Complementation of coenzyme Q-deficient yeast by coenzyme Q analogues requires the isoprenoid side chain Andrew M. James 1 , Helena M. Cocheme ´ 1,2 , Masatoshi Murai 3 , Hideto Miyoshi 3 and Michael P. Murphy 1 1 Medical Research Council Mitochondrial Biology Unit, Wellcome Trust ⁄ MRC Building, Cambridge, UK 2 Institute of Healthy Ageing and GEE, University College London, Darwin Building, London, UK 3 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Japan Introduction CoenzymeQ (CoQ) is composed of a head group that cycles between reduced ubiquinol and oxidized ubiqui- none forms and a hydrophobic isoprenoid tail that keeps the redox activity of the head group located within the lipid bilayer. The length of the isoprenoid tail varies between species, with Saccharomyces cerevisiae, rats and humans predominantly synthesizing forms of CoQ containing six (CoQ 6 ), nine (CoQ 9 ) and 10 (CoQ 10 ) isoprenoid units, respectively. CoQ is synthe- sized endogenously by a series of enzymes localized to Keywords coenzyme Q; diauxic shift; mitochondria; ubiquinone; yeast Correspondence A. M. James, Medical Research Council Mitochondrial Biology Unit, Wellcome Trust ⁄ MRC Building, Hills Road, Cambridge CB2 0XY, UK Fax: +44 1223 252905 Tel: +44 1223 252903 E-mail: aj@mrc-mbu.cam.ac.uk Website: http://www.mrc-mbu.cam.ac.uk (Received 18 December 2009, revised 2 February 2010, accepted 22 February 2010) doi:10.1111/j.1742-4658.2010.07622.x The ubiquinone coenzyme Q (CoQ) is synthesized in mitochondria with a large, hydrophobic isoprenoid side chain. It functions in mitochondrial res- piration as well as protecting membranes from oxidative damage. Yeast that cannot synthesize CoQ (DCoQ) are viable, but cannot grow on nonfer- mentable carbon sources, unless supplied with ubiquinone. Previously we demonstrated that the isoprenoid side chain of the exogenous ubiquinone was important for growth of a DCoQ strain on the nonfermentable sub- strate glycerol [James AM et al. (2005) J Biol Chem 280, 21295–21312]. In the present study we investigated the structural requirements of exoge- nously supplied CoQ 2 for growth on glycerol and found that the first dou- ble bond of the initial isoprenoid unit is essential for utilization of respiratory substrates. As CoQ 2 analogues that did not complement growth on glycerol supported respiration in isolated mitochondria, discrimination does not occur via the respiratory chain complexes. The endogenous form of CoQ in yeast (CoQ 6 ) is extremely hydrophobic and transported to mito- chondria via the endocytic pathway when supplied exogenously. We found that CoQ 2 does not require this pathway when supplied exogenously and the pathway is unlikely to be responsible for the structural discrimination observed. Interestingly, decylQ, an analogue unable to support growth on glycerol, is not toxic, but antagonizes growth of DCoQ yeast in the pres- ence of exogenous CoQ 2 . Using a DCoQ double-knockout library we iden- tified a number of genes that decrease the ability of yeast to grow on exogenous CoQ. Here we suggest that CoQ or its redox state may be a sig- nal for growth during the shift to respiration. Abbreviations CoQ, coenzyme Q with an isoprenoid side chain; CoQ 1–10 , coenzyme Q with a side chain of one to 10 isoprenoid units; DCoQ, yeast strains lacking the ability to synthesize endogenous CoQ 6 ; FCCP, carbonylcyanide-p-trifluoromethoxy-phenylhydrazone; I, fluorescence after the addition of ubiquinone; I 0 , initial fluorescence; Isc1, inositol sphingolipid phospholipase C; PP2A, protein phosphatase 2A; Pyr16, 1-pyrene hexadecanoic acid; YPD, yeast extract, peptone, glucose; YPG, yeast extract, peptone, glycerol; YPGG, yeast extract, peptone, glycerol with glucose. FEBS Journal 277 (2010) 2067–2082 ª 2010 The Authors Journal compilation ª 2010 FEBS 2067 the mitochondrial inner membrane, yet is a component of lipid bilayers throughout the cell [1]. In addition to its function as an electron carrier in the mitochondrial inner membrane, the reduced ubiquinol form of CoQ acts as a recyclable antioxidant that protects biological membranes from oxidative damage [2]. CoQ 10 levels in humans decrease in many pathological situations. For this reason it has been used as a therapy in diseases where oxidative damage is thought to be important, for example very high doses of CoQ 10 have been used with some beneficial effect in Parkinson’s disease [3]. As the requirement for high doses probably results from the extreme hydrophobicity of CoQ 10 and consequently its low bioavailability, efforts have been made to improve its water solubility by developing analogues with shorter and more hydrophilic hydrocarbon tails [4,5]. In the yeast S. cerevisiae, CoQ is not essential for via- bility, as strains lacking the ability to synthesize ubiqui- none (DCoQ) grow by fermentation on glucose. However, they do not grow on nonfermentable sub- strates unless they are supplemented with exogenous CoQ, in which case mitochondrial respiration is restored [6]. It has generally been thought that the redox active ubiquinone head group is the important moiety and that the hydrophobic tail merely anchors this activity in the membrane. Recently we have shown that when decylQ and idebenone, two artificial analogues of CoQ contain- ing a saturated 10-carbon alkane tail, are supplied exogenously they are unable to restore growth on non- fermentable substrates in DCoQ yeast [6]. As more hydrophilic (CoQ 2 ) or hydrophobic (CoQ 4 or CoQ 6 ) iso- prenoid analogues could restore growth on nonferment- able substrates [6], it would appear that the inability of decylQ to do this does not result from differences in pas- sive diffusion to mitochondria in yeast. Instead, it proba- bly arises from a selective protein interaction that can differentiate between an isoprenoid and a saturated alkane tail. This is important, as shorter alkyl ubiquinon- es, such as idebenone and decylQ, are easier to synthesize and have been used therapeutically [4,5]. Therefore, we set out to ascertain the structural requirements for the utilization of exogenous ubiquinone in yeast and to iden- tify proteins that might be involved in discriminating between alkane and isoprenoid ubiquinones. Results Complementation of cell growth in CoQ-deficient yeast by short-chain CoQ analogues shows a dramatic dependence on the isoprenoid side chain Yeast strains that cannot synthesize CoQ endoge- nously (DCoQ) are unable to grow on nonfermentable substrates, such as glycerol. However, when CoQ is supplied exogenously in the growth medium, the abil- ity to grow on nonfermentable substrates is restored. The utilization of glycerol as an energy source for growth requires the presence of CoQ in the mitochon- drial inner membrane to support respiration. That DCoQ strains grow when supplemented with exoge- nous CoQ indicates that some of the externally sup- plied CoQ is reaching the mitochondrial inner membrane within yeast. Previous work showed that CoQ 2 and CoQ 6 sup- ported growth when supplied exogenously to DCoQ yeast [6]. However, not all short-chain ubiquinone ana- logues could do this, as decylQ and idebenone, both of which contain a 10-carbon saturated side chain, failed to support growth on nonfermentable substrates once glucose was depleted (Fig. 1A). This suggested that the structure of the side chain was important for determin- ing whether DCoQ strains were able to grow on exoge- nously supplied ubiquinone. Each isoprenoid unit contains a double bond and a methyl group that could explain the differential reactivity. To identify the struc- tural basis of this interaction we utilized a series of ubiquinone analogues with varying similarities to CoQ 2 (shown in Fig. 1B) [7,8]. The analogues differed from CoQ 2 in a systematic way, with either the removal of a double bond, the deletion of a methyl group or the addition of a carbon atom. When we investigated whether each analogue supported growth in a DCoQ strain on the nonfermentable substrate glycerol, a consensus structural pattern emerged (Fig. 1C). Analogues in which the first (A1-Q 2 ) or sec- ond (A2-Q 2 ) methyl group or second double bond (A3-Q 2 ) were removed exhibited normal growth in glygerol-containing media, suggesting that none of these is required for growth (Fig. 1C). This was in stark contrast to the analogues in which the first dou- ble bond was removed (A4-Q 2 and A6-Q 2 ), as these failed to promote any growth on nonfermentable sub- strates in the DCoQ strain (Fig. 1C). The position of the double bond was also important, as inserting an extra carbon between the head group and the first dou- ble bond (A5-Q 2 ) abolished aerobic growth (Fig. 1C). Therefore, the presence of a double bond between C2 and C3 in the first isoprenoid unit of exogenously sup- plied CoQ is of critical importance for restoration of growth in DCoQ yeast strains. In summary, the high degree of selectivity for ubiq- uinones containing a double bond between C2 and C3 in the first isoprenoid unit suggests the presence of a specific interaction, presumably with a protein that is able to recognize subtle structural differences in the Yeast growth on exogenous ubiquinones A. M. James et al. 2068 FEBS Journal 277 (2010) 2067–2082 ª 2010 The Authors Journal compilation ª 2010 FEBS side chain of CoQ. Therefore, we set out to try and understand the nature of this interaction better. All CoQ 2 analogues restore respiration in isolated mitochondria from DCoQ yeast Growth on glycerol requires CoQ to pass electrons from glycerol-3-phosphate dehydrogenase to complex III of the mitochondrial respiratory chain. Therefore, we first considered the possibility that the different side chains affected the way in which the ubiquinone ana- logues interacted with the mitochondrial respiratory chain. To assess this, we tested their ability to restore respiration on glycerol-3-phosphate in isolated mito- chondria (Fig. 2). All the analogues could do this with equal efficacy (Fig. 2), suggesting that the inability of decylQ and the analogues A4-Q 2 , A5-Q 2 and A6-Q 2 to support growth on YPGG (yeast extract, peptone, glycerol with glucose; Fig. 1C) does not result from a failure of their mitochondria to pass electrons from glycerol-3-phosphate to O 2 . Short-chain ubiquinone analogues do not appear to require an intracellular transport pathway That DCoQ strains can grow on YPG (yeast extract, peptone, glycerol) when supplemented with exogenous CoQ indicates that a sufficient quantity of the supplied CoQ is reaching the mitochondrial inner membrane. As all of the analogues tested so far probably have hydrophobicities similar to that of CoQ 2 and support respiration to a comparable degree in isolated mito- chondria (Fig. 2), it is perhaps surprising that they do not all restore growth in intact cells (Fig. 1C). One simple explanation would be that decylQ does not reach the mitochondrial inner membrane within cells in sufficient quantities to facilitate the passage of elec- trons from glycerol to O 2 . This could arise via an Time (h) A 600 0 2 4 6 8 10 12 14 024487296 Wild-type Dcoq2 + 50 µ M CoQ 2 Dcoq2 + 50 µM decylQ Dcoq2 0 2 4 6 8 10 A 600 DCoQ CoQ 2 DecylQ A1-Q 2 A4-Q 2 A5-Q 2 A6-Q 2 A3-Q 2 A2-Q 2 AB O O MeO MeO A1-Q 2 O O MeO MeO A6-Q 2 O O MeO MeO A5-Q 2 O O MeO MeO A4-Q 2 O O MeO MeO A3-Q 2 O O MeO MeO A2-Q 2 O O MeO MeO DecylQ O O MeO MeO CoQ 2 O O MeO MeO CoQ 6 C Fig. 1. Structural requirements for exogenously supplied CoQ. (A) Growth curve of CENDcoq2 yeast in YPGG supplemented with CoQ 2 ana- logues. Growth to A 600  0.6 is glucose dependent, with growth above this requiring utilization of glycerol. (B) Structure of endogenous CoQ 6 and several hydrophilic CoQ 2 analogues. The circles highlight structural differences in relation to the parent CoQ 2 molecule. (C) Growth of CENDcoq2 yeast after 96 h in YPGG supplemented with 50 l M of each CoQ 2 analogue. Growth to A 600  0.6 is glucose dependent, with growth above this requiring utilization of glycerol. Values are the mean ± range of two independent experiments each carried out in duplicate. A. M. James et al. Yeast growth on exogenous ubiquinones FEBS Journal 277 (2010) 2067–2082 ª 2010 The Authors Journal compilation ª 2010 FEBS 2069 endogenous uptake pathway that recognizes the iso- prenoid structure of CoQ 2 , but cannot interact with the alkane tail of decylQ or any of the CoQ 2 analogues lacking the first double bond. That a CoQ transport pathway exists for endogenous CoQ 6 in yeast has been considered probable for some time, as the enzymes for CoQ 6 synthesis are located in the mitochondrial inner membrane, but CoQ 6 is found throughout the cell [1,9]. Spontaneous diffusion through the cytosol appeared unlikely for this dispersion, as CoQ 6 is very hydrophobic with a predicted octanol ⁄ water partition coefficient in the region of 10 14 [10]. Recently it has been shown that DCoQ yeast strains require at least four genes (tlg2, erg2, pep12 and vps45) from the endo- cytic pathway to grow on exogenously supplied CoQ 6 [11]. To test whether the endocytic uptake pathway was also required for the uptake of short-chain ana- logues such as decylQ and CoQ 2 in our experiments, we created two double-knockout strains and tested their ability to grow on CoQ 2 . Both Dcoq2Dtlg2 and Dcoq2Derg2 strains grew in the presence of CoQ 2 (Fig. 3A) as well as the even more hydrophobic ana- logue CoQ 4 (data not shown). The growth characteris- tics were similar to those seen in the Dcoq2 strain AB DE F C G3P + FCCP 2 min 100 nmol O myxo 1 5 15 10 20 [CoQ 2 ] (µM) [CoQ 2 ] (µM) [DecylQ] (µ M) [A1-Q 2 ] (µM) [A2-Q 2 ] (µM) [A3-Q 2 ] (µM) GHI [A4-Q 2 ] (µM) [A5-Q 2 ] (µM) [A6-Q 2 ] (µM) Respiration rate (nmol O·min –1 ·mg –1 protein) Respiration rate (nmol O·min –1 ·mg –1 protein) Respiration rate (nmol O·min –1 ·mg –1 protein) Respiration rate (nmol O·min –1 ·mg –1 protein) Respiration rate (nmol O·min –1 ·mg –1 protein) Respiration rate (nmol O·min –1 ·mg –1 protein) Respiration rate (nmol O·min –1 ·mg –1 protein) Respiration rate (nmol O·min –1 ·mg –1 protein) 0 50 100 150 200 250 0 5 10 15 20 0 50 100 150 200 250 0 5 10 15 20 0 50 100 150 200 250 0 5 10 15 20 0 50 100 150 200 250 0 5 10 15 20 0 50 100 150 200 250 0 5 10 15 20 0 50 100 150 200 250 0 5 10 15 20 0 50 100 150 200 250 0 5 10 15 20 0 50 100 150 200 250 0 5 10 15 20 Fig. 2. CoQ 2 analogues restore respiration in isolated CoQ-deficient mitochondria. (A) An example oxygen electrode trace of isolated CEN- Dcoq2 yeast mitochondria in mannitol buffer with glycerol-3-phosphate (G3P; 5 m M) and FCCP (1 lM). CoQ 2 was titrated successively as indicated by the arrowheads. That the oxygen consumption was mitochondrial in origin was confirmed by addition of the inhibitor myxothiazol (1 l M). (B)–(I) Uncoupled rates of mitochondrial respiration as a function of CoQ 2 (B), decylQ (C), A1-Q 2 (D), A2-Q 2 (E), A3-Q 2 (F), A4-Q 2 (G), A5-Q 2 (H) and A6-Q 2 (I). Data are the mean ± standard deviation of three independent experiments. Yeast growth on exogenous ubiquinones A. M. James et al. 2070 FEBS Journal 277 (2010) 2067–2082 ª 2010 The Authors Journal compilation ª 2010 FEBS (Fig. 3D), suggesting that the endocytic pathway is not required for the uptake of CoQ 2 to the mitochondria and, therefore, this pathway is unlikely to be responsi- ble for discriminating between decylQ and CoQ 2 . That a vesicle-based mechanism is not required for the uptake is reasonable as the octanol ⁄ PBS partition coef- ficients of CoQ 2 ( 10 4.5 ) and decylQ ( 10 5.5 ) are sev- eral orders of magnitude lower than that of CoQ 6 [6,10]. This suggests that CoQ 2 is sufficiently hydrophilic that it can passively equilibrate quite effectively within cells over the 48 h timeframe of the growth experi- ments. To test this we measured whether yeast sup- plied with ubiquinone exogenously contained a significantly lower concentration of decylQ than that of CoQ 2 or whether the accumulation of decylQ was slower than for CoQ 2 . To limit complications due to growth, a DCoQ culture was maintained in YPGG for 24 h, at which point they had consumed the available glucose and their growth had arrested. In the contin- ued absence of exogenous CoQ, the absorbance of this culture remained stable, with no evidence of growth for several days (Figs 1A and 3B). Upon the addition of a mixture of CoQ 2 and decylQ it took 3–6 h for growth to restart (Fig. 3B). To determine if there were differences in the accumulation of CoQ 2 and decylQ we measured the amount of the two ubiquinone ana- logues in the cell pellets. From 30 min up to 48 h after the addition of a mixture of 10 lm CoQ 2 and 10 lm decylQ to a yeast suspension, the ratio of decylQ to CoQ 2 in the pellet remained very similar (Fig. 3C). Although a difference was observed at a very early 2 min time point, the equilibration rate of both was relatively rapid in the context of the 48 h experiments where decylQ failed to restore growth. This suggests that the association of CoQ 2 and decylQ with yeast cells is similar, despite decylQ not being able to complement nonfermentative growth in DCoQ yeast. If more subtle differences in diffusion of decylQ to mitochondria within cells were responsible, it might be possible to restore growth with decylQ by adding a large excess of it to DCoQ yeast. Therefore we mea- sured the ability of DCoQ yeast to grow on glycerol with concentrations of decylQ up to 100 lm. No non- fermentative growth was observed with decylQ, even when its concentration was up to 50- and 250-fold higher than that required to observe such growth with CoQ 2 and CoQ 4 , respectively (Fig. 3D). However, there was a small, but significant, increase in the absorbance of the culture when concentrations of decylQ as low as 2 lm were added relative to the dim- ethylsulfoxide carrier alone, suggesting decylQ induced some change in the culture. The small increase was also observed with 10 lm of the ineffective CoQ 2 analogues A4-Q 2 , A5-Q 2 and A6-Q2, as well as with CoQ 1 , but it was not observed with 10 lm of CoQ 9 or CoQ 10 (data not shown). The lack of any concentra- tion dependence when an excess of decylQ was sup- plied exogenously suggests that its failure to complement nonfermentative growth in DCoQ yeast does not result from subtle differences in diffusion, particularly given that the hydrophobicity of decylQ is intermediate between CoQ 2 and CoQ 4 . To demonstrate that the physiochemical properties of decylQ and CoQ 2 are grossly similar, we measured their ability to diffuse between noncontiguous mem- branes by mixing two populations of vesicles. Both populations of vesicles contained equal concentrations of the very hydrophobic fluorophore, 1-pyrene hexa- decanoic acid (Pyr16), but only the second population contained ubiquinone. Ubiquinone collisionally quenches pyrene fluorescence if both are present in the same membrane system and are capable of physical interaction [12]. There is a linear relationship between ubiquinone concentration and I 0 ⁄ I – 1, where I 0 is the initial fluorescence and I is the fluorescence after the addition of ubiquinone, and for a typical ubiquinone in our hands the slope of this line is 24 mm )1 [12]. Using this value we would expect a decrease in relative fluorescence (I ⁄ I 0 ) from 1 to 0.83 upon the addition of ubiquinone-containing vesicles because the initial unquenched vesicle population (2 mL) is being diluted with a highly quenched second vesicle population con- taining ubiquinone (500 lL). This drop does not occur if the added vesicle population does not contain ubi- quinone (data not shown). We would then expect a further decrease in relative fluorescence to 0.51 if the ubiquinone is able to equilibrate between bilayers because 40 lm ubiquinone in 100% of vesicles will quench total fluorescence more effectively than 200 lm ubiquinone in 20% of vesicles. The exchange of ubi- quinone between the two populations of vesicles can be seen most easily using CoQ 4 , as there is a decrease in relative fluorescence from 0.77 to 0.57 over the course of the experiment (Fig. 3E). When the second population of vesicles was reconstituted with the rela- tively more hydrophilic analogues, CoQ 2 or decylQ, quenching was largely complete within seconds and after approximately 10 min relative fluorescence was  0.57 for both analogues, suggesting they are free to exchange between the phospholipid bilayers of the two vesicle populations (Fig. 3E). If the experiment was repeated with the very hydrophobic analogue, CoQ 9 , there was no evidence of movement between the two populations during the course of the 10 min A. M. James et al. Yeast growth on exogenous ubiquinones FEBS Journal 277 (2010) 2067–2082 ª 2010 The Authors Journal compilation ª 2010 FEBS 2071 incubation, as relative fluorescence remained stable at 0.82 (Fig. 3E). Therefore, both CoQ 2 and decylQ dif- fuse rapidly between the noncontiguous membranes of the two vesicle populations, taking only a few seconds to equilibrate. This is consistent with the rapid equili- bration of both CoQ 2 and decylQ within cells (Fig. 3C). Finally, we sought to confirm that decylQ and CoQ 2 both reach mitochondria in intact cells directly. DecylQ and CoQ 2 participate effectively in respiration by isolated mitochondria in the absence of endogenous CoQ 6 (Fig. 2). Therefore, if they can migrate to mito- chondria their redox state should be sensitive to com- pounds that manipulate the mitochondrial respiratory chain. To test this we incubated intact yeast cells with either decylQ or CoQ 2 for 3 h, after which we exposed them to the either cyanide (KCN) or carbonylcyanide- p-trifluoromethoxy-phenylhydrazone (FCCP) and ex- tracted the ubiquinone. KCN inhibits complex IV and leads to a reduced ubiquinone pool as electrons can no Ubiquinone concentration (µM) 0 1 2 3 4 5 0 1020304050 Ubiquinone concentration (µM) 0 1 2 3 4 5 012243648 Time (h) Growth (A 600 ) 0 5 10 15 20 25 0 12243648 CoQ 2 decylQ Pellet-associated ubiquinone normalised for growth (% of total Q in the pellet/A 600 unit) Time (h) Δcoq2Δerg2 Δcoq2Δtlg2 AB 0 1 2 3 4 5 6 7 8 0 5 10 15 20 50 100 CoQ 4 CoQ 2 decylQ CD Non-fermentative growth (A 600 ) Non-fermentative growth (A 600 ) 0 5 10 15 20 25 012 3 45 6 (h) (% of total Q in the pellet/A 600 unit) Ubiquinol (%) E CoQ 2 /decylQ Ubiquinone-loaded vesicles Normalised fluorescence (I/I 0 ) 0 10 20 30 40 50 60 70 80 CoQ 2 decylQ Ubiquinone FCCP KCN Time (min) F 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 0246810 CoQ 2 CoQ 4 CoQ 9 decylQ Yeast growth on exogenous ubiquinones A. M. James et al. 2072 FEBS Journal 277 (2010) 2067–2082 ª 2010 The Authors Journal compilation ª 2010 FEBS longer flow to O 2 , whereas FCCP uncouples mitochon- dria, increasing respiration and oxidizing the ubiqui- none pool as electrons rapidly flow from ubiquinone to O 2 in the absence of a membrane potential [6]. Although CoQ 2 responded as expected, surprisingly decylQ was not significantly reduced in the presence of KCN (Fig. 3F). In the absence of FCCP or KCN, the ratio of reduced to oxidized ubiquinone was similar to that in the presence of FCCP (data not shown). In summary, the presence of a sufficient concentra- tion of ubiquinone in the mitochondrial membrane is required for growth on glycerol. The above results sug- gest that CoQ 2 and decylQ are both sufficiently hydro- philic that they can rapidly equilibrate between noncontiguous membranes through the aqueous phase without this movement being mediated by a hydropho- bic phase such as a vesicle. This makes it unlikely that the difference between CoQ 2 and decylQ is due to dif- ferences in their ability to diffuse to mitochondria within cells. Paradoxically, decylQ was largely insensi- tive to a mitochondrial inhibitor that should have led to a reduction in decylQ in the presence of a substrate. The reason for this discrepancy remains unclear and will be discussed later. DecylQ is unlikely to be exported by yeast cells Above we dealt with the possibility that CoQ 2 is selec- tively transported to mitochondria within cells. The converse could equally have been true and decylQ could have been selectively excluded by yeast cells. This is because yeast can remove foreign molecules, primarily by using ATP binding cassette (ABC) trans- porters, as a mechanism for protecting themselves from toxins [13]. Selective exclusion of decylQ from the cell appears unlikely, as the amount of decylQ associated with the yeast cell pellet is similar to that of CoQ 2 (Fig. 3C), the putative export machinery cannot be overwhelmed by high concentrations of decylQ (Fig. 3D) and the structures that fail to restore growth are less similar than the ones that do restore growth, implying selective recognition of CoQ 2 rather than of decylQ (Fig. 1B). DecylQ is not toxic, but does confound growth complementation by CoQ 2 The previous experiments suggest that decylQ can migrate to mitochondria within cells. Therefore, it is possible that decylQ is in some way toxic to yeast cells and thereby prevents nonfermentative cell growth, despite complementing respiration (Fig. 2). To deter- mine if this was the case, we added increasing equimo- lar amounts of CoQ 2 and decylQ to DCoQ cultures and measured their growth in YPGG over 48 h (Fig. 4A). The addition of equimolar decylQ to CoQ 2 had no significant detrimental effect on growth com- pared with CoQ 2 alone. Therefore, decylQ appears to Fig. 3. CoQ 2 does not require a transport pathway to reach mitochondria in yeast. (A) Deletion of proteins in the endocytic pathway does not prevent exogenous CoQ 2 restoring growth. Dcoq2Dtlg2 and Dcoq2Derg2 double-knockout strains where grown in YPGG supplemented with CoQ 2 (0–50 lM) for 48 h. Data are the mean ± range of two independent experiments. The growth of the two strains in the absence of ubiquinone was subtracted to give the growth achieved on glycerol. (B) Time lag in the growth response of stationary phase yeast to exoge- nously supplied CoQ 2 . The BY4743Dcoq2 strain was grown in YPGG for 18 h, at which point it had reached A 600  0.8 and already been sta- tionary for several hours and would remain that way for a number of days (squares). At the point indicated by the arrow, a mixture of 10 l M decylQ and 10 lM CoQ 2 was added and the cultures were incubated for a further 48 h with A 600 measurements taken at intervals (circles). Data are the mean ± standard error of the mean of three independent experiments. (C) The equilibration of exogenously supplied CoQ 2 and decylQ with yeast cells is rapid. As in (B), but samples were taken at intervals and centrifuged before CoQ 2 and decylQ were extracted with hexane from both the supernatant and the pellet. They were separated by HPLC and quantitated using electrochemical detection. The values are expressed as the percentage of the total CoQ 2 or decylQ in the yeast pellet normalized for the growth (A 600 ) of the culture shown in (B). Data are the mean ± standard error of the mean of three independent experiments. (D) Even a 50-fold excess of decylQ above that required for CoQ 2 does not restore growth of the BY4743Dcoq2 strain. The BY4743Dcoq2 strain was grown in YPGG supplemented with decylQ (0–100 l M), CoQ 2 (0–20 lM) or CoQ 4 (0–20 lM) for 48 h. Data are the mean ± standard error of the mean of three independent experiments. The growth of the BY4743Dcoq2 strain in the absence of ubiquinone was subtracted to give the growth achieved on glycerol. (E) DecylQ and CoQ 2 move freely between noncontiguous phospholipid bilayers. An initial population of phosphatidylcholine vesicles containing 4 lM Pyr16 in 2 mL KP i buffer was mixed with 500 lL of a second population created containing 4 lM Pyr16 and either CoQ 2 , decylQ, CoQ 4 or CoQ 9 . A decrease in fluorescence indicated collisional quenching via a physical interaction between ubiquinone and Pyr16. The data are expressed as the relative amount of fluorescence at a given point in time (I) to initial fluorescence just before the addition of ubiquinone- containing vesicles (I 0 ). (F) The redox state of decylQ is largely insensitive to KCN. The BY4743Dcoq2 strain was grown in YPGG for 24 h at which point it had reached A 600  0.8 before either 10 lM decylQ or 10 lM CoQ 2 was added and the cultures were incubated for a further 3 h. Then, either 1 l M FCCP or 200 lM KCN was added, followed by a 2 min incubation at 30 °C. The cultures were centrifuged before CoQ 2 and decylQ were extracted with hexane from the pellet. They were separated by HPLC and quantitated using electrochemical detec- tion. The values are the percentage of the total CoQ 2 or decylQ found in the ubiquinol form. Data are the mean ± standard error of the mean of three independent experiments. A. M. James et al. Yeast growth on exogenous ubiquinones FEBS Journal 277 (2010) 2067–2082 ª 2010 The Authors Journal compilation ª 2010 FEBS 2073 be nontoxic. We next reasoned that because decylQ should diffuse to mitochondria (Fig. 3) and once there function effectively in oxidative phosphorylation (Fig. 1D), that decylQ might be unable to restore growth because CoQ 2 is required in trace amounts for some secondary function. To determine if this was the case, we added increasing amounts of decylQ (0–100 lm) in combination with 5 lm CoQ 2 to DCoQ cultures in YPGG and measured their growth over 48 h. This showed that trace amounts of CoQ 2 did not allow decylQ to complement growth (Fig. 4B). In fact, rather surprisingly, at higher concentrations decylQ largely inhibited the growth that would have been observed with 5 lm CoQ 2 alone (Fig. 4B). Interest- ingly, there was a strong correlation between the frac- tion of the total added ubiquinone present as CoQ 2 and growth (Fig. 4C; r 2 = 0.99). Thus, it would appear that decylQ in some way prevents CoQ 2 inter- acting with a protein that is required for nonfermenta- tive growth. This might occur via decylQ binding directly to the protein and preventing CoQ 2 interacting or growth could require an interaction with the reduced or the oxidized form of CoQ 2 and decylQ dis- turbs the normal CoQ 2 H 2 ⁄ CoQ 2 ratio. In summary, decylQ is not toxic, but can antagonize the stimulation of growth by CoQ 2 , especially when its concentration markedly exceeds that of CoQ 2 . This suggests that there may be a protein that regulates growth that can bind to CoQ 2 and that decylQ antago- nizes this interaction in some way. Screen for proteins influencing CoQ-dependent growth in yeast The data so far suggest that there is a protein that can distinguish between CoQ 2 and decylQ that is essential for growth on nonfermentable substrates. The nature of this CoQ–protein interaction is unclear, as we have been unable to identify an obvious secondary role for CoQ in growth on nonfermentable substrates from the literature. To investigate this interaction further we set up a screen to identify potential ORFs that might be involved in CoQ-dependent growth in yeast. The yeast 0 1 2 3 4 5 6 0 5 10 15 20 CoQ 2 CoQ 2 and equimolar decylQ Non-fermentative growth (A 600 ) CoQ 2 concentration (µM) 0 1 2 3 4 5 6 0 20 40 60 80 100 X µM decylQ + X µM CoQ 2 X µM decylQ + 5 µM CoQ 2 X µM decylQ Ubiquinone concentration (X µM) A B 0 0.5 1 1.5 2 2.5 0 0.1 0.2 0.3 0.4 0.5 Fraction of ubiquinone that is CoQ 2 ([CoQ 2 ] / [CoQ 2 +decylQ]) C Non-fermentative growth (A 600 ) Non-fermentative growth (A 600 ) Fig. 4. DecylQ is not toxic, but does interfere with growth comple- mentation by CoQ 2 . (A) Growth on exogenous CoQ 2 is neither stimulated nor inhibited by the presence of equimolar decylQ. The BY4743Dcoq2 strain was grown in YPGG supplemented with either CoQ 2 (0–20 lM) or equimolar CoQ 2 and decylQ (0–20 lM) for 48 h. Data are the mean ± standard error of the mean of three indepen- dent experiments. The growth of the BY4743Dcoq2 strain in the absence of ubiquinone was subtracted to give the growth achieved on glycerol. (B) Growth on trace exogenous CoQ 2 is inhibited by the presence of higher concentrations of decylQ. The BY4743Dcoq2 strain was grown in YPGG with either decylQ alone (0–100 l M), equimolar CoQ 2 and decylQ (0–100 lM) or CoQ 2 (5 lM) and a range of decylQ concentrations (0–100 l M) for 48 h. The growth of the BY4743Dcoq2 strain in the absence of ubiquinone was subtracted to give the growth achieved on glycerol. (C) Growth is related to the proportion of total ubiquinone that is CoQ 2 .Asin (B), the BY4743Dcoq2 strain was grown in YPGG with 5 l M CoQ 2 and a range of decylQ concentrations (0–100 lM) for 48 h. Yeast growth on exogenous ubiquinones A. M. James et al. 2074 FEBS Journal 277 (2010) 2067–2082 ª 2010 The Authors Journal compilation ª 2010 FEBS S. cerevisiae contains  6000 ORFs, of which  5000 can be deleted with the strain still viable. To do this we first created a double-knockout library unable to synthesize CoQ by crossing a strain in which a gene required for the endogenous synthesis of CoQ 6 (coq2) was deleted with a commercial library containing  4800 strains each harbouring a deletion in a single nonessential gene. To generate a stable haploid double-knockout library we utilized an approach developed by Tong and coworkers [14]. The stability arises because BY4743Dcoq2 contains a histidine synthesis gene under the control of a mating-type ‘a’ promoter (MFA1pr-HIS3) inserted into the middle of an arginine permease gene (CAN1). This allows mating-type ‘a’ progeny to be selected without cloning. For details on how the double-knockout library was created, see the Materials and Methods section. After its creation, the Dcoq2DORF strains in the double-knockout library were screened for their ability to grow in liquid YPGG when supplemented exoge- nously with 100 lm CoQ 2 . This identified 379 double- knockout strains that could not grow on YPGG containing CoQ 2 (Fig. 5A). Of these, 324 were not con- sidered further for three reasons. First, theDcoq2DORF strain had grown in liquid YPGG in the absence of CoQ2, suggesting they may not be DCoQ (Fig. 5B). Second, the corresponding DORF strain failed to grow on solid YPG, indicating that the ORF deleted in the strain was a gene essential for nonfermentative growth, e.g. a nuclear-encoded respiratory chain complex sub- unit (Fig. 5C). Finally, they did not produce viable progeny after mating and sporulation on the final selective plate. Therefore, there was no Dcoq2DORF strain to test for dependence on exogenous CoQ (Fig. 5D). The remaining 55 strains potentially har- boured a deletion that removes a gene that influences growth on exogenous CoQ. Consequently, these strains were cloned and analysed further to confirm whether they respond abnormally to exogenous CoQ. Confirmation of double-knockout strains with poor growth on exogenously supplied CoQ An inoculation from each of the 55 Dcoq2DORF strains identified from the screen were grown in 3 mL YPGG and 10 lm CoQ 4 . In addition, nine control Dcoq2DORF strains were selected to control for the mating and sporulation (see Materials and Methods). After 48 h their growth was measured and the 24 strains with decreased growth relative to the BY4743Dcoq2 parental strain and the nine Dcoq2- DORF control strains were cloned, as were their DORF counterparts from the original commercial library. All of these Dcoq2DORF strains grew by fermentation in YPD (yeast extract, peptone, glucose) to an A 600 of  10 (data not shown) and in YPGG without CoQ 4 to A B C D Fig. 5. Creation of the double-knockout library. (A, B) Double-knockout Dcoq2DORF strains grown in liquid glycerol media (YPG) supplemented with canavanine, G418 and cloneNAT with (A) or without (B) 100 l M CoQ 2 . (C) Parental single-deletion DORF strains grown on solid YPG. (D) Double-dele- tion Dcoq2DORF strains grown in solid glucose-based synthetic media lacking histi- dine and arginine and supplemented with canavanine, G418 and cloneNAT. The red squares indicate strains that failed to give information about whether the deleted ORF is or is not involved in CoQ-dependent growth. Unmarked strains have exogenous CoQ-dependent growth and are uninterest- ing, whereas black squares indicate the desired combination of phenotypes and these were analysed further. The top left black square is Srb8 and the bottom right is Kcs1. A. M. James et al. Yeast growth on exogenous ubiquinones FEBS Journal 277 (2010) 2067–2082 ª 2010 The Authors Journal compilation ª 2010 FEBS 2075 an A 600 of  0.8 (data not shown). Therefore, their utilization of glucose for growth was grossly normal and they were unable to make the transition to the uti- lization of glycerol as a carbon source in the absence of CoQ. This can also be seen in the slightly positive values obtained for growth in YPGG containing 10 lm CoQ 4 , as a failure to grow on glucose would lead to negative values (Table 1). These 24 Dcoq2DORF clones were grown in glass tubes with 3 mL YPGG and 10 lm CoQ 4 for 48 h at 30 °C. The growth of 21 of these fell below the range of the nine Dcoq2DORF control strains and the BY4743Dcoq2 parental strain. To ensure the defect in growth was related to an inability to utilize exogenous CoQ and not related to a general defect in mitochon- drial metabolism, the corresponding DORF strains were grown in glass tubes with 3 mL YPGG for 48 h at 30 °C. For five of the remaining ORFs, poor growth of the Dcoq2DORF strain could be sufficiently explained by similar poor growth of their DORF strain. This left 16 ORFs where the Dcoq2DORF strain could not grow as well when supplemented with exoge- nously supplied CoQ 4 as any of the nine control Dcoq2DORF strains yet contained mitochondria that were at least partially functional in their DORF strain (Table 1). Unlike the control Dcoq2DORF strains, for many of the potentially interesting Dcoq2DORF strains the counterpart DORF strain had reduced growth on glycerol (Table 1). However, when the growth of the Dcoq2DORF strain is expressed as a percentage of that of its corresponding single knockout, it is apparent that any decrease in growth on glycerol in the DORF strain is compounded by the introduction of a Dcoq2 deletion. In summary, we have identified 16 ORFs, the deletion of which dramatically decreased the ability of exogenous CoQ to support growth on glycerol in the absence of an ability to synthesize endogenous CoQ. Discussion Yeast lacking the ability to synthesize CoQ endoge- nously require exogenous CoQ to grow on nonfer- mentable substrates. Utilizing several very similar analogues of CoQ 2 we have shown that exogenous supplementation of decylQ and all analogues lacking the first double bond of the side chain failed to sup- port the growth of DCoQ yeast on glycerol. This sug- gests that there is very selective structural recognition of elements within the side chain, presumably by a protein, and that this interaction is required to com- plete the diauxic shift to nonfermentable substrates (Fig. 1). The most obvious level at which this struc- tural recognition could occur is mitochondrial respira- tion. However, isolated mitochondria lacking CoQ respired normally on glycerol-3-phosphate when sup- plemented with all CoQ 2 analogues tested (Fig. 2). This suggests that the selectivity does not arise from the respiratory chain complexes. The next possibility is that the protein of interest is involved in the transport of exogenous CoQ from the extracellular medium into the cell and on to mitochon- dria, and that decylQ and the analogues A4-Q 2 , A5-Q 2 and A6-Q 2 are not taken up correctly by this pathway. Deletion of two endocytic proteins shown to be involved in the transport of exogenous CoQ 6 to mito- chondria [11] failed to prevent growth of DCoQ yeast on glycerol (Fig. 3A), suggesting that this pathway is not an absolute requirement for the uptake of more hydrophilic CoQ analogues. Consistent with this, CoQ 2 and decylQ reached apparent equilibrium within minutes of being added to a culture of DCoQ yeast (Fig. 3C). Even though 2 lm CoQ 2 led to appreciable growth, 100 lm decylQ did not, suggesting that uptake of decylQ to mitochondria is not the factor limiting growth. Furthermore, mixing populations of vesicles containing fluorescent pyrenes with vesicles containing decylQ, CoQ 2 and CoQ 4 indicated rapid redistribution of decylQ and CoQ 2 over a timeframe of seconds and CoQ 4 within minutes (Fig. 3E). CoQ 2 and decylQ were also rapidly lost into the bulk aqueous phase during cell subfractionation or cell pellet washing steps (unpublished observations). Together this suggests that even though decylQ and CoQ 2 are hydrophobic, they are not hydrophobic enough to be retained within a membrane system over the 48 h course of the growth experiments and presumably equilibrate throughout all the lipid bilayers of the cell. The experiments outlined above suggest that enough CoQ 2 diffuses around blocks in the endocytic pathway used to transport CoQ 6 to support growth on nonfer- mentable substrates (Fig. 3A). However, even though it appears possible for CoQ 2 to diffuse to mitochon- dria, growth on glycerol with exogenously supplied CoQ 2 is significantly slower than that observed on endogenous CoQ 6 (Fig. 1). One reason for the dimin- ished growth on exogenous CoQ 2 relative to endoge- nous CoQ 6 could be because the concentration of CoQ in mitochondrial membranes appears significantly higher than that of other cellular membranes [11]. Because CoQ 2 could redistribute between bilayers (Fig. 3E) there may be no way of maintaining an ele- vated mitochondrial CoQ concentration, thereby resulting in suboptimal growth. Alternatively, it could arise from a decrease in substrate concentration for an enzyme because CoQ 2 cannot be accumulated and retained in a membrane system or via a decrease in Yeast growth on exogenous ubiquinones A. M. James et al. 2076 FEBS Journal 277 (2010) 2067–2082 ª 2010 The Authors Journal compilation ª 2010 FEBS [...]... DCoQ yeast strains This is despite all the CoQ analogues restoring respiration in isolated mitochondria This suggests the presence of a protein that is able to recognize subtle differences in the side chain of CoQ This protein is not part of the endocytic pathway used for the uptake of exogenous CoQ6 nor does it appear to be one of the mitochondrial respiratory complexes Growth is slowed by decylQ even... the protein recognizes decylQ itself One possibility is that during the diauxic shift, the ratio of CoQH2 ⁄ CoQ is an important growth signal and an excess of decylQ interferes with this redox couple The exact nature of the interaction remains unclear and will be the subject of future work Materials and methods Chemicals CoQ4, CoQ2 and decylQ were sourced from Sigma (St Louis, MO, USA) Other CoQ2 analogues. .. size of the CoQ2 and decylQ peaks Determination of the fraction of CoQ2 and decylQ that could be reduced by mitochondria was as above with some modifications The BY4 743Dcoq2 strain was grown in YPGG for 24 h, at which point it had reached A600  0.8 and become stationary As we were interested in their redox state, either 10 lm decylQ or 10 lm CoQ2 was added separately so they could not equilibrate The. .. acetylated by Nat4p 1.21 ± 0.06 37.8 Yeast growth on exogenous ubiquinones binding affinity of CoQ2 or CoQ4 for a hydrophobic pocket in a protein through the loss of isoprenoid units from the side chain of CoQ6 To identify proteins or pathways that might be involved in CoQ-dependent growth on nonfermentable substrates, we created a yeast double-knockout library (DORFDcoq2) and screened for strains whose... integrity Subunit 9 of the ubiquinol cytochrome c reductase complex, which is a component of the mitochondrial inner membrane electron transport chain; required for electron transfer at the ubiquinol oxidase site of the complex Aconitase, required for the tricarboxylic acid cycle and also independently required for mitochondrial genome maintenance; phosphorylated; component of the mitochondrial nucleoid;... 500 lL addition of the second vesicle population containing either CoQ2, decylQ, CoQ4 or CoQ9 was made after 90 s, such that the final ubiquinone concentration was 40 lm The data are expressed as the ratio of Yeast growth on exogenous ubiquinones fluorescence at a given point in time (I) to initial fluorescence just before this addition (I0) Acknowledgement This work was supported by the Medical Research... contain a functioning mitochondrial respiratory chain Although the ORFs deleted in these strains could be involved in CoQ uptake, they were not considered for further analysis, as they would not be responsive to exogenous CoQ in the final double-knockout library After creation of the doubleknockout Dcoq2DORF library,  4% of strains were not viable on the final selective plate, SD)Arg)His+Can+ Kan+Nat... g The supernatant was removed to another Eppendorf tube and snap frozen The residual supernatant was aspirated from the pellet and the Eppendorf tube was dried with tissue paper before the pellet was also snap frozen The pellet was not washed due to significant loss of decylQ and particularly CoQ2 into the washing medium over and above that which could be explained by the residual water space of the. .. min) The area under each peak was integrated and compared with standards containing known amounts (0–200 pmol) of CoQ2 and decylQ CoQ2H2 and decylQH2 were quantitated by assuming a similar peak intensity to their oxidized counterparts and rerunning of samples after slow oxidation under air suggested this was a valid assumption as loss of area from the CoQ2H2 and decylQH2 peaks was comparable with the. .. 48 h in YPGG with 10 lM CoQ4 Growth of the DORF strains was measured after 48 h in YPGG The BY4 743Dcoq2 strain grew to A600  0.8 in YPGG and this was subtracted from the A600 of each Dcoq2DORF and DORF strain to give the level of growth on glycerol All Dcoq2DORF colonies could utilize glucose and grew to A600  0.8 in YPGG without CoQ4 (data not shown) The ratio between Dcoq2DORF and DORF growth was . Complementation of coenzyme Q- deficient yeast by coenzyme Q analogues requires the isoprenoid side chain Andrew M. James 1 , Helena. (h) A 600 0 2 4 6 8 10 12 14 024487296 Wild-type Dcoq2 + 50 µ M CoQ 2 Dcoq2 + 50 µM decylQ Dcoq2 0 2 4 6 8 10 A 600 DCoQ CoQ 2 DecylQ A1 -Q 2 A4 -Q 2 A5 -Q 2 A6 -Q 2 A3 -Q 2 A2 -Q 2 AB O O MeO MeO A1 -Q 2 O O MeO MeO A6 -Q 2 O O MeO MeO A5 -Q 2 O O MeO MeO A4 -Q 2 O O MeO MeO A3 -Q 2 O O MeO MeO A2 -Q 2 O O MeO MeO DecylQ O O MeO MeO CoQ 2 O O MeO MeO CoQ 6 C Fig.