University of Nebraska - Lincoln
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Biochemistry -- Faculty Publications Biochemistry, Department of
2009
SDH5, a Gene Required for Flavination of
Succinate Dehydrogenase, Is Mutated in
Paraganglioma
Huai-Xiang Hao
University of Utah School of Medicine
Oleh Khalimonchuk
University of Nebraska-Lincoln, okhalimonchuk2@unl.edu
Margit Schraders
Radboud University Nijmegen Medical Centre
Noah Dephoure
Harvard University Medical School
Jean-Pierre Bayley
Leiden University Medical Centre
See next page for additional authors
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Hao, Huai-Xiang; Khalimonchuk, Oleh; Schraders, Margit; Dephoure, Noah; Bayley, Jean-Pierre; Kunst, Henricus; Devilee, Peter;
Cremers, Cor W.R.J.; Schi<man, Joshua D.; Bentz, Brandon G.; Gygi, Steven P.; Winge, Dennis R.; Kremer, Hannie; and Ru>er, Jared,
"SDH5, a Gene Required for Flavination of Succinate Dehydrogenase, Is Mutated in Paraganglioma" (2009). Biochemistry -- Faculty
Publications. 298.
h>p://digitalcommons.unl.edu/biochemfacpub/298
Authors
Huai-Xiang Hao, Oleh Khalimonchuk, Margit Schraders, Noah Dephoure, Jean-Pierre Bayley, Henricus
Kunst, Peter Devilee, Cor W.R.J. Cremers, Joshua D. Schi<man, Brandon G. Bentz, Steven P. Gygi, Dennis R.
Winge, Hannie Kremer, and Jared Ru>er
=is article is available at DigitalCommons@University of Nebraska - Lincoln: h>p://digitalcommons.unl.edu/biochemfacpub/298
SDH5, a Gene Required for Flavination
of Succinate Dehydrogenase,
Is Mutated in Paraganglioma
Huai-Xiang Hao,
1
Oleh Khalimonchuk,
1,2
Margit Schrader s,
5,6
Noah Dephoure,
7
Jean-Pierre Bayley,
8
Henricus Kunst,
5
Peter Devilee,
8,9
CorW.R.J.Cremers,
5
Joshua D. Schiffman,
3
Brandon G. Bentz,
4
Steven P. Gygi,
7
Dennis R. W inge,
1,2
Hannie Kremer,
5,6
Jared Rutter
1
*
Mammalian mitochondria contain about 1100 proteins, nearly 300 of which are uncharacterized. Given
the well-established role of mitochondrial defects in human disease, functional characterization of these
proteins may shed new light on disease mechanisms. Starting with yeast as a model system, we
investigated an uncharacterized but highly conserved mitochondrial protein (named here Sdh5). Both
yeast and human Sdh5 interact with the catalytic subunit of the succinate dehydrogenase (SDH) complex,
a component of both the electron transport chain and the tricarboxylic acid cycle. Sdh5 is required for
SDH-dependent respiration and for Sdh1 flavination (incorporation of the flavin adenine dinucleotide
cofactor). Germline loss-of-function mutations in the human SDH5 gene, located on chromosome
11q13.1, segregate with disease in a family with hereditary paraganglioma, a neuroendocrine tumor
previously linked to mutations in genes encoding SDH subunits. Thus, a mitochondrial proteomics
analysis in yeast has led to the discovery of a human tumor susceptibility gene.
M
itochondrial defects have been impli-
cated in a variety of human disorders,
including cancer (1, 2). Nearly one-
third of the mammalian mitochondrial proteome
is currently uncharacterized. Many of these un-
characterized proteins are evolutionarily conserved,
a strong indication that they perform fundamentally
important cellular functions (3). We initiated a
project to determine the function of one of these
proteins, named here Sdh5, using yeast as the
primary model system. The Sdh5 protein family
is highly conserved in eukaryotes and in some
prokaryotic species, including Rickettsia, which
is related to the bacterium that became the an-
cestral mitochondrion (fig. S1) (4, 5).
The mitochondrial localization of yeast Sdh5
(originally named EMI5/YOL071W) was suggested
by pro teomics studies (6), and we confirmed this
by fluorescence microscopy of a strain expressing
Sdh5–green fluorescent protein and subcellular frac-
tionation of a strain expressing Sdh5-GFP (green
fluorescent protein) (figs. S2 and S3). Both forms of
tagged Sdh5 were expressed from the native SDH5
promoter and were fully functional. We further
showed that Sdh5 resides in the mitochondrial matrix
(Fig. 1A) and is predominantly soluble (Fig. 1B).
Respiratory-deficient mutants of S. cerevisiae
are viable on fermentable carbon sources such as
glucose, but are inviable on nonfermentable carbon
sources such as glycerol. W e found that a yeast strain
with a deletion of SDH5 (sdh 5D) grew normally on
glucose medium but failed to grow on glycerol me-
dium, a phenotype that was rescued by an SDH5-
expressing plasmid (Fig. 1C). The sdh5D strain also
showed impaired oxygen consumption, similar to
the respiratory-deficient sdh1D strain (7) (Fig. 1D),
as well as the respiration-related phenotypes of
H
2
O
2
hypersensitivity and decreased chronological
life-span (figs. S4 and S5). The sdh5D respiratory
deficiency was not due to defective mitochondr ial
DNA (mtDNA), because the sdh5D strain com-
plemented the glycerol growth defect of a rho
0
(mtDNA-deficient) strain in mating tests (fig. S6).
Thus, the sdh5D strain is respiratory-deficient, de-
spite having a functional mitochondrial genome.
Silver staining of the final eluates from tan-
dem affinity purification of Sdh5-His
6
/HA revealed
two proteins specific for tagged Sdh5 as compared
to the wild type, including Sdh5 itself at ~22 kD
(Fig. 1E). The second migrated at ~70 kD and
was identified by mass spectrometry as Sdh1, the
catalytic subunit of th e succinate dehydrogenase
(SDH) complex. The presence of both Sdh5 and
Sdh1 in the final eluate was confirmed by im-
munoblot (Fig. 1E). The SDH complex is a com-
ponent of both the tricarboxylic acid (TCA) cycle
and the electron transport chain (ETC). In the
latter , the SDH complex is known as complex II.
It is a highly conserved heterotetramer, with Sdh1
1
Department of Biochemistry, University of Utah School of
Medicine, Salt Lake City, UT 84112, USA.
2
Department of
Internal Medicine, University of Utah School of Medicine,
Salt Lake City, UT 84112, USA.
3
Department of Oncological
Sciences and Huntsman Cancer Institute, University of Utah
School of Medicine, Salt Lake City, UT, 84112, USA.
4
Depart-
ment of Surgery, Division of Otolaryngology–Head and Neck
Surgery, University of Utah School of Medicine, Salt Lake City,
UT 84112, USA.
5
Department of Otorhinolaryngology, Donders
Centre for Brain, Cognition and Behaviour, Radboud University
Nijmegen Medical Centre, Nijmegen 6500 HB, Netherlands.
6
Centre for Molecular Life Sciences, Radboud University Nijmegen
Medical Centre, Nijmegen 6500 HB, Netherlands.
7
Department of
Cell Biology, Harvard University Medical School, 240 Longwood
Avenue, Boston, MA 02115, USA.
8
Department of Human Ge-
netics, Leiden University Medical Centre, Leiden 2333 ZA, Neth-
erlands.
9
Department of Pathology, Leiden University Medical
Centre, Leiden 2333 ZA, Netherlands.
*To whom correspondence should be addressed. E-mail:
rutter@biochem.utah.edu
Fig. 1. Sdh5 is required for
respiration and interacts with
Sdh1. (A) Mitochondria, mito-
plasts generated by hypotonic
swelling, and 1% Triton X-
100–solubilized mitochondria
from a strain expressing Sdh5-HA
were treated with (+) or without
(–) proteinase K and analyzed by
immunoblotting with an untreated
mitochondria control (UT). Mge1,
Tim10, and Fzo1 are matrix,
intermembrane space, and oute r
membrane proteins, respectively.
(B) Soluble and membrane frac-
tions of purified mitochondria (4)
as in (A) were immunoblotted.
Aco1, soluble matrix protein;
Sdh1, membrane-associated ma-
trix protein. (C) Serial dilutions of
WT and sdh5D strains containing
empty vector (EV) or a pl a s m i d
expressing Sdh5-HA were spotted
on glucose or glycerol medium
and grown at 30°C for 2 or 3 days,
respectively. (D)Oxygenconsump-
tion in WT, sdh5D,andsdh1D
strains grown to mid-log phase
in raffinose media (TSD, n =3
biological replicates). Similar results
were obtained in glucose medium.
(E) Tandem purification eluates (4)
from a WT strain and a strain
expressing Sdh5-His
6
/HA were resolved with SDS-PAGE and visualized by silver staining (top panel) or
immunoblot with antibodies to Sdh1 and HA (lower panels). (F) Immunoblot of purified mitochondria from
WT, sdh1D,orsdh2D strains expressing Sdh5-HA. Porin, mitochondrial loading control.
A
B
α-HA
(Sdh5)
α-Aco1
α-Sdh1
Soluble Membrane
C
α-Mge1
α-Tim10
α-Fzo1
α-HA
(Sdh5)
1 2 3 4 5 6 7
Treatment: Mitochondria Mitoplast 1% Triton UT
Proteinase K: - + - + - + -
WT+EV
sdh5
∆
+EV
glucose glycerol
sdh5
∆
+p
SDH5
α-Sdh1
α-HA
(Sdh5)
WT
sdh1
∆
sdh2
∆
α-Porin
α-Sdh2
Sdh1
75kD
50kD
37kD
25kD
15kD
20kD
Sdh5-His
6
/HA
α-
HA
(Sdh5)
α-
Sdh1
Sdh5: WT His
6
/HA
immunoblot silver staining
D
E
F
0
20
40
60
80
100
120
WT
sdh5
∆
sdh1
∆
Oxygen Consumpotion (% of WT)
www.sciencemag.org SCIENCE VOL 325 28 AUGUST 2009 1139
REPORTS
Copyright 2009 AAAS
and Sdh2 as catalytic subunits anchored to the
mitochondrial inner membrane by Sdh3 and Sdh4
(fig. S12) (8). Both sdh1D and sdh5D mutants
were respiratory-deficient and failed to grow on
glycerol medium, but grew weakly with eth anol
as the carbon source (Fig. 1D and fig. S7) and ex-
hibited ac etate hyperexcretio n, a phenotype shared
by only four other TCA cycle mutants (9). The
importance of the Sdh1-Sdh5 interaction was con-
firmed by the observation that Sdh5 (like Sdh2) is
completely degraded in the sdh1D strain (Fig. 1F).
In contrast, loss of SDH2 ledtoanincreaseinthe
Sdh5 protein level (Fig. 1F), presumably due to
enhanced Sdh1/Sdh5 complex formation in the
absence of Sdh2, the major Sdh1 partner.
The Sdh1-Sdh5 interaction is likely to be
functionally important because the sdh5D mutant
lacks SDH activity (Fig. 2A), as previously
observed for the sdh1D mutant (10). The activity
of malate dehydrogenase, another TCA cycle en-
zyme, was not affected by SDH5 deletion (Fig.
2A). Because SDH is complex II in the ETC, we
performed in-gel activity staining of ETC complexes
after separation by blue native–polyacrylamide
gel electrophoresis (BN-PAGE). As shown in Fig.
2B, complex II/SDH activity was absent in the
sdh5D mutant, whereas the activities of complexes
IV and V were normal. We then examined ETC
complex assembly and stability by Coomassie blue
staining after BN-PAGE and found that complex
II/SDH was specifically absent in sdh5D mitochon-
dria (Fig. 2C). The loss of complex II/SDH in the
sdh5D mutant was confirmed by anti-Myc immu-
noblot after BN-PAGE of wild-type (WT) and
sdh5D strains expressing Sdh3-Myc (Fig. 2D).
Sdh5 is not a stable component of complex II/SDH
because it migrates in a ~90-kD SDS-sensitive com-
plex durin g BN-PAGE (Fig. 2E), which is distinct
from complex II (~200 kD). This ~90-kD complex
is likely to be the Sdh5-Sdh1 (70-kD) heterodimer .
The levels of all four SDH subunits were sig-
nificantly decreased in the sdh5D mutant, probably
because of degradation in the absence of a stable
SDH complex (Fig. 2F, lane 1 versus lane 2). The
residual Sdh1 level was higher than that of the
other subunits, but much of it was i n the soluble
fraction, unassociated with the SDH complex (Fig.
2F). These data suggest that the SDH complex as-
sembles in the absence of Sdh5, but the complex is
nonfunctional and Sdh1 is not stably bound. As a
result, the unstable complex is more susceptible to
degradation and is disrupted by detergent extrac-
tion during the BN-PAGE procedure (the fraction-
ation shown in F ig. 2F was detergent-free).
Multiple cofactors are required for activity of the
SDH complex, including the flavin adenine dinucle-
otide (F A D) in Sdh1 (11). In other ETC complexes,
cofactors are important for complex assembly and
stability in addition to enzymatic activity (12). The
strongly fluorescent FAD is covalently attached to
Sdh1 and can be detected by in-gel fluorometry after
SDS-PAGE (13). Deletion of SDH5 caused a com-
plete loss of F AD cofactor attachment (flavination)
A
WT
sdh5
∆
Complex V
Complex IV
Complex III
Complex II
[kD]
669
67
140
232
440
Coomassie Stain
Malate Dehydrogenase Activity (% of WT)
0%
20%
40%
60%
80%
100%
120%
WT
sdh5
∆
WT
sdh5
∆
In-gel activity
Complex II
Complex V
V
2
V
Complex IV
F
Lane: 1 2 3 4 5 6
Fraction: total total sol mem sol mem
Strain: WT
sdh5
∆
WT
sdh5
∆
75%
40%
23%
47%
97%
total
sdh5
∆
/WT
α-
Sdh1
α-
Sdh2
α-
Myc
(Sdh3)
α-
Aco1
α-
Myc
(Sdh4)
Succinate Dehydrogenase Activity (% of WT)
0%
20%
40%
60%
80%
100%
120%
WT
sdh5
∆
[kD]
669
67
140
232
440
Sdh5-HA: + -
Sdh3-Myc: + +
Complex II
α-Myc
440
440
67
140
232
[kD]
Sdh5: WT TAP TAP
SDS: - - +
α-
TA P
α-
Porin
Sdh5
Complex
Porin
Complex
C
E
D
B
Fig. 2. Sdh5 is required for SDH activity and stability. (A) SDH and malate dehydrogenase
activity (4)fromWTandsdh5D mitochondria, normalized to total protein (TSD, n =3
biological replicates). (B) In-gel activity assay of ETC complexes after BN-PAGE of mito-
chondrial membranes from WT and sdh5D strains. V
2
, complex V dimer. (C) Coomassie-
stained BN-PAGE of mitochondrial membranes from WT and sdh5D strains. (D)Immunoblot
of BN-PAGE–separated complex II/SDH using an antibody to Myc to show Myc-tagged Sdh3
in WT and sdh5D mitochondria. (E)ImmunoblotofBN-PAGE–separated WT and Sdh5-TAP
mitochondria using an antibody to TAP, without and with 1% SDS pretreatment. Porin is
an ~440-kD control. (F) Immunoblot of mitochondria from WT and sdh5D strains in
which Sdh3 or Sdh4 was Myc-tagged, separated into soluble (sol) and membrane (mem)
fractions (4), or unfractionated (total). Aco1, soluble matrix protein. The indicated per-
centage is the amount remaining in sdh5D mitochondria relative to WT mitochondria.
Fig. 3. Sdh5 is neces-
sary and sufficient for
Sdh1 flavination. (A)
WT, sdh5D,andsdh1D
mitochondria were re-
solved by SDS-PAGE
and imaged (4)forco-
valent FAD (top panel)
or immunoblotted (low-
er panels). (B)Fluores-
cencegelimage(top
panel) and immuno-
blot (lower panels) as in
(A), with whole-cell ex-
tract from WT or flx1D
sdh5D strains contain-
ing EV, CEN plasmid
SDH5 (flx1D:~1copy
percell),or2m plasmid
SDH5 (O/E: ~10 copies
per cell). The bar graph
shows normalized FAD
fluorescence (TSD, n = 3 biological replicates) (bottom panel). (C) His-tagged yeast Sdh1 was expressed alone or
with Sdh5 or Sdh2 in E. coli, purified, and analyzed for FAD fluorescence as in (A) and by Coomassie blue staining.
B
C
A
co-exp: - Sdh5 Sdh2
fluorescence
Coomassie
Sdh1
Flavo-Sdh1
Lane: 1 2 3 4 5
Strain: WT WT
flx1
∆
flx1
∆
flx1
∆
SDH5- sdh5
∆
SDH5-
O/E O/E
Flavo-Sdh1
fluorescence
Flavoprotein
α-Sdh1
α-Porin
Flavo-Sdh1/total Sdh1
(%of WT)
0
25
50
75
100
125
12345
WT
sdh5
∆
sdh
1∆
α-Sdh1
Flavoprotein
Flavo-Sdh1
fluorescence
Flavoprotein
α-Porin
[kD]
75
50
37
25
20
28 AUGUST 2009 VOL 325 SCIENCE www.sciencemag.org1140
REPORTS
of Sdh1, although Sdh1 was still present (Fig. 3A).
The flavination of two other mitochondrial flavo-
proteins, visualized in the same gel, was unaffected
by either SDH5 or SDH1 deletion (Fig. 3A).
Sdh5 overexpression in WT cells did not in-
crease Sdh1 flavination (Fig. 3B, lane 1 versus lane
2), possibly due to flavination that was already
stoichiometric. A significant effect of Sdh5 over-
expression might require a state of reduced Sdh1
flavination as was observed in a strain with de-
creased mitochondrial FAD caused by deletion of
the mitochondrial FAD transporter Flx1 (13, 14).
The flx1D mutant displayed almost no Sdh1
flavination, but Sdh5 overexpression restored
flavination to about 50% of WT levels (Fig. 3B,
lanes 4 and 5). As shown in Fig. 3C, FAD in-
corporation was almost undetectable in Escherichia
coli–expressed Sdh1 but was increased dramat-
ically when Sdh5, but not Sdh2, was coexpressed.
These data demonstrate that Sdh5 is both nec-
essary and sufficient for Sdh1 flavination.
The amino acid sequence of yeast Sdh5 is 44%
identical (from residues 33 to 158 of 163) to its
human ortholog C11orf79, which we rename here
hSDH5 (fig. S1). We hypothesized that hSDH5 is
required for f lavination of SDHA (human Sdh1)
and thus for SDH activity . Previous genetic studies
have shown that mutations causing loss of SDH
activity are responsible for hereditary forms of a rare
neuroendocrine tumor called paraganglioma (PGL).
Of the four familial PGL syndromes (PGL1 to PGL4),
PGL1, PGL3, and PGL4 have been associated with
mutations in SDHD, SDHC,andSDHB, respec-
tively (15). The gene for PGL2 (Online Mendelian
Inheritance in Man number %601650) has not been
identified, but it maps to chromosome 11q13.1, the
genomic locus of the hSDH5 gene (16).
We sequenced hSDH5 in three affected indi-
viduals from different branches of the previously
described Dutch PGL2 lineage (17). In all three in-
dividuals, we found a single nucleotide c.232G>A
change in exon 2 (fig. S8), which causes a Gly
78
→
Arg
78
(G78R) mutation in the most conserved
region of the protein (fig. S1). None of 400 un-
affected control individuals carried the c.232G>A
DB
A
C
Flavo-SDHA
α-SDHA
α-GAPDH
Lane: 1 2 3 4 5 6 7 8 9
Sample: PGL tumor PGL2 tumor HEK HepG2 skM Liver
WT+EV
EV
sdh5
∆
y
SDH5
h
SDH5
h
SDH5
(G78R)
glucose glycerol
α-SDHA
Flavo-SDHA
Lane: 1 2 3 4 5 6 7 8 9
hSDH5: EV WT G78R EV WT G78R EV WT G78R
Fraction: lysate eluate unbound
α-Myc IP
α-Myc
(hSDH5)
α-GAPDH
E
fluorescence
Lane: 1 2 3 4 5
Strain: WT
sdh5
∆
Plasmid: EV EV y
SDH5
h
SDH5
h
SDH5
(G78R)
Flavo%: 100% 3% 96% 77% 6%
Flavo-Sdh1
α-Sdh1
Flavoprotein
α-Myc
(y/h Sdh5)
α-PGK
Fig. 4. AhumanSDH5 loss-of-function mutation in PGL2. (A) The heterozygous c.232G>A mutation
segregates with disease in the PGL2 lineage (17). Black symbols, affected persons; white symbols,
unaffected persons; +, heterozygous mutation; NT, not tested. Diamonds with the number 4 represent
four unaffected individuals. Individuals who are not affected because they carry the mutation on their
maternal chromosome 11 are marked by an m. One healthy maternal mutation carrier and five non-
affected paternal mutation carriers are not shown in the pedigree for privacy reasons. (B) Fluorescence gel image (top panel) and immunoblotting (lower panels) of samples
from human tumors, cell lines, and mouse tissues. Lanes 1 and 2, sporadic PGL tumors; lanes 3 to 5, PGL2 tumors (hSDH5 G78R); lanes 6 and 7, HEK293 and HepG2human
cell lines; lanes 8 and 9, normal mouse skeletal muscle (skM) and liver. (C) Lysate from HEK293 cells containing EV or expressing WT or G78R hSDH5-Myc were immu-
noprecipitated with agarose beads conjugated with antibody to Myc. Lysate, eluate, and unbound fraction were FAD-imaged (top panel) and immunoblotted (lower three panels).
(D) Serial dilutions of WT and sdh5D strains containing EV or plasmids expressing yeast Sdh5-Myc, WT human SDH5-Myc, or G78R hSDH5-Myc were spotted on glucose or glycerol
medium and grown at 30°C for 2 or 3 days, respectively. (E) Fluorescence gel image (top panel) and immunoblotting of whole-cell extract from the five strains in (D) (lower panels).
PGK, loading control. FAD fl uorescence was normalized to Sdh1 protein level and expressed as a percentage relative to WT (Flavo%, TSD, n = 3 biological replicates).
www.sciencemag.org SCIENCE VOL 325 28 AUGUST 2009
1141
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