Revealing human disease genes through analysis of the yeast mitochondrial proteome.
- SourceAvailable from: Kunitoshi Yamanaka[show abstract] [hide abstract]
ABSTRACT: p97/VCP/Cdc48 is one of the best-characterized type II AAA (ATPases associated with diverse cellular activities) ATPases. p97 is suggested to be a ubiquitin-selective chaperone and its key function is to disassemble protein complexes. p97 is involved in a wide variety of cellular activities. Recently, novel functions, namely autophagy and mitochondrial quality control, for p97 have been uncovered. p97 was identified as a causative factor for inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD) and more recently as a causative factor for amyotrophic lateral sclerosis (ALS). In this review, we will summarize and discuss recent progress and topics in p97 functions and the relationship to its associated diseases.Biochimica et Biophysica Acta 07/2011; 1823(1):130-7. · 4.66 Impact Factor
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ABSTRACT: OBJECTIVE: Mitochondria fulfill many tissue-specific functions in cell metabolism. We set out to identify differences in the protein composition of mitochondria from five tissues frequently affected by mitochondrial disorders. METHODS: The proteome of highly purified mitochondria from five mouse organs was separated by high-resolution 2D-electrophoresis (2DE). Tissue-specific spots were identified through nano-LC/ESI-MS/MS and quantified by densitometry in ten biological replicates. RESULTS: We identified 87 consistently deviating spots representing 48 proteins. The percentage of variant spots ranged between 4.2-6.0%; 21 proteins having tissue-specific isospots. Consistent tissue-specific processing/regulation was seen for Carbamoyl-phosphate-synthase, Aldehyde-dehydrogenase 2, ATP-synthase α-chain, and Isocitrate-dehydrogenase α-subunit. Thirty tissue-specific proteins were associated with mitochondrial disorders in humans. We further identified Alcohol-dehydrogenase, Catalase, Quinone-oxidoreductase, Cyclophilin-A and Upf0317, a potential biotin-carboxyl-carrier protein, which had not been annotated as "mitochondrial" in GO or MitoCarta databases. Their targeting to the mitochondria was verified by transfection of full-length GFP-tagged plasmids. CONCLUSION: Given the high evolutionary conservation of mitochondrial metabolic pathways, these data further annotate the mitochondrial proteome and advance our understanding of the pathophysiology and tissue-specificity of symptoms seen in patients with mitochondrial disorders. The generation of 2D-electrophoretic maps of the mitochondrial proteome using tissue specimens in the milligram range facilitates this technique for clinical applications and biomarker research.Proteomics 11/2012; · 4.43 Impact Factor
6 Cell Cycle Volume 8 Issue 24
is not conserved in higher eukaryotes.7
Presumably, Complex II requires other
factors for assembly and insertion of the
FAD, heme and iron-sulfur cofactors.
SDH5 is necessary and sufficient for one
of these events, specifically, FAD cofactor
insertion into SDHA. Another Complex
II assembly factor named SDHAF1 has
recently been described,8 although its pre-
cise role remains undetermined. SDH5
and SDHAF1 are the first two evolution-
arily conserved Complex II assembly fac-
tors to be described.
The molecular mechanism of covalent
FAD insertion into proteins is mysterious
and controversial. Other covalent cofac-
tors are inserted by specialized enzymes,
such as cytochrome c heme lyase for
heme attachment to apocytochrome c.9
Until now, no similar enzyme has been
identified for covalent FAD attachment.
Instead, an autocatalytic mechanism was
proposed based on suggestive data from
heterologously expressed bovine monoam-
ine oxidase A.10 In contrast, covalent FAD
attachment to Sdh1 appears to be depen-
dent on additional protein factors.11 Using
in vitro translated Sdh1 precursor and
differentially treated mitochondria, Sdh1
flavination was shown to require matrix
import, ATP and at least one additional
protein.11 We would suggest that Sdh5 is
the necessary matrix protein suggested by
these earlier experiments.
whether Sdh5 is the enzyme responsible
for the chemistry of FAD attachment or
simply a chaperone maintaining Sdh1 in
a conformation that is susceptible to auto-
catalytic FAD attachment. This is a very
difficult question and the answer remains
to be determined. Four observations from
the published work,4 however, would
We have recently described the studies
of one such protein that we named Sdh5.4
We showed that Sdh5 physically interacts
with the catalytic subunit of succinate
dehydrogenase (SDH), a component of
both the tricarboxylic acid cycle and elec-
tron transport chain. Sdh5 is necessary and
sufficient for FAD cofactor conjugation
(flavination) of Sdh1, which is required for
SDH activity. The human SDH5 ortholog
similarly interacts with human SDHA
(Sdh1 ortholog) and is able to comple-
ment the yeast sdh5∆ mutant phenotype
and rescue Sdh1 flavination, suggesting
functional conservation. Based on the
causal relationship of loss of SDH activity
with a rare neuroendocrine tumor called
paraganglioma (PGL), we sought to deter-
mine whether mutations in human SDH5
might be associated with some forms of
paraganglioma. Indeed, we discovered
that one familial form of paraganglioma,
PGL2, is caused by a G78R mutation in
hSDH5. The G78R mutant of hSDH5
was completely inactive in functional
studies and tumors derived from PGL2
patients exhibited a near complete loss of
SDHA flavination. The gene that we iden-
tified as SDH5 (C11orf79/FLJ20487) was
previously predicted as one of the possible
PGL2 genes as a result of elegant proteom-
ics and bioinformatics.5
The assembly of membrane bound
electron transport complexes is a com-
plicated multi-step process involving
assembly of multiple subunits as well as
insertion of cofactors such as FAD, heme
and metals. This complexity is illustrated
by cytochrome c oxidase (Complex IV)
assembly, which requires more than 30
accessory proteins.6 In contrast, only one
assembly factor had been reported for
Complex II, the yeast Tcm62 protein that
Mitochondria are unique, dynamic and
complex organelles that play vital roles
in many aspects of cellular function. As a
result, mitochondrial dysfunction is tightly
coupled with a variety of human diseases.
In fact, about 20% of predicted human
mitochondrial proteins have been impli-
cated in one or more hereditary diseases.1,2
The best current inventory of mammalian
mitochondrial resident proteins consists of
1098 proteins.3 Surprisingly, nearly 300
proteins in this compendium are essen-
tially uncharacterized. Among them, many
are highly conserved throughout eukarya,
indicating their functional importance.
Presumably, the quarter of the mitochon-
drial proteome that is uncharacterized,
especially those with high degree of con-
servation, contains many human disease
genes that await discovery.
Making the disease connection would
be greatly facilitated by an understanding
of the biochemical and physiological func-
tion of these proteins. Therefore, we initi-
ated a project to understand the function
of a subset of these proteins. We chose to
use yeast as the primary model system for
three reasons. First, the yeast S. cerevisiae
is a tractable and facile genetic and bio-
chemical system. Second, mitochondrial
biology is cell-autonomous and highly
conserved from yeast to humans. Finally,
yeast can survive the complete loss of
respiration, due to their ability to gener-
ate ATP from fermentation. This is very
important for genetic analysis of proteins
that are essential for respiration, deletion of
which would be lethal in other organisms.
The strategy we decided to employ was
to study these genes initially using yeast.
As it became appropriate, we intended to
confirm our findings in higher eukaryotes
and eventually move to human studies.
*Correspondence to: Jared Rutter; Email: email@example.com
Submitted: 09/21/09; Revised: 09/28/09; Accepted: 09/29/09
Previously published online: www.landesbioscience.com/journals/cc/article/10205
Comment on: Hao HX, et al. SDH5, a gene required for flavination of succinate dehydrogenase, is mutated in paraganglioma. Science 2009; 325:1139–42.
Revealing human disease genes through analysis of
the yeast mitochondrial proteome
Huai-Xiang Hao and Jared Rutter*
Department of Biochemistry; University of Utah School of Medicine; Salt Lake City, UT USA
www.landesbioscience.com Cell Cycle 7
of Sdh5 (surrounding the G78 position)
is highly basic in all species (Fig. S1). It
is possible that this region participates in
an interaction with the negatively charged
pyrophosphate moiety of FAD during the
Whatever the exact role of Sdh5 in
Sdh1 flavination, three observations from
the published work suggest that it plays
that role very specifically. First, in the
absence of Sdh1, the Sdh5 protein fails to
accumulate (Fig. S1F). The fact that loss
of Sdh1 leads to a complete loss of Sdh5,
presumably through protein destabiliza-
tion and degradation, suggests that Sdh1
is an obligate binding partner of Sdh5.
This intimate relationship implies a high
degree of specificity for Sdh5 function.
Second, Sdh5 overexpression in the flx1∆
mutant strain lacking the mitochondrial
FAD transporter enhanced Sdh1 flavina-
tion (Fig. S3B), but failed to rescue the
glycerol growth defect of the flx1∆ mutant
strain (Fig. S11), which is presumably
caused by deficiency of FAD occupancy in
other flavoproteins. Finally, covalent FAD
attachment to the two other mitochon-
drial proteins visualized in the fluorescent
gel assay was completely normal in the
suggest that Sdh5 does not act simply as
a general chaperone. First, in addition to
the myriad of native chaperones in both
the mitochondrial matrix of S. cerevisiae
and in E. coli, co-expression of Sdh2, a
clear Sdh1 binding partner, is completely
unable to support flavination of Sdh1 in
either yeast (Fig. S10) or bacteria (Fig.
S3C). Second, if hSDH5 were acting
simply as a chaperone to promote FAD
incorporation, it is likely that the hSDH5-
associated population of SDHA would
NOT be flavinated. We found, however,
that the hSDH5-associated SDHA has
exactly the same normalized FAD signal
as the hSDH5-unbound SDHA (Fig.
S4C). Third, we conducted an extensive
high-copy suppressor screen for rescue of
the sdh5∆ glycerol growth defect using
a yeast genomic library. We recovered
plasmids, but found no additional genes
that enabled bypass of SDH5. This is sug-
gestive of a fairly specific role for Sdh5
that cannot be compensated by increased
general chaperone activity (or by other
means). Fourth, the sequence of Sdh5 may
be suggestive of a direct relationship with
FAD. The most highly conserved region
sdh5∆ mutant and it was not affected by
Sdh5 overexpression (Fig. S3A and B).
In summary, we began this project with
an uncharacterized yeast mitochondrial
protein. Through careful elucidation of its
molecular function, we were able to gener-
ate a hypothesis about a possible link to
human disease. This hypothesis was found
to be true, enabling the identification of a
human tumor susceptibility gene.
Supplementary materials can be found at:
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Hao HX, et al. Science 2009; 325:1139-42.
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