Stefania Lo Feudo
email: stefania.lofeudo@unical.it

Anissa Allani
email: anissa.allani@enpc.fr

Gwendal Cumunel
email: gwendal.cumunel@enpc.fr

Pierre Argoul
email: pierre.argoul@enpc.fr

Domenico Bruno
email: d.bruno@unical.it

Experimental study of a tuned mass damper endowed with changeable stiffness and eddy currents damping

Keywords: AMA, courants de Foucault, transformée en ondelettes continue, control passif TMD, eddy currents, continuous wavelet transform, passive control

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

The Tuned Mass Damper (TMD) is a passive control device in which a mass-spring-dashpot system is attached to a primary structure, for example a tall building or a bridge (see [START_REF] Soong | Passive Energy Dissipation Systems in Structural Engineering[END_REF] for a list of actual applications). If the TMD is properly designed and properly tuned with the primary structure, it is able to reduce vibrations induced by external sources. Indeed, since an amount of vibration energy is transferred from the principal structure to the TMD, a dissipation energy mechanism is activated and the durability and the structural safety of the main structure can be greatly improved. Several criteria may be adopted in order to properly choose the TMD parameters. Among these, a class of optimization criteria is aimed to reduce the vibration energy of the system, which is referred as H 2 norm optimization [START_REF] Asami | Closed-form exact solution to 𝐻 2 optimization of dynamic vibration absorber attached to damper linear systems[END_REF]. Others strategies are based on the minimization of the transfer function maximum [START_REF] Nishihara | Closed-Form Solutions to the Exact Optimizations of Dynamic Vibration Absorbers (Minimizations of the Maximum Amplitude Magnification Factors)[END_REF], and on the system poles location [START_REF] Fujino | Design formulas for tuned mass dampers based on a perturbation technique[END_REF]. In addition, in the past decades several studies were conducted for different types of external excitation, especially concerning seismic loading. Indeed, a debate exists on the effectiveness of the damper masses and important studies were those carried out by [START_REF] Sadek | A method of estimating the parameters of tuned mass dampers for seismic applications[END_REF] and [START_REF] Soont | Parametric study and simplified design of tuned mass dampers[END_REF]. Also [START_REF] Hadi | Optimum Design of Absorber for MDOF Structures[END_REF] applied the H ∞ norm optimization to a MDoF structure endowed with a TMD. The authors found that for the case studied the control device was effective in reducing the vibration response, even though at the beginning of the excitation the response of the structure appeared quite similar to the uncontrolled case. Then, [START_REF] Lee | Optimal design theories and applications of tuned mass dampers[END_REF] analyzed the MDoF structure response with a single or multiples TMDs and [START_REF] Bekdaç | Estimating optimum parameters of tuned mass dampers using harmony search[END_REF] proposed a new procedure, called Harmonic Search, in which the parameter are obtained for a harmonic load, and then checked in case of earthquake. They obtained a considerable reduction in the maximum top story displacement, and remarked that optimal parameters are numerically small, which may be preferable since forces acting on the building from TMD are less, and owing to a likely cost reduction.

In this research study, the effectiveness of an experimental TMD model proposed by the authors in [START_REF] Feudo | Experimental Tuned Mass Damper Based on Eddy Currents Damping Effect and Adjustable Stiffness[END_REF] is further investigated. In particular, the TMD is used to control vibration of a three stories frame scale model, and placed on its top floor. The TMD, which mass is fixed for practical issues, has adjustable stiffness and damping coefficient, in order to allow the investigation of the proposed experimental model and to easily set the TMD with optimal parameters. The damping of the TMD is due to the eddy currents damping effect, which arises when a non-magnetic conductive material is subjected to a time-varying magnetic field [START_REF] Graves | Theoretical comparison of motional and transformer EMF device damping efficiency[END_REF]. Indeed, induced currents appear into the conductor and circulate in such a way to generate a magnetic field, which opposes the original change in magnetic flux. In addition, a Lorentz force contrasts the motion and, owing to the resistance of the conductor, energy is dissipated into heat and a vibrations reduction is achieved. This mechanism is adopted in several fields, such as in braking systems, vibration control of rotating systems, vibration isolation in levitation systems and also for the structural vibration suppression [START_REF] Sodano | Eddy Current Damping in Structures[END_REF]. One of the major advantages of adopting this damping mechanism is the lack of contact between the eddy current damper and the system to damp. Indeed, the material properties are unaffected by its addition, and the performance does not degrade over the time. For example, in order to control vibration of a beam, an eddy currents damper was analyzed by [START_REF] Bae | Vibration suppression of a cantilever beam using magnetically tuned-mass-damper[END_REF] and [START_REF] Wang | Feasibility study of a large-scale tuned mass damper with eddy current damping mechanism[END_REF], and also was recently used by the Gensler Architecture firm as a passive control device for the Shanghai Tower, which is 623 m tall. The TMD is placed on the 125th floor, it occupies five floors and it weighs about 1,000 tons. In particular, the TMD is made by 1,076-square-foot copper plate covered with 125 powerful magnets, mounted beneath a suspended mass damper, consequently such a system, according to the designers, is able to resist a 2500-year earthquake [START_REF] Gao | Construction Technology and Management Innovation[END_REF].

The present work is organized as follows. In the first sections, the TMD governing equations are briefly presented, together with the eddy current damping effect principle. Next, the proposed experimental TMD is described. Then, the TMD is tested and used in order to control vibrations of a frame scale model. In particular, the TMD is optimized starting from experimental results when a shock is applied on the structure top floor, and by using a numerical model in case of ground motion acceleration. Some remarks conclude the paper.

TMD governing equations

Let's consider the system represented in Figure 1, where a TMD is located on the top floor of a NDoF structure, where the floors may be considered as infinitely stiff and the masses as concentrated at the floor levels. Therefore, 𝐌 s , 𝐂 s and 𝐊 s are the mass, damping and stiffness matrices of the NDoF system, 𝑭(𝑡) is the vector of external forces, 𝑭 𝑇 (𝑡) the force exerted on the structure by the TMD, and 𝐌 s 𝑰𝑢̈𝑔(𝑡) the earthquake base acceleration, where 𝑰 is the unit vector. With the aim to control the predominant 𝑖 -mode and the relative displacement with respect to the ground of the 𝑛-th floor, 𝑢 𝑛 , the equations of motion may be expressed in the following form: [1] where m T , c T and k T are the TMD mass, damping coefficient and stiffness, respectively, u T is the TMD relative displacement, and [2] wherein Γ i,n * denotes the modal participation factor, and Φ is the modal matrix, which can be evaluated by solving the generalized eigenproblem K s Φ i = ω si 2 M s Φ i , where ω si are the modal frequencies. By passing to the frequency domain, and by applying the Fourier transform, it is also possible to define the dynamic stiffness matrix, G * (ω) as follows:

( ) ( ) ( ) ( ), i=1,...,n ( ( ) ( )) ( ( ) ( )) ( ( ) ( )) 0 si n n si n n si n n i n Ti n si n i n g T T g T T n T T n m u t c u t k u t f t f t m u t m u t u t c u t u t k u t u t             * * * * * * , , , , , , 2 2 2 * , ( ) ( ) , , , , , , ( ) ( )
T T T T s s s s s s T T s si si si i Ti i si n si n si n i n Ti n i n in in in in in in si n = , = , = , (t)= (t), (t)= (t), = , m c k f t f t g m c k f t f t m       TT m Φ M Φ c Φ C Φ k Φ K Φ Φ Φ Φ M I f F f F g Γ ΦΦ Φ Φ Φ Φ       * * * , , , 2 * 2                          T T T T T si n si n si n T T T T i c i c k i c k i c k m i c mk k G 9ème Colloque National AFPS 2015 -IFSTTAR 4 [4] , ( ) , ( ) ,
Finally, it is possible to introduce the normalized dynamic flexibility matrix, which can be evaluated as:

* *-1 (ω)= (ω) HG [5]

Optimization criterion

The TMD's performance, sensibility and robustness depend highly by its dynamical properties. Therefore, with the aim to properly set the TMD parameters, several optimization criteria were proposed in a large number of previous studies. In fact, the criterion to adopt is one of the most important design decisions, which may depend by several factors, as the nature of the external excitation, the type of structure, the technical and economic feasibility, etc. In particular, in this study, the chosen optimization criterion belongs to a class of criteria aimed to reduce the vibrational energy of the system. Indeed, such an approach was proved to be quite effective and robust, (Allani, 2015), and in general it leads to lower damping ratio values, which are preferable from a practical point of view. Therefore, the optimization criterion consists in minimizing a cost function which depends on the frequency response function (FRF) of the n-th floor, which is the floor to be controlled. In particular, the objective is to minimize the ratio between the square root of the squared absolute value of the FRF in the controlled case and in the uncontrolled case, i.e. when no TMD is used. Moreover, the above quantities are evaluated within a frequency range of interest, [ω 1 , ω 2 ], where ω 1 and ω 2 are the lower and upper frequency bound considered, respectively. According to (Allani, 2015), the criterion may be set in the following form: [6] where |u n (ω)/F n (ω)| and |u n (ω)/üg(ω)| are the moduli of the FRF referred to the cases of force acting on the top floor and ground acceleration, respectively, and |u n0 (ω)/F n (ω)| and |u n0 (ω)/üg(ω)| denote the same quantities of above, but evaluated when no TMD is placed on the structure. Therefore, for an uncontrolled structure, the criterion was setting up in such a way that it is equal to one. Hence, by keeping fixed for practical issues the TMD mass, m T , the design parameters to be chosen are the TMD stiffness and damping coefficient, k T and c T , respectively, which minimize the above cost functions.

Eddy currents damping effect

In this section, the governing equations related to eddy currents will be briefly recalled, according to the formulation used in [START_REF] Bae | Vibration suppression of a cantilever beam using magnetically tuned-mass-damper[END_REF] and reported by [START_REF] Feudo | Experimental Tuned Mass Damper Based on Eddy Currents Damping Effect and Adjustable Stiffness[END_REF]. When a conductive material moves through a stationary magnetic field, since it is subjected to a time-varying magnetic field, eddy currents are induced. By neglecting the surface charge in the conductive plate, the eddy currents density is given by = σ × () J v B [START_REF] Allani | Amortissement multimodal dans les grandes structures souples en vibration[END_REF] where σ is the material electrical conductivity, v is the relative velocity between the permanent magnet and the conductor, B is the magnetic flux density and v × B represents the electromotive force driving the eddy currents. Since the eddy currents generate another magnetic field, which polarity is opposite to those due to the

* * * ,, ( ) [ ( ), ( )] ( ) [ ( ) - ( ), - ( )]          T nT T i n si n in g T g uu f m u m u u P    F T T E T T 2 2 1 1 22 11 2 2 ω ω n n ω ω g n 22 ωω n0 n0 ωω ng C (k ,c )= , C (k ,c )= u( ω) u( ω) dω dω u( ω) F( ω) u( ω) u (ω) dω dω F( ω) u (ω)
9ème Colloque National AFPS 2015 -IFSTTAR 5 permanent magnet, a repulsive force appears. This force can be evaluated according to the Lorentz's Law and, if the velocity has a component only in the x direction, it takes the following form:

=  22 x z y x y x z V V ( × ) dV = σv [(-B -B ) + B B + B B ] d V F J B i j k [8]
where V is the conductor volume. Hence, according also to [START_REF] Wang | Feasibility study of a large-scale tuned mass damper with eddy current damping mechanism[END_REF], the electromagnetic force evaluated in the x direction does not depend on the B x component of the magnetic field. Therefore, the damping force in the x direction is proportional to the velocity vector with opposite direction, and a linear damping coefficient of the TMD may be defined as follows:

[9]

Experimental TMD

The proposed experimental TMD is made of a 0.15 kg copper mass, which is supported by two thin steel sheets. The device is designed in such a way to allow a manual adjustment of the natural frequency and of the inherent damping, and is placed on the top floor of a building scale model (Figure 1). The bottom of the mass is also connected to a slender cable fixed at the two ends. One of the cable's support position is adjustable, so that the amount of stretch in the slender cable can be manually adjusted. As long as the cable tensile stress is increased, the TMD stiffness and natural frequency rise. In this study, by considering the frequency bandwidth of interest, the support is moved over a range of 0.4 mm with a step size of 0.04 mm. The damping of the TMD is conversely given by a magnet located up to the mass. Indeed, since the conductive mass is subjected to a timevarying magnetic field, eddy currents are induced in the conductor. The distance between the magnet and the mass is adjustable, and the gap is moved from 3.35 mm to 5.35 mm, with a step size of 0.2 mm.

The evaluation of the TMD frequency and damping ratio is carried out by applying on experimental data the Continuous Wavelet Transform (CWT), which enables to estimate the instantaneous value of modal frequencies, damping and mode shapes. In particular, the CWT of an asymptotic signal is concentrated along some curves in the time-frequency domain called ridges, and the restriction of the CWT to each ridge is called skeleton of the wavelet. The skeleton gives the natural frequency of the signal, whereas the time evolution of the logarithm of the Wavelet transform amplitude gives information about damping, for further explanation please see [START_REF] Le | Continuous wavelet transform for modal identification using free decay response[END_REF]. Therefore, in order to characterize the TMD properties behavior also when the magnet distance and the stretch in the cable are changed, the response of the TMD alone was recorded in several free vibration tests. For each fixed value of tensile cable stress, the temporal response of the TMD was measured by changing the gap between the magnet and the mass, by carrying out each test three times. Then, the CWT was applied on data, which results were already presented in [START_REF] Feudo | Experimental Tuned Mass Damper Based on Eddy Currents Damping Effect and Adjustable Stiffness[END_REF] and will be here briefly reported. In Figure 2, the instantaneous values of TMD frequency and damping ratio, and the force -displacement trend are shown for a chosen TMD configuration. By looking within the domain of interest, which is delimitated by the solid red thick lines [START_REF] Le | Continuous wavelet transform for modal identification using free decay response[END_REF], it emerges that the frequency keeps almost constant during the test, whereas the damping ratio exhibits a non-linear behavior. However, this non-linearity seems to depend mostly on the intensity of the excitation (shock), than on the distance between the magnet and the mass. However, from Figure 2 it appears also that the damping force-displacement plot has a comparable behavior with respect to those of a linear oscillator. In Figure 3, results obtained in terms of modal parameters by changing the TMD configuration are summarized. In particular, the damped frequency and the damping ratio reported are the average values taken over the time interval for each test, and over the number of test (3) performed for each configuration. As a result, for a fixed tension in the cable, the frequency keeps constant by varying the distance of the magnet, and it varies linearly by displacing the cable support. Conversely, the damping ratio has nearly a nonlinear behavior with respect to the magnet position and the cable tensile stress.

Applications for vibration control of a frame scale model

The TMD is placed on the top of a three stories frame structure, which horizontal planes are made by aluminum plates supported by four thin aluminum beams placed on two opposite sides of the plates. The floors weights are m 1 = 2.43 kg, m 2 = 2.43 kg and m 3 = 3.76 kg, where the subscript 1 denotes the lower floor. In particular, the top floor hosts two fixed supports for the TMD, which allow to change its stiffness and damping and which are considered as a part of the principal structure. The instantaneous modal properties of the structural system, without the TMD, are evaluated by applying the CWT on the experimental signals obtained after an impact on the top floor. However, in order to implement a numerical model, the stiffness and the damping coefficient have to be evaluated. In this study, the Modal Assurance Criterion (MAC) is adopted, that is a model updating method able to measure the consistency, namely the degree of linearity, between two different modal vectors [START_REF] Allemang | The modal assurance criterion-twenty years of use and abuse[END_REF]. In particular, the experimental results are considered as the reference model, whereas the numerical results are obtained by iteration starting from the identification parameters, which are the unknowns of the problem, i.e. the stiffness and the damping coefficients. The MAC is bounded between zero and one, with zero representing no consistent correspondence. Therefore, for each set of unknowns, the mode shapes are evaluated and an objective function is minimized. In the standard formulation, the latter is defined as the relative error between modal frequencies and mode shapes obtained from a set of unknowns parameters (f num,i , ϕ num,i ) in the numerical case and in the reference solution (f exp,i , ϕ exp,i ), [START_REF] Gambarelli | A surrogate-assisted evolutionary algorithm for dynamic structural identification[END_REF]. In order to equally take into account the correspondence between the frequencies and the mode shapes, the objective function to minimize is defined according to [START_REF] Gentile | Operational modal analysis of curved cable-stayed bridges[END_REF] as:

[10] , 1 () 1 n exp,i num i obj i i exp,i ff J NMD nf         9ème Colloque National AFPS 2015 -IFSTTAR 7
where NMD i = √(1 -MAC)/MAC is the Normalized Modal Difference, which is adopted in order to estimate the average difference between two modal vectors, [START_REF] Mordini | The Finite Element Model Updating: A Powerful Tool for Structural Health Monitoring[END_REF]; a NMD value close to zero denotes a better correlation. The comparison between experimental and numerical results in term of modal frequencies, and mode shapes through the MAC and the NMD is reported in Table 1. A high correlation is found for the first mode, although the error slightly increases for the other two modes. A good correspondence exists in the structural response after a shock, as shown in Figure 4. In particular, the experimental signals are filtered in order to eliminate an unwanted low frequency component and a frequency component arising from the fixed support of the TMD. Indeed, since their mass is small with respect to the floor mass, they are supposed to be part of the top floor.

Free vibrations

The frame scale model free vibrations are recorded and analyzed when an impact force is applied on the top floor of the frame scale model. Then, the structural response is compared when no control device is used at all, and when the TMD is attached on the structure and set with optimal parameters. In particular, the optimal values of TMD frequency and damping are chosen by evaluating, for each combination of TMD properties here considered, the function C F reported in Eq. [6]. The combinations of damped frequency and damping ratio (f T , ξ T ) which corresponds to a minimum for C F is selected as the optimum configuration. However, this is not a numerical procedure, but an experimental optimization conducted among a limited range of dynamical properties, namely f T = 9.6 ÷ 11.7 Hz and ξ T = 1.40 ÷ 3.11 % . In Figure 5, the FRFs and the top floor response are reported in the uncontrolled and in the controlled cases. The TMD greatly reduces the first peak of the FRFs, which is also split into two. Moreover, although the TMD is tuned with only the first modal frequency and it does not affect greatly the others, a global response reduction may be observed in term of acceleration, velocity and roof displacement.

Numerical analysis under ground motion acceleration

The effectiveness of the proposed TMD is here numerically evaluated when the structure is subjected to a ground motion acceleration. To this end, three recorded accelerograms (El Centro NS 1940, L'Aquila AQV -EW 2009and Friuli GMN -EW 1976), and an artificial one were chosen and scaled in such a way that the first modal frequency of the uncontrolled structure falls in the earthquake frequency content, as shown in Figure 6. The value assumed by the cost function 𝐶 𝐸 presented in Eq. [6], which is valid under ground motion acceleration, is shown in Figure 7 for different TMD properties. As it could be seen, for each combinations of TMD properties, a structural response reduction in term of energy is obtained since 𝐶 𝐸 always keeps less than 1. An optimal combination of damping ratio and stiffness of the added mass can also be identified. In particular, as expected, the optimal TMD frequency value is in the neighborhood of the scale model first modal frequency, although it slightly varies with the accelerogram considered. In Figure 8, the response of the controlled and uncontrolled structure is compared when the TMD is set with the optimal parameters. As it could be seen, in the first instant of the earthquake, the top floor displacement is not influenced by the presence of the TMD. On the contrary, in the next part a high response reduction may be observed since a larger amount of energy can be transferred from the structure to the TMD.

Conclusion

In this study, an experimental TMD endowed with adjustable stiffness and damping, and based on the eddy currents damping effect, is studied. The CWT is applied on the experimental free vibration signals of the proposed TMD. As a result, it is obtained that the TMD frequency keeps constant during the dynamic test, whereas the damping ratio behaves non-linearly in the first instants. Moreover, the TMD frequency increases linearly with the cable tensile stress, whereas the damping ratio varies non-linearly by varying the gap between a circular magnet and the damper mass. The TMD is then attached on the top floor of a three stories frame scale model. Optimal parameters are found in order to control the structure under impulsive shock, and the TMD is show to be very effective. Finally, a numerical analysis is conducted in case of ground motion acceleration. It appears that, if the earthquake frequency content matches the structure mode for which the TMD is tuned, a response reduction may be observed. However, the TMD optimal parameters depend on the considered accelerogram, so that a statistical analysis of strong-motion accelerograms may be required.

Figure 1 .

 1 Figure 1. Experimental eddy currents Tuned Mass Damper

  pass from a set of differential equations, [1], to a set of algebraic equations through G * (ω)u(ω) = P(ω), where

Figure 2 .

 2 Figure 2. From left to right: CWT, damping ratio and damping force -displacement behavior of the TMD under an impulsive shock when the gap between magnet and copper mass is equal to 4,35 𝑚𝑚

Figure 4 .Figure 5 .

 45 Figure 4. Experimental and numerical top floor response

Figure 6 .

 6 Figure 6. Frequency content of the scaled accelerograms and frame scale model modal frequencies

Figure 7 .Figure 8 .

 78 Figure 7. Numerical variation of the optimization criterion depending on the TMD configuration

Table 1 .

 1 Model updating method results


Laurie-Anne Payet

Mélanie Leroux

John C Willison

Akio Kihara

Ludovic Pelosi

Fabien Pierrel
email: fabien.pierrel@univ-grenoble-alpes.fr

Mechanistic Details of Early Steps in Coenzyme Q Biosynthesis Pathway in Yeast

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

INTRODUCTION

Heterotrophic eukaryotic cells produce most of their energy via oxidative phosphorylation in mitochondria. The electrochemical gradient used for ATP synthesis is generated by the respiratory chain in which the redox lipid coenzyme Q (Q) transfers electrons between membrane-embedded multiprotein complexes responsible for proton pumping [START_REF] Genova | Functional role of mitochondrial respiratory supercomplexes[END_REF][START_REF] Wang | Understanding Ubiquinone[END_REF]. Q also functions as a membrane antioxidant and a cofactor of uncoupling proteins [START_REF] Bentinger | Coenzyme Q-biosynthesis and functions[END_REF]. Q is composed of a conserved fully-substituted benzoquinone ring which is attached to a polyisoprenyl tail of various length (six isoprenyl units in Saccharomyces cerevisiae, ten units in humans). The initial reaction of Q biosynthesis is catalyzed by the Coq2 protein [START_REF] Ashby | COQ2 is a candidate for the structural gene encoding para-hydroxybenzoate:polyprenyltransferase[END_REF][START_REF] Forsgren | Isolation and functional expression of human COQ2, a gene encoding a polyprenyl transferase involved in the synthesis of CoQ[END_REF], which conjugates the polyprenyl-pyrophosphate tail with 4-hydroxybenzoic acid (4-HB), the precursor of the benzoquinone ring of Q [START_REF] Olson | Biosynthesis of ubiquinone[END_REF]) (Figure S1A).

Multiple Coq proteins (Coq3-Coq9 in S. cerevisiae, Figure S1A) localized in the mitochondrial matrix then modify the aromatic ring of prenyl-4-HB in order to yield Q [START_REF] Tran | Endogenous synthesis of coenzyme Q in eukaryotes[END_REF][START_REF] Wang | Molecular genetics of ubiquinone biosynthesis in animals[END_REF]. Abrogation of Q biosynthesis results in the incapacity of S. cerevisiae to grow on media containing non-fermentable carbon sources [START_REF] Johnson | COQ9, a new gene required for the biosynthesis of coenzyme Q in Saccharomyces cerevisiae[END_REF] and is embryonically lethal in mammals [START_REF] Laredj | The molecular genetics of coenzyme Q biosynthesis in health and disease[END_REF][START_REF] Wang | Molecular genetics of ubiquinone biosynthesis in animals[END_REF]. Human primary coenzyme Q deficiency is caused by mutations in nine genes required for Q biosynthesis and results in clinically heterogeneous diseases which are often improved by Q supplementation [START_REF] Freyer | Rescue of primary ubiquinone deficiency due to a novel COQ7 defect using 2,4-dihydroxybensoic acid[END_REF][START_REF] Quinzii | Primary and secondary CoQ(10) deficiencies in humans[END_REF][START_REF] Salviati | Haploinsufficiency of COQ4 causes coenzyme Q10 deficiency[END_REF]. Q is also used as a dietary supplement and ongoing biotechnological efforts aim at improving Q biosynthesis in various microorganisms [START_REF] Cluis | Identification of bottlenecks in Escherichia coli engineered for the production of CoQ(10)[END_REF][START_REF] Lu | Identification and elimination of metabolic bottlenecks in the quinone modification pathway for enhanced coenzyme Q production in Rhodobacter sphaeroides[END_REF][START_REF] Moriyama | Production of CoQ10 in fission yeast by expression of genes responsible for CoQ10 biosynthesis[END_REF][START_REF] Xu | Programming Saposin-Mediated Compensatory Metabolic Sinks for Enhanced Ubiquinone Production[END_REF].

Although the synthesis of Q from 4-HB is reasonably well understood [START_REF] Gonzalez-Mariscal | Regulation of coenzyme Q biosynthesis in yeast: A new complex in the block[END_REF][START_REF] Wang | Understanding Ubiquinone[END_REF], the pathway that produces 4-HB in eukaryotes has remained elusive [START_REF] Kawamukai | Biosynthesis of coenzyme Q in eukaryotes[END_REF]. Tracer experiments performed fifty years ago suggested that animals derive 4-HB from tyrosine and phenylalanine (Phe), although Phe may first be converted into tyrosine [START_REF] Olson | Biosynthesis of ubiquinones in animals[END_REF][START_REF] Olson | Studies on coenzyme Q -Pattern of labeling in coenzyme Q9 after adminstration of isotopic acetate and aromatic amino acids to rats[END_REF]. Since then, neither enzymes nor intermediates have been assigned to the pathway from tyrosine to 4-HB. Bacteria are known to employ chorismatases like UbiC or XanB2 to synthesize 4-HB from chorismic acid, an intermediate of the shikimate pathway [START_REF] Siebert | Formation of 4-hydroxybenzoate in Escherichia coli: characterization of the ubiC gene and its encoded enzyme chorismate pyruvate-lyase[END_REF][START_REF] Zhou | The diffusible factor synthase XanB2 is a SUPPLEMENTAL REFERENCES[END_REF] which is widespread in microorganisms [START_REF] Bentley | The shikimate pathway--a metabolic tree with many branches[END_REF]. The yeast S. cerevisiae possesses the shikimate pathway and is thus believed to synthesize 4-HB from tyrosine and chorismic acid [START_REF] Clarke | New advances in coenzyme Q biosynthesis[END_REF] (Figure S1A). However, no homologs of UbiC or XanB2 have been identified in S. cerevisiae; thus a direct conversion of chorismic acid into 4-HB is hypothetical in this organism (Figure S1A). A recent study in the plant Arabidopsis thaliana demonstrated that Phe and tyrosine independently contribute to the synthesis of 4-HB, but only the pathway originating from Phe has been partially characterized and shown to involve -oxidation in peroxisomes [START_REF] Block | The Origin and Biosynthesis of the Benzenoid Moiety of Ubiquinone (Coenzyme Q) in Arabidopsis[END_REF]. Overall, the metabolic pathway from tyrosine to 4-HB appears to be common to eukaryotes, yet it remains uncharacterized to date.

We and others discovered that S. cerevisiae synthesizes Q not only from 4-HB but also from para-aminobenzoic acid (pABA) [START_REF] Marbois | paraaminobenzoic acid is a precursor in coenzyme Q(6) biosynthesis in Saccharomyces cerevisiae[END_REF][START_REF] Pierrel | Involvement of mitochondrial ferredoxin and para-aminobenzoic acid in yeast coenzyme Q biosynthesis[END_REF]. pABA is a precursor in folate metabolism and is synthesized from chorismic acid via two reactions catalyzed by Abz1 and Abz2 (Figure S1A) [START_REF] Botet | A chemogenomic screening of sulfanilamide-hypersensitive Saccharomyces cerevisiae mutants uncovers ABZ2, the gene encoding a fungal aminodeoxychorismate lyase[END_REF]. The use of pABA as a precursor of Q may be restricted to S. cerevisiae since Escherichia coli, A. thaliana and human cells do not incorporate pABA into Q [START_REF] Block | The Origin and Biosynthesis of the Benzenoid Moiety of Ubiquinone (Coenzyme Q) in Arabidopsis[END_REF][START_REF] Xie | Resveratrol and para-coumarate serve as ring precursors for coenzyme Q biosynthesis[END_REF]. pABA is a constituent of the widely used YNB synthetic growth medium and may thereby confer normal Q levels to S. cerevisiae cells deficient in 4-HB. In the present study, we used YNB culture medium depleted of pABA to study the pathway from tyrosine to 4-HB. By combining isotopic labelling, chemical analogues supplementation, and forward and reverse genetics, we have elucidated the first and last reactions of this pathway in S. cerevisiae and have identified the associated enzymes.

RESULTS

Tyrosine but not Phe is a precursor of Q in S. cerevisiae

We previously showed that addition of pABA or 4-HB to YNB medium depleted of pABA increased the Q content of S. cerevisiae cells, which demonstrated that endogenously synthesized pABA and 4-HB are limiting for Q biosynthesis [START_REF] Pierrel | Involvement of mitochondrial ferredoxin and para-aminobenzoic acid in yeast coenzyme Q biosynthesis[END_REF]. Addition of tyrosine to the growth medium increased the level of Q (Figure S1B), supporting that tyrosine is used as a precursor. Indeed, the mass spectrum of Q from cells grown in the presence of 15 N 13 C9-tyrosine was consistent with the incorporation of the 6 aromatic carbon atoms of tyrosine into Q (Figure S1C) and 78% of the Q pool was labelled (Figure 1A). In contrast to A. thaliana [START_REF] Block | The Origin and Biosynthesis of the Benzenoid Moiety of Ubiquinone (Coenzyme Q) in Arabidopsis[END_REF], S. cerevisiae did not convert 13 C9-Phe to Q (Figure 1A), demonstrating the absence of a pathway from Phe to 4-HB in yeast.

4-HPP is an intermediate in the biosynthesis of 4HB

We then evaluated the impact of mutations in the shikimate pathway (Figure S1A). We found that products of the shikimate pathway compete with exogenous 15 N 13 C9-tyrosine for Q biosynthesis since the labelling of Q increased in Δaro2 and Δtyr1 strains (Figure 1A). Aro8 and Aro9 are two aminotransferases that catalyze the last reaction of the biosynthesis of tyrosine, the conversion of 4-HPP into tyrosine (Figure S1A) [START_REF] Urrestarazu | Phenylalanine-and tyrosineauxotrophic mutants of Saccharomyces cerevisiae impaired in transamination[END_REF]. We assessed whether Aro8 and Aro9 may catalyze the reverse reaction in vivo, the deamination of tyrosine to 4-HPP. The labelling of Q from 15 N 13 C9-tyrosine diminished drastically in the Δaro8Δaro9 strain compared to WT (Figure 1A), supporting the idea that 4-HPP produced by Aro8 and Aro9 is an intermediate in the biosynthesis of 4-HB. Accordingly, inactivation of both branches of 4-HPP biosynthesis, as in Δaro8Δaro9Δtyr1 and Δaro8Δaro9Δaro2 cells, diminished Q levels (Figure 1B). These results establish that 4-HPP, originating from the shikimate pathway and from the deamination of tyrosine by Aro8 and Aro9, is an intermediate in the production of 4-HB that is used for Q biosynthesis.

The S241L mutation in hfd1 causes Q deficiency in Δaro2 cells Next, we reasoned that the inactivation of the shikimate pathway together with a disruption of the biosynthesis of 4-HB from exogenous tyrosine should result in Q deficiency.

We therefore used the Δaro2 strain in a forward genetic screen with lactate-glycerol (LG) medium, growth on which requires Q. After mutagenesis, we isolated a Δaro2-mutA clone that grew poorly on LG medium unless 4-HB was added (Figure S1D). The Q content of Δaro2-mutA cells was very low compared to that of Δaro2 cells and was increased by 4-HB but not by tyrosine (Figure 1C), a result consistent with the inactivation of the tyrosine to 4-HB pathway. Addition of pABA to the growth medium increased Q levels in Δaro2 and Δaro2-mutA cells (Figure S1E) but to levels slightly lower than those obtained with 4-HB (Figure 1C). Genetic analysis revealed that the mutation of a single gene caused the phenotype of Δaro2-mutA cells (see Supplemental Experimental Procedures). In order to identify the defective gene, the Δaro2-mutA strain was transformed with a S. cerevisiae genomic library and the resulting clones were tested for growth on LG medium. The growth was totally recovered with a plasmid carrying the HFD1 gene whereas plasmids expressing COQ2-MVD1 or ARO2 suppressed only partially the phenotype (Figure 1D). COQ2-MVD1 increased the Q content of Δaro2-mutA cells, whereas ARO2 was without an effect (Figure S1F). DNA sequencing of the HFD1 locus identified a C722T mutation in the Δaro2-mutA strain, causing a S241L mutation in the Hfd1 protein. This result establishes that the HFD1 gene complements the phenotype of the Δaro2-mutA strain, whereas COQ2-MVD1 and ARO2 behave as high copy suppressors, in agreement with the partial recovery of the respiratory growth (Figure 1D).

Hfd1 belongs to the aldehyde dehydrogenase family (Nakahara et al., 2012) and the importance of S241 for Hfd1 function is supported by its presence within a LELGGKSP sequence that is conserved in several family members (Figure S2A). The LELGGKSP sequence connects the catalytic and NAD binding domains of human ALDH2 and ALDH3A1 [START_REF] Lang | Vascular bioactivation of nitroglycerin by aldehyde dehydrogenase-2: reaction intermediates revealed by crystallography and mass spectrometry[END_REF][START_REF] Parajuli | Selective ALDH3A1 inhibition by benzimidazole analogues increase mafosfamide sensitivity in cancer cells[END_REF] and we found a similar topology in our homology model of Hfd1 (Figure S2B).

Inactivation of hfd1 results in Q deficiency

Δhfd1 cells were deficient in growth on LG medium unless it was supplemented with 4-HB (Figure 2A). Q levels were decreased ~7 fold in Δhfd1 cells compared to WT and increased strongly with 4-HB but not with tyrosine (Figure 2B). Q levels also increased with addition of pABA (Figure 2B). Expression of Hfd1, but not Hfd1-S241L, restored respiratory growth and the Q content of the Δhfd1 strain (Figures 2A and2B), confirming the deleterious effect of the S241L mutation. High copy COQ2-MVD1 increased the Q content of Δhfd1 cells, although not to WT levels, whereas ARO2 had no effect (Figure 2C). Only 7% of Q was labelled in Δhfd1 cells grown with 15 N 13 C9-tyrosine (Figure 2D), showing that exogenous tyrosine cannot efficiently supply Q biosynthesis in Δhfd1 cells contrary to WT cells (Figure 1A).

S. cerevisiae lacks a chorismatase to convert chorismic acid into 4-HB

Δhfd1 cells have a functional shikimate pathway since they are prototrophic for aromatic amino acids (data not shown). Nevertheless, they are deficient in Q (Figure 2B), suggesting that chorismic acid does not supply sufficient 4-HB or pABA for Q biosynthesis (Figure S1A). Indeed, we found that expression of the E. coli UbiC protein, which catalyzes the direct aromatization of chorismic acid into 4-HB, robustly increased Q in WT and Δhfd1 cells, but not in Δaro2Δhfd1 cells that do not produce chorismic acid (Figure 2B). As expected, 15 N 13 C9tyrosine did not label Q produced by Δhfd1 + ubiC cells (Figure 2D). These results demonstrate that S. cerevisiae lacks a functional chorismatase enzyme and explain that hfd1 cells are deficient in Q despite their ability to synthesize chorismic acid.

Endogenous pABA is the predominant precursor of Q in the absence of hfd1

The relative influx of pABA and 4-HB into the Q biosynthetic pathway is revealed in a Δcoq6 strain overexpressing Coq8, which does not synthesize Q but instead accumulates 3-hexaprenyl-4-aminophenol (4-AP) or 3-hexaprenyl-4-hydroxyphenol (4-HP) depending on whether the precursor used is pABA or 4-HB, respectively [START_REF] Ozeir | Coenzyme Q biosynthesis: Coq6 Is required for the C5-hydroxylation reaction and substrate analogs rescue Coq6 deficiency[END_REF][START_REF] Xie | Over-expression of the Coq8 kinase in Saccharomyces cerevisiae coq null mutants allows for accumulation of diagnostic intermediates of the Coenzyme Q6 biosynthetic pathway[END_REF]. 4-HP decreased in Δcoq6Δhfd1 cells compared to Δcoq6 cells (Figure 2E), reflecting a diminished influx of 4-HB caused by the deletion of hfd1. The major contribution to Q biosynthesis of 4-HB over pABA was restored by expression of HFD1 or ubiC which diminished 4-AP and increased 4-HP (Figure 2E). Taken together, our results demonstrate that the inactivation of hfd1 diminishes the pool of 4-HB available for Q biosynthesis whereas that of pABA remains unaffected. We conclude that Hfd1 acts upstream of 4-HB in the metabolic pathway from tyrosine, since E. coli UbiC, which produces 4-HB directly from chorismic acid, increased the levels of both Q (Figure 2B) and 4-HB, as indirectly assessed by measuring 4-HP (Figure 2E). Interestingly, expression of E. coli UbiC was also recently found to increases Q level in Schizosaccharomyces pombe [START_REF] Moriyama | Production of CoQ10 in fission yeast by expression of genes responsible for CoQ10 biosynthesis[END_REF].

Hfd1 oxidizes 4-Hbz to 4-HB Hfd1 was recently shown to catalyze the oxidation of hexadecanal to hexadecanoic acid as part of the degradation pathway of the sphingoid base dihydrosphingosine (Nakahara et al., 2012), which is converted to hexadecanal by Dpl1 in the endoplasmic reticulum [START_REF] Kihara | Sphingosine 1-phosphate is a key metabolite linking sphingolipids to glycerophospholipids[END_REF] (Figure S3A). Q levels were normal in Δdpl1 cells (Figure S3B), indicating that dihydrosphingosine degradation is independent from Q biosynthesis. Because of its aldehyde dehydrogenase activity and its partial localization at the mitochondrial outer membrane [START_REF] Zahedi | Proteomic analysis of the yeast mitochondrial outer membrane reveals accumulation of a subclass of preproteins[END_REF], we hypothesized that Hfd1 might catalyze the dehydrogenation of 4-Hbz to form 4-HB for mitochondrial Q biosynthesis (Figure S3C). 4-Hbz efficiently increased Q in WT but not in Δhfd1 cells (Figure 3A), showing that the use of 4-Hbz as a precursor of Q requires Hfd1.

Dehydrogenation of 4-Hbz or decanal was diminished in cellular extracts from Δhfd1 cells and was increased in Δhfd1+HFD1 (Figures S3D andS3E). The 4-Hbz dehydrogenase activity was mainly associated with the membrane fraction (Figure S3F) and mitochondria from WT or Δhfd1+HFD1 cells showed a robust oxidation of 4-Hbz into 4-HB, unlike those from Δhfd1 cells (Figure 3B). We further verified the capacity of Hfd1 to synthesize 4-HB from 4-Hbz in a heterologous system, the E. coli ΔubiC strain, which is impaired in 4-HB biosynthesis [START_REF] Siebert | Formation of 4-hydroxybenzoate in Escherichia coli: characterization of the ubiC gene and its encoded enzyme chorismate pyruvate-lyase[END_REF]. Addition of 4-HB complemented the Q deficiency of the E. coli ΔubiC strain (Figure 3C), whereas efficient complementation by 4-Hbz required the expression of a functional Hfd1 protein (Figure 3C). Finally, we purified Hfd1 fused to the maltose binding protein (Figure S3G). MBP-Hfd1 oxidized benzaldehyde and 4-Hbz (Figure 3D), and preferred NAD to NADP as a co-substrate (Figure S3H). Collectively, these data demonstrate that Hfd1 oxidizes 4-Hbz to 4-HB to supply Q biosynthesis in mitochondria.

4-Hbz is the last intermediate in the Tyr to 4-HB pathway

Next, we quantified 4-Hbz in yeast cellular extracts and found an increase upon addition of tyrosine to the culture medium (Figure S3I). 15 N 13 C9-tyrosine yielded a +7 m/z increment in 4-Hbz, demonstrating that all carbon atoms of 4-Hbz were labelled (Figure S3J). The labelling of the 4-Hbz pool was comparable in WT and Δhfd1 cells but decreased in Δaro8Δaro9 cells (Figure 3E). Together, these results establish that 4-Hbz is located downstream of 4-HPP in the pathway that converts tyrosine into 4-HB.

Human ALDH3A1 oxidizes 4-Hbz to 4-HB in S. cerevisiae Humans possess 19 aldehyde dehydrogenases [START_REF] Jackson | Update on the aldehyde dehydrogenase gene (ALDH) superfamily[END_REF] and a phylogenetic analysis revealed that Hfd1 clustered with the four members of subfamily 3 (Figure S4). Since the defect of Δhfd1 cells in dihydrosphingosine degradation was complemented by human ALDH3A2 and ALDH3B1 (Kitamura et al., 2013), it was of interest to verify if any of the four members of subfamily 3 may oxidize 4-Hbz to 4-HB. All four proteins were expressed at similar levels in yeast Δhfd1 cells (Kitamura et al., 2013) (Figure 4A), but only extracts containing ALDH3A1 oxidized 4-Hbz in vitro (Figure 4A). ALDH3A1 increased Q levels in Δhfd1 cells when 4-Hbz was added to the culture medium (Figure 4B), showing the ability of ALDH3A1 to oxidize 4-Hbz to 4-HB in vivo.

DISCUSSION

Hfd1 fulfills two independent functions in sphingolipid and Q metabolisms

Our results demonstrate that Hfd1 is required for coenzyme Q biosynthesis by oxidizing 4-Hbz to 4-HB. Hfd1 was also shown to oxidize aliphatic aldehydes like hexadecanal, an intermediate in a degradation pathway of the sphingoid base dihydrosphingosine (Nakahara et al., 2012). We believe that the defects of Δhfd1 cells in sphingolipid and Q metabolisms are independent. Indeed, Δdpl1 cells which cannot degrade dihydrosphingosine (Nakahara et al., 2012) have a normal Q content (Figure S3B), showing that a disruption of the degradation pathway of dihydrosphingosine does not perturb Q biosynthesis. Furthermore, human ALDH3A2 and ALDH3B1 complemented the hexadecenal dehydrogenase defect of Δhfd1 cells (Kitamura et al., 2013) but did not restore Q biosynthesis (Figure 4B), whereas ALDH3A1 complemented the Q defect (Figure 4B) but not the dihydrosphingosine degradation defect (Kitamura et al., 2013), demonstrating the independence of both cellular processes. Human ALDH3A1 and ALDH3A2 may have evolved restricted substrate specificities compared to Hfd1 which combines dehydrogenase activities on aliphatic aldehydes like hexadecenal and aromatic aldehydes like 4-Hbz. Hfd1 is dually localized in the endoplasmic reticulum and in mitochondria [START_REF] Zahedi | Proteomic analysis of the yeast mitochondrial outer membrane reveals accumulation of a subclass of preproteins[END_REF] and each pool may specifically contribute to a particular metabolism since hexadecanal is synthesized by Dpl1 in the ER whereas Q biosynthesis occurs in mitochondria [START_REF] Ikeda | Sphingosine-1-phosphate lyase SPL is an endoplasmic reticulum-resident, integral membrane protein with the pyridoxal 5 '-phosphate binding domain exposed to the cytosol[END_REF][START_REF] Tran | Endogenous synthesis of coenzyme Q in eukaryotes[END_REF](Figure 5).

The supply of aromatic precursors for Q biosynthesis in S. cerevisiae S. cerevisiae utilises 4-HB and pABA as aromatic precursors of Q [START_REF] Marbois | paraaminobenzoic acid is a precursor in coenzyme Q(6) biosynthesis in Saccharomyces cerevisiae[END_REF][START_REF] Pierrel | Involvement of mitochondrial ferredoxin and para-aminobenzoic acid in yeast coenzyme Q biosynthesis[END_REF]), yet their endogenous levels are limiting for Q biosynthesis since addition of either molecule to pABA-depleted growth medium increased the Q content of WT cells [START_REF] Pierrel | Involvement of mitochondrial ferredoxin and para-aminobenzoic acid in yeast coenzyme Q biosynthesis[END_REF]. Thus, the use of pABA-depleted medium was a prerequisite for the success of our forward genetic approach which revealed the requirement of Hfd1 for Q biosynthesis.

Inactivation of hfd1 diminished the influx of 4-HB but not pABA into Q biosynthesis (Figure 2E), but the endogenous pABA pool was insufficient to compensate the shortage of 4-HB since we showed that Δhfd1 cells were profoundly deficient in Q (Figure 3A). Endogenous pABA may thus be predominantly used for folate biosynthesis (Figure S1A). We observed a partial restoration of Q levels in Δhfd1 cells overexpressing COQ2-MVD1 (Figure 2C). Therefore, we think that endogenous pools of pABA and 4-HB may be more efficiently redirected towards Q biosynthesis upon overexpression of the 4-HB/pABA polyprenyltransferase Coq2. Altogether, we conclude that WT cells use 4-HB as the main precursor of Q under our growth conditions and that 4-HB is synthesized from the shikimate pathway and from exogenous tyrosine via a common intermediate, 4-HPP (Figure 5). Inactivation of either branch of 4-HPP synthesis maintained, and even increased Q levels in the case of Δtyr1 cells (Figure 1), reflecting compensatory mechanisms that might be linked to the extensive regulation of the shikimate pathway [START_REF] Braus | Aromatic amino-acid biosynthesis in the yeast Saccharomyces cerevisiae: a model system for the regulation of a eukaryotic biosynthetic pathway[END_REF].

The pathway from tyrosine to 4-HB and its potential conservation in higher eukaryotes Altogether, our results reveal several steps of the metabolic pathway from tyrosine to 4-HB and establish 4-HPP and 4-Hbz as the first and last intermediates, respectively. How the side chain of 4-HPP is shortened remains to be elucidated. However, contrary to what was previously hypothesized [START_REF] Clarke | New advances in coenzyme Q biosynthesis[END_REF][START_REF] Turunen | Metabolism and function of coenzyme Q[END_REF], β-oxidation is probably not involved, since it would form directly 4-HB but not 4-Hbz (Figure 5) [START_REF] Widhalm | A Familiar Ring to It: Biosynthesis of Plant Benzoic Acids[END_REF], whereas we have demonstrated that Q biosynthesis requires the oxidation of 4-Hbz by Hfd1. The hydration of 4-coumaric acid and a subsequent retro-aldol reaction to yield 4-Hbz is a more likely scenario for the shortening of the side chain [START_REF] Clarke | New advances in coenzyme Q biosynthesis[END_REF] (Figure 5) since S. cerevisiae was recently shown to incorporate 4-coumaric acid into Q, although not efficiently [START_REF] Xie | Resveratrol and para-coumarate serve as ring precursors for coenzyme Q biosynthesis[END_REF]. About half of 4-HB used for Q biosynthesis in the plant A. thaliana originates from tyrosine through an unknown metabolic pathway [START_REF] Block | The Origin and Biosynthesis of the Benzenoid Moiety of Ubiquinone (Coenzyme Q) in Arabidopsis[END_REF]. The final reaction may be the oxidation of 4-Hbz since aromatic aldehyde dehydrogenase activities with broad substrate specificities were reported in developing seeds of A. thaliana and in hairy roots of Daucus carota [START_REF] Ibdah | An Aldehyde Oxidase in Developing Seeds of Arabidopsis Converts Benzaldehyde to Benzoic Acid[END_REF][START_REF] Sircar | Characterization of p-hydroxybenzaldehyde dehydrogenase, the final enzyme of p-hydroxybenzoic acid biosynthesis in hairy roots of Daucus carota[END_REF]. Tyrosine also supports Q biosynthesis in animals [START_REF] Olson | Biosynthesis of ubiquinones in animals[END_REF]) and it will be important to verify whether mammalian 4-HB biosynthesis shares similarities with the yeast pathway. Tyrosine aminotransferase (TAT) catalyzes the deamination of tyrosine in the liver and is mutated in tyrosinemia type II [START_REF] Dietrich | Tyrosine aminotransferase: a transaminase among others[END_REF][START_REF] Scott | The genetic tyrosinemias[END_REF]. Measurements of Q levels in patients will indicate if TAT is required for Q biosynthesis. Animals may also rely on the oxidation of 4-Hbz to generate 4-HB because radiolabelled 4-Hbz was incorporated into Q when injected to rats [START_REF] Ranganathan | Regulation of biosynthesis of ubiquinone in rat[END_REF] and when added to rat kidneys in vitro [START_REF] Parson | Biosynthesis of benzoquinone ring of ubiquinone from p-hydroxybenzaldehyde + p-hydroxybenzoic acid in rat kidney azotobacter vinelandii + bakers yeast[END_REF]. Since a deficit in 4-HB biosynthesis is expected to cause Q deficiency, our work calls for patients with genetically unassigned Q deficiency to be screened for mutations in aldehyde dehydrogenase genes, especially ALDH3A1.

SIGNIFICANCE

Coenzyme Q is a redox lipid that is central for energetic metabolism of eukaryotes. The biosynthesis of Q from the aromatic precursor 4-hydroxybenzoic acid (4-HB) is conserved and requires multiple catalytic reactions and associated proteins. Mutations in corresponding genes cause primary coenzyme Q deficiencies and result in various severe human diseases which are often improved by coenzyme Q supplementation. However, the hydrophobicity of coenzyme Q and therefore its poor bioavailability is thought to limit the efficiency of the treatment. In eukaryotes, biosynthesis of 4-HB from tyrosine has been suspected for forty years, yet, the pathway from tyrosine to 4-HB has remained elusive to date. We partially solved the longstanding problem of the biosynthesis of the aromatic precursor of coenzyme Q by providing a molecular understanding of the first and last reactions of the pathway in the yeast S. cerevisiae, namely the deamination of tyrosine to 4-hydroxyphenylpyruvate by Aro8 and Aro9 and the oxidation of 4-hydroxybenzaldehyde to 4-HB by Hfd1. Whereas part of 4-HB is also provided by the shikimate pathway in S. cerevisiae, the importance of the tyrosine to 4-HB pathway for Q biosynthesis is likely to be more preponderant in higher eukaryotes which lack the shikimate pathway. We found that inactivation of hfd1 resulted in coenzyme Q deficiency which was efficiently complemented by the addition of exogenous 4-HB. Our demonstration that human ALDH3A1 is able to oxidize 4-hydroxybenzaldehyde to 4-HB represent the first stage in assessing the conservation of the tyrosine to 4-HB pathway from yeast to humans. Mutations in genes of the tyrosine to 4-HB pathway may lead to coenzyme Q deficiencies in humans. In such cases, Q levels might be restored in patients by supplementation with 4-HB, a hydrophilic compound that might prove more bioavailable than exogenous Q which is normally used to treat coenzyme Q deficiencies.

EXPERIMENTAL PROCEDURES

Plasmids and strains. Plasmids and strains used in this study are detailed in Supplemental Information.

Yeast culture conditions. Yeast strains were grown at 30 °C and 200 rpm shaking in YPD (1% (w/v) yeast extract, 2% (w/v) peptone and 2% (w/v) glucose) or in YNB-p (YNB without pABA and without folate, MP Biomedicals) supplemented with 10 mg/L folate, 10 µM FeCl3, 2% (w/v) carbon source (glucose, galactose or lactate-glycerol (LG)) and nutrients to cover the strains auxotrophies in quantities described in (Sherman, 2002). We note that exogenously added folate is unlikely to be imported in yeast [START_REF] Nguyen | Folate status of gut microbiome affects Caenorhabditis elegans lifespan[END_REF]. When necessary, aromatic amino acids were added at a final concentration of 20 mg/L for tryptophane, 50 mg/L for phenylalanine and 15-60 mg/L for tyrosine as indicated (Sherman, 2002). For labelling experiments, 30 mg/L 15 N 13 C9-tyrosine or 50 mg/L 13 C9-phenylalanine were added in replacement of the unlabelled amino acid. Only L-isomers of amino acids were used. YNB-p 2% galactose liquid cultures were performed by inoculating fresh medium with an aliquot of a 24 h culture grown in YNB-p 2% galactose 0.2% glucose. Bacto-agar (Euromedex) was added at 1.6% (w/v) for solid media.

Analysis of coenzyme Q content. Yeast cells were grown to OD600 = 1 in YNB-p galactose medium unless otherwise stated and cooled down on ice prior to collection by centrifugation and determination of the cells wet weight (Ozeir et al., 2015). Lipid extraction and Q analysis by HPLC-electrochemical detection (ECD) including MS detection or not was performed as previously described (Ozeir et al., 2015). Total Q was calculated from the ECD signal, unlabelled-Q and labelled 13 C6-Q were quantified by integrating the peaks obtained at the retention time of Q for the positive ions m/z 591.2 and 597.2, respectively. E. coli cells grown overnight in M9 medium containing 100 µg/mL ampicillin for plasmid selection as needed, were inoculated at OD600 = 0.05 in M9 medium containing 30 µM isopropyl β-D-1thiogalactopyranoside (IPTG) and supplemented or not with 4-HB or 4-Hbz. Cells were grown at 37 °C, 200 rpm shaking until OD600 = 0.8 and analyzed for Q content by HPLC-ECD as previously described [START_REF] Aussel | ubiJ, a New Gene Required for Aerobic Growth and Proliferation in Macrophage, Is Involved in Coenzyme Q Biosynthesis in Escherichia coli and Salmonella enterica Serovar Typhimurium[END_REF].

4-Hbz dehydrogenase activity assays.

For yeast extract activity assays, cell pellets (10 mL of yeast culture collected at OD600 = 1) were resuspended in 1 mL PIPES 100 mM pH 7.1 with 2 pellet-volumes of glass beads and vortexed during 10 min. Cell homogenates were pipetted to eliminate the glass beads and centrifuged at 15,000 rpm, 4 °C during 5 min; resulting supernatants, corresponding to soluble fractions were collected, and pellets, corresponding to membrane fractions were resuspended in 500 µL PIPES 100 mM pH 7.1. Cell homogenates collected prior to centrifugation corresponded to total extracts. Protein content was measured by the BCA method. 20 µL of each fraction was assayed for 4-Hbz dehydrogenase activity by incubation at 30 °C for 1 h in a total volume of 100 µL PIPES 50 mM pH 7.1, 500 µM NAD + , 100 µM 4-Hbz . Mitochondrial fractions (20 µL in PIPES 100 mM pH 7.1 mannitol 1.2 M, protein concentration ~1.5 µg/µL) were assayed similarly except that the assay buffer contained 0.6 M mannitol. The reaction was stopped by adding 2 µL pure formic acid. 4-HB and 4-Hbz were extracted as described elsewhere [START_REF] Villas-Bôas | Global metabolite analysis of yeast: evaluation of sample preparation methods[END_REF]. Briefly, 500 µL cold methanol was added to samples together with 10 µL of 10 µM vanillic acid (VA) used as an internal standard. The mixture was frozen in liquid nitrogen, stored if necessary at -80 °C, then thawed on ice and centrifuged at 770 g during 20 min at 4 °C. The supernatant was collected and evaporated under nitrogen at 60 °C. Samples were resuspended in water containing 0.25% (v/v) acetic acid and analyzed by HPLC (U3000 Dionex) on a NUCLEODUR® PFP column (Macherey-Nagel, 3 µm, 2 x 150 mm). Two mobile phases were used for elution at a flow rate of 0.25 mL/min: 0.25% (v/v) acetic acid in water (A) and methanol (B). The linear gradient profile was 0-2 min, 5% B; 2-12 min, 5-30% B; 12-18 min, 30% B; 18-20 min, 30-5% B; 20-24 min, 5% B. 4-HB, VA and 4-Hbz were detected by a diode array detector which recorded the absorbance at 260 nm (4-HB and VA) and 290 nm (4-Hbz). The recovery of VA was typically 75% and was used to correct 4-HB values for loss during extraction.

Mitochondrial preparation and Western blotting. Purification of mitochondria and immunodetection were conducted as described previously (Ozeir et al., 2015). Monoclonal anti-Flag M2 antibody (F1804, Sigma, dilution 1/3,000) and polyclonal anti-Anc2 antibody (dilution 1/1,500) were detected with HRP-coupled anti-mouse IgG and HRP-coupled protein A, respectively.

Extraction and GC-MS analysis of 4-Hbz. Cells were cultured until OD600 ~ 2 in 50 mL YNBp galactose supplemented or not with aromatic amino acids and 30 mg/L 15 N 13 C9-tyrosine, transferred into precooled 50 mL falcon tubes, and centrifuged for 1 min at 4,000 rpm, 4 °C.

The cell pellet was resuspended in 10 mL ice cold PBS and centrifugation was repeated. The supernatant was eliminated and the cells were vortexed in 5 mL acetonitrile/methanol/water (2/2/1, v/v) precooled at -20 °C. The suspension was stored at -80 °C until further use. The suspension was then allowed to thaw at room temperature for 30 min and was centrifuged for 2 min at 4,000 rpm, 20 °C. The supernatant was transferred into a 50 mL falcon tube using Pasteur pipette and was supplemented with 5 mL water, 1% (v/v) acetic acid, 2 g NaCl, and 100 ng VA as an extraction standard. The mixture was homogenized by vortexing, 10 mL ethyl acetate was added and vortex was repeated. After phase separation, the upper ethyl acetate phase was taken and the extraction was repeated with 10 mL ethyl acetate. The ethyl acetate phases were combined and dried in a water bath at 37 °C under a stream of nitrogen. The sample was dissolved in 1.2 mL ethanol and centrifuged for 2 min at 13,000 rpm at room temperature.

The resulting supernatant was transferred to Eppendorf tubes, dried under nitrogen and stored at -80 °C. On the day of the analysis, samples were dissolved in 50 µL of O-methoxyamine-HCl (20 mg/mL in pyridine) and incubated at 60 °C for 30 min; 50 µL of BSTFA-TMCS (99:1, Macherey Nagel) was then added and samples were further incubated at 60 °C for 30 min.

Samples were centrifuged at 13,000 rpm for 1 min. Derivatized samples were analysed by Gas Chromatography-Mass Spectrometry (GC-MS) using an Agilent 5973 Mass Selective Detector coupled to an HP6890 GC system equipped with an Optima-17-MS capillary column (30 m x 0.25 mm x 0.25 µm; Macherey-Nagel). The carrier gas was helium and the injection volume was 2.5 µl with an inlet temperature of 250 °C, with a split ratio of 50:1 for full scan detection and 20:1 for SIM, at an inlet temperature of 250 °C. The initial oven temperature was 75 °C for 3 min, then increased to 250 °C at a rate of 12 °C/min, held for 1 min then increased to 300 °C Bioinformatics methods. Sequence alignments were generated with Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/). The homology model of Hfd1 was constructed with Phyre2 [START_REF] Kelley | The Phyre2 web portal for protein modeling, prediction and analysis[END_REF] used in intensive mode. The program selected the aldehyde dehydrogenase 3A1 from Rattus norvegicus (PDB code 1AD3) as template. For the phylogenetic analysis, the protein sequences were retrived from NCBI and were aligned using the MUSCLE software (http://www.ebi.ac.uk/Tools/msa/muscle/). The resulting sequence alignment file was then processed with the latest version of the PHYLIP package (http://evolution.genetics.washington.edu/phylip), which comprises the PROTDIST program to compute a matrix distance and the DRAWGRAM program to plot the phylogenetic tree constructed with the Neighbor-Joining method.

Statistical methods. Results are presented as mean ± s.e.m. Data sets were compared by unpaired two-tailed t-test or an analysis of variance (ANOVA) using GraphPad Prism 6 software.

bifunctional chorismatase that links the shikimate pathway to ubiquinone and xanthomonadins biosynthetic pathways. Mol. Microbiol. 87, 80-93.

FIGURE LEGENDS

Figure 1: A genetic defect in the metabolism of tyrosine decreases Q. A, Quantification of 13 C6-Q and unlabelled Q in cells grown with 15 N 13 C9-Tyr or 13 C9-Phe. n=3 (Δaro8Δaro9), n=4 (WT, Δaro2 and Δtyr1). Mean + s.e.m. of total Q (upper error bar), means.e.m. of 13 C6-Q (lower error bar). The average percentage of 13 C6-Q is indicated on top of each bar, P value in comparison to WT + 15 N 13 C9-Tyr. B, Q content of cells grown in medium with aromatic amino acids. Mean ± s.e.m.; n=6. C, Q content of cells grown in glucose medium with aromatic amino acids (Tyr 15 mg/L) supplemented with 10 µM 4-HB or 60 mg/L tyrosine. Mean ± s.e.m.; n=2 (+ Tyr), n=3 (± 4-HB). D, Growth of strains containing the indicated YEp352 plasmids on medium without aromatic amino acids (-Aro) or with Phe, Trp and 15 mg/L Tyr, containing either glucose (Glu) or lactate-glycerol (LG) and supplemented or not with 10 µM 4-HB. Representative images from one of three independent experiments are shown. ns: no significant difference, *: P< 0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001, unpaired two-tailed t test (A, B), One-way ANOVA test (C). n, number of independent experiments. Hfd1 and human ALDH3A2 catalyze the oxidation of hexadecanal to hexadecanoic acid (Nakahara et al., 2012), which is likely to take place in the endoplasmic reticulum [START_REF] Kihara | Sphingosine 1-phosphate is a key metabolite linking sphingolipids to glycerophospholipids[END_REF][START_REF] Zahedi | Proteomic analysis of the yeast mitochondrial outer membrane reveals accumulation of a subclass of preproteins[END_REF]. In mitochondria, Hfd1 oxidizes 4-Hbz into 4-HB which is prenylated by Coq2 and further metabolized to Q (R, polyprenyl tail). 4-HPP originates both from exogenous tyrosine via Aro8 and Aro9 and from chorismic acid via Aro7 and Tyr1. The shortening of the side chain of 4-HPP may proceed via hydration of 4-coumaric acid and a subsequent retro-aldol reaction (grey arrows) but is unlikely to involve β-oxidation (dashed grey arrows) which would yield 4-HB and not 4-Hbz. Phylogenetic tree of Hfd1 and the 19 human aldehyde dehydrogenase proteins.

SUPPLEMENTAL EXPERIMENTAL PROCEDURES

Chemicals. 4-hydroxybenzoate (4-HB), 4-hydroybenzaldehyde (4-Hbz), para-aminobenzoic acid (pABA), benzaldehyde, decanal, decanoic acid, reduced and oxidized nicotinamide adenine dinucleotide (NADH and NAD + ), NAD phosphate (NADPH and NADP + ), methanol, acetonitrile, isopropanol, ethanol, n-heptane, trifluoroacetic acid, ethyl acetate, ethyl methanesulfonate (EMS), LC-MS grade formic acid and acetic acid were purchased from Sigma. 15 N 13 C9-L-Tyrosine and 13 C9-L-Phenylalanine were purchased from Cortecnet. 4-HB, 4-Hbz were prepared in water to a final concentration of 1 mM and stored at -20 °C. Tyrosine and phenylalanine stock solutions were prepared in water at 500 mg/L and 10 g/L, respectively, and were sterile filtered and stored at 4 °C. Decanal was prepared in ethanol at a final concentration of 10 mM and stored at -20 °C.

Yeast strains. Saccharomyces cerevisiae strains used in this study are listed in the Supplemental Table below. The Δaro8Δaro9 strain was constructed by crossing Δaro8 and Δaro9 strains and by selecting for the presence of both mutations in a haploid strain after sporulation of the diploid strain according to a published procedure (Sherman, 2002) and tetrad dissection on a Nikon 50i microscope equipped with a micromanipulator. The Δaro8Δaro9Δaro2 and Δaro8Δaro9Δtyr1 strains were obtained by transforming the Δaro8Δaro9 strain with the HIS3 gene amplified by PCR from Saccharomyces kluyveri using respectively, Aro2-his3 5' and Aro2-his3 3' primers or Tyr1-his3 5' and Tyr1-his3 3' primers (see Supplemental Table )  and by selecting His prototroph clones. The Δtyr1 strain was created similarly by transforming the BY4741 strain. The Δcoq6Δhfd1 strain was constructed by crossing Δcoq6 and Δhfd1 strains and by selecting for the presence of both markers associated to the respective deletions in a haploid strain after tetrad dissection. In all cases, the integration of the deletion cassette was verified by PCR amplification of the locus on the genomic DNA. Transformations were performed using the lithium acetate method [START_REF] Burke | Methods in Yeast Genetics[END_REF]. This study

Supplemental

This study

Δhfd1

MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 hfd1::kanMX4 Euroscarf Δcoq6 MATα coq6::LEU2 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 [START_REF] Gin | The Saccharomyces cerevisiae COQ6 gene encodes a mitochondrial flavin-dependent monooxygenase required for coenzyme Q biosynthesis[END_REF] Δcoq6Δhfd1 This study

Growth assay on plates. Overnight cultures in YNB-p 2% galactose were diluted in water to OD600 = 1 and ten-fold serial dilutions in sterile water were performed in 96 well plates. 5 µL of each dilution was spotted on YNB-p solid medium containing either glucose (Glu) or LG. Plates were imaged after incubation at 30 °C for three days (Glu) or five days (LG).

EMS mutagenesis and genetic analysis of Δaro2-mutA. EMS mutagenesis was carried out essentially as described [START_REF] Winston | EMS and UV mutagenesis in yeast[END_REF]. A culture of 10 mL of Δaro2 cells (MATa) in YNB-p LG medium at OD600 1.2, was centrifuged at 5,000 g for 30 sec. The cells were rinsed twice with water and resuspended in 2.5 mL 0.1 M sodium phosphate buffer pH 7.0. 80 µL EMS was added and the cells were incubated for 1 h at 30 °C, 200 rpm. The cells were harvested by centrifugation at 5,000 g for 30 sec, rinsed three times in 1 mL 5% (w/v) sodium thiosulfate and finally resuspended in 1 mL water. The cells were diluted 1,000 fold in water and 120 µL were spread on YPD plates. After two days, colonies (~1,000 / plate, ~50,000 colonies in total) were replica plated onto YNB-p glucose medium using sterile velvets and grown for 24 h at 30 °C. The cells were then replica plated onto YNB-p LG medium containing or not 10 µM 4-HB. After 4 days at 30°C, the plates were compared visually and clones that grew exclusively on YNB-p LG medium containing 4-HB were selected and their phenotype was verified by streaking on YNB-p LG medium containing or not 10 µM 4-HB. Only one clone, Δaro2-mutA, exhibited the desired phenotype. Δaro2-mutA was mated with the Δaro2 strain (MATα) and diploids were selected on YNB glucose medium lacking lysine and methionine. The diploid strain was grown in YPD medium and was then sporulated to produce meiotic progeny. Spores from 20 tetrads showed 2:2 segregation for respiratory growth on YNB-p LG medium implying mutation at a single locus. One clone unable to grow on YNB-p LG medium was transformed with a yeast genomic DNA library previously described [START_REF] Pierrel | Coa1 links the Mss51 post-translational function to Cox1 cofactor insertion in cytochrome c oxidase assembly[END_REF]. Transformants were screened for growth on YNB-p LG medium. Plasmids from positive clones (YEp352-ARO2, YEp352-HFD1 and YEp352-COQ2-MVD1) were recovered in E. coli TOP10 by transforming yeast genomic DNA and selecting on LB-agar medium containing ampicillin. Plasmids were then purified and sequenced.

Plasmid construction. Plasmids used in this study are listed in the Supplemental Table below. The centromeric plasmid pRS415 was used to construct pRS415-HFD1 by cloning a PCR product corresponding to the HFD1 gene with its own promoter and terminator obtained with the primers XhoI-hfd1 and NotI-hfd1 (see Supplemental Table ) and genomic DNA from the BY4741 strain as template. pRS415-HFD1-S241L was obtained similarly except that genomic DNA from the Δaro2-mutA strain was used as template in the PCR reaction. E. coli UBIC gene was amplified by PCR from the genomic DNA of the MG1655 strain using the primers 5EcoRI-ubiC and 3XbaI-ubiC. The fragment was cloned at the EcoRI and XbaI sites of pBADIK [START_REF] Py | Assembly of the type II secretion machinery of Erwinia chrysanthemi: direct interaction and associated conformational change between OutE, the putative ATPbinding component and the membrane protein OutL[END_REF] and was then subcloned into pRS426-M25C1 using the EcoRI and HindIII restriction sites to obtain pRS426-UBIC. pMAL-c2-HFD1 was obtained by ligating at the BamH1 and SalI sites of pMAL-c2 a digested PCR product obtained by amplifying the HFD1 gene from the genomic DNA of the BY4741 strain. pMAL-c2-HFD1-S241L was obtained similarly except that the PCR reaction was performed on the genomic DNA of the Δaro2-mutA strain. E. coli TOP10 cells were used for cloning and plasmid amplification, the plasmids were purified with a standard miniprep kit (EZ-10, Euromedex) and sequencing was used to confirm all cloning products. The chromosomal coordinates indicated for the inserts of the YEp352 plasmids are from the S288C strain (http://www.yeastgenome.org). Decanal dehydrogenase activity assay. The assay was similar to that described for 4-Hbz except that yeast extracts were incubated with 100 µM decanal instead of 4-Hbz for 10 min. The extraction procedure of decanoic acid (C10:0) was modified from a method described elsewhere [START_REF] Neuber | Method to simultaneously determine the sphingosine 1-phosphate breakdown product (2E)-hexadecenal and its fatty acid derivatives using isotope-dilution HPLC-electrospray ionization-quadrupole/time-of-flight mass spectrometry[END_REF]. Briefly, 500 µL of isopropanol/n-heptane/2 M trifluoroacetic acid (40:10:1, v/v/v) was added to the samples with 10 µL of 10 µM pentadecanoic acid (C15:0) used as an internal standard. After vortexing for 1 min, 200 µL of nheptane and 300 µL of water were added. After vortexing for 1 min, tubes were incubated on ice for 10 min and centrifuged at 400 g for 5 min. 200 µL of the upper phase was transferred into a new tube. The extraction was repeated with 200 µL of n-heptane and the upper phase was collected and pooled with the first one.

Supplemental

Supplemental Table:

After evaporation under nitrogen at 37 °C, samples were resuspended in ethanol for HPLC-MS analysis (U3000 Dionex and Thermofisher MSQ Plus mass spectrometer). Separation was performed on a C18 column (Betabasic-18, 5 µm, 4.6 x 150 mm, Thermo Scientific). Two mobile phases were used for elution at a flow rate of 0.5 mL/min: water (A) and 0.05% (v/v) acetic acid in methanol (B). The linear gradient profile was 0-4 min, 60% B; 4-11 min, 60-100% B; 11-21 min, 100% B; 21-22 min, 100-60% B; 22-27 min, 60% B. Commercial decanoic acid was used to optimize separation and MS detection. The MS spectrometer was operated in electrospray negative ion mode (cone voltage 70 V and 90 V for C10:0 and C15:0, respectively, 450 °C) and peaks obtained in single ion monitoring, m/z 171.3 (C10:0) and m/z 241.3 (C15:0), were used for quantification.

Purification and activity assay of MBP-Hfd1. E. coli Rosetta II cells containing pMAL-c2-HFD1 were cultured in 4 L LB medium supplemented with ampicillin (100 µg/mL) and 2% glucose until OD600 ~ 0.5, at which point 1 mM IPTG was added. The culture was shifted to 17 °C and incubated overnight at 200 rpm. Cells were harvested by centrifugation at 4,000 rpm, 4 °C, 20 min, then washed with cold water and centrifugation was repeated. The cell pellet was stored at -80 °C. Cells were resuspended in 20 mL buffer A (20 mM Tris-HCl pH 7.4, 200 mM NaCl, 1 mM EDTA), lysed by sonication (5 min on ice, 10 s on/off pulses) and centrifuged at 100,000 g for 45 min at 4 °C. The supernatant was deposited on amylose resin (2 mL, New England Biolabs) equilibrated with buffer A, the column was washed with 30 mL buffer A and the protein was eluted with buffer A containing 50 mM maltose. MBP-Hfd1 was concentrated by centrifugation with Centricon 50 (Millipore) and 2 mL were loaded onto a Sephadex 200 column equilibrated with 20 mM Tris-HCl pH 7.4, 200 mM NaCl. Fractions were analyzed by SDS-PAGE and those containing MBP-Hfd1 were pooled and concentrated with Centricon 50. 10% glycerol was added to the purified protein before storing at -80 °C. Purified MBP-Hfd1 (5-15 µg) was incubated with 500 µM NAD + and 100 µM substrate (4-Hbz or benzaldehyde) in PIPES 50 mM pH 7.1 at 30 °C, total volume 100 µL. The reaction was monitored by measuring the fluorescence of NADH produced by the reduction of NAD + (excitation at 356 nm and emission at 460 nm) in a Tecan infinite M200. A linear standard curve, obtained with 0, 1, 2.5, 5 and 10 nM NADH was used to quantify the production of NADH in the assay.

  at 10 °C/min and held for 5 min. The MSD transfer line heater, MS source and MS quad temperatures were set at 280 °C, 230 °C and 150 °C, respectively. The mass range used for full scan detection of 4-Hbz was m/z 204-234 with an abundance threshold set at 150. The mass ions and windows used for SIM were as follows: 4Hbz, 208 (M + -CH3), 215 ( 13 C7M + -CH3), 223 (M + ), 230 ( 13 C7M + ) from 5 to 12 min; VA, 223 (M + -CH3-TMS-H), 253 (M + -Si(CH3)2-H), 267 (M + -(CH3)3) and 297 (M + -CH3) from 12 min to 15 min. Standards containing 20 ng VA and 4-Hbz were analyzed in similar conditions and used for quantification. Sample loss during extraction was corrected by calculating the recovery of VA in the different samples (typically 80-95%) and quantities of 4-Hbz were normalized to OD values of the harvested cells. Labelling of 4-Hbz was calculated by averaging the ratios of the abundance of the unlabelled/labelled ion pairs 208/215 and 223/230.

Figure 2 :

 2 Figure 2: Cells lacking HFD1 are deficient in Q biosynthesis.A, Growth of WT and Δhfd1 strains containing the indicated pRS415 plasmids on medium without aromatic amino acids containing either glucose (Glu) or lactate-glycerol (LG) supplemented or not with 10 µM 4-HB. Representative images from one of three independent experiments are shown. B, Q content of cells containing an empty vector (-), pRS415-HFD1, pRS415-HFD1-S241L, or pRS426-UBIC grown in medium supplemented or not with 10 µM 4-HB, 10 µM pABA, 30 mg/L Tyr or aromatic amino acids (Aro) as indicated. Mean ± s.e.m.; n=4 (except for Δhfd1 with pABA, n=3). C, Q content of Δhfd1 cells containing the indicated YEp352 plasmids and grown in medium supplemented with 15 mg/L Tyr. Mean ± s.e.m.; n=3. D, Quantification of 13 C6-Q and unlabelled Q in cells grown in medium containing 30 mg/L 15 N 13 C9-Tyr (n=9). Mean + s.e.m. of total Q (upper error bar), mean -s.e.m. of 13 C6-Q (lower error bar). The average percentage of 13 C6-Q is indicated on top of each bar. E, Superimposition of HPLC-ECD chromatograms of lipid extracts of the indicated strains containing the pCOQ8 plasmid. Q4, internal standard; 4-AP, 3-hexaprenyl-4-aminophenol and 4-HP, 3-hexaprenyl-4hydroxyphenol (structures shown). Representative chromatograms from one of four independent experiments are shown. ns: no significant difference, *: P< 0.05, ****: P<0.0001, One-way ANOVA test (B), unpaired two-tailed t test (C). n, number of independent experiments.

Figure 3 :

 3 Figure 3: Hfd1 oxidizes 4-Hbz to 4-HB that is used for Q biosynthesis. A, Q content of cells grown in the presence or not of 10 µM 4-Hbz. Mean ± s.e.m.; n=4. B, 4-Hbz dehydrogenase activity measured on mitochondria purified from WT, Δhfd1 and Δhfd1 cells transformed with pRS415-HFD1. Mean ± s.e.m.; n=5. C, Q content of E. coli WT or ΔubiC cells containing pMAL-c2-HFD1 or pMAL-c2-HFD1-S241L grown in M9 medium supplemented or not with 10 µM 4-HB or 10 µM 4-Hbz. Mean ± s.e.m.; n=4. D, Dehydrogenase activity of MBP-Hfd1 with 4-Hbz or benzaldehyde (Bz). Mean ± s.e.m.; n=6 (4-Hbz), n=3 (Bz). E, Quantification of labelled and unlabelled 4-Hbz in cells grown in YNB-p galactose medium

Figure 4 :

 4 Figure 4: Human ALDH3A1 oxidizes 4-Hbz to 4-HB in S. cerevisiae. A, 4-Hbz dehydrogenase activity measured on total cellular extracts of Δhfd1 cells transformed with YEp352 (-), pNK10 (3xFLAG-ALDH3A1), pNK5 (3xFLAG-ALDH3A2), pNK12 (3xFLAG-ALDH3AB1) or pNK14 (3xFLAG-ALDH3AB2). Mean ± s.e.m.; n=3. Immunodetection of ALDH3 proteins with anti-FLAG antibody; Anc2 was used as a loading control. B, Q content of Δhfd1 cells described in 4A, grown in the presence or not of 10 µM 4-Hbz. Mean ± s.e.m.; n=3 . **: P<0.01, unpaired two-tailed t test. n, number of independent experiments.

Figure 5 :

 5 Figure 5: Reactions catalyzed by Hfd1 in Q biosynthesis and in the dihydrosphingosine degradation pathway in S. cerevisiae.Hfd1 and human ALDH3A2 catalyze the oxidation of hexadecanal to hexadecanoic acid(Nakahara et al., 2012), which is likely to take place in the endoplasmic reticulum[START_REF] Kihara | Sphingosine 1-phosphate is a key metabolite linking sphingolipids to glycerophospholipids[END_REF][START_REF] Zahedi | Proteomic analysis of the yeast mitochondrial outer membrane reveals accumulation of a subclass of preproteins[END_REF]. In mitochondria, Hfd1 oxidizes 4-Hbz into 4-HB which is prenylated by Coq2 and further metabolized to Q (R, polyprenyl tail). 4-HPP originates both from exogenous tyrosine via Aro8 and Aro9 and from chorismic acid via Aro7 and Tyr1. The shortening of the side chain of 4-HPP may proceed via hydration of 4-coumaric acid and a subsequent retro-aldol reaction (grey arrows) but is unlikely to involve β-oxidation (dashed grey arrows) which would yield 4-HB and not 4-Hbz.

Figure 1

 1 Figure 1

Figure 2

 2 Figure 2

Figure 3

 3 Figure 3

Figure 4

 4 Figure 4

  Figure 5

Figure S2 Figure

 S2 Figure S2

Figure S3 Figure

 S3 Figure S3

Figure S4 Figure

 S4 Figure S4

  bold correspond to the sequence of the HIS3 gene from Saccharomyces kluyveri.

Table :

 : Yeast strains used in this study

	Strain	Genotype	Source
	BY4741 / WT	Wild Type, MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0	Euroscarf
	Δaro2	MATa his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 aro2::kanMX4	Euroscarf
	Δaro2	MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 aro2::kanMX4	Euroscarf
	Δaro8	MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 aro8::kanMX4	Euroscarf
	Δaro9	MATa his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 aro9::kanMX4	Euroscarf
	Δaro8Δaro9	MATa his3Δ1 leu2Δ0 ura3Δ0 aro8::KanMX4 aro9::KanMX4	This study
		met15Δ0
	Δaro2-mutA	MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 aro2::kanMX4 hfd1-	This study
		C722T
	Δtyr1	MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 tyr1:: SkHIS3	This study
	Δaro8Δaro9Δaro2	MATa his3Δ1 leu2Δ0 ura3Δ0 aro8::KanMX4 aro9::KanMX4
		met15Δ0 aro2::SkHIS3
	Δaro8Δaro9Δtyr1	MATa his3Δ1 leu2Δ0 ura3Δ0 aro8::KanMX4 aro9::KanMX4 met15Δ0 Tyr1::SkHIS3

Table :

 : Plasmids used in this study

	YEp352-COQ2-MVD1	YEp352 containing the sequence 700373-703089 of	This study
		chromosome XIV encompassing the COQ2 and
		MVD1 genes
	YEp352-HFD1	YEp352 containing the sequence 490122-492366 of	This study
		chromosome XIII encompassing the HFD1 gene
	pCOQ8	pRS423 containing S. cerevisiae COQ8 under the	(Ozeir et al.,
		control of its own promoter	2015)
	pNK5	pAKNF316 encoding 3xFLAG-ALDH3A2	(Nakahara et al.,
			2012)
	pNK10	pAKNF316 encoding 3xFLAG-ALDH3A1	(Kitamura et al.,
			2013)
	pNK12	pAKNF316 encoding 3xFLAG-ALDH3B1	(Kitamura et al.,
			2013)
	pNK14	pAKNF316 encoding 3xFLAG-ALDH3B2	(Kitamura et al.,
			2013)
	plasmid	Description	source
	pRS415	Centromeric vector, lac promoter , Amp r , LEU2	(Sikorski	and
			Hieter, 1989)
	pRS415-HFD1	pRS415, Hfd1	This study
	pRS415-HFD1-S241L	pRS415, Hfd1-S241L	This study
	pBADKI-UBIC	pBADKI containing E. coli UBIC ORF	This study
	pRS426-M25C1	Episomal vector containing the MET25 promoter and	(Mumberg et al.,
		the CYC1 terminator, URA3, Amp r	1995)
	pRS426-UBIC	pRS426-M25C1 containing E. coli UBIC under the	This study
		control of the MET25 promoter and the CYC1
		terminator
	pMAL-c2-HFD1	pMAL-c2, Hfd1	This study
	pMAL-c2-HFD1-S241L	pMAL-c2, Hfd1-S241L	This study
	YEp352	Episomal vector, URA3, Amp r	ATCC
	YEp352-ARO2	YEp352 containing the sequence 226031-227968 of	This study
		chromosome VII encompassing the ARO2 gene

ACKNOWLEDGMENTS

We thank Laurent Loiseau for constructing the pBADKI-UBIC vector, Dr Joël Gaffé for assistance with the phylogenetic analysis, Dr Pavel Sindelar, Dr Vassilis Vassiliou and Ulrika Forsman for discussions. This work was supported by the Fondation pour la Recherche Médicale (FRM), grant number «DPM20121125553» to F. Pierrel.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures and four figures.

AUTHOR CONTRIBUTIONS

L.-A.P., M.L, L.P., J.C.W. and F.P. designed research, performed experiments, analyzed and interpreted the data; A.K. contributed reagents; F.P. supervised the project and wrote the manuscript; all authors discussed the results and commented on the manuscript.

SUPPLEMENTAL INFORMATION