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:
(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
DNA molecule consisting of a 3xHA-loxP-kanMX-loxP
cassette (conferring G418 resistance and transcriptional ter-
mination due to the TEF terminator [51]) flanked by short
homology regions to the end of the SDH1 locus. For this
purpose, two primers were constructed: S1-SDH1-HA
(5¢-TTTGGACGAA AAGGAATGTC CTTCCGTACC
TCCAACTGTA AGAGCCTACG CCGGTGCTGG ATC
CGGT-3¢); and S2-SDH1-HA (5¢-TGCAATTAAA GAAG
AGTATG ATATTCTTTT CCGTAAAATA CAATGAG-
GTT CAAAGCATAG GCCACTAGTG GATCTG-3¢). The
WT and EBY167-G418
S
strains were transformed using the
LiAc ⁄ SS Carrier DNA ⁄ PEG method as described in Gietz
& Woods [52]. PCR analysis confirmed replacement of the
stop codon of the SDH1 ORF by the 3xHA-loxP-kanMX-
loxP cassette in both the G418-resistant transformants,
EBY157-SDH1-HA (WT-HA)andEBY167-G418
S
-SDH1-
HA(flx1D-HA).
Construction of SDH1 promoter–lacZ fusions
in the WT strain

Genomic replacement of the SDH1 ORF by a PCR-ampli-
fied lacZ-loxP-kanMX-loxP reporter cassette was used to
fuse the promoter and the first 48 nucleotides of the SDH1
ORF to the E. coli lacZ gene, by using a modification of
the PCR targeting technique [51]. Plasmid pUG6lacZ was
used as a template to generate by PCR the DNA molecule
consisting of a lacZ-loxP-kanMX-loxP reporter cassette
flanked by short homology regions to the SDH1 locus [50].
For this purpose, appropriate oligonucleotides were con-
structed: S1-SDH1, 5¢-ATGCTATCGC TAAAAAAATC
AGCGCTCTCC AAGTTGACTT TGCTCAGATT CGT
ACGCTGC AGGTCGAC-3¢; and S2-SDH1, 5¢-TTAGT
AGGCT CTTACAGTTG GAGGTACGGA AGGACAT
TCC TTTTCGTCCA GCATAGGCCA 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 (WT-
lacZ) 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 region was
fused in-frame to the lacZ gene, an approach based on mei-
otic analysis was used. Haploid strains of opposite mating
types, WT-lacZ (MATa) and flx1D (MATa), were mixed
and incubated at 30 °C, and diploid cells were selected on
YEP-glucose plates. A colony from the selected diploids
was streaked on a sporulation plate (2% potassium acetate,
pH 6, 2% agar). After incubation for 3–4 days at 30 °C,

sporulated cells were suspended in zymolyase (1.5 mgÆmL
)1
in 0.5 m sorbitol) and incubated at room temperature for
few minutes, and the resulting tetrads were dissected using
a micromanipulator (Singer MSM System, series 200) and
incubated at 30 °C. Tetrad analysis was carried out by
replica plating the master plate containing the colonies
derived from ascospores, on different selective plates to test
the G418 resistance and b-Gal activity. PCR analysis was
also performed to identify the double mutant strain
EBY167-sdh1::lacZ-loxP-KanMX-loxP (flx1D-lacZ).
Construction of diploid strains
To obtain the diploid strains dEBY157-sdh1::lacZ-loxP-
kanMX-loxP (dWT-lacZ) and dEBY167-sdh1::lacZ-loxP-
kanMX-loxP (dflx1D-lacZ), the WT and flx1D-lacZ haploid
strains were transformed with the plasmid YCplac33 (con-
ferring ability to grow in a medium without uracil) and
crossed with a strain of opposite mating type. Specifically,
the transformed WT haploid strain (MATa) was crossed
with WT-lacZ, and the diploid strain, dWT-lacZ, was
selected on synthetic minimal solid medium (1.7 gÆ L
)1
yeast
nitrogen base, 5 gÆL
)1
ammonium sulfate, 18 gÆL
)1
agar)
supplemented with amino acid solution without uracil,
G418, and 2% glucose as carbon source. The transformed

haploid strain flx1D-lacZ (MATa) was crossed with flx1D
(MATa), and the diploid strains, dflx1D-lacZ, were selected
on slid medium supplemented with amino acid solution
without uracil and with 2% ethanol as carbon source. The
positively identified diploids were analyzed by PCR analysis
as previously described [53].
Media and growth conditions
Cells were grown aerobically at 30 °C with constant shak-
ing in rich liquid medium (YEP, 10 gÆL
)1
yeast extract,
20 gÆL
)1
Bacto peptone) supplemented with glucose, etha-
nol or glycerol (2% each) as carbon sources. The solid
YEP medium contained 18 gÆ L
)1
agar.
Isolation of mitochondria and oxygen uptake
measurements
Mitochondria were isolated from spheroplasts, and the
mitochondrial protein was determined as previously
described [13]. Oxygen uptake was measured at 25 °C with
a Gilson Oxygraph in a respiration medium (containing
0.6 m mannitol, 20 mm Hepes ⁄ Tris, pH 7.4, 10 mm potas-
sium phosphate, 2 mm MgCl
2
,1mm EDTA, 5 mgÆmL
)1
BSA), essentially as described previously [10,13].

Preparation of cellular extracts
Early or midexponential-phase (3 or 5 h of growth) or sta-
tionary-phase (24 h) cells were harvested by centrifugation
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 1113
(8000 g for 5 min) and washed with sterile water, resus-
pended in 250 lL of lysis buffer (10 mm Tris ⁄ HCl,
pH 7.6, 1 mm EDTA, 1 mm dithiothreitol, 0.2 mm phenyl-
methanesulfonyl fluoride, 1 · Roche protease inhibitor
cocktail), and vortexed with glass beads for 10 min at
4 °C. The liquid was removed and centrifuged at 3000 g
for 5 min to remove cell debris. The protein concentra-
tion 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 cova-
lently bound to proteins (i.e. a-FAD, a kind gift from R.
Brandsch, Freiburg, Germany) [13] or a specific monoclonal
antibody against the HA epitope (a-HA; Roche). a-FAD-
immunoreactive materials were visualized with the aid of a
secondary alkaline phosphatase conjugated anti-rabbit IgG,
whereas a-HA-immunoreactive materials were visualized
with the aid of a secondary peroxidase anti-rat IgG using a
chemiluminescence detection system (Pierce, Rockford, IL,
USA). Quantitative evaluations were carried by densitomet-
ric analysis using imagequant 5.2 software (Molecular
Dynamics, Sunnyvale, CA, USA). Some a-FAD-reactive

bands were excised from SDS ⁄ polyacrylamide gel, trypsin-
digested, and analyzed by ESI-MS ⁄ MS, as described in
Brizio et al. [56].
b-Gal assays
Quantitative determination of b-Gal activity in cellular
extracts done spectrophotometrically by measuring the pro-
duction of O-nitrophenolate at 420 nm, starting from O-ni-
trophenylgalactoside, as in Makuc et al. [57]. An on-plate
b-Gal assay was performed by treating whole cells with a
specific chromogenic substrate, 5-bromo-4-chloro-3-indolyl-
beta-d-galactopyranoside, and checking blue colonies, as in
Horwitz et al. [58].
Other enzymatic assays
Succinate dehydrogenase activity was measured as in Baf-
unno et al. [13]. PGI activity was spectrophotometrically
assayed by monitoring the absorbance at 340 nm due to
NADP
+
reduction in a coupled enzyme system with fruc-
tose-6-phosphate as substrate and glucose-6-phosphate
dehydrogenase as the indicator enzyme, as described in
Brocklehurst et al. [59]. d-Lactate dehydrogenase (Dld1p)
and glycerol-3-phosphate dehydrogenase (Gut2p) activities
were spectrophotometrically assayed at 600 nm, essentially
as in Chelstowska et al . [60].
Real-time RT-PCR assay
The RNeasy Midi Kit (Qiagen, Valencia, CA, USA) was
used to extract total RNA from WT and flx1D cells grown
for up to 5 h in YEP liquid medium supplemented with
either glucose or glycerol (2% each). Genomic DNA con-

tamination was eliminated by using an RNase-Free DNase
Kit (Qiagen). The total RNA amount was quantified by
monitoring absorbance at 260 nm, and RNA integrity was
electrophoretically verified by formaldehyde agarose gel
and by an absorption ratio (A
260 nm
⁄ A
280 nm
) > 1.95. Total
RNA (200 ng) was reverse-transcribed using the Enhanced
AMV Reverse Transcriptase Kit (Sigma) with 3.5 lm
anchored oligo(dT)
23
primer, according to the manufac-
turer’s instructions. Real-time PCR was performed using
SYBR Green PCR MasterMix (Applied Biosystems, Foster
City, CA, USA) and the ABI PRISM 7000 Sequence
Detection System (Applied Biosystems), essentially as
described in Roberti et al. [61]. The following primers,
designed with primer express 2.0 software (Applied Bio-
system), were used: SDH1.for (forward primer, 5¢-
GCCAATTCCT TGTTGGATCT TG-3¢) and SDH1.rev
(reverse primer, 5¢-TGGCAACCCA GGCTGTAAAG-3¢)
for detecting SDH1 expression; ACT1.for (forward primer,
5¢-TTCCATCCAA GCCGTTTTGT-3¢) and ACT1.rev
(reverse primer, 5¢-GGCGTGAGGT AGAGAGAAAC CA-
3¢) for detecting ACT1 expression. The resulting PCR
products were about 81 and 121 bp, respectively, for
SDH1 and ACT1. In each sample, the relative amount of
SDH1 mRNA was determined by normalizing SDH1

mRNA level to ACT1, used as an internal standard.
Acknowledgements
This work was supported by grants from MIUR
(FIRB 2003 project RBNE03B8KK: ‘Molecular recog-
nition in protein–ligand, protein–protein and protein–
surface interaction: development of integrated experi-
mental and computational approaches to the study of
systems of pharmaceutical interest’ to M. Barile) and
from Universita
`
degli Studi di Bari (Fondi di Ateneo
per la ricerca to M. Barile). T. A. Giancaspero was sup-
ported as a visiting scientist at the Institut fu
¨
r Moleku-
lare Biowissenschaften (J. W. Goethe-Universita
¨
t,
Frankfurt am Main, Germany) by a fellowship financed
by Universita
`
degli Studi di Bari (Bari, Italy) and by a
postgraduate fellowship (Assegno di Ricerca) financed
by FIRB 2003 project RBNE03B8KK. The authors
thank Professor P. Cantatore, Professor M. Roberti
and Dr C. Brizio (Universita
`
degli Studi di Bari, Bari,
Italy) for their critical reading of the manuscript, and
Dr F. Bruni (Universita

`
degli Studi di Bari, Bari,
Italy) for his valuable help with real-time RT-PCR
Regulatory role of Flx1p in SDH1 expression T. A. Giancaspero et al.
1114 FEBS Journal 275 (2008) 1103–1117 ª 2008 The Authors Journal compilation ª 2008 FEBS
experiments. The technical assistance of Mr V. Gian-
noccaro (Universita
`
degli Studi di Bari, Bari, Italy) and
Mrs D. Schnella (J. W. Goethe-Universita
¨
t, Frankfurt
am Main, Germany) is gratefully acknowledged.
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