Schizosaccharomyces pombe homologs of the Saccharomyces cerevisiae mitochondrial proteins Cbp6 and Mss51 function at a post-translational step of respiratory complex biogenesis.

Abstract

Complexes III and IV of the mitochondrial respiratory chain contain a few key subunits encoded by the mitochondrial genome. In Saccharomyces cerevisiae, fifteen mRNA-specific translational activators control mitochondrial translation, of which five are conserved in Schizosaccharomyces pombe. These include homologs of Cbp3, Cbp6 and Mss51 that participate in translation and the post-translational steps leading to the assembly of respiratory complexes III and IV. In this study we show that in contrast to budding yeast, Cbp3, Cbp6 and Mss51 from S. pombe are not required for the translation of mitochondrial mRNAs, but fulfill post-translational functions, thus probably accounting for their conservation.

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Schizosaccharomyces pombe homologs of the Saccharomyces cerevisiae
mitochondrial proteins Cbp6 and Mss51 function at a post-translational
step of respiratory complex biogenesis
Inge Kühla, Thomas D. Foxb, Nathalie Bonnefoya,⁎
aCentre de Génétique Moléculaire du CNRS, UPR 3404, FRC3115, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France
bDepartment of Molecular Biology and Genetics, Cornell University, Ithaca NY 14853, USA
a b s t r a c ta r t i c l e i n f o
Article history:
Received 28 October 2011
Received in revised form 31 January 2012
Accepted 3 February 2012
Available online 10 February 2012
Keywords:
Schizosaccharomyces pombe
Saccharomyces cerevisiae
Mitochondria
mRNA-specific translational activator
Protein stability
Translation–assembly coupling
Complexes III and IV of the mitochondrial respiratory chain contain a few key subunits encoded by the
mitochondrial genome. In Saccharomyces cerevisiae, fifteen mRNA-specific translational activators control
mitochondrial translation, of which five are conserved in Schizosaccharomyces pombe. These include homo-
logs of Cbp3, Cbp6 and Mss51 that participate in translation and the post-translational steps leading to the
assembly of respiratory complexes III and IV. In this study we show that in contrast to budding yeast,
Cbp3, Cbp6 and Mss51 from S. pombe are not required for the translation of mitochondrial mRNAs, but fulfill
post-translational functions, thus probably accounting for their conservation.
© 2012 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
1. Introduction
Mitochondria have their own genome and associated transcrip-
tion and translation machineries. The respiratory complexes, embed-
ded in the inner mitochondrial membrane are composed of subunits
encoded by both nuclear and mitochondrial genes. The translation
apparatus itself is also of dual genetic origin, with rRNAs and many
tRNAs encoded by the mitochondrial DNA (mtDNA), while depending
upon the species, most or, all of the protein components are encoded
by the nucleus and imported into mitochondria (Towpik, 2005;
Watanabe, 2010).
The control of mitochondrial translation in Saccharomyces cerevisiae
is very complex (Towpik, 2005). Synthesis of each of the 8 major pro-
teins encoded by the mtDNA requires mRNA-specific translational acti-
vator proteins encoded in the nucleus (Table 1). Genetic suppression
studies have shown that at least seven of the activators needed for the
translation of four mitochondrially encoded mRNAs function exclusive-
ly through the 5′-untranslated regions (UTRs) of their target mRNAs
(Table 1). In these cases, the requirement for the cognate activator can
be bypassed if its target ORF and 3′-UTR are fused to a 5′-UTR derived
from a different mRNA and under the control of another translational
activator. For example, Cbs1 and Cbs2 operate in this way on the CYTb
mRNA encoding apo-cytochrome b (Cytb) (Rödel, 1986; Rödel and
Fox, 1987). These activators are also indirectly required for the splicing
of the CYTb mRNA precursors, since several of their introns encode RNA
maturases that are essential for the excision of the introns that encode
them (Banroques et al., 1986).
In general, translation activators that function exclusively through
5′-UTR targets are not highly conserved in amino acid sequence
among budding yeast species, although the function of two of them
have been shown to be orthologously conserved among members of
that group (Costanzo et al., 2000). Translation activators are rate
limiting for the expression of COX1, COX2 and COX3 in S. cerevisiae
(Green-Willms et al., 2001; Perez-Martinez et al., 2009; Steele et al.,
1996), and play a role in the topological organization of gene expres-
sion at the surface of the inner membrane (Krause et al., 2004;
Naithani et al., 2003; Sanchirico et al., 1998). However, except for
the COX1 mRNA-specific activator Pet309, which has experimentally
verified orthologs in Schizosaccharomyces pombe (Ppr4) (Kühl et al.,
2011) and Neurospora crassa (CYA-5) (Coffin et al., 1997), no clearly
homologous proteins have been identified outside of the budding
yeast clade.
Translation of the S. cerevisiae COX1 mRNA also requires a more
complex activator, Mss51 (Perez-Martinez et al., 2003; Siep et al.,
Mitochondrion 12 (2012) 381–390
Abbreviations: aa, amino acids; bp, base pair; mt-mRNA, mitochondrial mRNA;
mtDNA, mitochondrial DNA; nt, nucleotides; ORF, open reading frame; UTR, untrans-
lated region.
⁎ Corresponding author at: Centre de Génétique Moléculaire, CNRS Bâtiment 26,
1 Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France. Tel.: +33 1 69 82 31
75; fax: +33 1 69 82 31 60.
E-mail address: bonnefoy@cgm.cnrs-gif.fr (N. Bonnefoy).
1567-7249/$ – see front matter © 2012 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
doi:10.1016/j.mito.2012.02.002
Contents lists available at SciVerse ScienceDirect
Mitochondrion
journal homepage: www.elsevier.com/locate/mito

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2000). In addition to acting upon the 5′-UTR of the COX1 mRNA
(Perez-Martinez et al., 2009), Mss51 interacts with the newly synthe-
sized Cox1 protein and is required for the synthesis of Cox1 from a
chimeric mRNA bearing the COX2 5′-UTR (Perez-Martinez et al.,
2003). Mss51 is present in early complex IV assembly intermediates
containing Cox1 and other assembly proteins and is presumably
required for assembly (Mick et al., 2007; Pierrel et al., 2007). Thus,
Mss51 appears to couple Cox1 synthesis to complex IV assembly in
an assembly-feedback loop by virtue of the fact that it cannot activate
translation when sequestered in assembly intermediates (Barrientos
et al., 2004; Fontanesi et al., 2010a; Mick et al., 2011; Perez-Martinez
et al., 2009; Shingu-Vazquez et al., 2010).
Similarly, two factors involved in complex III biogenesis also ap-
pear to have a dual function, promoting both synthesis and assembly
of cytochrome b: Cbp3, first described as an assembly factor
(Kronekova and Rödel, 2005; Wu and Tzagoloff, 1989), and Cbp6,
first proposed to be a translation factor (Dieckmann and Tzagoloff,
1985). Translation of the S. cerevisiae CYTb mRNA is reduced, but
not eliminated by nuclear cbp3 and cbp6 mutations (Dieckmann and
Tzagoloff, 1985; Gruschke et al., 2011). In addition, Cbp3 and Cbp6
have recently been shown to interact to form a complex that is asso-
ciated both with the exit tunnel of mitochondrial ribosomes and an
early assembly intermediate of respiratory complex III, containing
the assembly factor Cbp4 (Gruschke et al., 2011). These two functions
of the Cbp3/Cbp6 complex would allow coupled synthesis and assem-
bly of cytochrome b.
Interestingly, the S. pombe genome encodes proteins highly ho-
mologous to Mss51, Cbp3 and Cbp6 from S. cerevisiae, which are all
more conserved among fungi than other budding yeast translation ac-
tivators (Figure S1). It is of great interest to explore the function of
these proteins in S. pombe, since many aspects of its mitochondrial
gene expression system more closely resemble that of animals than
that of budding yeast. The mtDNA has two principal promoters
of different strengths, yielding two overlapping primary transcripts
that are generally processed by tRNA excision and cleavage down-
stream of a C-rich motif to generate the rRNAs and mRNAs containing
short UTRs (Schäfer, 2005). Finally, S. pombe is a petite-negative yeast,
whose respiratory physiology is closer to that of animal cells than
S. cerevisiae (Chiron et al., 2007)
In this study we have investigated the function of Mss51, Cbp3
and Cbp6 in S. pombe, and found that they only act at post-
translational steps in mitochondrial biogenesis. Since the functions
of Mss51, Cbp3 and Cbp6 conserved in these two widely divergent
species are post-translational, our findings suggest that their role in
respiratory complex assembly may be their ancestral role, while
their role in controlling budding yeast translation may be a special-
ized adaptation for facultative anaerobiosis.
2. Materials and methods
2.1. Strains, plasmids, media and genetic methods
All strains are described in Table 2 and were grown at 28 °C as indi-
cated. The wild type S. pombe strains used were NB205-6A (h- ade6-
M216 ura4-D18 his3∆ leu1-32 rho+[3 mitochondrial introns]) and
NB34-21A (h- ade6-M216 ura4-leu1-32 ptp1-1 rho+[3 mitochondrial
introns]) (Chiron et al., 2005). Plasmids used or constructed during
this work were derivatives of pGEM-T-easy (Promega #TM042),
pDUAL-FFH1 and pDUALYFH1 (Matsuyama et al., 2004, 2006),
pTG1754/NotI (Bonnefoy et al., 1996), or pFL61 (Minet et al., 1992).
Media and genetic methods were as reported previously (Bonnefoy et
Table 1
mt-mRNA specific factors acting in S. cerevisiae mitochondrial translation and their
conservation in fission yeast and humans.
S. cerevisiaeS. pombe
Target
mRNA
Translational
activator
PPR
motifs
Acts through:
5′UTRother sequences Homolog (PPR motifs)
VAR1
CYTb
Sov1
Cbs1
Cbs2
Cbp1
Cbp3
Cbp6
Pet309
Mss51
Pet111
Pet54
Pet122
Pet494
Atp22
Aep1/Nca1
Aep2/Atp13
3
−
−
3
−
−
13
−
5
−
−
−
2
2
4
?
+
+
+
+
+
+
+
+
+
+
+
+
?
+
?
−
−
?
Cytb protein
Cytb protein
?
Cox1 protein
−
−
−
−
?
?
−
−
−
−
−
Spcc4b3.171
Spbc947.14c2
Ppr4 (13)
Spac25b8.04c3
−
−
−
−
−
−
Ppr6 (5)
COX1
COX2
COX3
ATP6/8
ATP9
Cbp3, Cbp6 and Mss51 data are in bold.
Alignment parameters between the S. cerevisiae and S. pombe homologs using
the PIR server (http://pir.georgetown.edu/pirwww/search/pairwise.shtml) were the
following:
1: Z-score: 603.7; E-value: 8.1e-32; Identity : 35.2% (38.9% ungapped) in 284 aa overlap
(16-275:11-291).
2: Z-score: 137.3; E-value: 7.7e-06; Identity : 26.3% (27.0% ungapped) in 76 aa overlap
(23-96:77-152).
3: Z-score: 1035.8; E-value: 6.9e-56; Identity : 36.7% (39.7% ungapped) in 398 aa
overlap (1-371:37-431).
Data extracted from: Chen and Dieckmann, 1997; Dunstan et al., 1997; Ellis et al., 1999;
Fox, 1996; Gruschke et al., 2011; Islas-Osuna et al., 2002; Kühl et al., 2011; Lipinski et
al., 2011; Manthey and McEwen, 1995; Mick et al., 2011; Payne et al., 1993; Sanchirico,
1998; Zeng et al., 2007; for review, see Towpik, 2005.
Table 2
Strains used and constructed in this work.
Name Nuclear genotypeReference
S. pombe
NB205-6A
IK107-7
h- ade6-M216 ura4-D1.8 his3∆ leu1-32 [rho+]
h- ade6-M216 ura4-D1.8 his3∆ leu1-32
∆cbp3::kanR[rho+]
h- ade6-M216 ura4-D1.8 his3∆ leu1-32
∆cbp6::kanR[rho+]
h- ade6-M216 ura4-D1.8 his3∆ leu1-32
∆mss51::kanR[rho+]
h- ade6-M216 ura4-D1.8 his3∆ leu1-32
∆cbp6::kanRleu1+::cbp6-YFP-FlagHis6[rho+]
h- ade6-M216 ura4-D1.8 his3∆ leu1-32
∆mss51::kanRleu1+::mss51-Flag2His6rho+
h- ade6-M216 ura4-D1.8 his3∆ leu1-32
∆ppr4::kanR[rho+]
h- ade6-M216 ura4-D1.8 his3∆ leu1-32
∆ppr6::kanR[rho+]
h? ade7-50 leu1-32 [cytb-∆5411]
Chiron et al., 2005
This work
IK1-4
This work
IK3-1
This work
IK38-B
This work
IK42-6
This work
IK39-3
Kühl et al., 2011
IK91-4
Kühl et al., 2011
NB324-11D
N. Bonnefoy
S. cerevisiae
CW252MATx ade2-1, his3-11,15, trp1-1,
leu2-3,112 ura3-1 can1-1 [rho+∆i2]
MATx ade2-1, his3-11,15, trp1-1, leu2-3,112
ura3-1 can1-1 ∆cbp6 ::kanR[rho+∆i2]
MATx ade2-1, his3-11,15, trp1-1, leu2-3,112
ura3-1 can1-1 [rho+ Σi3]
MATx ade2-1, his3-11,15, trp1-1, leu2-3,112
ura3-1 can1-1 ∆cbp6 ::kanR[rho+ Σi3]
MATx his3∆1 cbs1::TRP1 [rho+5]
Saint-Georges
et al., 2002
This work
IK43-1
CW04
Banroques et al.,
1986
This work
IK44-5
MCC624
Costanzo and
Fox, 1988
Costanzo and
Fox, 1988
This work
MCC60R2-164
MATa ade2 his3∆1 leu2-3,112 ura3-52
cbs1::TRP1 [rho+5, MSUcbs1-26]
MATa ade2 his3∆1 leu2-3,112 ura3-52
cbs1::TRP1 ∆cbp6 ::kanR[MSUcbs1-26]
IK97-1
1541 bp deletion in the cytb gene derived from strain EB7 (Ahne et al., 1988).
2Intron-less genome with corrected CYTb sequence.
313 Intron-containing genome, W303 background.
4May contain the trp1-289 mutation.
58 Intron-containing genome, D273 background.
6Rearranged mitochondrial genome, with sequences coding the 5′UTR region of
COX3 down to -173 fused to sequences coding the CYTb 5′UTR starting at -6.
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al., 1996, 2000). S. pombe transformation (Okazaki et al., 1990) was im-
proved by (1) using single stranded salmon sperm DNA as carrier, (2)
regeneratingcellsincompleteliquidmediumovernight,and(3)plating
onto 5% glucose selective medium as described in Chiron et al. (2007).
Yeast genomic DNA was extracted as described (Hoffman and
Winston, 1987).
2.2. Deletion of the cbp3, cbp6 and mss51 S. pombe and S. cerevisiae
genes
For gene deletions, PCR fragments containing the kanRgene were
generated with hybrid oligonucleotides containing 75 to 80 bases
of homology with the recipient locus on both sides of the gene of
interest and transformed into NB205-6A or NB34-21A as described
in Chiron et al. (2007). [KanR] transformants able to grow in presence
of the drug G418 were streaked again on selective medium, and the
genomic DNA of single colonies was analyzed by PCR to look both
for the correct insertion of the deletion cassette and the absence
of the wild type sequences. Colonies carrying the deletion were
back-crossed to a wild type strain to verify the co-segregation of the
G418 resistance with the gene deletion.
In S. cerevisiae CBP6 and MSS51 were also deleted with the kanR
marker in the strains CW04 and CW252 using the classical PCR strate-
gies described in (Wach, 1996). CBP6 was also inactivated in a deriva-
tive of strain MCC60R2-16 (Costanzo and Fox, 1988) retaining only
the rearranged mitochondrial genome [MSUcbs1-2] (see Table 1) to
generate a double ∆cbs1 ∆cbp6 mutant containing the rearranged ge-
nome (IK97-1). In addition, the wild type open reading frames from
both genes and both yeasts were amplified by PCR, cloned into
pGEMT-easy and verified to be free of mutations, then transferred into
the S. pombe expression vector pTG1754/NotI or into the S. cerevisiae
expression vector pFL61 and used for complementation tests. These
showed that the corresponding wild type genes could complement
all four deletions; however, no cross-species complementation could
be detected.
2.3. Integration of FLAG versions of the cbp6 and mss51 genes
Plasmids containing different tagged S. pombe cbp6/mss51 genes
under the control of the nmt1 promoter (Matsuyama et al., 2006)
were purchased from the RIKEN consortium and first tested for
their ability to complement the corresponding mutants by transfor-
mation and selection for the ura4 marker to maintain the plasmid in
its replicative form. The tagged genes able to produce Cbp6-YFP-
FLAG-His6and Mss51-FLAG2-His6complemented the gene deletion,
but Cbp6-FLAG2-His6did not (data not shown). The plasmids that
showed a good complementation were cut by NotI and transformed
into the corresponding ∆cbp6 or ∆mss51 mutants to integrate the
tagged version into the leu1 locus (see Matsuyama et al., 2004).
2.4. Fluorescence microscopy
Fluorescence microscopy was performed on live cells in glucose
culture medium, using a Zeiss Axioplan 2 microscope linked to a Cool
Snap camera (Princeton Instruments). Mitotracker was obtained from
Molecular Probes and used in accordance with the manufacturer's
instructions.
2.5. Northern blot analyses
S. pombe cells were grown to exponential phase (100 Klett units or
1 OD600) in complete medium, total RNAs were extracted using the
hot phenol protocol (Ausubel et al., 1993) and run on a formaldehyde
gels before transfer onto Hybond-C extra membranes. The blots were
successively hybridized with different probes at 65 °C under standard
saline conditions. After overnight hybridization, the blots were
washed briefly several times with 6xSSPE before exposure for a few
hours, or up to 2 weeks. Mitochondrial probes were PCR fragments
labeled with dATP32using a random priming kit (Invitrogen).
2.6. S35labeling of mitochondrial proteins, pulse chase experiments
S. pombe cells were grown to early exponential phase in complete
5% raffinose medium containing 0.1% glucose. Mitochondrial proteins
were labeled at 30 °C by a 3 hour incubation of whole cells with35S
methionine and cysteine (Bioactif-Hartmann) in the presence of
10 mg/ml cycloheximide, which specifically blocks cytoplasmic trans-
lation. In pulse-chase experiments, cells were labeled in vivo for 2.5 h,
after which they were washed and resuspended in a chase buffer con-
sisting of complete 5% raffinose medium containing 0.1% glucose,
10 mg/ml cycloheximide, 3 mg/ml cysteine, and 10 mg/ml methio-
nine, and divided into four equal aliquots (we found that the chase
had to be carried out in the presence of cycloheximide even after
several washes, since removing the drug from the cold chase buffer
led to a strong background of radio-active amino-acid incorporation
into cytoplasmic proteins). The zero point was spun down directly
and frozen at -18 °C. The other tubes were incubated at 28 °C with
shaking, time points were generally taken after 1, 3 and 6 h. As a
control, cells were plated after 24 h in chase buffer, to verify that
they were still viable. For S. cerevisiae, galactose grown cells were
labeled in presence of 0.6 mg/ml cycloheximide for 20 min, washed
and chased with non-radioactive methionine and cysteine (3 and
10 mg/ml of cysteine and methionine respectively in complete
galactose medium) for 1 h in the absence of cycloheximide. S. pombe
and S. cerevisiae proteins were extracted as described in Gouget et al.
(2008), samples were run on 16% acrylamide–0.5% bisacrylamide
SDS gels and the dried gels were exposed to a film for 1 day, or up to
several weeks at -70 °C, or onto a phosphorimager screen for up to
several days at room temperature.
2.7. Purification of mitochondria and enzymatic activity measurements
Mitochondria were purified from S. pombe cells grown in complete
glucose medium (Chiron et al., 2007). For the measurement of enzy-
matic activities, the resuspension buffer was supplemented with 0.5%
BSA. Cytochrome c oxidase activity was measured on isolated mito-
chondria as described previously (Lemaire and Dujardin, 2008).
2.8. Antibody production: anti-Cytb
An S. pombe cytochrome b specific antibody was obtained from
Eurogentec after the immunization of two rabbits, simultaneously
with two synthetic peptides. These peptides were chosen based on the
results obtained when raising an anti-Cytb from S. cerevisiae (Nouet
et al., 2007) and by comparison of the S. pombe Cytb sequence to the
predicted structure for the S. cerevisiae Cytb (Fisher and Meunier,
2008).The peptidessynthesized
which is predicted to be in an inter-membrane space loop and
256CALPADPLKTPMS268which is expected to be in the matrix. Pre-
immune rabbit sera were verified to be free of background around the
expected band size (25–42 kDa) by western blotting of wild type
S. pombe mitochondria. The rabbits were then subjected to the AS-
DOUB-LX standard protocol from Eurogentec that included five immu-
nizations over 87 days, final bleeding was 15 weeks after the first
immunization. Working dilutions between 1/1 000–1/2 500 were
shown to give the best signals on non-heated samples.
were:
101LYYGSYKYPRTMT113,
2.9. Alkali carbonate treatment and western blots
Alkali carbonate treatment to isolate membrane and soluble mito-
chondrial fractions was performed as described previously (Lemaire
and Dujardin, 2008). Samples were run on 10 or 12% SDS-PAGE
383
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beforeWesternblotting.Primaryantibodieswere:anti-humanHsp60,
1/1 000, (Sigma H3524); anti-S. cerevisiae Arg8: 1/4 000 (Steele et al.,
1996);anti-S.pombeCox2,1/2500(GaisneandBonnefoy,2006);anti-
S. pombe Cytb, 1/1 000; anti-Flag, 1/1000 (Sigma F185); Secondary
antibodies were diluted 1/10 000 fold.
2.10. Blue-native gels and in gel activities
BN-PAGE was carried out according to (Schägger and von Jagow,
1991) modified as described in (Lemaire and Dujardin, 2008). The re-
spiratory complexes were separated on 5–8% or 5–10% polyacryl-
amide gradient gels. S. pombe mitochondria were solubilized with
2% digitonin. Western blots of blue native gels were performed as
reported by (Lemaire and Dujardin, 2008).
2.11. Cytochrome spectra
Low temperature cytochrome spectra of S. pombe cell pastes
were recorded using a Cary 400 spectrophotometer after addition of
sodium dithionite to fully reduce the cytochromes (Claisse et al.,
1970). The absorption maxima were 603, 560, 554 and 548 nm for
cytochromes aa3, b, c1 and c respectively. The S. pombe cytochrome c
peak always shows a 544 nm shoulder that disappears in a cyto-
chrome c mutant (N. Bonnefoy, unpublished).
3. Results
3.1. Only a few of the mRNA-specific translational activators from
budding yeast appear to be conserved in S. pombe
Blast searches for S. pombe homologs were run on the fifteen pro-
teins classified in S. cerevisiae as mitochondrial translation activators
specifically required for the synthesis of a given mitochondrial pro-
tein (see Table 1 and Introduction). Only five resulted in a clear
match in the S. pombe database: Pet309 with Spac8C9.06c and
Spac1093.01, Aep2 with Spcc11E10.04, Mss51 with Spac25B8.04c,
Cbp6 with Spbc947.14c, and Cbp3, recently classified as a translation
factor (Gruschke et al., 2011), with Spcc4B3.17. The function of the
homologs of Pet309 (Ppr4) and Aep2 (Ppr6) has been reported else-
where (Kühl et al., 2011). In this study we have focused on the homo-
logs of Cbp3, Cbp6 and Mss51; these are conserved in fungal genomes
(Figure S1–3). In addition there is a clear human sequence homolog
for Cbp3 (Figure S1); a potential Cbp6 homolog is also found in higher
eukaryotes including humans (Figure S2), however Cbp6 is a rather
small protein, which makes searches for sequence homologies be-
yond fungi less reliable. For Mss51, a possible human homolog has
also been detected, ZMNYD-17 (Mick et al., 2011; Perez-Martinez
et al., 2009, Figure S3). This has a conserved zinc finger MNYD domain
proposed to mediate protein–protein interactions (Matthews et al.,
2009).
3.2. In S. pombe Cbp3 and Cbp6 are important for complex III but not for
cytochrome b translation
In S. cerevisiae, Cbp3 and Cbp6 have been shown to form a com-
plex involved both in the translation of cytochrome b and the early
assembly of complex III. To investigate the function of the Cbp3 and
Cbp6 proteins in S. pombe, each gene was disrupted in several wild
type strains. Irrespective of the strain background, both deletion mu-
tants showed a similar stringent growth defect on galactose medium
(Fig. 1A and data not shown), which indicates a strong mitochondrial
respiratory defect in S. pombe (Chiron et al., 2007). In S. pombe, when
the electron transfer is blocked by antimycin A, the hydrolysis of ATP
by complex V becomes essential for viability, even on fermentable
medium as it is the only way to generate a membrane potential.
Neither ∆cbp3 nor ∆cbp6 mutants were sensitive to antimycin A on
glucose medium (Fig. 1A), demonstrating that they contain at least
some level of complex V activity, unlike ∆ppr6 cells, which are defec-
tive for complex V (Kühl et al., 2011).
Cox1
Cytb
Cox2
Atp6
Rps3
Cox3
*
Cox1
Cytb
Cox2
Cox3
Atp6
Rps3
Atp9
Wavelength (nm)
500
550 600
c1
b
aa3
c
cytb
wt
cbp6
cbp3
A
C
B
Galactose
cbp3
Glucose
Glucose +
Antimycin
ppr6
wt
Glucose
+ G418
cbp6
Fig. 1. A. Growth phenotype of the ∆cbp3, ∆cbp6 and ∆ppr6 mutants. Serial dilutions of
the mutants and isogenic wild type were spotted onto complete media containing
the indicated carbon sources and supplements and incubated for 4 days at 28 °C. B. Cy-
tochrome spectra of ∆cbp3, ∆cbp6 and ∆cytb mutants. Cells of the wild type and
the mutants were grown for 2 nights on glucose medium and low temperature
cytochrome spectra were recorded after the addition of dithionite to fully reduce the
cytochromes. Peaks for cytochromes aa3, b, c1 and c are indicated. C. In vivo35S labeling
of the ∆cbp3, ∆cbp6 and ∆cytb mutants. Mutant and wild type cells were labeled with
35S methionine/cysteine in the presence of cycloheximide that blocks cytoplasmic
translation. For ∆cbp3, the band marked with the asterisk is an artifact observed after
fast migrations. For ∆cbp6, the labeling presented is obtained after a long migration
time to separate the high molecular weight proteins. As a result of this Atp8 and 9
have run off the gel; however, Atp8 and 9 were clearly present in the mutant on
other gels. For reasons that are not clear, Atp6 is generally labeled better in electron
transport chain mutants than in the wild type.
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As expected from the respiratory growth defects, the cytochrome
spectra of the two deletion mutants differed from wild type
(Fig. 1B): both ∆cbp3 and ∆cbp6 cells showed strongly reduced
peaks of cytochrome b and c1 (specific for complex III) and reduced
cytochrome aa3 (specific for complex IV), like a ∆cytb mutant. Such
a decrease in the cytochrome aa3 peak has already been observed
in another S. pombe mutant affected in complex III, ∆abc1. This
suggests that this is a secondary consequence of the complex III
defect. Thus both Cbp3 and Cbp6 appear to be primarily required
for the biogenesis of complex III as in S. cerevisiae.
Since the S. cerevisiae ∆cbp3 and ∆cbp6 mutants are required
for efficient translation of cytochrome b (Gruschke et al., 2011), the
synthesis of mitochondrial proteins was investigated in ∆cbp3 and
∆cbp6 cells by in vivo labeling with35S methionine and cysteine in
the presence of cycloheximide. Both mutants produced mitochondrial
protein patterns similar to the wild type: Cytb, Cox1, 2 and 3 were
clearly visible, although Cox2 was less strongly labeled in both
mutants (Fig. 1C). As a control, ∆cytb cells were also labeled and
found to lack the Cytb protein as expected. Thus, unlike S. cerevisiae,
S. pombe strains lacking Cbp3 or Cbp6 appear to synthesize cyto-
chrome b at normal levels.
Since Cbp3 and Cbp6 appeared to have a similar phenotype, we
chose to pursue the study of Cbp6, in order to further understand its
role in S. pombe and S. cerevisiae. Like its S. cerevisiae counterpart
(Dieckmann and Tzagoloff, 1985), S. pombe Cbp6 is a mitochondrial
protein as shown by a Cbp6-YFP reporter integrated at the ectopic
leu1 locus under the control of the nmt1 promoter (Matsuyama et
al., 2006; Figure S4A, B, C). After carbonate treatment of purified mito-
chondria,thetaggedCbp6proteinwasgenerallydifficulttodetect, but
appeared to partition into the soluble fraction like Arg1 (Figure S4D).
This suggests that Cbp6 is not an integral membrane protein.
3.3. ∆cbp6 contains normal levels of mitochondrial mRNA but
destabilizes Cytb and prevents complex III assembly
In S. cerevisiae, the absence of a specific translation activator does
not strongly compromise the stability of its target RNA, but can pre-
vent the excision of RNA maturase encoding-introns from precursor
RNAs (Fox, 1996). We observed a similar phenotype for the S. pombe
∆ppr4 mutant, which is the Pet309 homolog. This mutant contains
substantial amounts of cox1 mRNA if the cox1 gene lacks introns, but
accumulates the cox1 precursor in an intron-containing background
(Kühl et al., 2011). We therefore investigated the state of the mito-
chondrially encoded cytb mRNA in the ∆cbp6 mutant in an intron-
containing background. The cox2 mRNA was also analyzed since35S
labeling experiments showed a slightly reduced level of Cox2 protein.
Both the cytb and cox2 mRNAs were present at normal levels in the
∆cbp6 mutant (Fig. 2A), showing that the effect on the Cox2 protein
is not a consequence of a lower mRNA level, and that the intron
encoded maturase from cytb is correctly synthesized leading to nor-
mal splicing in ∆cbp6 cells. This shows that Cbp6 is not required for
translation of the intronic region of the cytb precursor RNA.
To analyze the stability of newly synthesized mitochondrial pro-
teins in the ∆cbp6 mutant, a pulse-chase analysis was performed
(Fig. 2B). After 6 h of chase, cytochrome b was still stable in both
the mutant and the wild-type, while Cox1 and Cox3 appeared slightly
more unstable in the mutant. Longer chase experiments were difficult
to interpret because after 12 h of chase in presence of cycloheximide
all signals started to collapse in both strains. Given this behavior of
newly synthesized Cytb in S. pombe, we decided to perform a western
blot analysis rather than a pulse chase experiment to look at the
stability of Cytb. Thus, polyclonal antibodies recognizing an inter-
membrane space and a matrix peptide were raised and used to
probe Western blots of purified mitochondria (see Materials and
methods). Strikingly, Cytb was undetectable in the ∆cbp6 mutant
(Fig. 2C). Thus, virtually all of the Cytb protein synthesized in the
A
C
B
cytb
21S
lrRNA
cox2
Anti-CytbSp
232
669
440
III2
III2 IV1
III2 IV2
Rps3
Cox1
Cytb
Cox3
Atp6
Cox2
cbp6
wt
1
6
0
6
0
1
Chase (h):
Atp9
*
D
CytbSp
Hsp60
Cox2Sp
Hsp60
Fig. 2. A. Analysis of the steady state levels of some mtRNAs in the ∆cbp6 mutant. Total
RNA from the intron-containing NB205-6A wild type and isogenic ∆cbp6 mutant were
hybridized to cytb and cox2 probes as indicated. Loading controls are hybridizations
of the 21S ribosomal RNA and a UV photograph of the cytoplasmic large ribosomal
RNA. B. Pulse chase analysis of ∆cbp6 cells. Wild type and ∆cbp6 cells were labeled as
in Fig. 1C (time 0) and a chase was performed after washes and addition of non-
radioactive methionine and cysteine for 1 or 6 h before extraction of total proteins
and analysis by SDS-PAGE. C. Steady state level of mitochondrial proteins in the
∆cbp6 mutant. Purified mitochondria from the mutant and the wild type were ana-
lyzed by SDS-PAGE and Western blot. Proteins were hybridized to S. pombe antibodies
raised against Cox2 and Cytb. The loading control is Hsp60 that recognizes a mitochon-
drial matrix protein. D. Western blot analysis of respiratory complexes III and IV in
∆cbp6 cells. Blue-Native-PAGE analysis of purified mitochondria from the ∆ppr4,
∆cbp6 or ∆cytb mutant compared to the wild type NB205-6A was conducted and the
samples were analyzed by western blotting with the anti-Cytb antibodies. Positions
for dimers of complex III with, or without monomers or dimers of complex IV are
indicated.
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∆cbp6 mutant is degraded. The complex IV subunit Cox2 was easily
detectable in ∆cbp6 mitochondria, although its level is reduced
(Fig. 2C), consistent with the reduced cytochrome aa3spectral ab-
sorption (Fig. 1B). Taken together with the normal Cytb synthesis
observed in the experiment of Fig. 1C, these results indicate that in
S. pombe, Cbp6 is primarily required after the synthesis of Cytb, either
for its long term stabilization, or for its assembly into complex III.
Our results cannot formally exclude a partial effect on the stability
of cytochrome c oxidase subunits including Cox2, whether it is direct
or indirect. As a comparison, we found that Cox2 level is wild-type in
the ∆cytb mutant used in this work whereas Cytb is undetectable
(data not shown). However, a study of various mitochondrial mutants
from S. pombe (Ahne et al., 1984) shows that among 12 strains
harboring different cytb mutations, 11 show a decrease of cytochrome
c oxidase activity, which can reach only 3% of the wild-type level.
Thus, secondary effects of complex III defects on complex IV must
be analyzed individually.
Probing of BN-PAGE Western blots with the anti-Cytb antibody
showed that complex III was present as three bands in the wild-
type, probably corresponding to dimers of complex III with, or with-
out monomers or dimers of complex IV (Fig. 2D, lane 2). In both
∆cbp6 and control ∆cytb mitochondria, complex III was completely
lacking (lanes 3 and 4). As expected, the higher molecular weight
bands of complex III were absent in the ∆ppr4 mutant, which lacks
complex IV. These data are the first evidence for the existence of
respiratory supercomplexes containing complexes III and IV in
S. pombe mitochondria and confirm that complex III is not assembled
in ∆cbp6 mitochondria, despite a strong synthesis of Cytb.
3.4. S. cerevisiae Cbp6 does not function solely through a target in the
5′-untranslated region of the mRNA encoding Cytb
Since our data pointed towards a strictly post-translational role for
Cpb6 in S. pombe, we revisited the function of the relatively poorly
characterized S. cerevisiae Cbp6, heretofore considered a translation
activator of Cytb synthesis. Two other S. cerevisiae mRNA-specific
translation activators of Cytb synthesis, Cbs1 and Cbs2, have been
shown to function through targets in the 5′-untranslated region
(UTR) of the Cytb mRNA (Rödel, 1986; Rödel and Fox, 1987). This
was demonstrated by showing that in the absence of Cbs1 or Cbs2,
the Cytb coding sequence could be translated from chimeric mRNAs
bearing the 5′-UTRs of other mitochondrial mRNAs, leading to respi-
ratory growth. To test whether S. cerevisiae Cbp6 could be similarly
bypassed we placed a rearranged rho-mtDNA encoding a chimeric
mRNA, bearing the COX3 5′-UTR fused to the Cytb coding region
(Costanzo and Fox, 1988) in a ∆cbs1 ∆cbp6 double mutant. When
this strain was crossed to a rho+∆cbs1 single mutant, with a func-
tional CBP6 gene, the resulting diploids were respiratory competent,
demonstrating the expected bypass of the cbs1/cbs1 homozygous de-
letion (Fig. 3A). However, when the ∆cbs1 ∆cbp6 double mutant
strain containing the chimeric CYTb mRNA was crossed with a rho+
∆cbp6 single mutant harboring a functional CBS1 gene, the resulting
cbp6/cbp6 homozygous diploids remained respiratory defective
(Fig. 3A). Thus, at least part of the Cbp6 function could not be
bypassed by the presence of a heterologous 5′-UTR fused to the
Cytb coding sequence. This shows that the CYTb 5′-UTR cannot be
the sole target of Cbp6 in S. cerevisiae mitochondria.
3.5. S. cerevisiae Cbp6 is not essential for the synthesis of Cytb but is
required for its stability
The first study of Cbp6Sc reported that Cytb synthesis was reduced
but not abolished by a point mutation carried by the strain E158,
that had been isolated in an intron-containing mtDNA context
(Dieckmann and Tzagoloff, 1985). We sequenced the S. cerevisiae
CBP6 gene in the E158 mutant and found that the mutation changes
the initiation codon of CBP6 from ATG to ATA. Thus, in the mutant
E158, CBP6 may not be fully inactivated, since ATA has been shown
to serve as an alternative initiation codon in yeast (Chang et al.,
2010): this could explain the residual synthesis of Cytb in this mutant.
Therefore, we reinvestigated this question by deleting the CBP6 gene
in S. cerevisiae with mitochondria containing either the 13-intron
CYTb gene or the intronless CYTb gene. Growth on non-fermentable
medium was strictly abolished in both backgrounds (Fig. 3B). Next
we examined the synthesis and stability of the mitochondrially
encoded proteins by a short in vivo labeling followed by a one-hour
chase. The results in Fig. 3C show that in the absence of Cbp6Sc,
synthesis of Cytb is reduced but detectable in both mtDNA back-
grounds. However, Cytb made in the mutants was fully degraded
after the chase, in contrast to wild-type where no degradation
could be observed. Thus, in S. cerevisiae, Cbp6 is not essential for
the CYTb mRNA translation although it might be required for a
normal rate of synthesis. However, it is essential for the stability
of the Cytb protein. Thus, the functions of Cbp6 in S. pombe and in
S. cerevisiae appear to be largely similar, despite the absence of func-
tional cross-complementation.
3.6. Mss51 is a membrane associated mitochondrial protein important
for complex IV biogenesis
The role of S. cerevisiae Mss51 in the translational activation of the
COX1 mRNA through its 5′-UTR is firmly established, along with its
post-translational interaction with newly synthesized Cox1 protein
(Barrientos et al., 2004; Perez-Martinez et al., 2003, 2009). It is also
clear that in S. cerevisiae Mss51 is a mitochondrial protein. Strikingly,
the YFP fusion signal obtained for S. pombe Mss51 during the prote-
ome study by the RIKEN consortium (Matsuyama et al., 2006) was
a staining of both the plasma and nuclear membranes, and the
conclusion drawn was a localization of the protein in the endoplasmic
reticulum. Due to this unexpected result, we did not use the Mss51-
YFP-FLAG construct suspecting that it might not be functional, we
Fermentable
Non-
fermentable
wt
wt
cbp6
cbp6
i
i
glycerol
1
3
cbs1
CBP6
rho+
2
glucose
CBS1
cbp6
rho+
cbs1 cbp6
rhosup
1 x 2
1 x 3
3
2
1 x 2
1 x 3
1
0 1 0 0 0 1 1 1
Chase (h):
cbp6 cbp6 wt
wt
No mt intron 13 mt introns
Cox1
Var1
Cytb
Cox2
Cox3
Atp6
AB
C
Fig. 3. A. Suppression test of the ∆cbp6 mutation by a rearranged COX3::CYTb genome.
A ∆cbp6 ∆cbs1 strain (1) containing a rearranged suppressor mitochondrial genome
COX3::CYTb (rhosup) was crossed either with a ∆cbs1 rho+strain (2) or with a ∆cbp6
rho+strain (3) on a complete glucose medium (upper plate), and the diploids were
tested for growth on glycerol (lower plate). B. Effect of intron content on the growth
of ∆cbp6 cells. Serial dilutions of the intron-less (∆i) and intron-containing (Σi)
∆cbp6 mutants were spotted onto complete media containing fermentable (glucose)
or non-fermentable (glycerol) carbon sources and incubated for 2 and 3 days respec-
tively. C. In vivo35S pulse-chase analysis of the S. cerevisiae ∆cbp6 mutant. The ∆cbp6
mutant and the isogenic wild type were labeled with
presence of cycloheximide for 20 min (time 0) followed by a one-hour chase (1) in a
buffer devoid of cycloheximide and radioactive amino-acids.
35S methionine/cysteine in
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used the Mss51-FLAG2-His6construct, which we found could comple-
ment the mss51 deletion; this was integrated into ∆mss51 cells for
the cell fractionation experiments. The tagged Mss51 was detected
only in the mitochondrial fraction and like Cox2 was strongly resis-
tant to carbonate extraction (Fig. 4A), indicating that it is a membrane
protein. S. cerevisiae Mss51 is also membrane associated, but only
peripherally (Fontanesi et al., 2010b; Siep et al., 2000).
To investigate the function of Mss51 in S. pombe, the gene was dis-
rupted in different wild type strains. Whatever the strain background,
the deletion mutants showed a clear growth defect on galactose
medium (Fig. 4B). A very slight growth was observed upon longer
incubations (data not shown). Similarly to the ∆cbp3 and ∆cbp6 mu-
tants, ∆mss51 cells were resistant to antimycin A on glucose medium,
showing that they contain a functional complex V.
As expected from the respiratory growth defect, the cytochrome
spectra of the deletion mutant differed from wild type (Fig. 4C):
∆mss51 cells showed normal cytochrome b and c1 peaks, but cyto-
chromes aa3 were not detectable. The spectra from ∆ppr4 cells
appeared even more affected, with less remaining cytochrome b
and a complete lack of cytochromes aa3. Thus Mss51 appears to be
primarily required for the biogenesis of complex IV, although the
phenotype of its deletion is not as stringent as that of ∆ppr4.
3.7. In S. pombe Mss51 is required only at a post-translational step of
complex IV biogenesis
The synthesis of mitochondrial proteins was investigated in
∆mss51 and ∆ppr4 cells by in vivo labeling with35S methionine and
cysteine in the presence of cycloheximide. The ∆mss51 mutant pro-
duced mitochondrial protein patterns similar to the wild type: Cytb,
Cox1, 2 and 3 were clearly visible, although Cox2 was less strongly
labeled in both mutants, especially ∆mss51 (Fig. 5A). As expected,
∆ppr4 cells clearly lacked Cox1 (Kühl et al., 2011). Thus, in S. pombe,
Mss51 appears to be required at a post-translational step of complex
IV biogenesis; this contrasts with the role of Mss51 in S. cerevisiae,
where it is absolutely required for the synthesis of Cox1. As for Cbp6,
no functional cross-complementation could be obtained between
S. cerevisiae and S. pombe.
Given these surprising35S labeling results, we analyzed the mito-
chondrially encoded cox1 and cox2 mRNAs from a ∆mss51 strain
containing introns in cox1. Northern blots revealed no defect in
the accumulation of mature messengers (Fig. 5B). Thus, the reduced
labeling of the Cox2 protein in ∆mss51 cells is not due to a low level
of cox2 mRNA, and second, the intron encoded maturases of cox1
are synthesized as well as Cox1 itself in the absence of Mss51.
Next we looked at the stability of the Cox2 and Cox1 proteins in
∆mss51 cells. Cox2 was detectable in ∆mss51 purified mitochondria,
although its level was greatly reduced (Fig. 5C), consistent with the
reduced35S labeling (Fig. 5A). No antibodies against S. pombe Cox1
are available. Therefore, we undertook a pulse-chase experiment to
study the stability of newly synthesized mitochondrially-encoded
proteins in the ∆mss51 mutant (Fig. 5D). After 6 h of chase, Cox1
was clearly less stable in the ∆mss51 mutant than in the wild-type,
while Cox2 was poorly labeled in the mutant even before starting
the chase, as noted before (Fig. 5C).
To confirm the specificity of Mss51 function in S. pombe, we exam-
ined respiratory complex assembly by enzymatic activity measure-
ments. A strong, but not complete, decrease in complex IV activity
was observed in purified mitochondria from the ∆mss51 mutant
(Fig. 5E). Similarly, complex IV activity was barely detectable when
assayed in the gel after BN-PAGE (not shown). In contrast, the control
∆ppr4 mutant, that is unable to synthesize Cox1 (Kühl et al., 2011),
completely lacks complex IV activity (Fig. 5D). Thus, the low level
of Cox2 and the unstable Cox1 subunit of ∆mss51 cells seem to
partly assemble into complex IV to produce a weak residual activity,
consistent with the slightly leaky growth of ∆mss51 cells on galactose
medium.
4. Discussion
Mitochondrial genomes encode very few proteins, 8 in yeasts and
13 in mammals. This small number of proteins makes it possible to
specifically control the translation of each mt-mRNA. Over the years
budding yeast has been used as a model system to identify factors in-
volved in such translational control and its coupling to the assembly
of the newly-synthesized subunits into the final respiratory com-
plexes. It is fascinating that at present, so many factors have been
found to be required for the production of single mitochondrial pro-
teins, their binding with prosthetic groups, their insertion into the
membrane and within their enzymatic complex (Fontanesi et al.,
2008). The control of mitochondrial translation in S. cerevisiae re-
quires at least fifteen nuclear encoded proteins that activate the
translation of individual mRNAs, by acting generally, on the 5′UTR
of their target mRNAs (Table 1). This raises the question of whether
S. pombe uses equally complex mechanisms to regulate its mitochon-
drial biogenesis.
Searching the predicted proteome of S. pombe, we have only found
five sequence homologs of S. cerevisiae mRNA-specific translational
activators: Pet309, Mss51, Cbp6, Cbp3 and Aep2. Interestingly, in
S. cerevisiae most of these proteins fulfill a dual function. Pet309 and
Mss51 both activate translation of the COX1 mRNA through binding
the 5′ UTR. In addition, Pet309 is also required for the stability
of intron-containing COX1 mRNAs (Manthey and McEwen, 1995),
whereas Mss51 also binds the Cox1 protein until it is assembled.
mss51
leu1+::mss51-FLAG2His6
CMPS
FLAG
Arg8
Cox2
600
Wavelength (nm)
500
550
c1b
aa3
c
ppr4
wt
mss51
Glucose
Glucose +
Antimycin
mss51
ppr6
wt
Glucose
+ G418
Galactose
A
B
C
Fig. 4. A. Mitochondrial and membrane localization of Mss51 in S. pombe. The mss51-
FLAG2His6construct under the control of the nmt1 promoter was stably integrated
into the leu1 locus of ∆mss51 cells. Mitochondria (M) from these cells were purified
and either analyzed directly by western blotting together with the post-mitochondrial
supernatant (C), or subjected to alkaline carbonate extraction to generate pellet (P)
and supernatant (S) samples. Mss51 was detected with the FLAG antibody, control
antibodies were anti-Cox2 that reveals the mitochondrial fraction, while the anti-Arg8
produces in addition to a mitochondrial labeling a band in both the mitochondrial and
cytoplasmic fractions (antibodies against the S. cerevisiae Arg8 recognize the S. pombe
Arg1 protein). B. Growth phenotype of the ∆mss51 mutants. Serial dilutions of the
two mutants as well as the isogenic wild type and a control complex V deficient mutant,
∆ppr6 (Kühl et al., 2011) were spotted onto complete media containing the indicated
carbon sources and supplements and incubated for 4 days at 28 °C. C. Cytochrome
spectra of the ∆mss51 and ∆ppr4 mutants. Cells of the wild type and the mutants
were grown for 2 to 3 nights on glucose medium and low temperature cytochrome
spectra were recorded after the addition of dithionite to fully reduce the cytochromes.
Peaks for cytochromes aa3, b, c1 and c are indicated.
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Thus, as described in the Introduction, Mss51 participates in an
assembly-feedback regulation of Cox1 synthesis, a phenomenon
called Control by Epistasy of Synthesis that has been well described
in chloroplast biogenesis (CES, see Choquet and Wollman, 2008).
Cbp6 and Cbp3, were first proposed to be S. cerevisiae translation
and assembly factors, respectively (Dieckmann and Tzagoloff, 1985;
Wu and Tzagoloff, 1989), but have recently been reported to form a
complex that associates with the exit tunnel of the mitochondrial
ribosome (Gruschke et al., 2011). In addition, the Cbp3-Cbp6 complex
is also part of a none-ribosome-bound assembly intermediate of
the bc1 complex, containing newly synthesized cytochrome b and
the assembly factor Cbp4. This is consistent with our observations
that in S. cerevisiae, Cbp6 is important not only for translation but
also for the stability of the Cytb protein, and that replacing the 5′
UTR of cytochrome b by that of another mitochondrial gene does
not suppress the deletion of CBP6. Thus, Cbp6, like Mss51, has a
dual function and targets in S. cerevisiae. Finally, Aep2 is the least
well characterized; it interacts with the ATP9 5′UTR (Ellis et al.,
1999), but it is unknown whether it participates in assembly of the
Atp9 protein.
In this paper, we have focused on the function of three S. pombe
proteins, homologous to three S. cerevisiae proteins that function
both in mitochondrial translation and assembly of the respiratory
complexes: in mitochondria, these two steps have been shown to
play a key role in the regulation of mitochondrial gene expression.
Beyond the sequence conservation, do the fission yeast factors play
the same role as their budding yeast homologs? Whereas this seems
to be the case for Ppr4/Pet309 and Ppr6/Aep2 (Kühl et al., 2011),
we show here that the S. pombe Cbp3, Cbp6 and Mss51 genes carry
out only post-translational functions that may be similar to their S.
cerevisiae counterparts; they are not detectably required for cytb
or cox1 mRNA translation. These findings suggest that the stability/
assembly function of these three factors might represent their ancestral
roles, shared by the homologous genes from different organisms.
Budding yeast might have recruited these proteins for translational ac-
tivation asa lateradaptation,allowingthe coupling of protein synthesis
and complex assembly in response to its specific energetic needs.
This would suggest that in S. pombe assembly-feedback control
of synthesis mediated by Mss51 for Cox1 may not exist. In this
case, in S. pombe Mss51 would only have a role in protein stability,
insertion of prosthetic groups (e.g. in Cox1 or Cox2), or assembly of
complex IV. In S. pombe as well as in human cells, mitochondrial
translation might be regulated through general factors rather than
specific factors. For example mtEF-Ts and mtIF3 are mitochondrial
translation factors with a regulatory rather than protein synthesis
role in the general steps of translation, and they are conserved in
S. pombe and humans, but not in S. cerevisiae (Chiron et al., 2005).
In addition, we have discovered in S. pombe a negative regulator of
mitochondrial translation, Ppr5 (Kühl et al., 2011), which, like the
human PTCD1 (Rackham et al., 2009) that inhibits the stability of
the mitochondrial leucine tRNAs, might play an important function
in down-regulating translation of nearly all mt-mRNAs in S. pombe,
possibly in response to environmental conditions.
Alternatively, an assembly-feedback mechanism might occur in
S. pombe, but the actors could be different to S. cerevisiae. First, Cox1
could be a subunit subject to such control in S. pombe, but factors
other than Mss51, e.g. Ppr4 the homolog of Pet309, could mediate
the regulatory feedback loop, possibly by interacting with Mss51. In
humans, the COX1 translational activator TACO1 (Weraarpachai et
al., 2009) could play this role together with the possible Mss51 homo-
log, ZMYND17. A search for other components of the S. cerevisiae
Cox1 assembly feedback regulation does not retrieve reasonable
homologs of Coa3 and Cox14 in S. pombe (Mick et al., 2010); but
these are very small proteins, which makes sequence comparisons
rather unreliable. However, Coa1 which forms a complex with Mss51
and Cox1 in S. cerevisiae (Pierrel et al., 2007) is clearly conserved in
S. pombe, with an N-terminal extension that does not exist in the
S. cerevisiae protein and could take on the role of a missing component,
such as Cox14 or Coa3.
Second, the subunit controlled for complex IV in S. pombe could
be Cox2 rather than Cox1, since we obtained a much less efficient
labeling of Cox2 in the absence of Mss51. However, in our hands
the labeling of S. pombe Cox2 appears generally decreased in a wide
Rps3
Cox1
Cytb
Cox3
Atp6
Cox2
mss51
wt
1
6
0
6
0
1
Chase (h):
Cox2Sp
Arg8Sc
cox1
21S
lrRNA
cox2
Cytochrome c
oxidase activity
Cox1
Cytb
Cox2
Atp6
Cox3
Rps3
100
80
60
40
20
0
A
D
B
C
E
Fig. 5. A. In vivo35S labeling of ∆mss51 mutants. Mitochondrially-encoded proteins of
the mutant together with the wild type and a control strains lacking the cox1 transla-
tional activator Ppr4 (Kühl et al., 2011) were labeled with35S methionine/cysteine
and a typical result is shown. As for ∆cbp6 in Fig. 1C, Atp8/9 were clearly labeled in
other gels. B. Analysis of the steady state levels of the mtRNAs in the ∆mss51 mutant.
Total RNA from the intron-containing NB205-6A wild type and isogenic ∆mss51
mutants were hybridized to cox1 and cox2 probes as indicated. Loading controls are a
hybridization of the 21S ribosomal RNA and a UV photograph of the cytoplasmic
large ribosomal RNA. C. Steady state level of Cox2 in the ∆mss51 mutant. Purified mito-
chondria from the mutant and the wild type were analyzed by SDS-PAGE and Western
blot and hybridized to an antibody raised against S. pombe Cox2. The control antibody
is anti-Arg8 (see Fig. 4A). D. Pulse chase analysis of ∆mss51 cells. Wild type and ∆mss51
cells were labeled as in panel A (time 0) and a chase was performed after washes and
addition of non-radioactive methionine and cysteine for 1 or 6 h before extraction of
total proteins and analysis by SDS-PAGE. E. Cytochrome c oxidase activity in ∆mss51
mitochondria. Cyanide-sensitive cytochrome c oxidase activity was measured in
equal amounts of mitochondria of ∆mss51, ∆ppr4 (that lack Cox1) and the isogenic
wild type cells (wild type activity was set as 100%).
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variety of respiratory mutants such as the strains used in this work
(∆cytb, ∆cbp3/6, ∆ppr4), or other ∆ppr mutants (Kühl et al., 2011).
In addition labeling of mitochondrial proteins in S. pombe is much
less efficient than in S. cerevisiae. In our experiments we had to
label mitochondrial proteins for up to 3 h, this means that the results
of the protein labeling experiments are a mixture of synthesis and
turnover and not a simple measure of the neo-synthesis of the mito-
chondrial proteins. Thus additional experiments are needed to deter-
mine whether the effect of ∆mss51 on Cox2 labeling is specific and is
really synthesis or stability.
Finally, our data also raise the question of the exact function of
Cbp3 and Cbp6 in both S. pombe and S. cerevisiae and whether an
assembly-feedback process involving these factors could occur
for Cytb, linking it to the assembly of complex III. It is clear that in
S. cerevisiae CES regulation is not restricted to complex IV, as it has
also been described for the F0subunits of complex V, although the
mechanism and factors involved in this case are less clear (Rak and
Tzagoloff, 2009). It would be interesting to look at mutants that
fail to assemble complex III and determine whether this impacts
Cytb synthesis, and whether Cbp6 could play a role in this control
at least in S. cerevisiae. Cbp6 could also be required for the insertion
of heme into the newly synthesized Cytb, or act at a later step in
complex III assembly, explaining the defect in Cytb stability in its
absence.
5. Conclusion
Only one third of the specific translational activators found in
S. cerevisiae mitochondria appear conserved in sequence in the dis-
tant yeast S. pombe, and probably also in higher eucaryotes. These
conserved S. cerevisiae factors generally fulfill dual functions, e.g.
both in mRNA translation and early complex assembly. We show in
this study that three of the S. pombe sequence homologs act only
at a post-translational step, i.e. have a more limited role than their
S. cerevisiae counterparts although still active within the same
biogenesis pathway. This might also be true for the potential human
homologs of these factors. Such difference might be correlated to
the more compact structure of S. pombe mRNA and/or differences in
metabolism. Thus the question of how translation and its coupling
with assembly are mediated in organisms other than S. cerevisiae re-
mains open, suggesting that additional components or mechanisms
are yet to be discovered, probably through specific screens rather
than sequence homology searches.
Acknowledgments
We are grateful to Mauricette Gaisne for constant and skillful tech-
nical assistance, Cristina Panozzo for help with fluorescent imaging
and Sophie Marsy for sharing her expertise on Blue-Native Gels and
enzyme activity measurements. We thank Geneviève Dujardin and
Christopher J. Herbert for fruitful discussions and critical reading of
the manuscript, G.D. for the gift of antibodies. We thank B. Meunier
for advice on the choice of Cytb peptides and B. Schäfer for the gift
of the original ∆cytb strain. FLAG-tagged versions of the cbp6 and
mss51 genes were distributed by the RIKEN Bioresource Center. This
work was supported by a grant of the Agence Nationale pour la
Recherche (ANR) JCJC06-0163 to NB and a grant from the US National
Institutes of Health (GM29362) to TDF. IK was supported by the
ANR and by a grant FDT20091217787 from the Fondation pour la
Recherche Médicale.
Appendix A. Supplementary data
Supplementary data to this article can be found online at doi:10.
1016/j.mito.2012.02.002.
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