folded at physiological temperature and pH.

The mutation W155R, which can be fit to the category of functional mutations, shows only some destabilization as compared to the wild-type. Destabilization is expected from the destruction of a π interaction between a tryptophan and the contiguous arginine (Arg165) (Fig. 7), an interaction often observed in protein structure [START_REF] Levitt | Aromatic rings act as hydrogen bond acceptors[END_REF]. Additionally, introduction of another positively charged residue near Arg165 introduces further destabilization due to electrostatic repulsion. Observation of these properties is therefore consistent with the location of the tryptophan on the external surface of the β-sheet, with its aromatic moiety completely exposed to the solvent, which is highly suggestive of a role in recognition or interaction with a partner.

A significant perturbation was detected instead for the I154F mutant whose fold is substantially destabilised, as determined from the high number of peptides resulting from partial tryptic digestions and from a red shift in its fluorescence emission maximum. While this is reasonable since the mutation affects a buried residues which takes part of the hydrophobic core (Fig. 7) [START_REF] Musco | Towards a structural understanding of Friedreich's ataxia: the solution structure of frataxin[END_REF], the interesting observation is that the destabilization effect becomes evident only under stressing conditions. At room temperature, this mutant shows features very similar to those of the wild-type, suggesting that despite the mutation the protein can be correctly folded. Accordingly, I154F, as W155R, partly retains its iron binding capability, which nevertheless depends strongly on the specific three-dimensional scaffold. Correct folding would be essential to keep in spatial proximity the acidic residues found to be involved in iron binding, which are distributed along the first helix and in the first beta strand [START_REF] Nair | Solution structure of the bacterial frataxin ortholog, CyaY: mapping the iron binding sites[END_REF][START_REF] He | Yeast frataxin solution structure, iron binding, and ferrochelatase interaction[END_REF].

These observations are interesting because the severe effects of both mutations in FRDA patients would have possibly suggested a much stronger effect on the frataxin fold. What is then the mechanism that determines the pathology in heterozygous cases?

The effect seems to be more subtle and could occur at two different levels. On the one hand, because of a higher destabilization, the folding efficiency once the protein is Lines represent fits to the two state model, see Table 2 for parameters. The wild type Hfra(91-210) (○) denaturation curve with urea is compared to that of Hfra(91-210) W155R ( ) and Hfra(91-210) I154F (□). See Table 4 for parameters characterizing the urea denaturation.

  [D], y f and y u are the intercepts and m f and m u are the slopes of the pre-and posttransition baselines, [D] 1/2 is the denaturation agent concentration at the curve midpoint and m is from equation 2.

Figure 1 -

 1 Figure 1 -Spectroscopic characterisation of wild type Hfra(91-210) in different conformational states Panel A. Fluorescence spectroscopy. Emission spectra of the native form (25ºC), and the chemically (7M GdmCl) and thermally (80ºC) denaturated forms. Panel B. Far-UV CD spectroscopy. Spectra of the native and thermally denaturated forms.

Figure 2 -

 2 Figure 2 -Chemical denaturation curves for Hfra(91-210) at pH 7.9 Hfra(91-210) typical denaturation curves with ( ) GdmCl, (□) GuSCN and ( ) Urea.

Figure 3 -

 3 Figure 3 -Stern-Volmer plots of acrylamide and iodide quenching of frataxin tryptophan fluorescence. The figures show plots of F 0 /F versus concentration of acrylamide (A) and iodide (B) for the native wild type Hfra(91-210) ( ), W155RHfra(91-210) ( ) and I154F Hfra(91-210) ( ). See Table3for parameters.

Figure 4 -

 4 Figure 4 -Peptide maps resulting from partial tryptic digestions.Wild type (wt) and mutant variants (I154F and W155R) after being incubated with trypsin for 100 min at 37°C.

Figure 5 -

 5 Figure 5 -Comparison of chemical denaturation of FRDA Hfra(91-210) mutants, pH 7.0

Figure 6 -

 6 Figure 6 -Comparison of thermal denaturation of FRDA Hfra(91-210) mutants, pH 7.0 The wild type Hfra(91-210) (○) denaturation curve with urea is compared to that of Hfra(91-210) W155R ( ) and Hfra(91-210) I154F (□).

Figure 7 -

 7 Figure 7 -Ribbon representation of the Hfra structure. Two views differing by a 180 rotation around the y axis are displayed. The side chains of I145 and W155 are shown (top panel). The hydrophobic side chains of the residues which surround I145 and of R165 which packs against W155 are also marked (bottom panel).

6 a

 6 Difference between the [urea] 1/2 for the wild type and the mutant forms. b Δ(ΔG) = From Δ[urea] 1/2 x average of the three m values [18].

Figure 1 Figure 6 Figure 7

 167 Figure 1Correia et al(2006)

Table 1 -Parameters for Hfra thermal denaturation as a function of pH.

 1

	Tables
	pH	T m (ºC)	ΔH m (kJ.mol -1 )	(ºC) ΔT m	% refolding
	4.9	59.0 ± 0.1	275.1 ±1.7	-7.3 ± 0.1	20
	6.0 7.0 7.9 8.9 9.7	65.4 ± 0.2 62.5 ± 0.2 66.3 ± 0.1 62.1 ± 0.2 59.0 ± 0.1	371.8 ± 2.1 250.4 ± 1.3 373.5 ± 1.7 362.2 ± 3.3 282.2 ± 1.3	-0.9 ± 0.3 -3.8 ± 0.3 --4.2 ± 0.3 -7.3 ± 0.1	80 95 100 100 20

Table 2 -Parameters for the chemical denaturation of wild type hfra, pH 7.9

 2

	Denaturant	[D] 1/2 (M)	m kJ.mol -1 .M. -1 )	ΔG H2O (kJ.mol -1 )
	GuSCN GdmCl Urea	1.1 2.1 4.3	22.4 ± 1.4 16.8 ± 1.4 5.7 ±0.2	24.6 ± 1.7 35.3 ± 2.1 24.5 ± 1.3

Table 3 -Effect of FRDA mutations on quencher accessibility, Stern-Volmer constants using different quenchers.

 3

	Protein	K sv (M -1 )
		Acrylamide (close symbols)	KI (open symbols)
	Wild Type	11.2 ± 0.2	5.0 ± 0.2
	W155R	4.5 ± 0.1	3.2 ± 0.1
	I154F	10.5 ± 0.2	5.9 ± 0.2

Table 4 -Parameters for urea denaturation of wild type and mutant forms (I154F and W155R). Comparing the differences in stability.

 4

	Protein	ΔG H2O (kJ.mol -1 )	m (J.mol -1 .M -1 )	[Urea] 1/2 (M)	Δ[Urea] 1/2	a	Δ(ΔG) b (J.mol -1 )	T m (ºC)	ΔT m (ºC)
	Wild type 24.3 ± 1.3	5665 ± 264	4.3				66.3 ± 0.1	-
	W155R	21.4 ± 0.8	6209 ± 327	3.4	0.9		-7172	61.4 ± 0.4	-4.9
	I154F	24.7 ± 0.8	7687 ± 281	3.2	-1.1		-5866	50.7 ± 0.1 -15.

Biochemical Journal Immediate Publication. Published on 21 Jun 2006 as manuscript BJ20060345 Copyright 2006 Biochemical Society

Acknowledgements

This work was partly supported by a collaborative grant from the Conselho Reitores das Universidades Portuguesas (CRUP, Portugal to C.M.G.) and the Bristish Council (BC, UK to A.P.). A.P. acknowledges funding from the FRDA and MDA foundations. S. Martin (NIMR) is thanked for valuable assistance an comments during CD measurements. Paula Chicau (Amino Acid Analysis Service, ITQB) is gratefully acknowledged for skilled technical assistance on the HPLC separations of the limited proteolysis experiments.

Copyright 2006 Biochemical Society detached from the ribosome or migrates through the mitochondrial membrane could be reduced so that a lower concentration of 'functional' protein is released to the cell. On the other hand, the increased rate of proteolytic degradation also points out to a more efficient degradation of the mutant proteins. At the present stage, however, we do not have evidence of what the cellular fate of the mutant variant proteins upon their synthesis may be but much more efforts should be put to follow up these ideas. In vitro studies on mutant variants can therefore provide new experimental insights towards the understanding of the structural basis of the disease. The frataxin variants, which result to milder forms of the disease, are particularly interesting and are currently under investigation in our laboratories.


Arne Hammer

David Rw Hodgson

Martin J Cann
email: m.j.cann@durham.ac.uk

David R W Hodgson

REGULATION OF PROKARYOTIC ADENYLYL CYCLASES BY CARBON DIOXIDE

Keywords: adenylyl cyclase, cAMP, carbon dioxide, bicarbonate, Synechocystis

come

INTRODUCTION.

Inorganic carbon (Ci) is fundamental to the physiology of all organisms. Carbon dioxide and bicarbonate ions exist in a pH dependent equilibrium and are the major biologically active forms of Ci. CO 2 and HCO 3 -are vital to such diverse physiological processes as photosynthetic carbon fixation [START_REF] Badger | CO 2 concentrating mechanisms in cyanobacteria: molecular components, their diversity and evolution[END_REF], pH homeostasis [START_REF] Adrogue | Acid-base physiology[END_REF], and carbon metabolism [START_REF] Smith | Prokaryotic carbonic anhydrases[END_REF]. Study of Ci biology is essential to understand these vital physiological processes.

Relatively little is known of the signalling mechanisms through which prokaryotic and eukaryotic cells directly detect CO 2 /HCO 3 -fluctuations [START_REF] Hetherington | The biology of carbon dioxide[END_REF]. The identification of Ci activated signalling molecules and their role in physiology is fundamental to understanding the diverse roles of Ci in biology. Currently, no signalling enzymes directly activated by CO 2 are known.

The mammalian soluble adenylyl cyclase (sAC) synthesizes the second messenger adenosine 3', 5'-cyclic monophosphate and is stimulated by HCO 3 - [START_REF] Buck | Cytosolic adenylyl cyclase defines a unique signaling molecule in mammals[END_REF][START_REF] Chen | Soluble adenylyl cyclase as an evolutionarily conserved bicarbonate sensor[END_REF]. It was observed that HCO 3 -regulation of adenylyl cyclase (AC) was conserved in a cyanobacterial AC, CyaC of Spirulina (Arthrospira) platensis, which had significant sequence homology in the AC domain to sAC [START_REF] Chen | Soluble adenylyl cyclase as an evolutionarily conserved bicarbonate sensor[END_REF]. More recently, an active site Asp→Thr polymorphism in the Class III AC family has been proposed as a marker for HCO 3 -responsiveness [START_REF] Cann | A defined subset of adenylyl cyclases is regulated by bicarbonate ion[END_REF]. On this basis proposed HCO 3 -responsive ACs are predicted to be widespread among the genomes of prokaryotes and eukaryotes [START_REF] Cann | Bicarbonate stimulated adenylyl cyclases[END_REF]. To date, Ci regulation of AC has been confirmed in prokaryotes as diverse as

Anabaena PCC 7120, Mycobacterium tuberculosis, Stigmatella aurantiaca, and Chloroflexus aurantiacus [START_REF] Cann | A defined subset of adenylyl cyclases is regulated by bicarbonate ion[END_REF][START_REF] Kobayashi | Conservation of functional domain structure in bicarbonate-regulated "soluble" adenylyl cyclases in bacteria and eukaryotes[END_REF]. An implicit assumption is made in the literature that the activating Ci ligand for AC is HCO 3 -on the basis that the ionic form is more likely to bind in the active site than CO 2 . Identification of the activating carbon ligand for AC is essential to validate or question the relevance of significant recent literature in the field.

The photosynthetic cyanobacteria are an excellent model for investigating Ci signalling through AC as hypothesized HCO Asp→Thr polymorphism associated with a putative HCO 3 -responsiveness [START_REF] Cann | A defined subset of adenylyl cyclases is regulated by bicarbonate ion[END_REF][START_REF] Kaneko | Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions[END_REF]. We expressed the AC domain of Slr1991 as a purified recombinant protein (Figure 1a). The purified wild type protein had a significant AC specific activity in the presence of both Mg 2+ -ATP (154 ± 2.0 pmol cAMP mg -1 min -1 , n=8) and Mn 2+ -ATP (5816 ± 87 pmol cAMP mg -1 min -1 , n=8) under optimal conditions (pH 9.5, 40 °C, 0.3 mM ATP, 8 µM protein).

The Slr1991 120-337 protein had a pH optimum of 9.5 and a temperature optimum of 40

°C. The enthalpy of activation (E A ) derived from the linear arm of an Arrhenius plot using Mn 2+ -ATP was 33.5 ± 1.4 kJ mol -1 (n=6). We investigated whether Slr1991 120-337 was regulated by Ci with a view to determining the identity of the activating species, CO 2 or HCO 3 -. Slr1991 120-337 specific activity was stimulated two-fold by 20 mM total Ci (1.2 mM CO 2 /18.8 mM HCO 3 -) at pH 7.5 compared to Cl -. Stimulation was independent of cation and robust to 95% confidence intervals (Figure 1b). A previous report had not observed stimulation of Slr1991 by Ci at pH 7.5 [13]. We noted that an extended assay period (40 mins) was required to observe robust Ci activation of Slr1991 at pH 7.5. Although Masuda and Ono do not report assay time, this is the most likely cause of the discrepancy.

We determined the kinetics of activation of Slr1991 120-337 by Ci (Table I). Slr1991 120- 337 showed Michaelis-Menten kinetics in the presence of both Cl -and Ci. The K M value for Mn 2+ -ATP was greater in the presence of Ci than Cl -but V max values were proportionately greater for Ci than Cl -. The overall result was that Ci increased turnover rate (k cat ). A dose response curve with increasing Ci was performed at a reduced pH 6.5 to eliminate problems with enzyme inhibition at >20 mM total Ci at pH 7.5 in the presence of Mn 2+ -ATP (Figure 2).

The experiment revealed a maximum eight-fold stimulation with an apparent E.C. 50 for Ci of 52.7 ± 1.0 mM (n=6) (20.4 mM CO 2 /32.3 mM HCO 3 -).

We investigated the response of Slr1991 120-337 to total Ci at varying pH to gain insight into whether the enzyme is responsive to CO 2 and/or HCO 3 -The experiment was performed using Mg 2+ -ATP as substrate as Mg 2+ co-factor is more soluble than Mn 2+ in the presence of Ci at alkaline pH. Intriguingly, relative stimulation (Ci:NaCl) varied from 1.1 at pH 8.5 (0.3 mM CO 2 /39.1 mM HCO 3 -/0.6 mM CO 3 2-) to 2.4 at pH 6.5 (15.5 mM CO 2 /24.5 mM HCO 3 -) (Figure 3a). This is consistent with a role for CO 2 as opposed to HCO 3 -as the activating carbon species but may also be due to altered protonation status of the enzyme limiting the ability of Slr1991 to respond to HCO 3 -at elevated pH. We therefore sought direct evidence for regulation of Slr1991 120-337 by CO 2 and/or HCO 3 -by analysis under conditions of Ci disequilibrium when a single predominant carbon species, CO 2 or HCO 3 -, is present at a defined pH. We exploited the fact that acquisition of the equilibrium between CO 2 and HCO 3 is significantly lowered at reduced temperature in the absence of carbonic anhydrase and is a well established method for identifying the Ci substrate for CO 2 /HCO 3 -fixing enzymes [14].

We followed the acquisition of the CO 2 /HCO 3 -equilibrium by measuring the pH of a weakly buffered (5 mM) MES solution on addition of 20 mM CO 2 or 20 mM NaHCO 3 in the presence or absence of carbonic anhydrase at 0 °C (Figure 3b). On the basis of this data we defined conditions for assaying AC under conditions of disequilibrium using 20 mM CO 2 or HCO 3 -as a 10 second assay period at 0 °C after addition of Ci. Under these conditions, Ci is predominantly in the form added to the assay (CO 2 or HCO 3 -) and has not significantly advanced toward the equilibrium determined by assay pH (clamped with 100 mM MES).

Control experiments demonstrated that under the conditions used for the assay final pH was equivalent when either CO 2 , HCO 3 -, or Cl -were added demonstrating that any observed stimulation was due to addition of Ci and not a change in assay pH (data not shown).

We assayed Slr1991 120-337 under conditions of Ci disequilibrium and observed, surprisingly, that CO 2 but not HCO 3 -stimulated the enzyme (Figure 3c). We investigated whether this highly significant result was unique to Slr1991 or of more general significance.

The CyaB1 595-859 protein of Anabaena PCC 7120 was previously shown to respond to HCO 3 -/CO 2 but the activating species not proven [START_REF] Cann | A defined subset of adenylyl cyclases is regulated by bicarbonate ion[END_REF]. Consistent with the findings for Slr1991 120-337 , CyaB1 595-859 was also stimulated by CO 2 but not HCO 3 -under conditions of Ci disequilibrium (Figure 3d).

This data demonstrates that for at least two randomly selected prokaryotic ACs, Slr1991 and CyaB1, the activating carbon species is dissolved CO 2 and not the more ably binding HCO 3 -species. These enzymes therefore represent the first identified signalling molecules demonstrated to respond directly to CO 2 . HCO 3 -regulation of AC has been proposed but not proven for enzymes from species as diverse as Spirulina platensis, Cryptococcus neoformans, Candida albicans, Chloroflexus aurantiacus, and mammals [START_REF] Chen | Soluble adenylyl cyclase as an evolutionarily conserved bicarbonate sensor[END_REF][START_REF] Kobayashi | Conservation of functional domain structure in bicarbonate-regulated "soluble" adenylyl cyclases in bacteria and eukaryotes[END_REF]15,16]. An urgent examination of these systems is required to prove whether the ACs defined from these species respond to HCO 3 -or to CO 2 as described here.

Biochemical Journal Immediate Publication. Published on 30 Mar 2006 as manuscript BJ20060372

Figure 1 .

 1 Figure 1. Adenylyl cyclase activity of purified recombinant Slr1991 120-337 . A, Purification of recombinant Slr1991 120-337 (SDS-PAGE analysis, Coomassie Blue staining). 1.5 µg of protein was applied and molecular mass standards (in kDa) are indicated. B, Slr1991 120-337 specific activity (n=8) in presence of 20 mM total Ci/salt (0.6 µM protein, 20 µM Mn 2+ -ATP, pH 7.5).

Figure 2 .

 2 Figure 2. Response of wild Slr1991 120-337 to Ci. Slr1991 120-337 specific activity (n=6) at increasing total Ci (1.5 µM protein, 20 µM Mn 2+ -ATP, pH 6.5, Na + as cation, total salt made up to 200 mM with NaCl).

Figure 3 .

 3 Figure 3. Activation of adenylyl cyclase by CO 2 . A, The ratio of the specific activities of Slr1991 120-337 when assayed in the presence of 40 mM total Ci or NaCl at varying pH (8 µM protein, 1 mM Mg 2+ -ATP, 20 mM Mg 2+ ). The inset shows the percentage of total Ci made up by CO 2 and HCO 3 -over the pH range tested. B, Change in pH of a 5 mM MES solution (starting pH 6.4) on addition of 20 mM NaHCO 3 (arrow) in the presence (squares) or absence (triangles) of 132 U carbonic anhydrase at 0 °C. C, cAMP produced by Slr1991 120-337 under conditions of Ci disequilibrium (50 µM Slr1991 120-337 protein, 0 °C, 10 secs, 20 mM CO 2 /NaHCO 3 /NaCl, 100 mM MES pH 6.5, 150 µM Mn 2+ -ATP). D, cAMP produced by CyaB1 595-859 under conditions of Ci disequilibrium (38 µM CyaB1 595-859 protein, 0 °C, 10 secs, 20 mM CO 2 /NaHCO 3 /NaCl, 100 mM MES pH 6.5, 150 µM Mn 2+ -ATP, n=6).

  Figure 3c

Biochemical Journal Immediate Publication. Published on 30 Mar 2006 as manuscript BJ20060372 Copyright 2006 Biochemical Society RESULTS AND DISCUSSION.

  The cya1 (slr1991; http://www.kazusa.or.jp/cyano/Synechocystis) gene of the unicellular cyanobacterium Synechocystis PCC 6803 encodes an enzyme consisting of a single FHA (Forkhead Associated) domain and a Class III AC domain that contains an

	MATERIALS AND METHODS.
	Recombinant proteins -DNA corresponding to amino acids 120-337 of slr1991 was isolated
	by PCR from genomic DNA of Synechocystis PCC 6803, subcloned into pQE30, and fitted
	with an N-terminal MRGSH 6 GS affinity tag. Constructs were confirmed by double-stranded
	sequencing. Slr1991 120-337 protein was expressed in Escherichia coli M15 [pREP4] cells at 25
	°C, for 3 h with 300 µM isopropyl β-D-1-thiogalacto-pyranoside. Pelleted cells were washed
	(50 mM Tris-HCl 8.5, 1 mM EDTA), resuspended (50 mM Tris-HCl pH 8.5, 250 mM NaCl,
	10 mM 1-thioglycerol), lysed by sonication (1 x 150 s), and protein purified from the
	supernatant with Ni 2+ -nitrilotriacetic acid (NTA, Qiagen) as previously described [10].
	CyaB1 595-859 protein was generated as previously described [10]. Primer sequences are
	available on request.
			3	-responsive ACs are widespread in these
	organisms and Ci has clearly defined roles in their physiology. Here we demonstrate that the
	single Class III AC, Slr1991 (Cya1), of the unicellular cyanobacterium Synechocystis PCC
	6803 is activated by Ci. Furthermore, we demonstrate, surprisingly, that the activating ligand
	for this enzyme is CO 2 and not HCO 3	-. A previously characterized proposed HCO 3	-regulated
	AC, CyaB1 of Anabaena PCC 7120, is also proven to respond to CO 2 rather than HCO 3	-. This
	work provides the first evidence for AC as a CO 2 activated signalling molecule. This original
	finding prompts an immediate reassessment of the true activating carbon species in reported
	HCO 3	-responsive ACs.

Adenylyl cyclase assays -AC assays were performed at 40 °C in a final volume of 100 µl and typically contained 50 mM buffer, 2 mM MnCl 2 , 2 mM [2,8-3 H]-cAMP (150 Bq), and [α 32 P]ATP (25 kBq) as substrate, if not stated otherwise

[START_REF] Salomon | A highly sensitive adenylate cyclase assay[END_REF]

. Protein concentrations were adjusted to maintain substrate utilization at less than 10%. Kinetic constants were determined over a concentration range of substrate of 1-100 µM (Mn 2+ -ATP). The following buffers were used at pH 6.5 (2-[N-morpholino]ethanesulfonic acid; MES), pH 7.0-7.5 (3-[Nmorpholino]propanesulfonic acid), pH 8.0-8.5 (Tris-[hydroxymethyl]aminoomethane hydrochloride, and pH 9.0 (2-[N-cyclohexylamino]ethanesulfonic acid). Enzyme, buffer, and substrate were all prepared at the appropriate pH for the required assay. CO 2 was quantitated by titration against NaOH. Assay pH was stable over a period of at least 40 minutes. All errors correspond to the standard error of the mean. If absent, errors are smaller than the symbol used to depict the data point.

Table I .

 I Kinetic parameters for Slr1991 120-337 . 0.6 µM protein was assayed at pH 7.5 in the presence of 20 mM salt (n=6).

		Addition
	Parameter	Cl -	HCO 3	-
	V max (nmol cAMP mg -1 min -1 ) 0.74 ± 0.01 1.13 ± 0.03
	K M [ATP] (µM)	11.4 ± 0.7 16.2 ± 1.3
	Hill Slope	1.01 ± 0.01 1.03 ± 0.03
	k cat (min -1 )	0.018	0.027

Biochemical Journal Immediate Publication. Published on 30 Mar 2006 as manuscript BJ20060372 Copyright 2006 Biochemical Society

Cooper, T. G. and Filmer, D. (1969) The active species of "CO 2 " utilized by ribulose diphosphate carboxylase. J. Biol. Chem. 244, 1081-1083


Arne Engelsberg
email: engelsba@neuroscience.fu-berlin.de

Franziska Kobelt

Dietmar Kuhl

The N-terminus of the serum and glucocorticoid inducible kinase Sgk1 specifies mitochondrial localization and rapid turnover

Keywords: Sgk1, mitochondria, half-life, localization, topology, orientation

abbreviations used GFP -green fluorescent protein, DAPI -4',6-diamidino-2-phenylindole, OMM -outer mitochondrial membrane

Introduction

The serum and glucocorticoid inducible serine/threonine-kinase Sgk1 consists of 431 amino acids and belongs to a family of structurally related enzymes termed AGC kinases. Among these, Sgk1 shares about 50% sequence similarity on the amino acid level with protein kinase B/Akt, protein kinase C, ribosomal protein S6 kinase and cAMP-dependent protein kinase [START_REF] Webster | Characterization of sgk, a novel member of the serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum[END_REF].

Sgk1 has been described to regulate a variety of cellular processes. In vitro assays suggested that Sgk1 might control signalling pathways and cell survival by phosphorylation of glycogen synthase kinase 3, B-raf kinase and forkhead family member FOXO3a [START_REF] Sakoda | Differing roles of Akt and serum-and glucocorticoid-regulated kinase in glucose metabolism, DNA synthesis, and oncogenic activity[END_REF][START_REF] Zhang | Serum-and glucocorticoid-inducible kinase SGK phosphorylates and negatively regulates B-Raf[END_REF][START_REF] Brunet | Protein kinase SGK mediates survival signals by phosphorylating the forkhead transcription factor FKHRL1 (FOXO3a)[END_REF]. In addition, the action of Sgk1 has been implicated in the control of a variety of plasma membrane transport proteins including sodium channels ENaC and SCN5A, chloride channel ClC2, the epithelial calcium channel TRPV5, glucose transporter SGLT1, the potassium channels Kv1.3, ROMK, KCNE1, and glutamate transporter EAAT1 [START_REF] Debonneville | Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na(+) channel cell surface expression[END_REF][START_REF] Diakov | A novel pathway of epithelial sodium channel activation involves a serum-and glucocorticoid-inducible kinase consensus motif in the C terminus of the channel's alpha-subunit[END_REF][7][START_REF] Palmada | Serum and glucocorticoid inducible kinases functionally regulate ClC-2 channels[END_REF][START_REF] Embark | Regulation of the epithelial Ca2+ channel TRPV5 by the NHE regulating factor NHERF2 and the serum and glucocorticoid inducible kinase isoforms SGK1 and SGK3 expressed in Xenopus oocytes[END_REF][START_REF] Dieter | Regulation of glucose transporter SGLT1 by ubiquitin ligase Nedd4-2 and kinases SGK1, SGK3, and PKB[END_REF][START_REF] Henke | Regulation of the voltage gated K+ channel Kv1.3 by the ubiquitin ligase Nedd4-2 and the serum and glucocorticoid inducible kinase SGK1[END_REF][START_REF] Yun | The serum and glucocorticoidinducible kinase SGK1 and the Na+/H+ exchange regulating factor NHERF2 synergize to stimulate the renal outer medullary K+ channel ROMK1[END_REF][START_REF] Embark | Regulation of KCNE1dependent K(+) current by the serum and glucocorticoid-inducible kinase (SGK) isoforms[END_REF][START_REF] Boehmer | Regulation of the glutamate transporter EAAT1 by the ubiquitin ligase Nedd4-2 and the serum and glucocorticoid-inducible kinase isoforms SGK1/3 and protein kinase B[END_REF]. In the kidney, Sgk1 contributes to sodium homeostasis by mediating the aldosterone induced upregulation of ENaC in the plasma membrane of distal tubule cells. Accordingly, Sgk1 knockout mice display an impaired sodium retention during dietary sodium restriction [START_REF] Wulff | Impaired renal Na(+) retention in the sgk1knockout mouse[END_REF]. Sgk1 was first identified as an immediate early gene expressed in a rat mammary tumor cell line following stimulation with serum or glucocorticoids [START_REF] Webster | Characterization of sgk, a novel member of the serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum[END_REF][START_REF] Webster | Immediate-early transcriptional regulation and rapid mRNA turnover of a putative serine/threonine protein kinase[END_REF]. Depending on the specific tissues or cell lines examined, Sgk1 was shown to be induced by diverse factors, such as changes in osmolarity [START_REF] Waldegger | Cloning and characterization of a putative human serine/threonine protein kinase transcriptionally modified during anisotonic and isotonic alterations of cell volume[END_REF], aldosterone [START_REF] Naray-Fejes-Toth | Sgk is an aldosterone-induced kinase in the renal collecting duct. Effects on epithelial na+ channels[END_REF], and various growth factors, including fibroblast growth factor, platelet derived growth factor [START_REF] Mizuno | The ERK MAP kinase pathway mediates induction of SGK (serum-and glucocorticoid-inducible kinase) by growth factors[END_REF], follicle stimulating hormone [20] and transforming growth factor β [START_REF] Waldegger | h-sgk serine-threonine protein kinase gene as transcriptional target of transforming growth factor beta in human intestine[END_REF]. Activation of the kinase requires at least two phosphorylation events in the activation loop and the C-terminal hydrophobic motif [START_REF] Park | Serum and glucocorticoid-inducible kinase (SGK) is a target of the PI 3-kinase-stimulated signaling pathway[END_REF][START_REF] Kobayashi | Activation of serum-and glucocorticoid-regulated protein kinase by agonists that activate phosphatidylinositide 3-kinase is mediated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and PDK2[END_REF] and is further limited by a short half-life, resulting in low levels of protein expression in many tissues and cell lines. In line with these obervations Sgk1 is rapidly ubiquitinated and degraded by the proteasome [START_REF] Brickley | Ubiquitin modification of serum and glucocorticoid-induced protein kinase-1 (SGK-1)[END_REF].

Interestingly, although lysine residues at the N-terminus are not subject to ubiquitination, a variant of Sgk1 lacking the N-terminal 60 amino acids has a much longer half-life [START_REF] Brickley | Ubiquitin modification of serum and glucocorticoid-induced protein kinase-1 (SGK-1)[END_REF]. Another level of regulation was attributed to the subcellular localization of Sgk1. Various studies suggested that Sgk1 is translocated from the cytoplasm to the nucleus in a stimulus and cell cycle dependent manner via binding to importin-α [START_REF] Buse | Cell cycle and hormonal control of nuclear-cytoplasmic localization of the serum-and glucocorticoid-inducible protein kinase, Sgk, in mammary tumor cells. A novel convergence point of anti-proliferative and proliferative cell signaling pathways[END_REF][START_REF] Maiyar | Importin-alpha mediates the regulated nuclear targeting of serum-and glucocorticoid-inducible protein kinase (Sgk) by recognition of a nuclear localization signal in the kinase central domain[END_REF][START_REF] Leong | Expression of the serum-and glucocorticoid-inducible protein kinase, Sgk, is a cell survival response to multiple types of environmental stress stimuli in mammary epithelial cells[END_REF]. In the nucleus, Sgk1 is thought to phosphorylate specific targets such as FOXO3a [START_REF] Brunet | Protein kinase SGK mediates survival signals by phosphorylating the forkhead transcription factor FKHRL1 (FOXO3a)[END_REF]. In contrast, two recent reports showed an association of Sgk1 with the plasma membrane in the rat kidney and in different cell lines, and that the N-terminus of the protein plays a major role in membrane targeting [START_REF] Alvarez De La Rosa | Distribution and regulation of expression of serum-and glucocorticoid-induced kinase-1 in the rat kidney[END_REF][START_REF] Brickley | Ubiquitin modification of serum and glucocorticoid-induced protein kinase-1 (SGK-1)[END_REF].

Experimental

Molecular Cloning

Sgk1-GFP fusion protein variants and variants with a C-terminal FLAG-tag were generated by PCR using a Pwo polymerase (Roche Biochemicals). Primers were extended with restriction sites and fragments were cloned in frame into pEGFP-N3 (BD Clontech), or in case of the FLAG-tagged variants into pcDNA3.1(+) (Invitrogen). The 5' primers contained a Kozak sequence surrounding the ATG start codon. For variants starting with amino acids 17, 33 and 60, we made use of the internal ATGs coding for the respective methionines. In variants starting at position 48, tyrosine 47 was replaced with methionine to obtain a start codon. All constructs were confirmed by DNA sequencing.

Cell Culture and Transfections

Human embryonic kidney HEK293 cells were maintained in DMEM medium (Biochrom, Berlin, Germany), supplemented with 5% fetal calf serum (Biochrom) and 2 mM L-glutamine. For pulse-chase experiments and immunoblot analysis, 2 x 10 6 cells were resuspended in 500 µl of serum-free medium and, after addition of 20 µg of the respective plasmid-DNA, electroporated at 170 V and 960 µF in 4 mm gap electroporation cuvettes (Bio-Rad, Munich, Germany) using a Bio-Rad Gene Pulser II. Transfected cells were plated on a 10 cm diameter dish. For confocal microscopy, 50,000 cells were grown on 12 mm coverslips in 24-wells and transfected with 100

Pulse Chase Experiments and Immunoprecipitation

Transfected HEK293 cells were incubated for 45 minutes in methionine-and cysteine-free DMEM medium, and then pulse-labelled for 10 minutes with 200 µCi [ 35 S]methionine/cysteine labelling mix (Hartmann, Braunschweig, Germany). Incorporation of label was stopped by addition of complete medium supplemented with 1.5 mM nonradioactive methionine and 0.5 mM nonradioactive cysteine to a final volume of 5 ml. After 0 minutes, 15 minutes, 60 minutes, and 4 hours, cells from 1 ml suspension were pelleted and lysed in 50 mM Tris pH7.5, 5 mM MgCl 2 , 0.5% NP-40, 1 mM PMSF and 1.5 µg/ml aprotinin for 30 minutes at 4°C. Lysates were depleted of nuclei and cell debris by centrifugation for 10 minutes at 14,000 rpm in a microcentrifuge and precleared with normal rabbit serum. GFP fusion proteins were recovered by immunoprecipitation with anti-GFP rabbit serum (gift by Ralf Schülein) and Protein A-sepharose (Sigma, Steinheim, Germany), followed by five washes in 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 0.5% NP-40. Sepharose-beads were boiled in reducing sample buffer and subjected to PAGE. Gels were dried and signals were obtained with 2,5-diphenyloxazol fluorography.

Subcellular Fractionation

Fractions were prepared from livers and kidneys of Sgk1 knockout mice and their wild-type littermates [START_REF] Wulff | Impaired renal Na(+) retention in the sgk1knockout mouse[END_REF]. Organs were homogenized in TES-buffer (10 mM Tris, 250 mM sucrose, 1 mM EDTA, pH 7.4) in a Dounce homogenizer by 12 strokes with a tight fitting pestle. After centrifugation at 500 g for 10 minutes, crude mitochondria were pelleted from postnuclear supernatant at 10,000 g for 20 minutes. The pellet was resuspended in TES, layered on a discontinuous sucrose gradient consisting of 1 M and 1.5 M sucrose and centrifuged at 80,000 g for one hour. Highly purified mitochondria were harvested at the 1 M/1.5 M interphase and washed twice in TES. Cytosolic and microsomal fractions were prepared by ultracentrifugation of the postmitochondrial supernatant for 1 hour at 100,000 g in a TLA 110 rotor (Beckman). The pellet served as microsomal fraction. For the purification of nuclei and plasma membranes from the 500 g-pellet, the pellet was resuspended in 120 mM Tricine, 150 mM KCl, 30 mM MgCl 2 , pH 7.5 supplemented with 1 mM DTT and 0.1 % Triton X-100, and nuclei and plasma membrane sheets were pelleted by centrifugation through a 1.75 M sucrose cushion in a Beckman SW-Biochemical Journal Immediate Publication. Published on 16-Jun-2006 as manuscript BJ20060386

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© 2006 Biochemical Society 32Ti rotor for 2 hours at 38,000 g. In some experiments, mice were injected with 100 µg/kg body weigth dexamethasone (Sigma) 4 hours before the preparation.

In vitro protein import into mitochondria

Coupled in vitro transcriptions and translations were performed in the presence of [ 35 S]methionine according to the manufacturer's instructions using the Promega TnT kit (Promega, Mannheim, Germany). Reticulocyte lysates containing the radiolabled Sgk1 variants were cleared of low molecular weight components by passage through Sephadex G-50 and added for the indicated periods of time at room temperature to purified C57BL/6 kidney mitochondria in import buffer consisting of 250 mM sucrose, 80 mM KCl, 10 mM Mg(OAc) 2 , 2,5 mM EDTA, 2 mM KH 2 PO 4 , 2.5 mM ATP, 5 mM NADH, 2.5 mM disodiumsuccinate, 50 mM Hepes pH 7.3, 0.2 mg/ml BSA. Mitochondria were washed twice in ice-cold import buffer and subjected to PAGE. For the analysis of membrane association and orientation, in vitro translated proteins were added to 1 ml mouse kidney postnuclear supernatant from C57BL/6 mice supplemented with 2.5 mM ATP, 5 mM NADH, and 2.5 mM disodiumsuccinate, where import in mitochondria occurred for 20 minutes at 30°C. Mitochondria were pelleted at 10,000 g for 15 minutes, and were washed once with TES.

Membrane association, sodium carbonate extraction and protease protection assays

For protease protection assays, mitochondria were incubated with 200 µg/ml proteinase K (Roth, Karlsruhe, Germany) for 15 minutes at room temperature with or without 0.5 % Triton X-100. The reaction was stopped by addition of 5 mM PMSF.

Mitochondria were sonicated and then separated in soluble and membrane fractions ultracentrifugation for one hour at 300,000 g in a Beckman TLA 100.3 rotor. For sodium carbonate extractions, a membrane pellet was resuspended in 100 mM Na 2 CO 3 , incubated for one hour at 4°C under agitation and again pelleted for one hour at 300,000 g. Samples were separated by PAGE and radiolabeled proteins were visualized by fluorography.

Immunoblots

Transfer of proteins onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) or onto PVDF membranes for anti-Sgk1 and anti-FLAG M2 blots (Immobilon P, Millipore) was conducted using a tank blot chamber (Biorad, Munich, Germany). After blocking in 5% non-fat dry milk in PBS containing 0.05% Tween20 (PBS-T), antibodies were applied in PBS-T as Biochemical Journal Immediate Publication. Published on 16-Jun-2006 as manuscript BJ20060386

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© 2006 Biochemical Society follows: Anti-Sgk1 (Sigma) 1:50,000, anti-Sgk1 (Cell Signaling Technology, Danvers, MA.) 1:2,000, anti-Sgk1 [START_REF] Alvarez De La Rosa | Distribution and regulation of expression of serum-and glucocorticoid-induced kinase-1 in the rat kidney[END_REF] 1:5000, anti-Sgk1 [START_REF] Murray | Exploitation of KESTREL to identifiy NDRG family members as physiological substrates for SGK1 and GSK3[END_REF] 1:2000, anti-Sgk1 (this publication, see below) 1:10,000, anti-FLAG M2 (Sigma) 1:10,000, anti-Hsp60 (Stressgen, San Diego, CA.) 1:4,000, anti-Cytochrome C (Santa Cruz, Heidelberg, Germany) 1:5,000, anti-Smac/Diablo (ProSci Inc., Poway, CA.) 1:1,000, anti-Bip/Grp78 (Stressgen) 1:500, anti-GAPDH (Chemicon, Temecula, CA.) 1:2,500,000, anti-Na/K-ATPase α-subunit (Upstate) 1:10,000, and anti-Histone H1 (Santa Cruz) 1:1,000. For detection of Sgk1 with a sheep anti-Sgk1 antibody [START_REF] Murray | Exploitation of KESTREL to identifiy NDRG family members as physiological substrates for SGK1 and GSK3[END_REF], membranes were blocked with Rotiblock reagent (Carl Roth, Karlsruhe, Germany) instead of dry milk. Membranes were washed, incubated with peroxidase-conjugated secondary antibodies (Vector labs), washed again and subjected to chemiluminescence detection.

We gave rise to an anti-Sgk1 antibody by immunization of a rabbit five times with 500 µg of Sgk1 amino acids 290-431 fused to an N-terminal His-tag. The protein was expressed in E.coli and purified using Ni-NTA spin columns (Qiagen, Hilden, Germany). IgGs were purified from the serum using Protein A-sepharose.

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Results

A signal peptide between amino acids 17 and 32 targets Sgk1 to mitochondria Previous studies indicated that Sgk1 is localized to the cytosol, the nucleus and the plasma membrane in response to different external stimuli. We used the PSORTII prediction algorithm [START_REF] Nakai | PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization[END_REF] to calculate the probability of Sgk1 localization to different cellular compartments. In this analysis, Sgk1 has a 43,5% probability to be a cytosolic protein and a 34,8% probability to localize to mitochondria. According to TargetP V1.0 [30], and MitoProt II 1.0a4 [START_REF] Claros | Computational method to predict mitochondrially imported proteins and their targeting sequences[END_REF], two programs that predict mitochondrial localization, Sgk1 is likely to be imported into mitochondria and a predicted mitochondrial signal peptide might be cleaved at amino acid position 33 (Figure 1A). Analysis using the Kyte-Doolittle plot suggested that the N-terminus of Sgk1 might contain a transmembrane segment (Figure 1B).

To experimentally examine the subcellular localization of Sgk1, we transfected HEK293 cells with a series of Sgk1 variants that harboured a C-terminally fused green fluorescent protein (GFP) for analysis with confocal laser scanning microscopy. We focussed on the N-terminus of the kinase, because the first 60 amino acids were previously proposed to be responsible for plasma membrane association [START_REF] Brickley | Ubiquitin modification of serum and glucocorticoid-induced protein kinase-1 (SGK-1)[END_REF] and the mitochondrial targeting signal was predicted to reside in this region. When we transiently transfected HEK293 cells with full length Sgk1-GFP, we obtained a reticulo-tubular staining pattern that is characteristic for mitochondria in this cell line. To corroborate mitochondrial localization of Sgk1-GFP, we cotransfected mito-DsRedII as mitochondrial marker and stained nuclei with 4',6-diamidino-2-phenylindole (DAPI). Our data demonstrate that GFP-and DsRedII fluorescence signals were overlapping (Figure 2A). To narrow down the region responsible for organelle targeting, we next generated and analyzed Nterminal deletion variants starting with amino acids 17, 33, and 48 (Figures 2C,E,G). Our analysis shows that amino acids 1-16 are dispensable for protein targeting as a Sgk1 variant containing amino acids 17-431 is still localized to mitochondria (Figure 2C). In contrast, Sgk1 © 2006 Biochemical Society throughout the cell (Figure 2I), whereas Sgk1 1-32-GFP localized to mitochondria (Figure 2J).

Taken together, our data demonstrate that the region between amino acids 17 and 32 is necessary and sufficient for mitochondrial localization of Sgk1. These findings are in good accordance with the amino acid composition of Sgk1 in this region (Figure 1). Here, negatively charged residues are rare and arginine is overrepresented as is the case in most mitochondrial targeting sequences [30]. In a few cells, Sgk1-GFP signals were not restricted to mitochondria.

However, these cells showed a strong overexpression of the transgene. In no instance were any Sgk1-GFP variants enriched in the nucleus even when cells were serum-stimulated or exposed to heat shock (data not shown). Moreover, none of the fusion protein variants were detectable at the plasma membrane.

Sgk1 is localized to mitochondria in vivo

To confirm a mitochondrial localization of Sgk1 in vivo, we performed subcellular fractionation experiments from mouse tissue. We first tested five commercial and non commercial antibodies for specificity of Sgk1 binding in Western blot analyses. Although all antibodies readily detected overexpressed Sgk1 in cell lines, we were not able to detect Sgk1 expression in any mouse tissue including kidney and liver (Supplementary Figure 1). We therefore increased levels of Sgk1 protein expression by administration of dexamethason. Specificity of the Sgk1 signal was verified by direct comparison with corresponding tissue from knockout animals. Four hours after intraperitoneal application of dexamethasone we fractionated mouse livers from Sgk1 knockouts and their wild-type littermates into cytosol, nuclei and plasma membranes, microsomes, and mitochondria by differential centrifugation and processed them for immunoblot analyses. The purity of the fractions was examined with antibodies directed against GAPDH, Histone H1, Na/K-ATPase, BiP/Grp78, cytochrome C and the α-subunit of the Na/K-ATPase (Figure 3). We obtained robust Sgk1 signals from the mitochondrial fraction and weak signals from the cytosolic and microsomal lane (Figure 3). The signals originated from Sgk1 as they were absent in the knockout tissue fractions.

Sgk1 is localized to the cytosolic surface of the outer mitochondrial membrane

Mitochondrial proteins can be localized to the inner or outer mitochondrial membrane (OMM), the intermembrane space or the matrix. To overcome the low endogenous Sgk1 protein expression levels, we investigated the localization and orientation of in vitro translated radiolabeled Sgk1 after import into intact mouse kidney mitochondria. Figure 4 shows that full Biochemical Journal Immediate Publication. Published on 16-Jun-2006 as manuscript BJ20060386

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© 2006 Biochemical Society length Sgk1 is imported by purified mitochondria in a time-dependent manner. In constrast, the ∆1-59 variant, lacking the mitochondrial targeting sequence, completely remained in the supernatant after pelleting the organelles. When mitochondria were disrupted and separated into a soluble and a membrane fraction after protein import, Sgk1 was detectable only in the membrane fraction even after treatment with 100 mM sodium carbonate (Figure 5A, upper panel). Under these conditions, peripheral membrane proteins are released to the supernatant as is the case for cytochrome C, which is attached to the inner mitochondrial membrane mainly by electrostatical force (Figure 5A, lower panel). These experiments indicate that Sgk1 is an integral mitochondrial membrane protein. Based on the observation that Sgk1 can be phosphorylated by cytosolic kinases, we speculated that Sgk1 might be localized to the OMM with the kinase domain and the C-terminus facing the cytosol. To test this assumption, imported Sgk1 was subjected to protease protection assays. As control for correct protein import, we used the in vitro translated and radiolabeled mitochondrial matrix enzyme fumarate hydratase (Figure 5D). Proteinase K treatment of mitochondria led to complete digestion of Sgk1. To demonstrate that mitochondria remained intact during protease treatment, we performed Western blot analyses with antibodies directed against the intermembrane space protein Smac/Diablo and the matrix protein Hsp60. Figure 5C shows that both proteins were protected unless Triton X-100 was added. Cytochrome C is extremly resistent to digestion by proteinase K [START_REF] Uren | Mitochondrial release of pro-apoptotic proteins: electrostatic interactions can hold cytochrome c but not Smac/DIABLO to mitochondrial membranes[END_REF] and served as loading control (Figure 5C, lowest panel). The degradation of Sgk1 suggests a largely cytoplasmic orientation at the OMM. From these studies, we conclude that Sgk1 is targeted and anchored via amino acids 16-33 to the OMM and faces the cytosol with the Cterminus containing the catalytic domain.

Half-life of Sgk1 is determined by amino acids 17-47

It has been documented previously [START_REF] Kobayashi | Activation of serum-and glucocorticoid-regulated protein kinase by agonists that activate phosphatidylinositide 3-kinase is mediated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and PDK2[END_REF][START_REF] Brickley | Ubiquitin modification of serum and glucocorticoid-induced protein kinase-1 (SGK-1)[END_REF] that Sgk1 protein levels are very low even after overexpression in cell lines because of rapid ubiquitin mediated degradation by the proteasome [START_REF] Kobayashi | Activation of serum-and glucocorticoid-regulated protein kinase by agonists that activate phosphatidylinositide 3-kinase is mediated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and PDK2[END_REF][START_REF] Brickley | Ubiquitin modification of serum and glucocorticoid-induced protein kinase-1 (SGK-1)[END_REF]. The short life span is conferred by the first 60 amino acids of Sgk1, but is not due to ubiquitination of lysine-residues within this region [START_REF] Brickley | Ubiquitin modification of serum and glucocorticoid-induced protein kinase-1 (SGK-1)[END_REF]. To more precisely determine the region responsible for proteasomal degradation, we performed pulse-chase experiments using Sgk1-GFP deletion variants. After a 10 minute pulse with [ 35 S]methionine/cysteine, transfected HEK293 cells were immediately lysed or chased for 15 minutes, 60 minutes, and 4 hours. Sgk1 fusion proteins were recovered by immunoprecipitation with an anti-GFP serum and subjected to autoradiography (Figure 6). GFP alone does not show any apparent degradation within a 4

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© 2006 Biochemical Society hour chase, nor does Sgk1 60-431, as previously described [START_REF] Brickley | Ubiquitin modification of serum and glucocorticoid-induced protein kinase-1 (SGK-1)[END_REF]. In contrast, full length Sgk1 and Sgk1 1-60 are rapidly degraded. Fine mapping of the regions responsible for the fast turnover showed that amino acids 17-32 and 33-47 are sufficient for targeting Sgk1 for proteasomal degradation (Figure 6). Therefore, the short half-life of Sgk1 and mitochondrial targeting are conveyed by the identical peptide sequence.

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Discussion

Here we demonstrate that Sgk1 is anchored via the N-terminus to the OMM, that mitochondrial targeting is conferred by amino acids 17-32, that the C-terminus containing the kinase domain faces the cytosol, and that mitochondrial targeting is inseparably linked to the rapid degradation of Sgk1 by the proteasome. Using an in vitro import assay, confocal microscopy of Sgk1-GFP deletion variants in HEK293 cells, and biochemical fractionation of murine liver tissue, we provide three independent experimental lines of evidence that Sgk1 is predominantly localized to mitochondria and resides at the OMM. Mitochondrial localization is mediated by amino acids 17-32 which resemble a bona fide mitochondrial targeting signal. However, this region is not a classical mitochondrial presequence, since Sgk1 is not imported into the mitochondrial matrix and is not cleaved by the mitochondrial processing peptidase. Several OMM proteins with cytoplasmic orientation are targeted by a non-cleavable N-terminal signal anchor consisting of a hydrophobic transmembrane domain and a net positive charge at the corresponding C-terminal flanking segment [START_REF] Suzuki | Characterization of rat TOM70 as a receptor of the preprotein translocase of the mitochondrial outer membrane[END_REF][START_REF] Kanaji | Characterization of the signal that directs Tom20 to the mitochondrial outer membrane[END_REF]. Similarly, the N-terminal region of Sgk1 is hydrophobic and is followed by three positively charged residues (Figure 1). Although the carbonate extraction data support that Sgk1 is an integral component of the OMM, a stretch of ten hydrophobic amino acids as indicated by the Kyte-Doolittle plot is generally considered to be too short for a membrane spanning segment. However, even if not integral, the experiments indicate that this segment at least confers an unusually strong peripheral membrane association. The cytoplasmic orientation of Sgk1 meets the requirement to be accessible for activation by cytosolic kinases [START_REF] Park | Serum and glucocorticoid-inducible kinase (SGK) is a target of the PI 3-kinase-stimulated signaling pathway[END_REF][START_REF] Kobayashi | Activation of serum-and glucocorticoid-regulated protein kinase by agonists that activate phosphatidylinositide 3-kinase is mediated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and PDK2[END_REF].

Our data provide strong evidence that Sgk1 is primarily localized to the outer mitochondrial membrane, but do not exclude other sites of localization. Previous reports describe that Sgk1 is found in association with membranes [START_REF] Brickley | Ubiquitin modification of serum and glucocorticoid-induced protein kinase-1 (SGK-1)[END_REF][START_REF] Alvarez De La Rosa | Distribution and regulation of expression of serum-and glucocorticoid-induced kinase-1 in the rat kidney[END_REF]. Our data indicated that these membranes might have been of mitochondrial origin. Moreover, our data support the observation of Brickley and coworkers that a variant of Sgk1 lacking the first 60 residues does not exhibit membrane association, is not ubiquitinated, and consequently, has a prolonged half-life [START_REF] Brickley | Ubiquitin modification of serum and glucocorticoid-induced protein kinase-1 (SGK-1)[END_REF]. Finally, the Nterminus of Sgk1 does not contain a classical targeting signal, and targeting of the kinase to different membrane systems and compartments might depend on the specific cell lines or culture conditions used. For example, the Bcl-2 protein can associate with both, the OMM and microsomal membranes via a C-terminal hydrophobic domain [START_REF] Janiak | Assembly of Bcl-2 into Microsomal and Outer Mitochondrial Membranes[END_REF].

At the plasma membrane, Sgk1 was proposed to interact and phosphorylate the E3 ubiquitin ligase Nedd4-2 and thereby controlling plasma membrane insertion of ENaC sodium transporters [START_REF] Debonneville | Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na(+) channel cell surface expression[END_REF]. However, using surface plasmon resonance analysis, a direct interaction of Biochemical Journal Immediate Publication. Published on 16-Jun-2006 as manuscript BJ20060386

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Sgk1 with Nedd4-2 could not be confirmed [START_REF] Henry | Affinity and specificity of interactions between Nedd4 isoforms and the epithelial Na+ channel[END_REF]. Moreover, consistent with our results, a recent report described activation of ENaC by Sgk1 independent of Nedd4-2 [START_REF] Diakov | A novel pathway of epithelial sodium channel activation involves a serum-and glucocorticoid-inducible kinase consensus motif in the C terminus of the channel's alpha-subunit[END_REF].

Using confocal fluorescence microscopy, we also never observed significant localization of Sgk1 to the nucleus, not even after serum stimulation or heat shock, two stimuli that were reported to lead to nuclear translocation of Sgk1 [START_REF] Buse | Cell cycle and hormonal control of nuclear-cytoplasmic localization of the serum-and glucocorticoid-inducible protein kinase, Sgk, in mammary tumor cells. A novel convergence point of anti-proliferative and proliferative cell signaling pathways[END_REF][START_REF] Maiyar | Importin-alpha mediates the regulated nuclear targeting of serum-and glucocorticoid-inducible protein kinase (Sgk) by recognition of a nuclear localization signal in the kinase central domain[END_REF][START_REF] Leong | Expression of the serum-and glucocorticoid-inducible protein kinase, Sgk, is a cell survival response to multiple types of environmental stress stimuli in mammary epithelial cells[END_REF]. Consistent with this, we demonstrate that endogenous Sgk1 is not present at detectable levels in the nuclear fraction of mouse liver tissue. However, we would like to point out that in these experiments pretreatement of mice with steroid hormones was necessary to induce detectable levels of Sgk1 protein expression. In mammary epithelial and tumor cells, steroid hormone treatment was shown to lead to a translocation of Sgk1 from the nucleus to a perinuclear compartment which contains mitochondria [START_REF] Buse | Cell cycle and hormonal control of nuclear-cytoplasmic localization of the serum-and glucocorticoid-inducible protein kinase, Sgk, in mammary tumor cells. A novel convergence point of anti-proliferative and proliferative cell signaling pathways[END_REF][START_REF] Leong | Expression of the serum-and glucocorticoid-inducible protein kinase, Sgk, is a cell survival response to multiple types of environmental stress stimuli in mammary epithelial cells[END_REF]. Moreover, different cell lines and tissues are likely to differ in their capacity to import Sgk1 into the nucleus.

We here show that amino acids 17-32 are essential for organelle targeting and determine the half-life of Sgk1 protein. This makes it likely that degradation of Sgk1 is facilitated rapidly upon arrival at its mitochondrial destination. A corollary of this finding is that a large number of studies using an active variant of Sgk1, lacking the N-terminal 60 residues, need to be critically revisited. This long-lived variant, which has often been used in kinase assays for the detection or validation of kinase targets, is not directed to its physiological subcellular localization and is therefore prone to interact with and phosphorylate nonphysiological protein targets. Moreover, our data indicate that the use of Sgk1 variants with N-terminal tags should be avoided, because these are likely to alter the subcellular localization. In this context it is interesting to note that Helms and colleagues [START_REF] Helms | Hormone-regulated transepithelial Na+ transport in mammalian CCD cells requires SGK1 expression[END_REF] reported that a ∆1-60 variant showed less biological activity than full length Sgk1 on ENaC function. C. Aliquots of the protease protection assay were subjected to Western blot analyses with antibodies directed against the matrix protein Hsp60, and the intermembrane space proteins Smac/Diablo and cytochrome C (CytC). CytC is resistent to proteinase K treatment under the conditions used, even when the membranes are permeabalized by Triton X-100 [START_REF] Uren | Mitochondrial release of pro-apoptotic proteins: electrostatic interactions can hold cytochrome c but not Smac/DIABLO to mitochondrial membranes[END_REF], and therefore served as loading control. Hsp60 and Smac/Diablo remain intact in the absence of Triton X-100 demonstrating the integrity of the organelles. D. The capability of the preparation to correctly import proteins is shown by the import of in vitro translated fumarate hydratase (FH) in parallel. Imported FH migrates slightly faster than the in vitro translation product (ivt) because of the removal of the signal peptide by the processing peptidase after import into the matrix. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 16-Jun-2006 as manuscript BJ20060386 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. © 2006 Biochemical Society In order to gain further insight into the regulation and the range of cellular functions of Sgk1 the goals of the present study were to examine the intracellular localization of Sgk1 and to determine the region responsible for localization and degradation.

  ng Mito-DsRedII (BD Clontech, Palo Alto, CA.) and 200 ng of GFP or the indicated Sgk1-GFP fusion protein variants using the FUGENE transfection reagent (Roche Biochemicals, Mannheim, Germany) according to the manufacturers instructions. Confocal Laser Scanning Microscopy 48 hours after transfection, HEK293 cells were washed with PBS, fixed with 4% paraformaldehyde and nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) before mounting. Microscopy was performed with a Zeiss LSM510 inverted laser scanning microscope (Carl Zeiss, Jena, Germany) using the following configuration: DAPI: λ exc. = 364 nm, BP λ em. = Biochemical Journal Immediate Publication. Published on 16-Jun-2006 as manuscript BJ20060386 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. © 2006 Biochemical Society 385-470 nm, GFP: λ exc. = 488 nm, BP λ em. = 505-550 nm, DsRed2: λ exc. = 543 nm, LP λ em. = 560 nm. Images were processed with the Zeiss LSM image browser software.

  33-431 (Figure 2E) and Sgk1 48-431 (Figure 2G) show a fluorescence pattern indistinguishable from that of GFP alone (data not shown). To investigate if the C-terminal regions of Sgk1 contribute to protein targeting, we fused the same deletion variants but ending at amino acid position 60 of Sgk1 to GFP. Confocal analyses of HEK293 cells transfected with the variants Sgk1 1-60-GFP (Figure 2B), Sgk1 17-60-GFP (Figure 2D), Sgk1 33-60-GFP (Figure 2F) and Sgk1 48-60-GFP (Figure 2H) revealed no difference between these and the long variants arguing against an influence of the C-terminal parts of Sgk1 on protein targeting. Additionally, we analyzed the variants Sgk1 1-16-GFP and Sgk1 1-32-GFP. Sgk1 1-16-GFP was distributed Biochemical Journal Immediate Publication. Published on 16-Jun-2006 as manuscript BJ20060386A 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 influence of Sgk1 on a broad spectrum of cellular functions and its activation by several different stimuli indicates that Sgk1 plays a central role in cell signalling. Our data provide evidence that Sgk1 acts at the OMM. This finding may lay the basis for a further analysis of the function of Sgk1. The low protein expression levels of Sgk1 however renders a thorough biochemical examination of this molecule difficult. Detailed analysis of the molecules involved in the degradation of Sgk1 will clearly provide more insight into the biology of this kinase. Biochemical Journal Immediate Publication. Published on 16-Jun-2006 as manuscript BJ20060386 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. © 2006 Biochemical Society

Figure 1 .

 1 Figure 1. Amino acid sequence and hydropathy of the Sgk1 N-terminus. A. The N-terminal 60 amino acids of murine Sgk1. Residues are shown in the one-letter code. Starting amino acids for the constructs used in this study are in bold, in some constructs tyrosine 47 (Y47) was replaced by methionine. Grey box highlights a predicted mitochondrial targeting sequence, and arrowhead indicates a potential cleavage site for the mitochondrial processing peptidase at amino acid position 33. Black bar indicates a potential transmembrane segment as predicted by the Kite-Doolittle analysis. B. Kite-Doolittle hydropathy plot of Sgk1. The N-terminus of Sgk1 contains a potential transmembrane segment.

Figure 2 .

 2 Figure 2. Amino acids 17-33 target Sgk1 to mitochondria in HEK293 cells. HEK293 cells were transfected with the mitochondrial marker Cytochrome C oxidase subunit VIII coupled to DsRedII and either full length Sgk1-GFP (A) or Sgk1-GFP deletion variants (B-J). Cells were grown on coverslips, formaldehyde-fixed and nuclei were stained with DAPI. Left panels show GFP fluorescence signals (green), middle panels show DAPI (blue) and mitochondrial staining (red) and right panels show merged images. Yellow color indicates colocalization.

Figure 3 .

 3 Figure 3. Sgk1 localizes to mitochondria in vivo.The distribution of the Sgk1 protein in subcellular fractions from liver tissue was analyzed four hours after intraperitoneal administration of 100 µg/kg body weight dexamethasone. To be able to identify Sgk1 signals, Sgk1 knockouts (-/-) and their wild-type littermates were analyzed in parallel. Signals were obtained predominantly from the mitochondrial fraction and only weakly from the cytosolic and microsomal fraction. Lower panels show immunoblots with antibodies against the indicated organelle markers to demonstrate purity of the fractions. 20 µg protein were loaded per lane for detection of Sgk1 and 10 µg were loaded for detection of organelle markers. For the detection of Sgk1 we employed the antibody obtained from Sigma. PM: Plasma membrane.

Figure 4 .

 4 Figure 4. Sgk1 associates with purified mitochondria in a time dependent manner.Radiolabeled in vitro translated full length Sgk1 or Sgk1 ∆1-59 were exposed to highly purified mitochondria from mouse kidneys for the indicated times. Mitochondria were washed, lysed,

Figure 5 .

 5 Figure 5. Sgk1 associates with the outer mitochondrial membrane. A. Membrane association of imported radiolabeled Sgk1 was assessed by preparation of mitochondrial (mitos) soluble (s/n) and membrane (mem) fractions. Radiolabeled proteins were visualized by autoradiography (AR). The peripheral membrane protein cytochrome C (CytC) was released by Na 2 CO 3 -treatment and visualized by Western blot (WB, lower panel). Asterisk: this signal sometimes occurred after in vitro translation and is most likely a degradation product (also in B). B. After import of in vitro translated radiolabled Sgk1, mitochondria were treated with 200 µg/ml proteinase K (PK) in the presence or absence of Triton X-100 or left untreated. Sgk1 was subject to complete digestion indicating a localization to the cytoplasmic face of the OMM. ivt: an aliquot of the in vitro translation product.

Figure 6 .

 6 Figure 6. Rapid turnover of Sgk1 is determined by amino acids 17-47. Sgk1-GFP fusion protein variants or GFP alone were transfected into HEK293 cells and subjected to pulse-chase analysis. After a pulse of 10 minutes, proteins were chased for 0 minutes, 15 minutes, 1 hour, or 4 hours, and the respective fusion proteins were recovered by immunoprecipitation with an anti-GFP antiserum. All variants containing amino acids 17-47 were rapidly degraded.

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Acknowledgements

We thank Burkhard Wiesner from the confocal imaging unit of the Institute for Molecular Pharmacology, Berlin, for help with laser scanning microscopy, Ralf Schülein for providing anti-GFP rabbit serum, and Cecilia Canessa and James Murray for providing anti-Sgk1 antibodies.

We also thank Volker Haucke for discussions.