Báo cáo khoa học: Succinate dehydrogenase flavoprotein subunit expression in Saccharomyces cerevisiae – involvement of the mitochondrial FAD transporter, Flx1p ppt

Succinate dehydrogenase flavoprotein subunit expression in Saccharomyces cerevisiae – involvement of the mitochondrial FAD transporter, Flx1p Teresa A. Giancaspero 1 , Robin Wait 2 , Eckhard Boles 3 and Maria Barile 1 1 Dipartimento di Biochimica e Biologia Molecolare ‘‘E. Quagliariello’’, Universita ` degli Studi di Bari, Italy 2 Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College London, UK 3 Institut fu ¨ r Molekulare Biowissenschaften, J.W. Goethe-Universita ¨ t, Frankfurt am Main, Germany Several mitochondrial dehydrogenases and oxidases require FMN and FAD for their activity [1,2]. Thus, intramitochondrial flavin cofactor availability is poten- tially a crucial regulator of oxidative terminal metabo- lism. Consistent with this, some patients suffering from riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency (RR-MADD) exhibit profound disorders in mitochondrial biochemistry that are reversed by treat- ment with high doses of riboflavin [3]. Mammals obtain flavin cofactors from dietary ribo- flavin, which enters their cells via plasma membrane riboflavin transporters, although these have not yet been characterized at the molecular level [1,4]. In Sac- charomyces cerevisiae, the product of the MCH5 gene was recently identified as a plasma membrane riboflavin transporter [5], although this organism, in common with other yeasts and plants, is able to synthesize riboflavin de novo and export it into the culture medium [3–9]. In previous publications, we proposed that mainte- nance of flavin cofactor levels inside mitochondria requires the activity of mitochondrial riboflavin trans- port system(s) and two enzymes, riboflavin kinase (EC 2.7.1.26) and FAD synthetase (EC 2.7.7.2), which catalyze the synthesis of FMN and FAD respectively [10–13]. In this scenario, the lumiflavin-sensitive flavin transporter, Flx1p, is responsible for FAD export from S. cerevisiae mitochondria (SCM) [13]. Alternatively, on the basis of the cytosolic localization of the FAD synthetase, encoded by FAD1 [14], other authors sug- gested that Flx1p is involved in mitochondrial FAD import in exchange with FMN [15]. FLX1 deletion or mutation results in a respiration- deficient phenotype, in which the activities of the mitochondrial FAD dependent-enzymes, lipoamide dehydrogenase and succinate dehydrogenase (SDH), are reduced [13,15]. Measurement of the mitochondrial Keywords flavin; Flx1p; mitochondrial FAD transporter; post-transcriptional control; succinate dehydrogenase flavoprotein subunit Correspondence M. Barile, Via Orabona, 4, 70126 Bari, Italy Fax: +39 0805443317 Tel: +39 0805443604 E-mail: m.barile@biologia.uniba.it (Received 30 July 2007, revised 27 December 2007, accepted 4 January 2008) doi:10.1111/j.1742-4658.2008.06270.x The mitochondrial FAD transporter, Flx1p, is a member of the mitochon- drial carrier family responsible for FAD transport in Saccharomyces cerevi- siae. It has also been suggested that it has a role in maintaining the normal activity of mitochondrial FAD-binding enzymes, including lipoamide dehydrogenase and succinate dehydrogenase flavoprotein subunit Sdh1p. A decrease in the amount of Sdh1p in the flx1D mutant strain has been deter- mined here to be due to a post-transcriptional control that involves regula- tory sequences located upstream of the SDH1 coding sequence. The SDH1 coding sequence and the regulatory sequences located downstream of the SDH1 coding region, as well as protein import and cofactor attachment, seem to be not involved in the decrease in the amount of protein. Abbreviations FCCP, carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone; Flx1p, mitochondrial FAD transporter; HA, hemagglutinin; PGI, phosphoglucoisomerase; RR-MADD, riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency; SCM, Saccharomyces cerevisiae mitochondria; SDH, succinate dehydrogenase; Sdh1p, succinate dehydrogenase flavoprotein subunit; WT, wild-type; a-FAD, polyclonal antibody against FAD covalently bound to protein; a-HA, monoclonal antibody against hemagglutinin epitope; b-Gal, b-galactosidase. FEBS Journal 275 (2008) 1103–1117 ª 2008 The Authors Journal compilation ª 2008 FEBS 1103 flavin content in wild-type (WT) and flx1D mutant yeast strains suggested that the impairment in flavo- enzyme activity was not strictly correlated with flavin cofactor availability, but seemed to be associated with a significant decrease in levels of the SDH flavoprotein subunit (Sdh1p) [13]. These data thus imply a role for Flx1p in the control of Sdh1p levels. Whether this reg- ulation is achieved via modulation of rates of protein expression or degradation is, however, unclear. We have therefore investigated Sdh1p biogenesis by using both epitope tagging and lacZ reporter strate- gies, and have demonstrated that Flx1p controls Sdh1p expression, presumably at the post-transcriptional level. Results FLX1p controls SDH activity by regulating the amount of flavinylated Sdh1p We previously showed that deletion of FLX1, the mitochondrial FAD transporter gene, results in a res- piration-deficient phenotype in which cells are unable to form colonies on glycerol-containing or pyruvate- containing agar, and exhibit reduced growth rates in YEP liquid media with these carbon sources [13]. Polarographic measurements of oxygen consumption induced by addition of succinate to WT and flx1D mutant mitochondria are reported in Fig. 1. WT SCM utilized succinate with a rate equal to 100 ngatoms OÆmin )1 Æmg protein )1 (Fig. 1A). Respiration was com- pletely inhibited by malonate, an inhibitor of SDH [16,17], and this inhibition was reversed by exogenous NADH, with a rate equal to 135 ngatoms OÆ min )1 Æmg protein )1 , but was blocked by the complex III inhibi- tor antimycin A. Succinate respiration in flx1D SCM was reduced by 40% (to 59 ngatoms OÆmin )1 Æmg pro- tein )1 ), but NADH oxidase activity (121 ngatoms OÆmin )1 Æmg protein )1 ) was similar to that in WT SCM (Fig. 1B). As both succinate and NADH oxidation involve common electron carriers downstream of ubi- quinone reduction, the defect in succinate metabolism in the flx1D mutant could be located either in com- plex II (SDH) or in the succinate transporter. To exclude the possibility that succinate transport limits the rate of the overall process of succinate mitochon- Fig. 1. Polarographic measurements of the succinate-dependent oxygen uptake rate in SCM. SCM (0.1 mg) isolated from WT (A) and flx1D (B) cells grown until the stationary phase in YEP liquid medium supplemented with glycerol were incubated in respiration medium as reported in Experimental procedures. The arrows indicate when the additions were made. The numbers along the trace refer to the oxygen uptake rate expressed as ngatoms OÆmin )1 Æmg protein )1 . In the table, the mean (± SD) of the oxygen uptake rates induced by succinate and NADH and the normalization of the succinate versus NADH-dependent oxygen uptake rate, determined in three experiments performed with different mitochondrial preparations, are reported. Statistical evaluation was carried out according to Student’s t-test (*P < 0.05). In (C), 1 min before succinate addition, either phenylsuccinate (s) or malonate (d) were added at the reported concentrations. Regulatory role of Flx1p in SDH1 expression T. A. Giancaspero et al. 1104 FEBS Journal 275 (2008) 1103–1117 ª 2008 The Authors Journal compilation ª 2008 FEBS drial metabolism, we applied control strength analysis, essentially as described in Pastore et al. [18] and refer- ences therein, using the impermeable inhibitor phenyl- succinate (Fig. 1C). Over the concentration range 0.1–0.5 mm, the overall process of succinate respiration was not reduced. By increasing the phenylsuccinate concentration, and therefore reducing the succinate transporter activity, we obtained a significant reduction in succinate respiration. Conversely, the SDH inhibitor malonate reduced the oxygen consumption rate at con- centrations below 0.25 mm. Thus, we conclude that in SCM isolated from glycerol-grown WT cells, the rate- limiting step of respiration was the SDH complex. To prove the specificity of SDH impairment, use was made of glycerol-3-phosphate (5 mm) and d-lac- tate (5 mm), which yield electrons to the respiratory chain via other two flavoenzymes, i.e. glycerol-3-phos- phate–ubiquinone oxidoreductase and d-lactate–cyto- chrome c oxidoreductase, encoded by the genes GUT2 and DLD1, respectively [19,20]. Glycerol-3-phosphate and d-lactate respiration rates measured in WT SCM were found to be equal to 88 ± 24 and 63 ± 10 nga- toms OÆmin )1 Æmg protein )1 . Similar respiration rate values were determined in flx1D SCM (107 ± 40 and 63 ± 20 ngatoms OÆmin )1 Æmg protein )1 , respectively, for glycerol-3-phosphate and d-lactate). We also measured SDH activity directly in both sol- ubilized SCM [13] and cellular extracts, and showed that SDH activity was eight-fold to 10-fold higher in cells grown on glycerol or ethanol than in cells grown on glucose (Fig. 2A). However, no change in the activ- ity of the constitutive enzyme phosphoglucoisomerase (PGI) [21] was observed (Fig. 2A). A statistically sig- nificant reduction of SDH activity was found in the flx1D mutant as compared to the wild-type, ranging from about 30% (P < 0.05) in early exponential phase in ethanol to about 70% (P £ 0.01) in glycerol (Fig. 2A). No change in the enzymatic activities of the mitochondrial flavoenzymes Gut2p and Dld1p was found (data not shown). The lower SDH activity observed in flx1D SCM is hypothesized to be due to decreased levels of the flavo- protein subunit Sdh1p [13]. This was confirmed by probing cellular extracts with an antibody against the flavin moiety of covalently flavinylated proteins (a-FAD). Following western blotting analysis, a band Fig. 2. (A) Succinate dehydrogenase (SDH) activity in cellular extracts. WT (a) and flx1D (b) cells were grown for up to 3 h in YEP liquid medium supplemented with different carbon sources. SDH (black bars) and PGI (white bars) enzymatic activities were mea- sured in cellular extracts as described in Experimental procedures. (B) Level of fla- vinylated Sdh1p. Proteins from WT (a) and flx1D (b) cellular extracts were separated by SDS ⁄ PAGE and transferred onto nitrocellu- lose membrane. Covalently flavinylated Sdh1p (FAD-Sdh1p, black bars) was detected with a-FAD, and its amount was densitometrically evaluated. The a-FAD-reac- tive band migrating at the same molecular mass as the ESI-MS ⁄ MS-identified chaper- one Hsc82p (i.e. 83 kDa) was used as an internal standard (FAD-83p, white bars). The values are the mean (± SD) of four (A) and three (B) experiments performed with different cellular extract preparations. Statis- tical evaluation was carried out according to Student’s t-test (* P < 0.05; ** P £ 0.01). T. A. Giancaspero et al. Regulatory role of Flx1p in SDH1 expression FEBS Journal 275 (2008) 1103–1117 ª 2008 The Authors Journal compilation ª 2008 FEBS 1105 migrating at 69 kDa (theoretical molecular mass 67 kDa) was revealed, corresponding to flavinylated Sdh1p (FAD-Sdh1p); an aspecific a-FAD-crossreactive band (FAD-83p) was observed at 83 kDa, identified by ESI-MS ⁄ MS as the constitutive molecular chaper- one Hsc82p (theoretical molecular mass 80.7 kDa). Densitometric analysis of these a-FAD-crossreactive bands (Fig. 2B) revealed a significant reduction in FAD-Sdh1p that paralleled the reduction in enzymatic activity. No change was observed in the amount of the internal standard FAD-83p. Biogenesis and mitochondrial import of HA-tagged Sdh1p in a WT-HA strain and an flx1D-HA strain The level of the flavinylated Sdh1p in functional com- plex II could potentially be regulated at several points between transcription and cofactor addition inside mitochondria [22–26]. To investigate these processes, we constructed a novel yeast strain, WT-HA, in which Sdh1p was fused to three consecutive copies of an influenza HA epitope (YPYDVPDYA). The HA-tag was inserted at the C-terminal end of Sdh1p, so as not to disrupt the N-terminal mitochondrial targeting sequence. Both the NCBI tool orf finder (http:// www.ncbi.nlm.nih.gov/gorf/gorf.html) and the bestorf gene prediction program from Softberry Inc. (http:// www.softberry.com) predicted a single 680 amino acid translation product from the recombinant SDH1-HA gene sequence. Its theoretical molecular mass is 74.4 kDa. The growth properties on YEP plates of the novel strain are shown in Fig. 3A. WT-HA cells exhib- ited a respiration-deficient phenotype, as they were able to grow well on a fermentable carbon source (glu- cose), more slowly on ethanol, and not at all on A BC WT-HA WT-HA WT-HA Fig. 3. (A) Growth properties of the WT-HA strain and detection of Sdh1-HAp. The 3xHA-loxP-kanMX-loxP cassette (1669 bp) was genomi- cally fused in frame to the 3¢-end of the SDH1 ORF of a WT strain (first line) to obtain a new strain (WT-HA, second line), as described in Experimental procedures. In (A) WT-HA, flx1D-HA, WT and flx1D strains were streaked on YEP solid medium supplemented with different carbon sources. The plates were incubated at 30 °C for up to 2 days. In (B), proteins from cellular extracts (EC), mitochondria (SCM) and postmitochondrial supernatant (SN postSCM) (1.5 lg each) prepared from WT-HA cells grown for up to 3 h in YEP liquid medium supple- mented with glycerol were separated by SDS ⁄ PAGE and transferred onto a poly(vinylidene difluoride) membrane. Sdh1-HA proteins were detected with a-HA. In (C), proteins from SCM and EC and rat liver mitochondria (RLM) (15 lg each) were separated by SDS ⁄ PAGE and transferred onto a nitrocellulose membrane. Covalently flavinylated proteins were detected with a-FAD. Regulatory role of Flx1p in SDH1 expression T. A. Giancaspero et al. 1106 FEBS Journal 275 (2008) 1103–1117 ª 2008 The Authors Journal compilation ª 2008 FEBS glycerol. In YEP liquid medium supplemented with these nonfermentable carbon sources, they exhibited a reduced growth rate (data not shown). In cellular lysates of glucose-grown cells, Sdh1-HAp was detected after SDS ⁄ PAGE as a single band of about 70 kDa, which increased in abundance about 10-fold when glycerol or ethanol was the carbon source (data not shown). Two additional a-HA- reactive bands were detected under these growth conditions, with molecular masses of 74 and 66 kDa (Fig. 3B). The correct delivery of the recombinant protein to mitochondria (Fig. 3B) was indicated by the observa- tion that HA-tagged proteins were fourfold to eight- fold enriched in the mitochondrial fraction as compared to cellular extracts and were absent in postmitochondrial supernatants. As it has been reported that cofactor attachment requires correctly folded Sdh1p [23], it is possible that the C-terminal HA-tag may inhibit flavinylation. The inability of the recombinant protein to constitute a functional SDH complex was indicated by the respira- tion-deficient phenotype of the WT-HA strain (Fig. 3A) and by the lack of enzymatic SDH activity in the cellular extracts of engineered cells (data not shown). Immunoblotting analysis with a-FAD (Fig. 3C) revealed only a faint band at 70 kDa in mitochondria from WT-HA strains, which appeared to migrate a little more slowly than the major band rec- ognized by a-FAD in mitochondria from WT cells and thus may represent a nonspecific reaction. The 70 kDa migrating protein in this position was identified as the mitochondrial heat shock protein Ssc1p (theoretical molecular mass 70.6 kDa) by ESI-MS ⁄ MS. As both the band detected by a-HA (Fig. 3B) and the one rec- ognized by a-FAD in WT cells are four-fold enriched in mitochondria as compared to cellular extracts, the recombinant Sdh1-HAp is probably flavinylated poorly or not at all. Thus, Sdh1-HAp is a useful reagent for the investigation of apoprotein synthesis and import independently of flavin cofactor attachment or avail- ability. Digitonin titration experiments, performed as in Barile et al. [27], proved that the 70 kDa HA-tagged protein was released roughly like cytochrome c oxidase activity, whereas the 66 kDa and 74 kDa proteins followed kynurenine hydroxylase release (data not shown). This suggests that the 70 kDa HA-tagged pro- tein is localized in the inner mitochondrial membrane, whereas the 66 and 74 kDa proteins are localized in the outer membrane. The uncoupler carbonyl cyanide p-(trifluorometh- oxy)-phenylhydrazone (FCCP) collapses the membrane potential generated by the respiratory chain and there- fore inhibits import of proteins into the mitochondrion [28]. WT-HA cells were incubated either in the absence or presence of FCCP (25 lm) for 3, 5 or 24 h, and the HA-tagged proteins were monitored by SDS ⁄ PAGE and immunoblotting. As expected, three a-HA-reactive bands with molecular masses of about 74, 70 and 66 kDa were detected (Fig. 4A). After 3 h of growth, each band represented about 30% of the total Sdh1- HA proteins. In the presence of FCCP (Fig. 4A, lane 2), a 60% reduction of the total amount of Sdh1- HA proteins was observed, the 70 kDa band, which presumably represents the mature Sdh1-HAp, being the most significantly reduced. The relative amount of the 74 kDa band was unaffected by FCCP; it probably represents an extramitochondrial form of precursor Sdh1-HAp. The intensity of the 66 kDa band was not changed by FCCP treatment, and it may be an N-ter- minally cleaved form generated in the outer mitochon- drial compartment. After 5 h of growth, the intensity of the 70 kDa band increased two-fold, and this increase was prevented by FCCP (Fig. 4A, lane 4). After 24 h of growth, the abundance of the 70 kDa form was decreased even in the absence of FCCP (Fig. 4A, lane 5), presumably because of degradation of nonflavinylated protein. The 74 kDa band also decreased, whereas the 66 kDa band remained con- stant. Thus, the 66 kDa cleaved form seems to be more stable than the intramitochondrial mature pro- tein. No a-HA-reactive bands were detectable in cells treated for 24 h with FCCP (Fig. 4A, lane 6). No change in the amount of FAD-83p, used as an internal standard, was found under these experimental condi- tions (Fig. 4A). To determine how Flx1p controls the level of Sdh1p, we used an flx1D-HA yeast strain, which carries both the FLX1 gene deletion and the SDH1-HA gene. These cells were incubated in the absence or presence of FCCP (25 lm) for 3, 5 or 24 h, and the HA-tagged proteins were detected by SDS ⁄ PAGE and immuno- blotting as above (Fig. 4B). In the flx1 D-HA mutant after 3 and 5 h of growth, both the 74 kDa precursor and the 70 kDa mature Sdh1-HA proteins were detect- able, but not the 66 kDa, putative cleaved form (Fig. 4B, lanes 1 and 3). At 24 h, neither a-HA-reac- tive bands nor the internal standard, FAD-83p, were detected, presumably because generalized protein degradation correlated with the flx1D-HA growth defect (Fig. 4B, lane 5). The total amount of Sdh1- HAp was reduced as compared to the WT-HA strain (by 86%, 90%, and 100%, respectively at 3, 5 and 24 h in the experiments reported in Fig. 4). In four replicate experiments using different cellular extracts of T. A. Giancaspero et al. Regulatory role of Flx1p in SDH1 expression FEBS Journal 275 (2008) 1103–1117 ª 2008 The Authors Journal compilation ª 2008 FEBS 1107 glycerol-grown WT-HA and flx1D-HA cells, the total amount of Sdh1-HAp was reduced in 73% and 81% (means), respectively, at the 3 h and 5 h growth points (P £ 0.01; Fig. 4C). Extracts from ethanol-grown cells exhibited a smaller but still significant reduction (45% and 40% at 3 h and 5 h of growth, respectively; P < 0.05; Fig. 4C). The 70 kDa mature Sdh1-HAp form was efficiently generated and was more abundant than the full-length precursor in the flx1D-HA cellular extracts at both 3 h and 5 h. Thus, its abundance seems to be solely limited by the rate of precursor synthesis. On treatment with FCCP, the 74 kDa precursor band was almost the only a-HA-crossreactive band detectable. Its amount was decreased by 78–80% in the flx1D-HA mutant strain as compared to the total amount of protein found in the WT-HA strain (Fig. 4A,B, lanes 2 and 4). These results are consistent with the proposal that Flx1p con- trols Sdh1-HAp expression, rather than import and processing of the precursor protein. Flx1p controls SDH1 expression To substantiate the hypothesis that Flx1p controls SDH1 expression, independently of cofactor attachment, in a new yeast strain, namely WT-lacZ , SDH1 ORF was genomically replaced by the lacZ gene coding for b-galactosidase (b-Gal) of Escherichia coli (gene reporter strategy), as described in Experimental procedures. This transformed strain exhibits the same respiration-deficient phenotype as the WT-HA strain, as it was able to grow as well as the WT cells on glucose, more slowly on ethanol, and not at all on glycerol (Fig. 5A). In YEP liquid medium supplemented with these nonfermentable carbon sources, growth was reduced but not abolished (data not shown). The b-Gal activity was 40 ± 7 lmolÆmin )1 Æmg pro- tein )1 in cellular extracts of glucose-grown WT-lacZ cells up to 5 h. The activity increased about six-fold and nine-fold at the 3 h time point when cells were grown on glycerol and ethanol, respectively, and reached a plateau after 5 h of growth on glycerol, whereas it still increased when ethanol was the carbon source (Fig. 5Ba). As a control, to show that altered SDH1 expression was not a secondary effect of growth rate, the activity of the constitutive enzyme PGI was measured in the same extracts (Fig. 5Bb) and showed no difference between fermentable and nonfermentable carbon sources. We also constructed a double mutant, flx1D-lacZ, containing both the FLX1 gene deletion and the repor- ter gene. This strain exhibited the same respiration- deficient phenotype as the flx1D and the WT-lacZ strains (Fig. 5A). The b-Gal activity in extracts of WT-HA 100 * ** ** * 80 60 40 20 0 FAD-83p Sdh1-HAp FCCP Growth time (h) p m cl Strain A C B Strain Lanes FAD-83p Sdh1-HAp FCCP Growth time (h) Growth time (h) 3 Gl y cerol α-HA-Sdh1-HAp amount (%) Ethanol 5 3 5 p m Lanes 1 – + – + – + 24 5 3 – + – + – + 24 5 3 2 3 4 5 6 1 2 3 4 5 6 Fig. 4. Detection of Sdh1-HAp in cellular extracts from WT-HA and flx1D-HA cells incubated in the absence or presence of the uncoupler FCCP. Glycerol-grown WT-HA (A) and flx1D-HA (B) cells were incubated in the presence (+) or absence ()) of FCCP (25 l M) for 3, 5 and 24 h. Proteins from cellular extracts (10 lg) were separated by SDS ⁄ PAGE and transferred onto a poly(vinylidene difluoride) membrane, and the Sdh1-HA proteins were detected with a-HA. The a-FAD-reactive band (FAD-83p) was used as an internal standard. In (C), the total Sdh1- HAp amount of protein in flx1D-HA cellular extracts is reported as a percentage of that detected in WT-HA cellular extracts. The values are the means (± SD) of four experiments performed with different cellular extract preparations. Statistical evaluation was carried out according to Student’s t-test (* P<0.05; ** P £ 0.01). Regulatory role of Flx1p in SDH1 expression T. A. Giancaspero et al. 1108 FEBS Journal 275 (2008) 1103–1117 ª 2008 The Authors Journal compilation ª 2008 FEBS Growth time (h) 3 5 Glucose Glycerol Ethanol 24 3 5 24 3 5 24 NDNDND ND Growth time (h) 3 0 20 40 60 80 100 120 140 160 0 70 140 210 280 350 0 100 200 300 400 500 800 900 1000 a, WT-lacZ Glucose Glycerol Ethanol b b ** ** * 5 Glycerol PGI (%) PGI (µmol·min –1 ·mg protein –1 ) β-Gal (µmol·min –1 ·mg protein –1 ) 0 20 40 60 80 100 β-Gal (%) Ethanol 24 3 5 24 NDND A B C Fig. 5. (A) Growth properties of WT-lacZ and flx1D-lacZ strains. WT-lacZ, flx1D-lacZ , WT and flx1D strains were streaked on YEP solid medium supplemented with different carbon sources. The plates were incubated at 30 °C for up to 5 days. (B, C) b-Gal and PGI activities in WT-lacZ and flx1D-lacZ strains. b-Gal (a) and PGI (b) activities were measured in WT-lacZ (B) and flx1D-lacZ (C) cellular extracts obtained from cells grown for up to 3, 5 or 24 h in YEP liquid medium supplemented with different carbon sources. In (C), values are reported as percent- age of the activities measured in the WT-lacZ cellular extracts. The values are the means (± SD) of three experiments performed with differ- ent cellular extract preparations. ND, not determined. Statistical evaluation was carried out according to Student’s t-test (*P< 0.05; ** P £ 0.01). T. A. Giancaspero et al. Regulatory role of Flx1p in SDH1 expression FEBS Journal 275 (2008) 1103–1117 ª 2008 The Authors Journal compilation ª 2008 FEBS 1109 glucose-grown flx1D-lacZ and WT-lacZ cells was simi- lar, indicating no significant differences in basal SDH1 expression (data not shown). However when flx1D- lacZ cells were grown on glycerol for 3 or 5 h, SDH1 expression was reduced to 50% (P < 0.01). A 35% reduction (P < 0.05) was observed when cells were grown for up to 3 h in ethanol. Extending the growth time restored b-Gal activity. No significant differences in PGI activity were detected in the same extracts (Fig. 5Cb). To exclude the possibility that the reduction in lacZ expression levels was caused by the absence of func- tionally active Sdh1p, we constructed diploid heterozy- gous SDH1 ⁄ sdh1 strains (dWT-lacZ and dflx1D-lacZ). The dWT-lacZ strain was able to grow on nonferment- able carbon source, as expected for a recessive disrup- tion mutation [29]. The SDH1 expression level, measured as b-Gal activity, was significantly reduced in dflx1D-lacZ as compared to dWT-lacZ cells, the reduction being more severe in glycerol than in ethanol (Fig. 6A). No significant change in PGI activity was detected in these extracts (Fig. 6B). These results are consistent with the control of SDH1 expression by Flx1p via a mechanism that involves regulatory regions located upstream of the SDH1 ORF. To understand how this control is exerted, SDH1 mRNA level was measured by real-time RT-PCR experiments, with ACT1 mRNA being used as an internal control for gene expression. As expected [22,26], the relative amount of SDH1 mRNA was 5.5 times higher in glycerol-grown WT cells than in glucose-grown WT cells (Fig. 7). No change in the rel- ative amount of SDH1 mRNA was found in the flx1D mutant strain in comparison to the WT strain in both the carbon sources used. As changes in Sdh1p amounts were not paralleled by changes in the SDH1 mRNA level, we expected that the 5¢-UTR, defined as in de la Cruz et al. [25], rather than the promoter region is involved in Flx1p–SDH1 crosstalk. Discussion We have investigated the relationship between defects in flavin cofactor homeostasis and the function of mitochondrial FAD-binding enzymes. Correlation of these has been demonstrated in human pathologies, including deficiencies of the flavoprotein subunit of respiratory chain complex II [30] and in RR-MADD [31,32], in which polypeptides involved in fatty acyl- CoA and amino acid metabolism are impaired [3]. The molecular mechanism underlying these defects is unknown, but one possibility is that low levels of intramitochondrial FAD causes accelerated breakdown of FAD-binding enzymes [31,33]. Previously, we pro- posed that riboflavin cofactors may play a direct role in transcriptional or translational regulation in RR- MADD [3]. The hypothesis that riboflavin deficiency alters the affinity of transcription factors for DNA or modulates translational efficiency has also been pro- posed for HepG2 and in Jurkat lymphoid cells [34]. Saccharomyces cerevisiae provides a useful model for the alterations of flavoprotein biochemistry typical of 3 Growth time (h) PGI (%) β-Gal (%) Glycerol Ethanol 5 24 3 5 24 ND ND ND ** 100 80 60 40 20 0 0 40 80 120 160 ** * A B ND Fig. 6. b-Gal and PGI activities in the diploid strains dWT-lacZ and dflx1D-lacZ. b-Gal (A) and PGI (B) activities were measured in cellu- lar extracts obtained from dWT-lacZ and dflx1D-lacZ cells grown for up to 3, 5 or 24 h in YEP liquid medium supplemented with differ- ent carbon sources. The enzymatic activities, measured in dflx1D- lacZ, are reported as percentage of the activities measured in the dWT-lacZ cellular extracts. The values are the means (± SD) of three experiments performed with different cellular extract prepara- tions. ND, not determined. Statistical evaluation was carried out according to Student’s t-test (* P<0.05; ** P £ 0.01). Regulatory role of Flx1p in SDH1 expression T. A. Giancaspero et al. 1110 FEBS Journal 275 (2008) 1103–1117 ª 2008 The Authors Journal compilation ª 2008 FEBS RR-MADD, as the activity of the flavoenzymes lipo- amide dehydrogenase and SDH can be reduced by mutation or deletion of the genes encoding the ribofla- vin membrane transporter (MCH5) [5], FAD synthe- tase (FAD1) [14], and the mitochondrial FAD transporter (FLX1) [13,15,35]. The reduced activity of SDH in FLX1 mutant ⁄ deleted yeast strains was explained by an accelerated breakdown of apoprotein in the absence of mitochon- drial FAD, whose origin is still a matter of debate [15,35]. Previous studies reported that FAD synthetase, Fad1p, was present only in the cytoplasm fraction and not in mitochondria, so it was hypothesized that Flx1p is responsible for FAD import into mitochondria in exchange with FMN [14,15]. We proposed an alterna- tive hypothesis, in which FAD synthetase is present inside mitochondria and Flx1p is involved in FAD export from the organelle [13]. Nevertheless, Flx1p seems not to be required for maintaining cytosolic FAD levels, at least under the experimental conditions used, as the activities of Gut2p and Dld1p (which reside on the outer face of the inner mitochondrial membrane) are unaffected by FLX1 gene deletion. Direct measurements of flavin cofactor levels in sphe- roplasts confirm this conclusion (data not shown). In the present study we have investigated how Flx1p enables mitochondrial succinate respiration and con- trols levels of Sdh1p, using epitope-tagged SDH1. Our data suggest that Sdh1-HAp is correctly imported and processed, but cannot be flavinylated either in the WT- HA strain or in the flx1D-HA strain. These experi- ments also showed that the availability and attachment of flavin cofactors are not involved in the regulation of Sdh1p reduction. Using their differential sensitivity to the uncoupler FCCP, we were able to distinguish pre- cursor and mature forms of Sdh1-HAp. Accumulation of the natural precursor of Sdh1p in the purified outer membrane has been previously reported in a proteomic study, using cells grown on nonfermentable carbon sources [36]. We also postulated that an unexpected N-terminal cleavage product, presumably located in the outer mitochondrial compartments, is generated from a putative misfolded precursor by the mitochon- drial quality control system [37,38]. In the flx1D-HA mutant strain, this cleaved form is not detectable, sug- gesting that import is favored over cleavage. This is consistent with the reduced expression of precursor Sdh1-HAp, which prevents its accumulation in the outer membrane. Reporter gene experiments demonstrated that regu- lation of Sdh1p expression is exerted via the regulatory regions located upstream of the SDH1 ORF, and that regulatory sequences downstream of the SDH1 gene are not strictly required for the regulation of protein expression. Thus, the reduced level of Sdh1p in an flx1D mutant strain is due to decreased precursor Sdh1p expression, rather than to its accelerated break- down. To rationalize the mechanism by which Flx1p modu- lates Sdh1p expression, we can speculate that, in a sort of ‘retrograde’ crosstalk, Flx1p coordinates cofactor status inside mitochondria with apoprotein synthesis occurring outside, presumably on mitochondria-bound polysomes [36]. In this pathway, Flx1p might function either as a ‘nutrient sensor’ [39,40] or as a flavin trans- porter (whatever the flavin transported is, FMN [15] or FAD [13]), triggering a downstream cytosolic sig- naling pathway. The finding that apoprotein expression may be regu- lated by vitamins or vitamin-derived cofactors is not Strain SDH1 mRNA relative amount WT 0 0.2 0.4 0.6 0.8 Glucose WT Glycerol Fig. 7. Relative quantification of SDH1 mRNA level in WT and flx1D cells by real-time RT-PCR. Total RNA extracted from WT and flx1D cells, grown for up to 5 h in YEP liquid medium supple- mented with glucose or glycerol as carbon sources, were reverse- transcribed and used in real-time RT-PCR assays, as described in Experimental procedures. SDH1 mRNA level was normalized to ACT1 mRNA level, used as an internal standard, in order to correct for differences in mRNA quantity between samples. The SDH1 mRNA relative amount values reported are the means (± SD) of four independent real-time RT-PCR reactions performed with two different total RNA preparations. Statistical evaluation was carried out according to Student’s t-test. T. A. Giancaspero et al. Regulatory role of Flx1p in SDH1 expression FEBS Journal 275 (2008) 1103–1117 ª 2008 The Authors Journal compilation ª 2008 FEBS 1111 surprising. This regulation might be exerted at a tran- scriptional level by modulating the activity of specific transcription factors as described for Vhr1p for biotin [41,42], Pdc2p for thiamine diphosphate [43], and Rip140 for pyridoxal 5¢-phosphate [44], or at a post-transcriptional level by stabilizing or melting RNA secondary structure (i.e. via riboswitches or via the internal ribosome entry site) with regulatory conse- quences. This control has been reported for biotin [45] and more recently for vitamin B 12 , which binds specific responsive elements in the 5¢-UTR of methionine syn- thetase mRNA [46]. Sequence analysis of the 5¢-UTR of this mRNA also reveals the presence of two upstream ORFs involved in regulating the translational efficiency of the main ORF [47]. Translational effi- ciency may also be regulated by vitamin ⁄ cofactors via phosphorylation of translation initiation factors, as suggested for riboflavin in riboflavin-deprived cells [34]. Real-time RT-PCR experiments showed no change in SDH1 mRNA level in the flx1 D mutant strain as compared to the WT strain. This suggested that regu- lation of SDH1 expression is exerted post-transcrip- tionally, via a mechanism that involves the 5¢-UTR of SDH1 mRNA. Searching for cis-acting elements in the regulatory region located upstream of the SDH1 ORF with bioinformatic tools [48,49], we found 12 highly conserved motifs (six with an unknown function). None of these were found in the 5¢-UTR, and no upstream ORFs were found using the NCBI tool orf finder. Then, either allosteric rearrangements of the 5¢-UTR upon nutrient ⁄ protein binding or differential phosphorylation of translation initiation factors might be evoked to explain regulation of SDH1 mRNA translation on the outer mitochondrial surface [36]. Owing to the high energy required to synthesize apo- proteins, a translational response to flavin cofactor level would be more ‘economic’ than the degradation of translational products. Such a control might also underlie the riboflavin-dependent restoration of com- plex II deficiencies in humans [30]. Experimental procedures Materials All reagents and enzymes were obtained from Sigma- Aldrich Corp. (St Louis, MO, USA), Fermentas Inc. (Glen Burnie, MD, USA), Carl Roth GmbH+Co.KG (Kar- lsruhe, Germany) and Calbiochem (San Diego, CA, USA). Zymolyase was obtained from ICN Biomedicals (Aurora, OH, USA). Bacto yeast extract and yeast nitrogen base were obtained from Difco (Lawrence, KS, USA), and anti-HA and anti-rat peroxidase conjugated IgG were obtained from Roche (Basel, Switzerland) and Jackson Immunoresearch (West Grove, PA, USA), respectively. Yeast strains The wild-type S. cerevisiae strain (EBY157, WT), derived from the CEN.PK yeast series and the flx1D mutant strain (EBY167A, flx1D), constructed as described in Bafunno et al. [13], were used as recipient strains to obtain the new strains reported in Table 1. Genomic HA-tagging of SDH1 Three consecutive copies of the HA epitope were fused to the 3¢-end of the SDH1 ORF in the genome of both the WT and EBY167-G418 S strains, by using a modification of the PCR targeting technique [50]. EBY167-G418 S was pre- viously obtained by transforming the flx1D mutant strain with the plasmid pSH47 to remove the kanMX marker in the FLX1 locus, according to Gu ¨ ldener et al. [51]. Plasmid pUG6-HA was used as a template to generate by PCR a Table 1. Genotypes of S. cerevisiae strains used in this study. Strain Genotype Haploid EBY157 (WT) ura3-52 MAL2-8 c SUC2 FLX1 SDH1 EBY167 (flx1D) ura3-52 MAL2-8 c SUC flx1::loxP-kanMX-loxP SDH1 EBY157-SDH1-HA (WT-HA) MATa ura3-52 MAL2-8 c SUC2 FLX1 SDH1-3xHA- loxP-kanMX-loxP EBY167-G418 S -SDH1-HA (flx1D-HA) MATa ura3-52 MAL2-8 c SUC2 flx1D SDH1-3xHA-loxP-kanMX-loxP EBY157-sdh1D (WT-lacZ) MATa ura3-52 MAL2-8 c SUC2 FLX1 sdh1::lacZ-loxP-kanMX-loxP EBY167-sdh1D (flx1D-lacZ ) MATa ura3-52 MAL2-8 c SUC2 flx1D sdh1::lacZ-loxP-kanMX-loxP Diploid dEBY157-sdh1D (dWT-lacZ ) MATa ⁄ a ura3-52 ⁄ ura3-52 MAL2-8 c ⁄ MAL2-8 c SUC2 ⁄ SUC2 + YCplac33URA3 FLX1 ⁄ FLX1 SDH1 ⁄ sdh1::lacZ-loxP-kanMX-loxP dEBY167-sdh1D (dflx1D-lacZ ) MATa ⁄ a ura3-52 ⁄ ura3-52 MAL2-8 c ⁄ MAL2-8 c SUC2 ⁄ SUC2 + YCplac33URA3 flx1::loxP-kanMX loxP ⁄ flx1::loxP-kanMX-loxP SDH1 ⁄ sdh1::lacZ-loxP-kanMX-loxP Regulatory role of Flx1p in SDH1 expression T. A. Giancaspero et al. 1112 FEBS Journal 275 (2008) 1103–1117 ª 2008 The Authors Journal compilation ª 2008 FEBS [...]... Tzagoloff A (1995) Cloning and characterization of FAD1 , the structural gene for flavin adenine dinucleotide synthetase of Saccharomyces cerevisiae Mol Cell Biol 15, 26 4–2 71 15 Tzagoloff A, Jang J, Glerum M & Wu M (1996) FLX1 codes for a carrier protein involved in maintaining a proper balance of flavin nucleotides in yeast mitochondria J Biol Chem 271, 739 2–7 397 16 Dibrov E, Fu S & Lemire BD (1998) The Saccharomyces. .. dinucleotide synthesis in isolated rat liver mitochondria caused by imported flavin mononucleotide Arch Biochem Biophys 305, 44 2–4 47 13 Bafunno V, Giancaspero TA, Brizio C, Bufano D, Passarella S, Boles E & Barile M (2004) Riboflavin uptake and FAD synthesis in Saccharomyces cerevisiae mitochondria: involvement of the Flx1p carrier in FAD export J Biol Chem 279, 9 2–1 02 Regulatory role of Flx1p in SDH1 expression. .. mRNAs encoding succinate dehydrogenase peptides in Saccharomyces cerevisiae: sequence elements in the 5¢untranslated region of the Ip mRNA play a dominant role Mol Biol Cell 6, 112 5–1 143 23 Robinson KM & Lemire BD (1996) Covalent attachment of FAD to the yeast succinate dehydrogenase flavoprotein requires import into mitochondria, presequence removal, and folding J Biol Chem 271, 405 5–4 060 24 Lemire BD &... of S cerevisiae mating type by PCR Trends Genet 6, 236 FEBS Journal 275 (2008) 110 3–1 117 ª 2008 The Authors Journal compilation ª 2008 FEBS T A Giancaspero et al 54 Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72, 24 8–2 54 55 Laemmli UK (1970) Cleavage of structural proteins during the. .. processing, and post-processing stability of various mitochondrial acyl-CoA dehydrogenases and of electron transfer flavoprotein and the site of holoenzyme formation J Biol Chem 267, 1792 5–1 7932 34 Manthey KC, Rodriguez-Melendez R, Hoi JT & Zempleni J (2006) Riboflavin deficiency causes protein and DNA damage in HepG2 cells, triggering arrest in G1 phase of the cell cycle J Nutr Biochem 17, 25 0–2 56 35... species inhibit the succinate oxidation-supported generation of membrane potential in wheat mitochondria FEBS Lett 516, 1 5–1 9 19 Larsson C, Pahlman IL, Ansell R, Rigoulet M, Adler L & Gustafsson L (1998) The importance of the glycerol 3-phosphate shuttle during aerobic growth of Saccharomyces cerevisiae Yeast 14, 34 7–3 57 20 Lodi T, Alberti A, Guiard B & Ferrero I (1999) Regulation of the Saccharomyces cerevisiae. .. on the cellular uptake and trafficking of riboflavin Adv Drug Deliv Rev 55, 146 7–1 483 5 Reihl P & Stolz J (2005) The monocarboxylate transporter homolog Mch5p catalyzes riboflavin (vitamin B2) uptake in Saccharomyces cerevisiae J Biol Chem 280, 3980 9–3 9817 6 Perl M, Kearney EB & Singer TP (1976) Transport of riboflavin into yeast cells J Biol Chem 25, 322 1–3 228 7 Koser SA (1968) Vitamin Requirements of. .. CTAGTGGATC-3¢ The WT strain was transformed according to Gietz & Woods [52] PCR analysis and on-plate b-Gal assays confirmed replacements of the reporter cassette in the G418-resistant transformant EBY157-sdh1::lacZ-loxP-kanMX-loxP (WTlacZ) strain Construction of SDH1 promoter–lacZ fusion in the flx1D mutant strain To isolate an flx1D mutant strain in which the regulatory region placed upstream of the SDH1 coding... budding yeast, regulates biotin-dependent expression of VHT1 and BIO5 J Biol Chem 281, 1351 3–1 3524 Mojzita D & Hohmann S (2006) Pdc2 coordinates expression of the THI regulon in the yeast Saccharomyces cerevisiae Mol Genet Genomics 276, 14 7–1 61 Huq MD, Tsai NP, Lin YP, Higgins L & Wei LN (2007) Vitamin B6 conjugation to nuclear corepressor RIP140 and its role in gene regulation Nat Chem Biol 3, 16 1–1 65... remove cell debris The protein concentration of the extracts was assayed according to Bradford [54] Western blotting of Sdh1p Proteins from cellular extracts were separated by SDS ⁄ PAGE [55] and transferred as in Bafunno et al [13] The immobilized proteins were incubated with a 2000-fold dilution of either a polyclonal antibody against FAD covalently bound to proteins (i.e a -FAD, a kind gift from R Brandsch, . Succinate dehydrogenase flavoprotein subunit expression in Saccharomyces cerevisiae – involvement of the mitochondrial FAD transporter, Flx1p Teresa. role in maintaining the normal activity of mitochondrial FAD- binding enzymes, including lipoamide dehydrogenase and succinate dehydrogenase flavoprotein subunit