Doctoral
Thesis
in
Biochemistry
University, Sweden, 2012
at
Stockholm
Early steps in the biogenesis of the bc1 complex in yeast
mitochondria – The role of the Cbp3-Cbp6 complex in
cytochrome b synthesis and assembly
Early steps in the biogenesis of the
bc1 complex in yeast mitochondria
The role of the Cbp3-Cbp6 complex in cytochrome b synthesis and assembly
Steffi Gruschke
©Steffi Gruschke, 2012
ISBN 978-91-7447-534-0, pp. 1-54
Printed in Sweden by Universitetsservice AB, Stockholm, 2012
Distributor: Department of Biochemistry and Biophysics, Stockholm Univeristy
Abstract
The inner membrane of mitochondria harbors the complexes of the respiratory
chain and the ATP synthase, which perform the key metabolic process of
oxidative phosphorylation. These complexes are composed of subunits from
two different genetic origins: the majority of constituents is synthesized on
cytosolic ribosomes and imported into mitochondria, but a handful of proteins,
which represent core catalytic subunits, are encoded in the organellar DNA and
produced on mitochondrial ribosomes. Despite the endosymbiotic origin of
mitochondria, the organellar translation machinery differs significantly from
that of its bacterial ancestor. How mitochondrial ribosomes work and translation
is organized in the organelle is hardly understood. The tunnel exit region of a
ribosome is the site where the nascent chain emerges and is exposed to a
hydrophilic environment for the first time. In cytosolic ribosomes, the proteins
forming the rim of the tunnel exit constitute a platform where biogenesis factors
that help the newly synthesized proteins to mature can bind. Using a proteomic
approach and baker’s yeast as a model organism, I defined the composition of
the mitochondrial ribosomal tunnel exit region and analyzed whether this site
serves a similar purpose as in cytosolic ribosomes. This study not only provided
insights into the structural composition of this important site of mitochondrial
ribosomes, but also revealed the positioning of Cbp3 at the tunnel exit, a
chaperone required specifically for the assembly of the bc1 complex. In my
subsequent work I found that Cbp3 structurally and functionally forms a tight
complex with Cbp6 and that this complex exhibits fundamental roles in the
biogenesis of cytochrome b, the mitochondrially encoded subunit of the bc1
complex. Bound to the ribosome, Cbp3-Cbp6 stimulates translation of the
cytochrome b mRNA (COB mRNA). Cbp3-Cbp6 then binds the fully
synthesized cytochrome b, thereby stabilizing and guiding it further through bc1
complex assembly. The next steps involve the recruitment of another assembly
factor, Cbp4, to the Cbp3-Cbp6/cytochrome b complex and presumably
acquisition of the two redox-active heme b cofactors. During further assembly
Cbp3-Cbp6 is released, can again bind to a ribosome and activate further rounds
of COB mRNA translation. The dual role of Cbp3-Cbp6 in both translation and
assembly allows the complex to act as a regulatory switch to modulate the level
of cytochrome b synthesis in response to the bc1 complex assembly process.
v
List of Publications
I.
II.
III.
IV.
Gruschke S, Gröne K, Heublein M, Hölz S, Israel L, Imhof A,
Herrmann JM, Ott M. Proteins at the polypeptide tunnel exit of the
yeast mitochondrial ribosome (2010) J Biol Chem. 25, 1902219028
Gruschke S, Kehrein K, Römpler K, Gröne K, Israel L, Imhof A,
Herrmann JM, Ott M. Cbp3-Cbp6 interacts with the yeast
mitochondrial ribosomal tunnel exit and promotes cytochrome b
synthesis and assembly (2011) J Cell Biol. 193, 1101-1114
Gruschke S, Römpler K, Hildenbeutel M, Kehrein K, Kühl I,
Bonnefoy N, Ott M. The Cbp3-Cbp6 complex coordinates
cytochrome b synthesis with bc1 complex assembly in yeast
mitochondria (2012) J Cell Biol. 199, 137-150
Hildenbeutel M, Hegg E, Gruschke S, Meunier B, Ott M. The
timing of heme incorporation into yeast cytochrome b. Manuscript
vi
Additional Publications
I.
II.
Gruschke S and Ott M. The polypeptide tunnel exit of the
mitochondrial ribosome is tailored to meet the specific
requirements of the organelle (2010) Bioessays. 12, 1050-1057
Hildenbeutel M, Gruschke S, Römpler K, Meunier B, Dujardin G,
Ott M. Qcr7 – more than a structural subunit of the yeast bc1
complex. Manuscript
vii
Abbreviations
ALA
Aminolevulinic acid
BN PAGE
Blue Native polyacrylamide gel electrophoresis
COB
Cytochrome b (gene and mRNA)
Cryo-EM
Cryo electron microscopy
IMM
Inner mitochondrial membrane
IMS
Intermembrane space
MAP
Methionine aminopeptidase
mtDNA
Mitochondrial DNA
MTS
Mitochondrial targeting signal
nt
Nucleotides
OMM
Outer mitochondrial membrane
ORF
Open reading frame
PDF
Peptide deformylase
PPR
Pentatricopeptide repeat
SRP
Signal recognition particle
TE
Tunnel exit
TF
Trigger factor
UTR
Untranslated region
viii
Contents
Abstract ............................................................................................................... v
List of Publications ............................................................................................. vi
Additional Publications ..................................................................................... vii
Abbreviations ................................................................................................... viii
Introduction ......................................................................................................... 1
Functions of mitochondria ............................................................................... 1
Respiratory chain complexes are of dual genetic origin .................................. 3
Mitochondria in disease and aging .................................................................. 6
The structure and composition of mitochondrial ribosomes ........................... 7
The tunnel exit of ribosomes ........................................................................... 8
Interaction partners of bacterial ribosomes ..................................................... 9
Interaction partners of mitochondrial ribosomes ........................................... 11
Translation in yeast mitochondria ................................................................. 13
Assembly of the yeast bc1 complex ............................................................... 24
Aims of the thesis .............................................................................................. 29
Summaries of the Papers ................................................................................... 30
Conclusions and future perspectives ................................................................. 36
Sammanfattning på svenska .............................................................................. 40
Deutsche Zusammenfassung ............................................................................. 42
Acknowledgements ........................................................................................... 44
References ......................................................................................................... 47
ix
Introduction
Functions of mitochondria
Key metabolic processes of eukaryotic cells take place within mitochondria.
These important organelles harbor the enzymes of the TCA cycle, which
represents the central hub in metabolism, are the sites of catabolic pathways like
-oxidation, amino acid degradation and the urea cycle and participate in
biosynthesis of steroid hormones and essential redox-active prosthetic groups
like heme and iron/sulfur (Fe/S) clusters (1). Furthermore, mitochondria are
crucial for apoptosis, the controlled form of cell death, and for thermogenesis
not only in newborn mammals and hibernating animals as long assumed, but
also in adult humans (2-5). The most prominent function of these organelles,
however, is nevertheless the production of ATP by oxidative phosphorylation
(1). Mitochondria evolved when an archaeal cell established a symbiotic
relationship with aerobic bacteria (6). As a consequence of their evolutionary
origin, mitochondria are enclosed by two membranes, the outer and the inner
membrane, the latter surrounding the mitochondrial matrix. The aqueous
compartment between the two membranes is the intermembrane space. Whereas
many functions of mitochondria are exerted by enzymes of the mitochondrial
matrix, the respiratory chain complexes and the ATP synthase that mediate
oxidative phosphorylation reside in the inner mitochondrial membrane. Four
multisubunit complexes make up the respiratory chain and transport electrons
from NADH and succinate to the final electron acceptor O2 (Figure 1A). Some
of the energy of these redox reactions is used to establish a proton gradient
across the inner mitochondrial membrane that drives the synthesis of ATP by the
ATP synthase complex. Complexes I (NADH dehydrogenase) and II (succinate
dehydrogenase) transfer electrons from NADH and succinate, respectively, to
the lipid-soluble electron carrier ubiquinone. During NADH oxidation by
complex I, protons are pumped across the inner membrane from the matrix to
the intermembrane space. Reduced ubiquinone (ubiquinol) diffuses in the inner
membrane to complex III (cytochrome c reductase, bc1 complex) where
electrons are transferred to the soluble cytochrome c in the intermembrane
space by a complex mechanism called the Q cycle (Figure 1B) (7).
1
Figure 1: Oxidative phosphorylation in mitochondria. A: The inner membrane of
mitochondria harbors the complexes of the respiratory chain and the ATP
synthase. Complex I (NADH dehydrogenase) and complex II (succinate
dehydrogenase) transfer electrons from NADH and succinate, respectively, to
ubiquinone (Q). Reduced ubiquinone (ubiquinol) delivers electrons to complex
III (bc1 complex), which transports them further to cytochrome c (dark blue).
Reduced cytochrome c (light blue) gets oxidized by complex IV (cytochrome
oxidase) and electrons are transferred to the final acceptor oxygen to form H 2O.
The activities of complexes I, III and IV establish an electrochemical gradient
across the inner membrane, which is used by the ATP synthase to produce ATP
from ADP and inorganic phosphate (Pi). The path of electrons is depicted in
blue arrows, proton movement is shown in red arrows. B: The Q cycle. Every
molecule of ubiquinol (QH2) at the IMS side of the membrane donates one
electron via the iron-sulfur cluster (FeS) of the Rieske subunit and the heme
group in cytochrome c1 to cytochrome c in the IMS, which is thereby reduced.
The second electron of the same ubiquinol is transferred via the two heme
groups of cytochrome b (bL and bH) to a ubiquinone molecule (Q) at the
opposite side of the membrane. The oxidation of two molecules ubiquinol
releases four protons into the IMS, leads to the generation of two molecules
reduced cytochrome c and produces (under consumption of two protons from
the matrix) one molecule QH2 at the matrix side of the membrane. IMM, inner
mitochondrial membrane. IMS, intermembrane space. OMM, outer
mitochondrial membrane.
2
Again, protons are translocated from the matrix to the intermembrane space
when electrons pass complex III (Figure 1A). Reduced cytochrome c then
moves to complex IV (cytochrome oxidase), which transfers the electrons to the
final acceptor O2, generating H2O. In addition to the consumption of protons
from the matrix to generate H2O, complex IV pumps protons across the inner
membrane (Figure 1A). The concerted action of complexes I, III and IV thus
generates an electrochemical proton gradient across the inner membrane, which
consists of the difference in proton concentration and the charge separation
between the matrix and the intermembrane space. The ATP synthase (also called
complex V) uses the energy of this gradient to generate ATP from ADP and
inorganic phosphate. As protons passively flow back into the matrix, a ring-like
structure within the membrane-embedded part of the enzyme (Fo) rotates. This
rotation is transmitted into conformational changes within the subunits of the
hydrophilic part (F1) that catalyze ATP synthesis (Figure 1A) (8).
Respiratory chain complexes are of dual genetic origin
About two billion years ago, mitochondria derived in an endosymbiotic event
from aerobic bacteria. In the course of mitochondrial evolution there has been
an extensive transfer of ancestral bacterial genes to the nucleus of the cell. This
required the invention of intricate pathways to import proteins that are
synthesized on cytosolic ribosomes into mitochondria. Despite this huge
genome remodeling, a few genes were kept in the mitochondrial DNA (Figure
2). Although few in number, all of the 13 mitochondrially encoded proteins in
humans and seven of eight proteins in yeast represent core catalytic subunits of
respiratory chain complexes and the ATP synthase (Figure 3), which makes
them essential for oxidative phosphorylation.
3
Figure 2: Biogenesis of respiratory chain complexes. Mitochondrial proteins
that are encoded in the nucleus are translated on cytosolic ribosomes and
typically contain N-terminal targeting signals (MTS) for import into the organelle.
The binding of cytosolic chaperones to these proteins keeps them in an importcompetent state. Evolved from a bacterial ancestor, mitochondria contain their
own genetic system including mitochondrial DNA (mtDNA) and ribosomes. The
complexes of the respiratory chain are localized in the inner mitochondrial
membrane and are assembled from proteins encoded in both the nuclear (blue
arrow) and the organellar DNA (pink arrow). Modified from (9).
Even though the molecular reason for the retention of a handful of genes in the
mitochondrial DNA is not clear, cells apply a huge effort to maintain the
mitochondrial genetic system. In simple organisms like Saccharomyces
cerevisiae more than 250 proteins are involved in the expression of
mitochondrial genes and the assembly of the respiratory chain (10). The distinct
functions and sites of action of these factors, however, for the most part remain
to be elucidated.
4
Figure 3: The respiratory chain is a mosaic of components derived from two
different genetic systems. In baker's yeast, two ribosomal RNAs (15S and 21S)
and eight polypeptides are encoded in the mitochondrial DNA. Except the
soluble component Var1, which is a part of the small subunit of the
mitochondrial ribosome, all proteins are hydrophobic membrane proteins. They
form core catalytic subunits (shown in pink) of the respiratory chain complexes
cytochrome c reductase (bc1 complex, complex III), cytochrome oxidase
(complex IV) and ATP synthase (ATPase) and have to be assembled with a
number of nuclear encoded polypeptides (blue) in a precisely coordinated
fashion. The topologies of the mitochondrially encoded subunits are depicted.
COB, cytochrome b gene. IMM, inner mitochondrial membrane. IMS,
intermembrane space. Modified from (9).
5
The dual genetic origin of subunits complicates the biogenesis of respiratory
chain complexes. As mentioned above, the majority of components is encoded
in the nucleus and imported into mitochondria after their synthesis. The
molecular mechanisms underlying protein import into mitochondria are well
documented (11,12). In contrast, how protein synthesis is mediated and
translation is organized in mitochondria is only poorly understood. Moreover, to
allow formation of a functional respiratory chain, subunits of both genetic
systems have to be assembled in a precisely coordinated manner. The need to
adjust two expression systems located in different cellular compartments
requires tight regulation and communication, but not much is known to date
how exactly this is accomplished.
Mitochondria in disease and aging
A functional impairment of the respiratory chain due to mutations in structural
genes or factors mediating its biogenesis can lead to severe human diseases.
During the last decade, increasing numbers of sporadic and inherited mutations
in mitochondrial genes have been reported that cause neuro- and
muscledegenerative disorders (13,14). Proteins of the mitochondrial import
machinery as well as assembly factors for respiratory chain complexes and
components of the mitochondrial ribosome have been described to be affected
in such patients. Studies of homologous proteins using the model organism S.
cerevisiae have revealed basic mechanisms of biogenesis pathways of the
respiratory chain. For instance, this strategy has proved to be successful in the
discovery of a molecular cause for the neurodegenerative disease Leigh
syndrome (15,16). Understanding general principles of respiratory chain
biogenesis can therefore provide the basis for the treatment of human diseases.
Mitochondrial dysfunction is not only implicated in human diseases, but it also
has a high impact on the process of aging in mammals. Various mammalian
tissues show an age-related increase in mitochondrial DNA mutations and
deletions leading to an impairment of respiratory chain function (17-19). It was
long debated whether the steady decline of functional mtDNA is the cause of
age-related symptoms or only coincidental. Furthermore, it was speculated what
molecular reason in the first place leads to the generation of the mtDNA
aberrations, for example if they are caused by the accumulation of oxidative
6
damage (20). The pioneering generation and study of the so-called mutator
mouse (21,22) brought valuable insights into these questions. This animal
model expresses a version of the mitochondrial DNA polymerase  that lacks its
proofreading activity and the mice show a progressive aging phenotype and an
increased number of mtDNA mutations. Interestingly, despite the functional
impairment of the respiratory chain mutator mice show only a slight increase in
oxidative damage (23). It might therefore be that the age-related decline of
functional mtDNA is rather caused by random point mutations introduced into
mitochondrial genomes by the inherent error-proneness of the mitochondrial
DNA polymerase  and that oxidative damage only adds up to this (24). These
studies have supported the view that the mitochondrial genetic system is a
central player in the process of aging.
The structure and composition of mitochondrial ribosomes
Mitochondria evolved from a bacterial ancestor and still contain their own
genome and the translation system to synthesize the few encoded proteins.
Because of powerful in vitro approaches and high-resolution structural studies,
the molecular mechanisms of bacterial protein synthesis are understood in great
detail (25-27). In contrast, no in vitro-translation system exists for
mitochondrial ribosomes and high-resolution structural data allowing the
identification of individual ribosomal proteins are not available yet. Thus, it is
not clear how exactly mitochondrial protein synthesis is executed, which factors
are involved and how translation in general is organized.
The central components of the mitochondrial translation machinery are
mitochondrial ribosomes. It has long been assumed that mitochondrial
ribosomes closely resemble the bacterial particles. However, this holds true only
for certain aspects as millions of years of evolution have led to modifications
making mitochondrial ribosomes strikingly different from their ancestors.
Features that are shared by bacterial and mitochondrial ribosomes are for
example catalytic properties, as the RNA moieties and proteins that contribute
to decoding of the mRNA and peptide bond formation are highly conserved.
Both types of ribosomes furthermore exhibit sensitivity to similar antibiotics. In
addition, factors involved in translation initiation, elongation and termination
7
share a high degree of similarity (28). In contrast, mitochondrial ribosomes
differ from their bacterial counterparts in size, structure and composition. While
eubacterial ribosomes consist of 55 proteins and three ribosomal RNAs,
mitochondrial ribosomes are composed of only two rRNAs and 70 to 80
proteins, depending on the species (29). Moreover, studies of the mammalian
mitochondrial ribosome have revealed that many helices and loops within the
rRNA of bacterial ribosomes are absent in the mitochondrial counterparts and
that there is an overall significantly large increase in protein mass in the porous
structure, especially on the surface of the ribosome (30). This dramatic gain in
protein mass is on the one hand caused by the modification of conserved
proteins with N- and C-terminal extensions (Figure 4) and on the other hand by
the addition of mitochondria-specific ribosomal proteins. The reason for this
huge remodeling of mitochondrial ribosomes is not clear, but it has been
discussed that the additional protein parts could compensate for the loss of
rRNA moieties and thus help to stabilize the structure of the ribosome (31,32).
Alternatively, the additional proteins and domains could introduce novel
organizational or regulatory features that are specific for the requirements of the
organellar translation machinery (33,34). For example, the process of translation
initiation in mitochondria differs from that in bacteria as mitochondrial
messenger RNAs do not contain Shine-Dalgarno sequences used to load the
mRNA correctly onto the ribosome in bacteria (see below). In fact, it has been
suggested that mitochondrial ribosomes themselves, employing mitochondriaspecific ribosomal proteins of the small subunit, might recognize translatable
mRNAs directly (35).
The tunnel exit of ribosomes
An important site of ribosomes in general is the polypeptide tunnel exit, where
the newly synthesized nascent chain emerges from the ribosome and for the first
time encounters a hydrophilic environment. This site of cytosolic ribosomes
serves as a platform to organize the biogenesis of newly synthesized proteins as
a variety of factors bind there that influence the fate of the translated protein. In
bacterial ribosomes, the tunnel exit is mainly formed by four proteins, L22, L23,
L24 and L29. Interestingly, although these proteins are conserved in
mitochondria, this region of mitochondrial ribosomes was significantly altered
8
in the course of evolution (Figure 4). Cryo-EM reconstruction analyses showed
that many protuberances and additional protein mass are found at the tunnel exit
of mitochondrial ribosomes, although the resolution of these structures was not
high enough to identify individual proteins (30,36). If these alterations have
only structural reasons or whether they introduce novel organizational features
to the translation system and the coordination of protein biogenesis in
mitochondria, is currently not known.
Figure 4: The proteins forming the rim of the tunnel exit of mitochondrial
ribosomes were altered during evolution. A: Schematic view onto the bottom of
the large subunit of bacterial ribosomes. Distinct proteins are forming the rim of
the tunnel exit (TE) region. B: Homologs of the conserved bacterial tunnel exit
proteins can also be found in mitochondrial ribosomes from yeast. During
evolution these proteins were altered and as a consequence contain in addition
to the conserved regions (grey boxes) N- and C-terminal extensions (NE-/CEdomain). The NE-domains include the mitochondrial targeting signal (MTS),
which is necessary for the import into mitochondria. The CE-domains of Mrp20
and Mrpl40 exhibit a high propensity to form coiled-coil structures. Numbers
indicate amino acid residues. From (37).
Interaction partners of bacterial ribosomes
In bacteria, a variety of proteins interacts with the ribosomal polypeptide tunnel
exit to support maturation of the emerging protein. Those interactors bind to the
conserved proteins that form the rim of the tunnel exit (L22, L23, L24, L29;
Figure 4A) and can be classified into three categories: 1. processing enzymes,
2. molecular chaperones, and 3. components of the translocation machinery.
9
Members of the first category are PDF (peptide deformylase) and MAP
(methionine aminopeptidase). These factors deformylate and remove the start
methionine co-translationally from nascent chains in a concerted action. To
allow efficient N-terminal enzymatic maturation of the growing polypeptide,
PDF and MAP can simultaneously bind to the tunnel exit of ribosomes, the
former in proximity to L22 (38) and the latter potentially in vicinity to L24 (39).
After N-terminal processing, newly synthesized proteins in bacteria can either
fold (if they are soluble) or be inserted into or translocated across the inner
membrane. Folding of soluble proteins is assisted by the chaperone trigger
factor. Its binding to the ribosomal tunnel exit proteins L23 and L29 positions
trigger factor optimally to efficiently contact nascent chains (40,41). Trigger
factor cooperates in the folding of soluble proteins with the heat shock protein
DnaK (42). In case that a protein is inserted into the inner membrane or
transported to the periplasm, the presence of a hydrophobic signal sequence
within the nascent chain is recognized by a member of the third interactorfamily, the signal recognition particle (SRP) (43). Although SRP also uses L23
as a docking site on the ribosome (44), simultaneous binding of trigger factor
and SRP can occur (45). The ribosome-SRP-complex is targeted to the
cytoplasmic side of the inner bacterial membrane where the interaction of SRP
with its membrane-bound receptor FtsY induces a release of the ribosome that is
passed on to the membrane insertion machinery, the SecYEG translocon (46)
(Figure 5, left). A co-translational insertion of proteins into or translocation
across the inner membrane is mediated by the SecYEG complex that directly
contacts the tunnel exit of the ribosome at L23 and L29 (47,48). The binding of
all these biogenesis factors to the polypeptide tunnel exit region is a conserved
phenomenon and found also in other organisms. For example, the L23 homolog
of eukaryotic ribosomes interacts with the mammalian SRP (49) as well as the
yeast Sec61 translocon (50).
10
Figure 5: Interactions of ribosomes with membranes. Left: In bacteria, upon
translation initiation the dissociated small and large subunit of the ribosome (1)
have to be joined and protein synthesis starts. Synthesis of a membrane protein
(2) on free cytosolic ribosomes is recognized by the signal recognition particle
(SRP) (3). The ribosome-nascent chain-SRP complex is guided to the inner
membrane (4) where SRP interacts with its membrane-associated receptor. The
ribosome is transferred to the SecYEG translocon to allow co-translational
membrane insertion of the protein being synthesized (5). Binding of SRP to its
receptor at the membrane dissociates the chaperone trigger factor (TF) from the
ribosome. Right: Mitochondrial ribosomes of S. cerevisiae are permanently
associated to the membrane, even when no translation occurs and the subunits
do not dissociate completely. Ribosomes interact with components of the
protein insertion machinery like Oxa1 and Mba1. These factors mediate the cotranslational insertion of hydrophobic membrane proteins encoded in the
mitochondrial DNA. IM, inner membrane. IMM, inner mitochondrial membrane.
IMS, intermembrane space. SRP, signal recognition particle. TF, trigger factor.
Modified from (9).
Interaction partners of mitochondrial ribosomes
In contrast to the well understood interactions that take place on bacterial and
eukaryotic cytosolic ribosomes, the knowledge about factors that bind to
mitochondrial ribosomes is very limited (Figure 5). Due to the absence of high
11
resolution cryo-EM structures of mitochondrial ribosomes, one can only
speculate about the composition of the tunnel exit region of those particles.
Homology studies, however, show that the conserved set of proteins forming the
rim of the tunnel in bacterial ribosomes is also present in mitochondrial
ribosomes, although altered in structure and size (Figure 4B). It is therefore
possible that a similar set of biogenesis factors binds to the organellar
translation particles. Although the presence of N-terminal processing enzymes
has also been demonstrated in mitochondria of eukaryotes (51-53), it is not clear
whether homologs of PDF and MAP bind to mitochondrial ribosomes. The
matrix of mitochondria contains molecular chaperones of the DnaK family, the
mitochondrial Hsp70 proteins (54). Those proteins, together with their
nucleotide exchange factors, have been shown to contact nascent chains (55,56),
but a direct interaction with mitochondrial ribosomes has not been demonstrated
so far. In contrast, a trigger factor-like protein was not found in mitochondria.
As mitochondrial ribosomes mainly synthesize hydrophobic membrane
proteins, a dynamic interaction with the membrane as described for bacteria is
not mandatory. Possibly for that reason, mitochondrial ribosomes are
permanently associated with the inner mitochondrial membrane, even in the
translationally inactive state (34,57) (Figure 5, right). This might also explain
why no SRP-like factor is present in mitochondria (58).
Membrane insertion of mitochondrially encoded proteins as it is understood so
far is much simpler than in bacteria as only a limited number of polypeptides
are synthesized by mitochondrial ribosomes. Homologs of the SecYEG
complex are absent in the organelles (58). The process of protein insertion into
the inner mitochondrial membrane is presumably mediated by Oxa1, a protein
of the YidC/Alb3/Oxa1 family (59). Oxa1 contains a C-terminal domain that is
critical for binding the mitochondrial ribosome (60,61). The docking site of
Oxa1 on the ribosome is again the tunnel exit region, where it binds in the
vicinity to Mrp20, the homolog of the bacterial L23, and Mrpl40, the L29
homolog (62). This positioning allows immediate contact of Oxa1 to nascent
chains as they exit the ribosome (59). Oxa1 cooperates in the insertion process
with the peripheral membrane protein Mba1 (63). This protein has also been
shown to interact with mitochondrial ribosomes (Figure 5, right) and serves as
a receptor to align the site of synthesis of mitochondrially encoded proteins with
the insertion site of the inner membrane (57). Recently, a third component of the
mitochondrial insertion machinery, Mdm38, was found to physically interact
12
with Mba1 (64). Mdm38 could be crosslinked to Mrp20, supporting a binding
close to the tunnel exit of mitochondrial ribosomes (65). However, as Mdm38
also plays an important role in the ion homeostasis in mitochondria (66,67), its
exact molecular function is not clear so far.
Taken together, many components of bacterial and cytosolic insertion
machineries are missing in mitochondria. Instead, mitochondria contain
additional factors like Mba1 and Mdm38 that support insertion of
mitochondrially encoded proteins into the inner membrane. These observations
highlight the many alterations during evolution that led to the general
differences between the organellar system and its ancestor. Whether processes
like membrane insertion are surprisingly simple in mitochondria or additional
components of such pathways still await identification remains to be elucidated.
Translation in yeast mitochondria
Despite their common ancestry, the bacterial and mitochondrial translation
systems differ from each other. This is not only reflected in the structure of the
ribosomes and the organization of early steps of protein biogenesis as described
above, but also in the general process of translation and the factors involved
therein. In the following, the features that distinguish protein synthesis in
mitochondria from that in bacteria are described and the concept of translational
activation used in mitochondria is introduced. Because my work deals mainly
with the biogenesis of the mitochondrially encoded protein cytochrome b, the
main focus will be on the mechanism how this subunit of the bc1 complex is
synthesized in baker’s yeast.
Differences between bacterial and mitochondrial translation
During prokaryotic translation initiation, the Shine-Dalgarno sequence of
bacterial mRNAs base pairs with a complementary sequence in the 16S rRNA
of the small ribosomal subunit and thereby helps loading the mRNA correctly
onto the ribosome (68). Although it has been shown that nucleotides in the 15S
rRNA of the small subunit of mitochondrial ribosomes are complementary to
13
sequences in mitochondrial mRNAs in yeast (69), those regions on the mRNAs
do not fulfill a Shine-Dalgarno-like function as they were dispensable for
translation (70,71). It is not clear to date which mechanism is used to initiate
translation in mitochondria. One possibility is a scenario similar to the process
in the cytosol of eukaryotes where the mRNA is scanned for initiation codons
(72). This mechanism requires the interaction of the small ribosomal subunit
with the 5’-cap structure of the mRNA. However, mitochondrial mRNAs lack
5’-cap structures (73) and usually contain multiple AUG codons, so it is rather
unlikely that initiation of translation in mitochondria is accomplished like that
(71). Experiments with isolated bovine mitochondrial ribosomes and all
necessary initiation components showed that efficient formation of an initiation
complex was only possible when the mRNA contained the start AUG codon at
or very close to the 5’-end (74). Similarly, removal of the start AUG did not
allow initiation of translation at alternative AUG codons within downstream
sequences of the mRNA in yeast mitochondria (75). Changing the start AUG to
AUA in COX2 and COX3 mRNAs without providing alternative downstream
AUGs showed that synthesis of Cox2 and Cox3 was reduced, but translation
was still initiated at the mutant AUA codon (76,77).
The concept of translational activators
Gene expression in mitochondria is controlled rather on a translational than a
transcriptional level. Messenger RNAs in yeast mitochondria contain 5’- and 3’untranslated regions (UTRs) that serve as recognition sites for so-called
translational activator proteins. These factors mediate translation of a single
mRNA, for which they are specific (Figure 6). Translational activators have
been identified by the pioneering work of Tom Fox, Alexander Tzagoloff,
Gottfried Schatz and coworkers, already in the 1970ies and 1980ies. The
cytochrome oxidase subunits Cox2 and Cox3 served as the prime examples for
the concept of specific translational activation in mitochondria. Early studies
showed that the nuclear gene products of PET111 and PET494 are required for
the specific expression of the mitochondrial COX2 and COX3 gene, respectively
(78). In subsequent years, yeast genetic screens revealed that mutants lacking
one translational activator (and therefore one mitochondrially encoded protein)
could be rescued by the remodeling of sequences within the mitochondrial
genome that allowed regaining respiratory growth. Those rearrangements led to
14
the creation of fusion genes or the exchange of regulatory regions with the
result that affected transcripts contained 5’-UTRs of other genes. This made
their expression independent from their authentic, missing translational
activator but dependent on the factor controlling synthesis of the other gene
(79). Translational activators are normally expressed at very low levels, so their
availability limits the rate of mitochondrial translation (80-82).
Figure 6: Translational control in yeast mitochondria. The mitochondrial
genome (mtDNA) of S. cerevisiae encodes two ribosomal RNAs (15S and 21S
rRNA), eight proteins as well as the RNA part of RNase P and 24 tRNAs (not
shown). The majority of the genes is transcribed as polycistronic precursor
mRNAs that have to be matured to release the functional mRNAs. The mRNAs
in yeast mitochondria contain 5'- and 3'-untranslated regions (UTRs) flanking
the respective open reading frame (ORF). Especially the 5'-UTR is the target of
specific translational activators (X) whose action is required to activate
translation of their one client mRNA in a yet unknown way. A number of proteins
have been shown to act as translational activators of specific mRNAs and are
depicted. For ATP8 and VAR1 no translational activators have been identified
so far.
A number of proteins has been identified that are required for the translation of
six of the eight messenger RNAs in yeast (Figure 6). Translation of the
cytochrome b mRNA depends on three nuclear encoded proteins, Cbs1, Cbs2,
and Cbp6 and is described in greater detail below. COX1 mRNA translation
requires Pet309 and Mss51 (83-86). The latter factor has a second, post15
translational role for Cox1 biogenesis, which is described later in more detail
(87,88). Pet111 is the only protein needed for translation of the COX2 mRNA
(89-92). In contrast, after identification of Pet494 as a translational activator for
COX3, two other proteins were described to fulfill this function, Pet54 and
Pet122 (70,78,79,93,94). Three subunits of the ATP synthase, all of which are
components of the membrane-embedded Fo part of the enzyme are encoded in
the mitochondrial DNA of yeast. No translational activator has been described
yet for the ATP8 mRNA. The only translational activator for ATP6 is Atp22
(95,96). Interestingly, ATP22 deletion mutants specifically lack Atp6 but show
normal translation rates of ATP8 although both proteins are derived from one
bi-cistronic transcript, suggesting the presence of a still missing translational
activator for ATP8. Three proteins influence translation of the ATP9 mRNA:
Aep1 (97,98) which acts as a translational activator, Aep2 (97,99) that is either
required for the stabilization of the ATP9 transcript or stimulating its translation,
and Atp25 (100) which has a dual role in translation and assembly of the ATP
synthase. The only soluble protein encoded in the mitochondrial DNA of S.
cerevisiae is Var1, a component of the small ribosomal subunit. How translation
of the VAR1 messenger is accomplished and whether it involves specific
translational activators is, like in the case of ATP8, not unraveled yet.
The specific activation of mitochondrial translation and the importance of the
5’-UTR of the mRNAs have been extensively described and are well
established in yeast and related fungi (101). However, the majority of
translational activators has no homologs in mammalian mitochondria (102). The
reason for this might be that most of the 13 mRNAs harbor none or only very
short 5’-UTRs (103). It is therefore not clear how translation is controlled in the
mammalian system. However, a possible homolog of one of the translational
activators for COX1, Pet309 exists in mammals, the protein LRPPRC (104).
Like Pet309, LRPPRC belongs to the class of PPR proteins, which harbor
pentatricopeptide repeats, a motif involved in protein-RNA interactions (84). In
contrast to the COX1-specific action of Pet309, however, the molecular function
of LRPPRC is not yet clear. Initially it was suggested that the protein does not
act in translation, but on a post-transcriptional level by stabilizing specifically
the COX1 and COX3 messengers (105). Very recently, a more general role of
LRPPRC in stabilization of mitochondrial mRNAs was reported (106).
Importantly, a recent study identified the first “true” translational activator in
mammalian mitochondria. TACO1 is specifically required for translation of the
16
COX1 mRNA (107). Although there is a TACO1 homolog present in yeast, it
has no essential role in mitochondrial translation (107). This suggests that the
general mechanisms to activate protein synthesis differ between yeast and
higher eukaryotes, however, the concept of specific translational activation of
distinct mRNAs might be conserved.
Synthesis of cytochrome b
Cytochrome b is the only subunit of the respiratory bc1 complex that is encoded
in the mitochondrial genome. Translation of its mRNA (COB mRNA) is
dependent on several factors (81) and involves excessive preceding maturation
of the transcript. The COB gene is co-transcribed with the adjacent tRNAGlu
(108). The resulting bi-cistronic precursor transcript thus has to be matured in a
complex process to yield the tRNA and the mature COB mRNA (Figure 7). The
tRNAGlu is excised from the initial transcript in a two-step process mediated by
RNase P (109) and 3’-endonuclease (110). The RNA part of the RNase P is
encoded in the mitochondrial genome and not only required to release the
tRNAGlu, but also other tRNAs from precursor transcripts (111). The COB gene
furthermore contains introns, some of which encode maturases (112), proteins
required for the excision of introns. Maturases can either act in cis and excise
introns within the transcript which encoded them (113) or in trans by processing
other intron-containing precursor RNAs (114,115). Independent from their
mode of action, synthesis of maturases depends on the translation of the
precursor transcript as the open reading frames encoding them are in frame with
the upstream cytochrome b exons (116). Additionally, the protein Cbp2 has been
shown to be involved in the excision of one intron of the COB mRNA precursor
(117-119).
17
Figure 7: Schematic representation of early steps in the synthesis of
Glu
cytochrome b. The COB gene is co-transcribed with the adjacent tRNA from a
promoter upstream of the tRNA gene. Processing of the initial bi-cistronic
Glu
transcript by RNase P and 3' endonuclease releases the tRNA and the preCOB mRNA with a 5'-UTR of 1098 nucleotides. Further modification of this
transcript, involving the Pet127-dependent degradation of the mRNA in 5'-3'direction until a region shielded by Cbp1 is reached, generates the mature 5'end of the COB mRNA (954 nt). A CCG triplet near the 5'-end is essential for the
Cbp1-mediated stabilization of the transcript. The terminal intron of the pre-COB
mRNA is excised with the help of Cbp2, while the other intron is removed by
intron-encoded maturases. The mature COB mRNA is finally translated on
mitochondrial ribosomes in dependence of the translational activators Cbs1,
Cbs2, and Cbp6. Different yeast strains harbor COB variants of different
lengths; here a short form consisting of three exons is depicted (E1, E2, E3).
The numbers mark positions within the 5'-UTR relative to the start AUG at +1.
IMM, inner mitochondrial membrane. IMS, intermembrane space. mtDNA,
mitochondrial DNA. UTR, untranslated region.
18
After cleavage of the tRNA from the precursor, the unprocessed COB transcript
(pre-COB) is further modified by a reaction involving Pet127. This protein
exerts or stimulates the 5’-3’ exonucleolytic degradation of the 5’-end of preCOB until a region is reached that is shielded by Cbp1 from nucleolytic
degradation (120). Yeast cells lacking Cbp1 therefore show strongly decreased
amounts of pre-COB mRNA and no matured COB mRNA (80,121). Whereas
Pet127 does not act specifically on the COB mRNA (122), the Cbp1-mediated
stabilization of the transcript requires the 5’-UTR of cytochrome b, as CBP1
deletion mutants can be suppressed by a gene rearrangement that generates a
COB transcript containing the 5’-UTR of the ATP synthase subunit Atp9 (121).
Accordingly, Cbp1 is required for stabilization of an mRNA containing the 5’UTR of COB, but the open reading frame of Atp9 (71). The sequence within the
long 5’-UTR of COB mRNA that is essential for Cbp1-mediated stabilization of
the transcript was step by step narrowed down to a single CCG triplet near the
5’-end (71,123-125). A single-base change within the CCG triplet leading to
degradation of the pre-COB transcript could be rescued by a single amino acid
substitution within Cbp1, pointing to a direct interaction between this protein
and the COB messenger (125).
In addition to Cbp1 and Cbp2, three other proteins are specifically required for
the synthesis of cytochrome b, which act as classic translational activators.
Yeast cells lacking either the product of the CBS1 or the CBS2 gene cannot
translate COB mRNA and accumulate the unprocessed pre-COB transcript
(126,127). Both proteins could therefore be involved in either splicing of the
precursor transcript or translation of the mRNA per se. The former hypothesis
was disproved by Muroff and Tzagoloff who showed that Cbs1 and Cbs2 are
needed for cytochrome b synthesis even in an artificially designed strain where
the COB gene does not contain any introns (128). The inability to excise introns
and accumulate the unprocessed pre-COB mRNA is thus presumably a
secondary effect in cbs1 and cbs2 cells as synthesis of the maturases encoded
within introns requires translation of the pre-transcript. Similar to the case of
Cbp1, the COB 5’-UTR seems to dictate the dependence on Cbs1 and Cbs2 for
translation. The same gene rearrangement that was able to rescue the CBP1
deletion strain by fusing the promoter and upstream leader sequence
(“encoding” the 5’-UTR) of ATP9 to the COB open reading frame (atp9::COB)
was able to suppress both CBS1 and CBS2 knockouts (126). Concomitantly, a
cob::COX3 mRNA could support the synthesis of the cytochrome oxidase
19
subunit Cox3 only in the presence of Cbs1, whereas translation of the authentic
COX3 mRNA was independent of Cbs1 (129). Ten years after identification of
Cbs1 and Cbs2, the region within the roughly 1000 nucleotides long COB 5’UTR necessary for recognition by the two translational activators could be
narrowed to the sequence -232 to -4 relative to the start AUG at +1 (71).
However, a direct interaction between either Cbs1 or Cbs2 and the 5’untranslated region of COB mRNA was not shown yet. Thus it is not clear
whether they act directly or indirectly through yet unknown components.
The last factor involved in cytochrome b translation characterized so far is
Cbp6. Opposed to cbs1 and cbs2 cells, the pre-COB transcript is processed
and matured similar to the wild type in the absence of Cbp6 (130). Despite this,
cytochrome b can only accumulate to hardly detectable level in the mutant. This
could be due to proteolytic degradation, but the authors at that time favored the
idea that Cbp6 stimulates translation, for example by facilitating the binding of
the mRNA to the ribosome or the formation of an initiation complex (130). In
contrast to the translational activators mentioned afore, the mutant phenotype of
cbp6 could not be suppressed by the atp9::COB gene rearrangement (131).
This suggests a mode of action for Cbp6 distinct from Cbs1 and Cbs2 and
points to an additional function of Cbp6 in cytochrome b biogenesis.
Possible functions of translational activators
Translational activators have been studied for more than three decades but it is
still unclear what their exact molecular function is. Based on the literature, four
different ways how translational activators act during translation can be devised:
1. Stabilization of the mRNA, 2. Support of translation initiation, 3.
Organization of translation, and 4. Regulation of translation. As there is
experimental evidence for all of these possibilities, translational activators
might not share one common function but rather exert their roles at different
steps of the translation process and sometimes have more than one function.
Stabilization of the mRNA
Several cases have been described where translational activators have a role in
stabilizing the transcripts. One example is Cbp1 that shields the mature and preCOB mRNA from nucleolytic degradation (120). Pet309 was shown to be
20
required for the stabilization of COX1 transcripts that contain introns (83).
However, as the levels of COX1 mRNA from an intronless strain were not
affected by the absence of Pet309, the protein does not seem to be essential to
support COX1 mRNA stability. Other examples are Pet111 with an
accumulation of COX2 mRNA to about one third of the wild type in a PET111
deletion strain (91) or Aep2, which is either implicated in stabilization or
maturation of the ATP9 transcript (97,99). Another protein that affects stability
of the ATP9 mRNA is Atp25. This protein is cleaved after its synthesis and the
two halves are functionally separable. Wheras the N-terminal part presumably
participates in assembly of the Atp9 ring, the C-terminal half of Atp25 stabilizes
the ATP9 transcript (100). Interestingly, some studies suggested that the
instability of the transcripts is only a secondary effect. According to this, the
block of translation caused by the absence of the respective translational
activator would make the mRNAs prone to degradation. However, an inability
to translate an mRNA does not generally lead to degradation of transcripts.
Thus, a role in conferring stability to mRNAs is one possible function of
translational activators.
Support of translation initiation
The most classical function of translational activators is believed to be the
support of translation initiation. As Shine-Dalgarno-like sequences are absent in
mitochondrial messengers, translational activators could help initiating
translation by assisting in loading the mRNA correctly onto the ribosome.
Interactions of translational activators with both the 5’-UTR and mitochondrial
ribosomes would support such an idea and have been proposed based on genetic
studies (132-135). In some cases, a direct interaction between the specific 5’UTR and translational activators has been suggested as amino acid residues
within the proteins could rescue base modifications in the mRNA (89,94,125).
Furthermore, translational activators can facilitate initiation of translation by
helping to deliver the mRNA to the membrane-bound mitochondrial ribosomes.
Many translational activators are peripheral or integral membrane proteins and
by binding the transcripts could localize them to the membrane and the
translation machinery (136). In this respect, one study described that productive
synthesis of Cox2 and Cox3 is only possible if mRNAs comprised their
authentic regulatory regions (137). The authors suggested the presence of
targeting information within the 5’-UTRs that ensures proper, translational
21
activator-mediated alignment of the mRNA with translation sites at the inner
mitochondrial membrane.
Organization of translation
Another intriguing idea for the function of translational activators is their
participation in organization of mitochondrial protein synthesis. Experimental
data support this in some general aspects of translational organization: For
example, Pet309 was found to be present in a complex of about 900 kDa that
also contained Cbp1 (138). Pet309 was independently shown to be in contact
with Nam1, a general mRNA metabolism factor (139). A third study revealed a
Nam1 interaction with the mitochondrial RNA polymerase in yeast (139,140).
From all these findings a model was proposed in which transcription, mRNA
maturation and protection is linked to translation at the inner mitochondrial
membrane (138). Moreover, the efficient assembly of cytochrome oxidase
requires, in addition to the general balance of nuclear and mitochondrial gene
expression, the adjusted synthesis of the three subunits of mitochondrial origin.
Considering how specific translational activation is exerted within
mitochondria, this may seem intricate. However, evidence that it is realized in
the organelle stems from the finding that the translational activators for COX1,
COX2 and COX3 interact with each other and therefore might generate sites at
the inner mitochondrial membrane dedicated for the assembly of cytochrome
oxidase (139).
Regulation of translation
Respiratory chain complexes and the ATP synthase are composed of subunits of
two genetic origins and the assembly of these complex machineries is a highly
intricate event. To allow efficient assembly of the respiratory chain, the
expression of mitochondrially and nuclear encoded subunits has to be
synchronized. Another well documented function of translational activators
therefore is the regulation of mitochondrial protein synthesis in response to the
efficiency of respiratory chain assembly (Figure 8). In recent years, studies of
cytochrome oxidase in yeast have revealed how regulation of mitochondrial
protein synthesis is accomplished and how the levels of mitochondrially
encoded subunits are adjusted to an efficient assembly process. A similar
situation was also shown for the ATP synthase. The coupling of synthesis and
assembly seems to be a conserved process as it has also been described for the
22
biogenesis of chloroplast photosystems and there been termed “control of
epistatic synthesis” (CES) (141).
Figure 8: Schematic representation of the feedback regulation of mitochondrial
protein synthesis. Translation in mitochondria requires specific translational
activators (TA). A TA can have a dual role in activating translation of its client
mRNA and assembly of the encoded protein into a respiratory chain complex.
Such a TA can mediate a regulatory feedback loop by sensing the efficiency of
complex assembly. Binding of the TA to the newly synthesized protein
sequesters it in an assembly intermediate. The TA is therefore not available for
activating translation, which causes reduction of the overall synthesis of the
mitochondrially encoded protein (-). Further assembly of the respiratory chain
complex releases the TA, which can again bind to the ribosome and stimulate
translation (+). By this, the level of a mitochondrially encoded protein is adjusted
to efficient assembly of the respiratory chain. IMM, inner mitochondrial
membrane. IMS, intermembrane space.
Assembly of cytochrome oxidase is initiated from Cox1, the central subunit of
complex IV. During its biogenesis this protein is equipped with two redoxactive cofactors, two heme molecules and one copper ion. Unassembled Cox1
equipped with those redox cofactors is potentially harmful for cells as it may
give rise to reactive oxygen species (142). The precise adjustment of Cox1
synthesis to levels that can successfully be incorporated into cytochrome
oxidase is therefore crucial for the integrity of cells. In recent years it was found
that Cox1 translation in yeast is subject to a complex feedback regulatory circle
(143). The key player of this feedback loop is the dually functioning protein
Mss51 that acts as a translational activator for COX1 mRNA as well as a Cox1
assembly factor by binding the newly synthesized protein (88). Mss51 is
23
sequestered in assembly sub-complexes that are unable to be resolved when
further assembly is blocked and thereby is precluded from activating new
rounds of COX1 translation. Aside of Mss51, several other factors participate in
the feedback regulation mechanism, namely Cox14, Coa3/Cox25, Ssc1, Coa1
and Coa2 (87,144-148). Cox14 and Coa3/Cox25 presumably play a role as
negative regulators of COX1 synthesis and are important for efficient
sequestration of Mss51 (144,149).
The Fo part of the ATP synthase comprises a proton channel through which
protons flow back from the IMS into the mitochondrial matrix driven by the
electrochemical gradient across the membrane. If the Fo part is not sealed by the
hydrophilic F1 unit, the membrane potential would be dissipated by passive
proton flow through the Fo part. In 2009, Rak and Tzagoloff found that
translation of the ATP8/ATP6 bi-cistronic mRNA is dependent on F1 (96).
Mutants lacking the F1 assembly factors Atp11 or Atp12 or the two main
structural F1 subunits  and  display reduced synthesis rates of Atp6 and Atp8.
Overexpression of Atp22, the translational activator of ATP6, was able to
suppress the phenotype of such mutants on Atp6 and partially also Atp8
synthesis, showing the importance of Atp22 in this regulatory circuit (96). This
reduction in synthesis of Atp6, which together with Atp9 forms the proton
channel, in response to the inability to assemble F1 is physiologically important
and prevents the dissipation of the membrane potential in the absence of F 1.
Although the F1-dependent assembly of Fo mechanistically differs from the
feedback-regulated expression of COX1 of cytochrome oxidase by the dual
functioning of Mss51, it provides another example of how organellar translation
is adjusted to the level of cytoplasmic protein synthesis.
Assembly of the yeast bc 1 complex
Respiratory chain complexes and the ATP synthase are composed of subunits
from two genetic systems and the assembly of these complex machineries is a
highly intricate process. Thus, it requires assistance of a number of so-called
assembly factors. These proteins are not part of the functional enzyme
complexes, but help to mediate and coordinate subunit interactions during
assembly and/or the incorporation of cofactors. In the case of cytochrome
oxidase, many proteins are involved in several steps during the assembly
24
process. In the following, the assembly of the bc1 complex and the participation
of the so-far characterized assembly factors are described in more detail.
One protein of the bc1 complex, cytochrome b, is encoded in the mitochondrial
genome and, in yeast, assembled together with nine nuclear encoded subunits
(150). Seven of these nine proteins are only accessory subunits that do not
participate in the catalytic reactions of the enzyme complex. Electron transport
is mediated by the redox-active prosthetic groups of the catalytic subunits
cytochrome b, cytochrome c1 and the Fe/S Rieske protein in a reaction
described as the Q cycle (7) (Figure 1B). Interestingly, the fully functional
cytochrome c reductase complex in bacteria is composed exclusively of these
three catalytic proteins (151), and the function of the accessory subunits in the
mitochondrial complex is largely unknown.
The general assembly line of the bc1 complex has been studied primarily by
Blue Native polyacrylamide gel electrophoresis (BN PAGE). Employing
mutants lacking individual bc1 complex subunits revealed a clear order in the
incorporation of proteins into the growing complex (152,153) (Figure 9).
Figure 9: Scheme of the step-wise assembly of the bc1 complex. Assembly
starts with membrane insertion of the mitochondrially encoded subunit
cytochrome b. After that, nine nuclear encoded proteins are added in a
coordinated fashion. To the cytochrome b-Qcr7-Qcr8 intermediate, a
subcomplex consisting of cytochrome c1 and the two core proteins Cor1 and
Cor2 is added as well as the accessory subunit Qcr6 to form the 500 kDa
complex. Into this assembly intermediate the last three nuclear encoded
subunits are incorporated, forming the functional bc1 complex. The five proteins
shown in bold in the upper part of the figure are assembly factors that ensure
efficient and successful assembly. Mzm1 and Bcs1 are known to play a role in
the incorporation of the Rieske protein (Rip1) into the complex, but the distinct
points during assembly when Cbp3, Cbp4 and Bca1 are required are not
known. The topology of the proteins is depicted according to (153). IMM, inner
mitochondrial membrane. IMS, intermembrane space. Modified from (154).
25
The assembly starts with the synthesis and membrane insertion of cytochrome
b. The nuclear encoded subunits are then added in a step-wise process involving
the formation of specific sub-complexes and assembly intermediates. The first
two subunits that are added to cytochrome b are Qcr7 and Qcr8. In parallel,
another module is formed containing the two core proteins Cor1, Cor2 and the
catalytic subunit cytochrome c1. This trimeric complex is then joined with the
cytochrome b-Qcr7-Qcr8 intermediate followed by the addition of the small,
acidic, intermembrane space subunit Qcr6 to generate the so-called 500 kDa
complex. Finally, the two accessory proteins Qcr9 and Qcr10 as well as the
third catalytic subunit, the Rieske Fe/S protein (Rip1) are incorporated to form
the mature bc1 complex. The fully assembled bc1 complex exists as a
homodimer in the inner mitochondrial membrane (150,155). It is unclear when
during assembly dimerization occurs. The dimer is further associated with
cytochrome oxidase complexes, either in their monomeric or their dimeric form
(156-158). This presumably stabilizes the complexes in the membrane and
moreover facilitates electron transfer by tight spatial proximity (159).
Three redox-active hemes are present in the bc1 complex, two heme b moieties
in cytochrome b and one heme c in cytochrome c1. A heme b residue that is
covalently linked to its surrounding protein backbone (exploiting cysteinemediated thioether linkages) defines the heme as c-type. Heme b biosynthesis is
a complicated process that takes place within mitochondria and in the cytosol. It
starts with the formation of aminolevulinic acid (ALA) from the amino acid
glycine and the citric acid cycle intermediate succinyl-CoA and ends with the
insertion of ferrous iron into protoporphyrin IX by ferrochelatase. Both of these
steps are exerted by enzymes of the mitochondrial matrix. Interestingly, many
hemylated proteins or protein domains reside outside this cellular compartment,
which implies the existence of a mechanism to allow the heme molecule to
traverse the inner mitochondrial membrane. It is not clear how exactly this is
accomplished. Furthermore, even for inner mitochondrial membrane proteins
like cytochrome b there is no experimental data that describes when, by which
factor or from which side of the inner membrane acquisition of heme occurs. In
contrast, hemylation of cytochrome c1 has been described to a certain degree.
The maturation of this protein is a complex process, requiring insertion of the
protein in an Nin-Cout topology into the inner mitochondrial membrane,
proteolytic cleavage and covalent attachment of the heme cofactor to the protein
(160). However, in the case of cytochrome c1 it is clear that hemylation occurs
26
prior to the processing step by proteases of the intermembrane space (161).
Heme is anchored to the apoprotein preferably by the Cyt2 heme lyase,
although it is not clear whether the factor itself mediates formation of the
thioether bond or just serves as a chaperone keeping apo-cytochrome c1 in a
conformation that enables hemylation (162).
Apart from Cyt2, several other non-subunit proteins have been described to play
a crucial role in the assembly of the bc1 complex. The functionally bestcharacterized ones are Bcs1 and the only recently identified Mzm1 (163). The
AAA-ATPase family member Bcs1 mediates translocation of the folded Fe/S
domain of the Rieske protein Rip1 across the inner mitochondrial membrane
and its incorporation into the 500 kDa complex assembly intermediate (164167). Bcs1 is assisted in these processes by Mzm1, which stabilizes Rip1 in the
matrix prior to membrane insertion (163). The interaction of Mzm1 with Rip1 is
mediated by the very C-terminal part of Rip1 (168). However, opposed to Bcs1,
the presence of Mzm1 is not mandatory for bc1 complex function as respiration
is only impaired when mzm1 cells are grown at elevated temperature (163).
Despite this, the protein is conserved in metazoans, suggesting that it might
have a similar role in bc1 complex assembly in higher eukaryotes (163).
Another only recently found assembly factor of complex III is the fungi-specific
Bca1 (169). The absence of Bca1 does not, similar to Mzm1, affect assembly
and function of complex III per se. It was proposed that Bca1 acts during bc1
complex assembly prior to the insertion of the Rip1 subunit (169).
In addition to the assembly factors Bcs1, Mzm1, and Bca1 the loss of two other
proteins independently leads to a strong impairment of bc1 complex assembly,
namely Cbp3 (170) and Cbp4 (171). Mutants lacking either CBP3 or CBP4
show a similar phenotype, including the absence of spectroscopically detectable
cytochrome b and other structural bc1 complex subunits (170,171). Cbp3 has
been suggested to act post-translationally as the mutant exhibits normal
maturation of the COB mRNA, but cytochrome b still fails to accumulate (170).
In 2001, Shi et al. characterized certain Cbp3 mutants for their ability to support
bc1 complex assembly and found that two regions within the protein are
functionally important (172). However, these regions seemed to affect complex
assembly in different ways, as structural complex III subunits were able to
accumulate to different extents in the mutants tested. It was furthermore
demonstrated that Cbp3 and Cbp4 can be co-purified with each other and are
27
present in high molecular mass complexes of similar size in BN PAGE analyses,
suggesting that they might act at the same step/s during bc1 complex assembly
(173). The exact molecular functions of these proteins, however, remained
unknown.
28
Aims of the thesis
Mitochondrial ribosomes are the central machineries of the gene expression
system in mitochondria. Two billion years of evolution led to extensive
remodeling of these particles so that they today differ substantially from their
bacterial ancestors. The process of translation and its organization in
mitochondria is in contrast to cytosolic protein synthesis only poorly
understood. During translation in bacteria and the cytosol of eukaryotes, the
tunnel exit region of the ribosome serves as a platform to organize early
maturation steps of the newly synthesized proteins. Many processing enzymes,
biogenesis factors and components of the membrane insertion machinery bind
to this site. The tunnel exit of mitochondrial ribosomes might serve an
equivalent purpose. However, it is not clear whether translation is similarly
organized in mitochondria or if the altered structure of mitochondrial ribosomes
introduces novel organizational features to the organellar system.
Using baker’s yeast as a model system, the aim of this thesis was to characterize
the tunnel exit region of mitochondrial ribosomes. Because there are no highresolution cryo electron microscopy structures of mitochondrial ribosomes
available that allow the identification of individual proteins, this work was
intended to provide insights into the molecular composition of the tunnel exit
region of mitochondrial ribosomes. Moreover, I aimed to identify factors that
bind to mitochondrial ribosomes and might exhibit functions in the membrane
insertion process of proteins encoded in the organellar DNA or serve as
biogenesis factors for respiratory chain complexes. A general description of the
interactome of mitochondrial ribosomes was supposed to be followed by the
comprehensive molecular characterization of distinct candidates.
29
Summaries of the Papers
Paper I: Proteins at the polypeptide tunnel exit of the yeast
mitochondrial ribosome
Mitochondrial ribosomes synthesize proteins encoded in the mitochondrial
genome. Those proteins are mainly hydrophobic membrane proteins that form
the central core subunits of the complexes of the respiratory chain. An important
site of every ribosome is the tunnel exit where the nascent chain emerges and
for the first time encounters a hydrophilic environment. In cytosolic ribosomes,
this region serves as a binding platform for proteins that mediate early steps in
the biogenesis of newly synthesized proteins. In contrast to bacterial and
eukaryotic cytosolic ribosomes, virtually nothing is known about the
composition and interaction partners of mitochondrial ribosomes. In this study,
we undertook a comprehensive approach to identify the composition of the
tunnel exit region of yeast mitochondrial ribosomes. The conserved proteins
that make up the rim of the polypeptide tunnel exit in bacteria, L22, L23, L24,
and L29, are also present in mitochondria (namely Mrpl22, Mrp20, Mrpl40 and
Mrpl4, respectively), although they contain, in addition to the conserved part,
N- and C-terminal extension domains. We constructed yeast strains that
harbored His7-tagged versions of the respective mitochondrial homolog in their
genome. Isolated mitochondria of these strains were incubated with chemical
crosslinking reagents to covalently link the His7-tagged protein to components
that bind to or are present in the vicinity of the mitochondrial ribosomal tunnel
exit. The obtained crosslink products were then purified via metal affinity
chromatography and subsequently identified by mass spectrometry. This method
revealed that Mba1, a factor that cooperates with Oxa1 in the insertion of
mitochondrially encoded proteins into the inner mitochondrial membrane, binds
in close proximity to Mrpl22 and Mrpl4. The Mba1 docking site on
mitochondrial ribosomes therefore does not overlap with the previously shown
contact point for Oxa1 (Mrp20 and Mrpl40; (62)), indicating that a
simultaneous binding of the two components to the translation machinery is
generally possible. Furthermore, this study led to the identification of three
ribosomal proteins, namely Mrpl13, Mrpl27 and Mrpl3, which are specific for
30
mitochondrial ribosomes and are located at the tunnel exit region. These results
substantiate the notion that mitochondrial ribosomes were dramatically
modified during evolution and that the tunnel exit region is no exception in this
respect. Especially at this site of the ribosome such a unique organization might
not only have structural reasons but could also introduce novel features to the
translation machinery that evolved in adaptation to the specific requirements of
the organelle.
Paper II: Cbp3-Cbp6 interacts with the yeast
mitochondrial ribosomal tunnel exit and promotes
cytochrome b synthesis and assembly
In the studies that led to Paper I, we applied a biochemical approach to identify
interaction partners of the yeast mitochondrial ribosome. The results obtained
included a protein named Cbp3, which we found as a crosslink partner of the
tunnel exit protein Mrpl4 and we chose to characterize it in more detail in this
study. Cbp3 was previously described as an assembly factor for the bc1 complex
of the respiratory chain (170). Here we show that its positioning in close
proximity to the polypeptide tunnel exit of the mitochondrial ribosome allows
Cbp3 to directly interact with newly synthesized cytochrome b. In the absence
of Cbp3, cytochrome b is rapidly degraded, indicating that Cbp3 exhibits an
essential stabilizing function for this mitochondrially encoded subunit of the bc1
complex. To address the question whether translation of the cytochrome b
mRNA (COB mRNA) also is affected in cells lacking Cbp3 we used a yeast
strain with a mitochondrial genome where the cytochrome b open reading frame
was exchanged to a recoded version of Arg8 (cob::ARG8m mtDNA). The
produced mRNA, importantly, still contains the 5’ and 3’ untranslated regions
(UTR) of cytochrome b that are the target of translational regulation. Thus, this
strain allows analyzing translation of an mRNA with COB UTRs independent
from the assembly of the respiratory chain, because Arg8 is a soluble protein of
the mitochondrial matrix involved in arginine biosynthesis. Using this strain, we
show that Cbp3 is specifically required for efficient translation of mRNAs that
contain the 5’- and 3’-untranslated regions of the cytochrome b mRNA. Cbp3
forms a tight complex with Cbp6, which has been described as a translational
activator for the cytochrome b mRNA (130). We furthermore provide evidence
31
that Cbp3 and Cbp6 do not exhibit separate functions, but rather stabilize each
other and that only the Cbp3-Cbp6 complex can bind to mitochondrial
ribosomes and fulfill two important roles in the biogenesis of cytochrome b. We
find that the Cbp3-Cbp6 complex binds to the tunnel exit of mitochondrial
ribosomes when no COB mRNA or its translation product is present. This
binding is a pre-requisite for efficient translation of the incoming cytochrome b
mRNA and represents the first function of the Cbp3-Cbp6 complex. As soon as
cytochrome b emerges from the ribosome, the Cbp3-Cbp6 complex fulfills its
second role and shields the newly synthesized protein. The trimeric Cbp3Cbp6/Cytb complex is released from the ribosome and Cbp4, another assembly
factor of the bc1 complex, is recruited. This non-ribosome-bound form of the
Cbp3-Cbp6 complex therefore likely represents an assembly intermediate of the
respiratory chain bc1 complex. By allowing efficient synthesis of cytochrome b
only when the Cbp3-Cbp6 complex is present at the tunnel exit of the ribosome,
it is ensured that the newly synthesized protein is shielded properly and can
successfully be channeled into the further assembly pathway.
Paper III: The Cbp3-Cbp6 complex coordinates
cytochrome b synthesis with bc1 complex assembly in yeast
mitochondria
In this study we continued our analysis on the Cbp3-Cbp6 complex and its role
during the biogenesis of the bc1 complex. Given the dual function of Cbp3Cbp6 in translation of the cytochrome b mRNA (COB mRNA) and assembly of
cytochrome b, we asked whether Cbp3-Cbp6 might have a more general role in
regulating cytochrome b synthesis in response to the efficiency of the assembly
process. We therefore generated yeast mutants that lacked individual structural
subunits of the bc1 complex or one of the known assembly factors. Such
mutants indeed showed decreased cytochrome b levels to varying extent.
However, such reduction could have different reasons: 1. the synthesized
cytochrome b could be degraded because of the failure to assemble into a
functional bc1 complex, 2. the translation of the COB mRNA could be directly
impaired in the absence of certain subunits or assembly factors or, 3. the rates of
cytochrome b synthesis could be decreased in response to a blocked assembly
process. As these three possibilities cannot be distinguished unambiguously in
32
cells with a wild type mitochondrial genome because of the overlap of
cytochrome b synthesis and assembly, we used yeast strains with engineered
mtDNAs. A direct impairment of COB mRNA translation was analyzed in
mutants harboring the cob::ARG8m mtDNA introduced in Paper II and led to the
finding that only Cbp3 and Cbp6, but no other assembly factors or structural
subunits of the bc1 complex are directly required for translation. To address
whether cytochrome b synthesis is regulated in response to the efficiency of bc1
complex assembly, we constructed a novel mtDNA by inserting a cytochrome b
gene into the cob::ARG8m genome (cox2::COB cob::ARG8m mtDNA).
Importantly, cytochrome b is not translated from its authentic mRNA, but from
an mRNA containing the 5’ and 3’-UTR of COX2. This strain is able to grow
normally on non-fermentable carbon sources and allows uncoupling translation
of a COB UTR-containing mRNA from assembly of cytochrome b. By directly
monitoring the Arg8 levels in assembly mutants we found that translation of the
cob::ARG8m mRNA indeed is modulated when cytochrome b fails to assemble.
Interestingly, Arg8 synthesis was only affected when early or intermediate, but
not late assembly steps were blocked, indicating that cytochrome b expression is
not in general reduced when a functional bc1 complex fails to assemble.
Furthermore in this work we investigated kinetics of the assembly process and
show that cytochrome b assembles through a series of four intermediates into
the bc1 complex. The Cbp3-Cbp6/Cytb/Cbp4 complex identified in Paper II is
the first intermediate that is formed. Upon addition of the first two nuclear
encoded subunits Qcr7 and Qcr8, Cbp3-Cbp6 is released from cytochrome b
whereas Cbp4 stays attached and intermediate II is generated. Incorporation of
the resulting nuclear encoded subunits completes assembly through
intermediates III and IV to the functional bc1 complex. If the process is blocked
at early or intermediate steps, assembly intermediates pile up and the Cbp3Cbp6-containing intermediate I accumulates. This leads to sequestration of
Cbp3-Cbp6, making the complex unavailable to stimulate new rounds of
translation and thereby causing the reduction in cob::ARG8m expression in such
mutants. Simultaneous overexpression of CBP3 and CBP6 allows overcoming
this feedback regulatory mechanism and regaining full ARG8m synthesis in the
assembly mutants. These data show that the efficiency of bc1 complex assembly
is sensed by the Cbp3-Cbp6 complex to modulate cytochrome b expression. In
the case of other respiratory chain complexes a regulated synthesis of
mitochondrially encoded subunits in response to complex assembly has
previously been demonstrated (87,96). Our work now shows this for the bc1
33
complex and thus provides the missing part to demonstrate that regulatory
circuits are a common feature for the biogenesis of respiratory chain complexes
of dual genetic origin.
Paper IV: The timing of heme incorporation into yeast
cytochrome b.
Cytochrome b is the only subunit of the eukaryotic bc1 complex that is encoded
in the mitochondrial genome. It is equipped with two redox-active heme b
moieties that are essential for the electron transfer reactions executed by this
respiratory chain complex. Heme biosynthesis is an intricate process that
involves both cytosolic and mitochondrial enzymes. The final step of this
pathway is the incorporation of ferrous iron into the protoporphyrin IX ring,
which is catalyzed by ferrochelatase in the mitochondrial matrix. It is not clear
when and how during cytochrome b biogenesis the protein is equipped with its
cofactors or which accessory proteins are involved in this process. In Paper III,
we described how cytochrome b is assembled through four distinct
intermediates into the functional bc1 complex. The first and the second
assembly intermediate contain assembly factors that could potentially act in
hemylation of cytochrome b. The Cbp3-Cbp6 complex binds to cytochrome b as
soon as it is fully translated and the recruitment of Cbp4 gives rise to assembly
intermediate I. Upon addition of the first two nuclear encoded structural bc1
complex subunits, Cbp3-Cbp6 is released from cytochrome b, whereas Cbp4
stays attached. This complex represents intermediate II. In this study we used
variants of cytochrome b that harbor point substitutions of individual histidine
residues implicated in heme coordination (H183T and H197F) to analyze when
during cytochrome b biogenesis the hemes are incorporated. Both mutants are
respiratory deficient. We find that a mutant that cannot acquire heme bL located
near the intermembrane space (IMS) side of the inner membrane is able to
assemble cytochrome b into supercomplexes. In contrast, the H197F mutant that
lacks heme bH on the opposite side of the membrane does not even reach the
state of intermediate I. Rather, this cytochrome b version is bound by the Cbp3Cbp6 complex but fails to recruit Cbp4. Furthermore, analyzing the heme
content of purified intermediate I by HPLC revealed that this complex contains
hemylated cytochrome b. These results show two important aspects of
34
cytochrome b biogenesis: First, our study proves that intermediate I of the bc1
complex, which serves as the pool of assembly-competent cytochrome b in wild
type mitochondria, already carries heme. Second, we reveal that there is a clear
order of incorporation of the heme moieties into cytochrome b, because
assembly can only proceed normally if heme bH is attached properly. This might
indicate that hemylation occurs from the intermembrane space side of the inner
mitochondrial membrane. Alternatively, the first heme (bH) could be
incorporated into cytochrome b from the matrix side and the second heme (bL)
from the IMS side. Furthermore our data implies that Cbp4 fulfills a crucial role
in the hemylation process by acting as a proofreading factor allowing further
assembly only when heme bH is incorporated.
35
Conclusions and future perspectives
The mitochondrial complexes of the respiratory chain and the ATP synthase
mediate the production of ATP, the energy currency of living cells, by oxidative
phosphorylation. These complexes are not only composed of subunits of nuclear
origin but also comprise a few proteins that are synthesized within
mitochondria. My work has provided new insights into the processes of
mitochondrial protein synthesis and assembly of the respiratory chain.
Mitochondrial ribosomes that translate the mRNAs encoded in the
mitochondrial genome differ remarkably from their bacterial ancestors. In
bacteria and the cytosol, the tunnel exit of ribosomes provides a binding site for
factors that mediate maturation of newly translated proteins. The analysis of
factors that bind to this site of mitochondrial ribosomes (Paper I) revealed the
presence of Mba1 at this region. Mba1 cooperates with Oxa1 in the insertion of
mitochondrially encoded proteins into the inner membrane. Furthermore, a
protein required specifically for the assembly of the bc1 complex (Cbp3)
interacts with this site of the ribosome. These findings show that the tunnel exit
region of mitochondrial ribosomes on the one hand serves as a platform to bind
general biogenesis factors like Mba1 and Oxa1 and on the other hand provides
binding sites for specific biogenesis factors for distinct respiratory chain
complexes. This implies that protein synthesis in mitochondria in some aspects
is organized similarly to the bacterial and eukaryotic cytosolic system and in
other aspects differs remarkably.
The biogenesis of the respiratory chain complex cytochrome oxidase has been
studied extensively over the past years and requires the action of a large number
of factors involved in translation, complex assembly or cofactor acquisition. In
contrast, the knowledge about the molecular events leading to the production of
the bc1 complex is still scarce. The list of accessory proteins involved in
assembly of this complex is growing recently and my work contributed
important aspects to this process. We could show that one of the translational
activators of the COB mRNA, Cbp6, and the complex III assembly factor Cbp3
form a structurally and functionally inseparable complex that exhibits
fundamental roles in the biogenesis of cytochrome b (Paper II). Cbp3-Cbp6
36
binds to mitochondrial ribosomes and stimulates, in a yet unknown manner,
efficient translation of the COB mRNA. Upon translation of the mRNA, the
Cbp3-Cbp6 complex binds to the fully synthesized cytochrome b and is released
from the ribosome. Our preliminary data indicate that at this stage heme
incorporation into cytochrome b occurs, with Cbp3-Cbp6 stabilizing the protein
in a conformation that allows efficient cofactor acquisition (Paper IV). At the
next step Cbp4 is recruited and the first intermediate of bc1 complex assembly is
formed. Addition of nuclear encoded structural bc1 complex subunits in a
coordinated fashion gradually releases the assembly factors from cytochrome b
and forms the dimeric complex III.
Paper IV of this work just gives a starting point to disclose the long standing
question of cytochrome b hemylation. Although we could answer that this
process happens very early during cytochrome b biogenesis, it still remains
unclear from which side of the membrane and by which factor/s it is
accomplished.
The dual subunit origin of respiratory chain complexes and the ATP synthase
requires regulated and coordinated expression of two genetic systems. It has
been shown for cytochrome oxidase and for the ATP synthase that
mitochondrial translation is modulated in response to the efficiency of assembly
of the respective complex. With demonstrating a coordination of cytochrome b
synthesis with bc1 complex assembly mediated by the Cbp3-Cbp6 complex
(Paper III), my work provides the missing part to prove that such regulatory
circuits are a common feature to adjust mitochondrial protein synthesis to the
success of respiratory chain assembly.
Several open questions, however, await clarification in future research. The
molecular mechanism by which translational activators fulfill their function has
not been unraveled for any of the mitochondrially encoded proteins. Moreover,
how can the Cbp3-Cbp6 complex that binds at the tunnel exit of mitochondrial
ribosomes stimulate translation of the COB mRNA? Possible scenarios would
be a crosstalk between Cbp3-Cbp6 and the other two translational activators of
COB, Cbs1 and Cbs2 or the transmission of conformational changes within the
ribosome by binding of Cbp3-Cbp6 upon translation.
The finding that mitochondria-specific ribosomal proteins are present at the
tunnel exit region of mitochondrial ribosomes (Paper I) suggests the intriguing
37
idea that the great evolutionary remodeling of mitochondrial ribosomes not only
has structural relevance but introduces novel organizational features specific for
the translation system in organelles. According to this, distinct ribosomal
proteins might serve as binding sites for certain translational activators or
assembly factors, optimizing the synthesis of one specific translation product.
The findings I gathered during my studies of the biogenesis of the mitochondrial
bc1 complex give room for speculations about the organization of mitochondrial
protein synthesis on a higher level. In particular, the results could suggest that
distinct mitochondrial ribosomes are dedicated to specifically support the
biogenesis of one mitochondrially encoded client protein (9). In this respect,
mitochondria-specific ribosomal proteins could serve as individual binding sites
for specific translational activators, thereby priming the ribosome for synthesis
of one translation product. Furthermore, the mitochondria-specific ribosomal
proteins at the tunnel exit region could recruit specific assembly factors required
to support maturation of the respective translation product and its assembly into
the respiratory chain complex. By this it would be ensured that synthesis of one
specific protein is optimally organized to allow efficient assembly of the
respiratory chain (Figure 10).
Figure 10: Hypothetical model of dedicated mitochondrial ribosomes.
Mitochondrial ribosomes get primed for the synthesis of one specific translation
product by the binding of a certain translational activator 1 (left).
Simultaneously, factors are recruited to the tunnel exit of the ribosome that
specifically guide assembly and co-factor acquisition of the translated protein.
The addition of the mRNA to the primed ribosome complex, for example by
interaction of a translational activator 2 with translational activator 1, initiates
translation (right). This organization ensures efficient maturation and assembly
of the newly synthesized protein into a respiratory chain complex. IMM, inner
mitochondrial membrane. IMS, intermembrane space. Modified from (9).
38
In addition to my own findings that ectopically expressed cytochrome b is
synthesized efficiently, but fails to accumulate to wild type level (Paper III), a
model of dedicated ribosomes is also supported by the work of others. For
example, in one study mitochondrial genomes were constructed that allowed
synthesis of Cox2 or Cox3 from an mRNA containing the 5’-UTRs of the only
soluble mitochondrially encoded protein Var1 (var1::COX2 and var1::COX3)
instead of the authentic sequences (137). This setting led to robust synthesis of
those two subunits of cytochrome oxidase, but they as well failed to accumulate.
These and my own results could indeed be explained in a scenario involving
dedicated ribosomes. If incorrect biogenesis factors would be present at a
ribosome primed for the synthesis of another mitochondrially encoded protein,
the protein translated from the foreign ORF would not be properly matured and
assembled, resulting partly in its degradation. Thus, the ectopic expression of
proteins encoded in the mitochondrial DNA from non-authentic messengers
would not be as optimally organized as the endogenous synthesis and
maturation pathway. However, the authors of the other study chose an
alternative interpretation of their data and suggested that 5’-UTRs contain
targeting information that ensures proper, translational activator-mediated
alignment of the mRNA with translation sites at the inner mitochondrial
membrane (137). Although there are other studies that used similar yeast strains
to ectopically express mitochondrial genes, many of them lack information to
which degree the proteins could accumulate (71,90,121,126,129). It will be
exiting to test the hypothesis of dedicated ribosomes in future experiments and
unravel how exactly mitochondrial translation is organized to allow efficient
assembly of the respiratory chain.
39
Sammanfattning på svenska
Det inre membranet i mitokondrier innehåller de proteinkomplex som ingår i
andningskedjan och ATP-syntas, som katalyserar den viktiga metaboliska
processen, oxidativ fosforylering. Komplexen består av subenheter från två
olika genetiska ursprung: A) Majoriteten av beståndsdelarna syntetiseras på
cytosoliska ribosomer och importeras till mitokondrierna och B) proteiner som
representerar centrala katalytiska subenheter kodas i mitokondrie-DNA och
produceras på mitochondriella ribosomer. Trots sitt endosymbiotiska ursprung
skiljer sig mitokondriernas translationsmaskineri signifikant från dess
bakteriella förfader. Hur mitokondriella ribosomer fungerar och hur translation
är organiserad i organellen är dock fortfarande inte helt klarlagt. ’Tunnel exit
region’ i en ribosom är platsen där ’nascent chain’ kommer ut och exponeras till
en hydrofil omgivning för första gången. I cytosoliska ribosomer, finns
proteiner som skapar en ’rim of the tunnel exit’ och bildar en plattform där
biogenetiska faktorer kan binda och hjälpa till i mognaden av ny syntetiserade
proteiner. Genom att använda proteomic strategi och bagerijäst som en
modelorganism har jag definierat sammansättningen av den mitokondriella
ribosom ’tunnel exit’ och analyserat om den här platsen fungerar på ett liknande
sätt som i cytosoliska ribosomer. Denna studie gav inte bara insikt i den
strukturella sammansättningen av mitokondriella ribosomer, utan visade även
placeringen av Cbp3 (en chaperon som krävs speciellt för ihopsättning av bc1
komplexet) vid ’tunnel exit’. Vidare fann jag att Cbp3 strukturellt och
funktionellt bildar ett tätt komplex med Cbp6 och att detta komplex har en
grundläggande roll i den biogenes av cytokrom b, den mitokondriellt kodade
subenheten i bc1 komplexet. Bundet till ribosomen, stimulerar Cbp3-Cbp6
translation av cytokrom b-mRNA (COB-mRNA). Cbp3-Cbp6 binder helt
syntetiserade cytokrom b, stabiliserar och styr därigenom den fortsatta
ihopsättning av bc1 komplexet. Nästa steg involverar rekryteringen av ännu en
ihopsättnings faktor (Cbp4) till Cbp3-Cbp6/cytokrom b-komplexet och
förmodligen insättning av två redoxaktiva heme b faktorer. Under den fortsatta
ihopsättning släpper Cbp3-Cbp6 från cytokrom b, kan åter binda till ribosomen
och aktivera ytterligare rundor av COB mRNA translation. Den dubbla rollen
hos Cbp3-Cbp6 i både translation och ihopsättning tillåter komplexet att fungera
40
som en reglerande omkopplare för att modulera nivån av cytokrom b-syntes
som svar på hur effektiv bc1 komplex som bildats.
41
Deutsche Zusammenfassung
Die mitochondriale Innenmembran beherbergt die Komplexe der Atmungskette
und ATP-Synthase, die einen der wichtigsten metabolischen Prozesse, die
oxidative Phosphorylierung, durchführen. Die Komplexe der Atmungskette und
ATP-Synthase bestehen aus Untereinheiten, die von zwei unterschiedlichen
genetischen Systemen bereitgestellt werden: Während Mitochondrien selbst
eine kleine Anzahl an Proteinen, die zentrale katalytische Untereinheiten der
Atmungskettenkomplexe sind, herstellen, werden die verbliebenen
Komplexkomponenten durch Ribosomen im Zytosol synthetisiert. Trotz des
endosymbiontischen Ursprungs von Mitochondrien unterscheidet sich die
organelläre Translationsmaschinerie deutlich von der ihrer bakteriellen Ahnen.
Der Tunnelausgang eines Ribosoms ist die Stelle, an der die naszierende Kette
während der Translation austritt und zum ersten Mal einer hydrophilen
Umgebung ausgesetzt wird. In zytosolischen Ribosomen bilden die Proteine um
den Tunnelausgang eine Plattform für die Bindung von Biogenese-Faktoren, die
die Maturierung des neusynthetisierten Proteins unterstützen. Mittels eines
proteomischen Ansatzes und dem Modellorganismus der Bäckerhefe habe ich
untersucht, ob der Tunnelausgang mitochondrialer Ribosomen einem ähnlichen
Zweck dient und welche Faktoren dort binden. Diese Studie verlieh einerseits
Einblicke in den Aufbau mitochondrialer Ribosomen und enthüllte andererseits
die Bindung von Cbp3, eines Chaperons für die Assemblierung des bc1Komplexes, an den Tunnelausgang. Im weiteren Verlauf meiner Arbeit konnte
ich zeigen, dass Cbp3 strukturell und funktionell mit Cbp6 interagiert. Der
Cbp3-Cbp6-Komplex übt fundamentale Funktionen in der Biogenese von
Cytochrom b, der mitochondrial kodierten Untereinheit des bc1-Komplexes,
aus. In seiner Ribosomen-gebundenen Form stimuliert Cbp3-Cbp6 die
Translation der Cytochrom b-mRNA (COB-mRNA). Cbp3-Cbp6 bindet dann
das vollständig synthetisierte Cytochrom b, stabilisiert und geleitet es durch
weitere Schritte der Komplexassemblierung. Diese weiteren Schritte beinhalten
die Rekrutierung eines anderen bc1-Komplex-Assemblierungsfaktors, Cbp4,
sowie die Inkorporation der beiden redoxaktiven Häm-Gruppen in Cytochrom
b. Während der weiteren Assemblierung dissoziiert Cbp3-Cbp6 von Cytochrom
b, kann wieder an das Ribosom binden und neue Runden der COB-mRNATranslation aktivieren. Die duale Rolle von Cbp3-Cbp6 während der Biogenese
42
von Cytochrom b ermöglicht es dem Komplex, als ein Schalter zur Modulation
der mitochondrialen Cytochrom b-Expression entsprechend den momentanen
Ansprüchen der Zelle an den Komplex III-Assemblierungsstatus zu fungieren.
43
Acknowledgements
A lot of people have accompanied me during the last 4 ½ years of my time as a
PhD student and helped making this work possible and I would like to thank all
of them.
First of all an enormously big “thank you” goes to Martin Ott. Despite my
request for several weeks of consideration time (I now found the old email
again and admit that I really was so bold to write “several weeks” – sorry for
always having denied this ) you gave me the ability to do my PhD thesis in
your group. It was a terrific time and I learned a great deal from you, not only
scientifically. Thanks for your constant support, input and motivation!
I would like to thank our collaboration partners Alex Imhof and Lars Israel
from the LMU in Munich, Germany, who did all the mass spectrometric
analysis and thereby laid the basis for the rest of my studies. I thank Inge Kühl
and Nathalie Bonnefoy, Brigitte Meunier and Geneviève Dujardin from
CNRS in Yif-Sur-Yvette, France for their excellent expertise in manipulating the
yeast mitochondrial genome and exploiting this technique to create the
cox2::COB cob::ARG8m and the cytochrome b heme mutant strains as well as
for several nice get-togethers in Paris and Kaiserslautern. I am very grateful to
Alex Tzagoloff from Columbia University in New York, USA, who shared a lot
of unpublished data and material and inspired many ideas concerning the Cbp3
projects on more than one occasion. Although it wasn’t a collaboration in the
common sense, I would like to thank Per Ljundal, Claes Andréasson and
their whole groups at the Wenner Gren Institute at Stockholm University for
vivid, stimulating and constructive discussions during our Thursday seminars,
which I enjoy very much!
Many thanks to the complete Hannes Herrmann group how it existed in April
2008 when I started my PhD. The decision to leave my family and friends
almost 500 km behind (which at that point seemed a huge distance) for 3 years
or more and move from Leipzig to Kaiserslautern was not an easy one. But all
of you gave me such a warm welcome that in the end it wasn’t so hard at all
choosing to leave Leipzig to work with you guys and join your team. I never
regretted that!
44
Some people from the time back in Kaiserslautern should be mentioned
especially: Andrea – thanks for all your support, in the everyday lab work as
well as everyday life matters and for all the fun we had in the lab! Simone –
you took care of pretty much everything and helped in every possible way with
organizational, bureaucratic matters and much more. Thank you so much for
that! Martin P., Kirsten, Kerstin G., Steffi H., Kathi and Ramon – it was
always a pleasure working with you and I am happy to have you as friends. We
shared so many great moments inside and outside the lab and I hope, despite the
fact almost all of us are distributed across Europe, we will manage to keep our
friendship in the future. Kerstin K. – thanks for your friendship, for all the fun
we had and bread we shared at Southside and for being a great roommate. I
hope we keep in touch! Janne – thanks for all the critical discussions during the
seminars, your input and for opening my mind to the mysterious and exciting
world of cysteines .
In September 2011 the whole Martin Ott group moved to the Department of
Biochemistry and Biophysics at Stockholm University and I am grateful for
the warm welcome we experienced from everyone here. I thank all the people,
who helped “squeezing” me into the system, so that I have the great honor to be
awarded with a PhD from Stockholm University. Many thanks to Stefan for
always having an open door and ear, to Gunnar for being my co-supervisor and
to Elzbieta for being my mentor. Unfortunately, working on the 5th floor and
spending not even 1 ½ years at the department does not contribute to getting to
know everyone at DBB better. But still I would like to thank all present
members of DBB for creating a cheery working environment. Special thanks
go to Beata, Pedro, Candan, Catarina and Diogo – you guys have become
good friends to me and I really enjoyed being with you on many occasions and
experiencing great food and lots of fun on all of them ! I hope we will keep in
touch in the future. Ljubica, Amin, Dominik and Manfred – thank you for
being great office mates! Another big thanks to Dominik and his friend Fredrik
for managing the thankless task of proofreading my summary in Swedish .
Some people always or at least for very long have been part of my life and
hopefully this will stay like that in the future. I thank my family for their love,
constant support and for all the precious moments we had and will have
together in the future. Danke Mutti, Roland, Paps, Karin, Christoph, Oma
und Opa (ich weiß du bist bei mir, siehst von irgendwo her auf mich herab und
bist wahnsinnig stolz auf mich!) für eure Liebe, Unterstützung in allen
45
erdenklichen Lebenslagen und dafür, dass ihr immer für mich da seid! For a
little bit more than three years now I have a second family in the Odenwald.
Karin, Dieter, Boris, Aline, Karin, Wilfried und dem ganzen Rest des
Hildenbeutel/Eckel-Clans – danke, dass ihr mich so herzlich und warm in eure
Familie aufgenommen habt, für eure Unterstützung in allen Belangen und die
Freude, die ihr bereitet. I thank all my close friends from back home for the
times we shared, the fun we had together and for all what friends are for,
because this is exactly what they gave me! Nicki – there is not much to say... I
am glad that I met you! Thank you for everything! Nadine, Sandra and
Franzi, our friendship goes back till school times, I am happy and proud that
we managed to keep it over the long distances between us and am convinced
this won’t change in the future!
Save the best for the last: Markus. Thanks for your love, support, friendship
and everything else you give me. You bring joy to my world every day and I
cannot express how much I am looking forward to our future together!
46
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