- The document examines the role of membrane potential in the biogenesis of cytochrome c oxidase subunit II, a mitochondrial gene product. - Using an in vitro mitochondrial translation system, the authors find that accumulation of unprocessed subunit II precursor (pre-II) occurs when mitochondrial gene products are synthesized under conditions that prevent formation of a normal membrane potential. - The majority of pre-II generated in this way is inserted into the lipid bilayer, as judged by resistance to sodium carbonate extraction, indicating that membrane potential is required for a step in subunit II biogenesis other than precursor insertion into the membrane.

1. W E JOURNALOF BIOLOGICAL CHEMISTRY
01989 by The American Societyfor Biochemistryand Molecular Biology,Inc.
Vol. 264, No. 17, Issue of June 15,pp. 10114-10118,1989
Printed in U.S.A.
A Role for Membrane Potential in the Biogenesis of Cytochromec
Oxidase Subunit11, a Mitochondrial Gene Product*
(Receivedfor publication, January 17, 1989)
GeorgeH. D. Clarkson and Robert0.PoytonS
From the Department of Molecular, Cellular,and DevelopmentalBiology, University of Colorado at Boulder,
BouMer, Cokrado80309-0347
Subunit I1 of yeast cytochromec oxidase is synthe-
sized on mitochondrial ribosomesas a precursor con-
taining a transient NHa-terminal presequence and is
inserted into the mitochondrial innermembrane from
the matrix side. Usingan optimizedin vitro mitochon-
drial translation system (McKee,E. E., and Poyton,R.
0. (1984) J. Biol. Chem. 259, 9320-9331), we have
examined the requirementfor an electrochemical po-
tential (Ab,+) across the inner mitochondrialmembrane
during subunitI1biogenesis.When mitochondrial gene
products are synthesized under conditions that prevent
formation of a normal Ai,+, accumulation of unproc-
essed subunitI1 (pre-11) occurs. The majority of pre-I1
generated inthis way is inserted into the lipidbilayer,
as judgedbyresistanceto extraction with 0.1 M
Na2COs.Therefore, it appears that aAb* is required
for the normal biogenesis of subunit 11, and that the
Ab,+ is requiredfor a function other than the insertion
of pre-I1 into the lipid bilayerof the inner membrane.
Most mitochondrial polypeptides are encoded by nuclear
genesand areimported into mitochondria fromthe cytoplasm.
However, a few are mitochondrial gene products andare
synthesized on endogenous ribosomes (1).The majority of
mitochondrially synthesized polypeptides are integral mem-
brane components and areassociated with nuclear gene prod-
uctsin heterooligomeric proteins of the electron transport
chain (2). Therefore, the biogenesis of these respiratory com-
plexes entails assembly of polypeptides that are synthesized
on opposite sides of the mitochondrial inner membrane.
Recent studies have revealed many of the mechanistic
details pertainingto the importof cytoplasmicallysynthesized
mitochondrial precursor polypeptides (reviewedin Refs. 3-5).
These include a requirement for an electrochemical potential
(Ai,+) across the inner membrane for the initial interaction
of precursor polypeptides with this bilayer (6-8) andthe
involvement of nucleoside triphosphates in productive inter-
action of precursors with the mitochondrial surface and in
completion of precursor translocation (9-11). Incontrast,
much less is known about the mechanism(s) of insertion of
mitochondrial gene products. Indeed, the energetic require-
ments associated with the biogenesis of these polypeptides
are thusfar undefined.
We have investigated the role of a A;,+ in the biogenesis
*This workwas supported by Public Health Service Research
Grant GM 30228 from the National Institutes of Health. The costs
of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked “aduer-
tisement” in accordancewith 18U.S.C. Section 1734 solelyto indicate
this fact.
$To whom correspondence should be addressed. Tel.:303-492-
3823.
of subunit I1 of yeast cytochrome c oxidase. This subunit is
synthesized on mitochondrial ribosomes as a precursor con-
taining a transient NHs-terminalpresequence (12-15) and is
inserted into the inner membrane from the matrix side. Pre-
sequence removal is most probably catalyzed by a protease
located in the intermembrane space (16),andapparently
occurs co-translationally, since the precursor form is not
normally detected in pulse-labeled cells or isolated mitochon-
dria (12, 17). The mature polypeptide is predicted to be
anchored in the inner membrane viatwo hydrophobic do-
mains, with both termini protruding intothe intermembrane
space (19).
In this paper, we demonstrate that the precursor form of
subunit I1accumulates when mitochondrial translation prod-
ucts aresynthesized under conditions that prevent formation
of a normal A;,+. Furthermore, we show that themajority of
the precursor generated in this way is tightly associated with
the inner membrane. Our results indicate that 1) normal
biogenesis of subunit I1 is dependent on Ai,+ and 2) the
Ai,+ is implicated in a function other than the insertion of
pre-I1 (unprocessed subunit 11) into the lipid bilayer of the
inner membrane. Together with previous findings, these re-
sults also indicate that theAi,+ is involved in the biogenesis
of mitochondrial polypeptides that are synthesized on oppo-
site sides of the inner membrane.
MATERIALSANDMETHODS
Isolation of Mitochondria-The wild type Saccharomycescereuisiae
strain D273-10B (MATa, ATCC No. 24657)wasgrown at 30 “C in
semi-synthetic galactose medium (20) and harvested in mid-logarith-
mic phase (AKlett = 200-220). Mitochondria were isolated as de-
scribed (18), except that cells were spheroplasted using Zymolyase
20T (3mg/g wet weight cells), and therecovery period after cell wall
removal was increased to 60 min. For storage, isolated mitochondria
were diluted 2-fold with ice-cold 0.6 M sorbitol, 40% (v/v) glycerol
adjusted to pH 7.2 with KOH, frozen in liquid NP, and stored in
aliquots at -80 “C.Frozen mitochondria were thawed on ice, diluted
10-fold with 0.6 M mannitol adjusted topH 7.2 with KOH, and
pelleted by centrifugation for 10 min at 13,000 X gmaXand 2 “2.
Mitochondria that have been “rescued” in this way perform protein
synthesis that is insensitive to the presence of ribonuclease A, at
rates greater than 90%of those exhibited by freshly isolated organ-
elles. The results presented in this paper were obtained with such
rescued mitochondria.
Conditionsfor in VitroMitochondrial Translation-The optimized
protein-synthesizing medium (PSM)’described earlier (18)was used.
Inhibitors were added from 100 X stock solutions. Oligomycin, val-
inomycin, and CCCP were dissolved inethanol and used at final
concentrations of 30 wg/ml, 1.25+g/ml, and 20 pM, respectively;stock
solutions of KCN (100 mM) and aurintricarboxylic acid (ammonium
salt) (50mM) were made in distilled water. Mitochondria were prein-
cubated in PSM for 10 min before radiolabeling of mitochondrial
’The abbreviations used are: PSM, protein-synthesizing medium;
CCCP, carbonyl cyanide m-chlorophenylhydrazone; SDS-PAGE, SO-
dium dodecyl sulfate-polyacrylamide gel electrophoresis.
10114

2. Biogenesis of Cytochrome c Oxidase Subunit 11
translation products was initiated by adding [35S]methionine(0.02-
0.025 mCi/ml) to the reaction mixture. Conditions for "run-off" of
nascent labeledpolypeptides and for preparation of samples for SDS-
PAGE were as described (18).
Measurement of Mitochondrial Protein Synthesis-Aliquots of
translation mix (20 pl) were spotted onto 2.3-cm diameter Whatman
3MM discs and allowed to air dry. The discs were then dropped into
ice-cold 10%trichloroacetic acid, and after a minimum of 5 min, the
solution was brought to a rolling boil for 10 min and then cooled.
After discarding the trichloroacetic acid solution, the discs were
washed twice each with distilled water, 95% ethanol, and acetone (2
min/wash) and airdried.
Gel Electrophoresis-SDS-polyacrylamide slab gel electrophoresis
was performed in a discontinuous buffer system (21). Resolving and
stacking gels contained 16and 3%polyacrylamide, respectively,with
an acry1amide:bisacrylamide ratio of 31.6:l.
Immunoprecipitation-IgG from anti-subunit I1 serum (12) was
prepared by ammonium sulfate fractionation (22). This material was
adsorbed to protein A-Sepharose CL-4B beads (Sigma) as described
(23). The coated beads were washed twice with immunobuffer (10
mM NaPOs, 15 mM NaC1, 1%Tritonx-100, 0.2% SDS, 1 mM
phenylmethylsulfonyl fluoride, pH 7.4) and diluted to give a 15%(w/
v) suspension. Samples of radiolabeled mitochondria were dissociated
in immunobuffer at 0.6 mg mitochondrial protein/ml and rockedwith
an equal volume of suspended beads for 2 h at room temperature.
The beads were reisolated by centrifugation, washed three timeswith
immunobuffer,once with distilled water, and immune complexeswere
dissociated by suspending the beads in protein dissociation buffer
(18)and boiling for 2 min.
Miscellaneous-Published procedures were used for carbonate ex-
traction of labeled mitochondria (24), liquid scintillation counting
(18), and proteindetermination using bovine serum albumin as
standard (25). SDS-polyacrylamide gels were processed for fluorog-
raphy using ENHANCE'" (Du Pont) according to manufacturer's
directions.
Materials-Zymolyase 20T was from ICN ImmunoBiologicals(Li-
sle,IL). Sorbitol was a product of Pfanstiehl Laboratories (Waukegan,
IL). All inhibitors and biochemicals, as well as pyruvate kinase (type
111),bovine serum albumin (essentially fatty acid-free),and SDS were
from Sigma. ~-[~~S]Methionine(>1100 Ci/mmol) was from Du Pont-
New England Nuclear. All other reagents were readily available
commercially.
RESULTS
Previously, we developed an optimized " P S M for in vitro
synthesis of yeast mitochondrial translation products (18).
This medium containsarespiratorysubstrate,permitting
synthesis of matrix ATP via oxidative phosphorylation, as
well as an exogenous ATP-regenerating system. To investi-
gate the role of a A;,+ insubunit I1 biogenesis, isolated
mitochondria were suspended in PSM and pulse-labeled in
the presence of various energy poisons. In theseexperiments,
oligomycin wasused to inhibitthe FIFo-ATPaseand thus the
generation of a A;,+ via hydrolysis of matrix ATP.Similarly,
respiration-induced energization of the inner membrane was
blocked using cyanide (which inactivates cytochrome c oxi-
dase)or dissipated with either CCCP, a protonophore, or
valinomycin, a potassium ionophore.
Since, in this optimized system, mitochondrial protein syn-
thesis is dependent to a large extent on ATP derived from
oxidative phosphorylation (18), addition of energy poisons
should inhibit translation. As shown in Fig. 1,this is indeed
the case. Addition of oligomycin, together with cyanide,
CCCP, or valinomycin, causes significant inhibition of both
therateandextent of protein synthesis. Incontrast, the
presence of ethanol (the solvent for oligomycin, CCCP, and
valinomycin) causes only minimal inhibition of protein syn-
thesis. In the absence of oligomycin,additions of KCN, CCCP,
or valinomycin are less inhibitory.'
Radiolabeled translationproducts synthesized under the
G. H. D. Clarkson and R. 0. Poyton, unpublished observations.
10115
control
+ethanol
CCCPtoligo.
KCN+oligo.
Val.toligo.
0oV""""20 40 60 00
Time (min.)
FIG.1. Effects of energypoisons on protein synthesis in
isolated yeast mitochondria. Mitochondria were incubated for 10
min in PSM containing the additions indicated, then pulse-labeled
for 60 min with [%]methionine. At each time point, duplicate ali-
quots (10pg of mitochondrial protein) were taken from each reaction,
and theamount of trichloroacetic acid-precipitable radioactivity was
determined. The maximum value obtainedin the control reaction
(2.7 X IO7 cpm/mg mitochondrial protein) was defined as 100%
protein synthesis. Oligo., oligomycin (30 pg/ml final concentration);
CCCP, carbonyl cyanide m-chlorophenylhydrazone (20pM final con-
centration); ual., valinomycin (1.25 pg/ml final concentration). Po-
tassium cyanide (KCN)was included at l mM final concentration.
above conditions were resolvedby SDS-PAGE and visualized
by fluorography (Fig. 2A). In thepresence of oligomycin and
cyanide (lane 7), oligomycinand CCCP (lane8),or oligomycin
and valinomycin (lane 9), an extra polypeptide is observed
which exhibits the same electrophoretic mobility as pre-I1
accumulated in the presence of aurintricarboxylic acid (12,
14),a putative inhibitorof the protease that catalyzes removal
of the subunit I1 presequence (lune 10).This band is faintly
detectable in mitochondria labeled in the presence of CCCP
(lane5)or valinomycin (lane6)alone,but not in mitochondria
incubated with ethanol (lune Z),oligomycin (lane 3),or cya-
nide (lane4).The identity of the extraband observed in lanes
5-9 of Fig. 2A was examined by subjecting detergent-solubi-
lized mitochondria to immunoprecipitation with anti-subunit
I1 IgG(Fig.2B).The immunoprecipitate obtained from trans-
lation products labeled in the presence of aurintricarboxylic
acid contains both pre- and mature subunit I1 (but none of
the other translation products),indicating that our IgG prep-
aration effects specific recovery of both forms of subunit I1
(lane IO).The immunoprecipitates shown in Fig. 2B, lanes 5-
9, illustrate thattheextra band generated by these drug
treatments is also recognized by anti-subunit I1IgG. From
the co-migration of this band with pre-I1 and its cross-reac-
tivity with subunit-specific anti-subunit I1 IgG, we conclude
that itrepresents unprocessed pre-11.
Interestingly, we have never observed complete inhibition
of pre-I1processing in the presence of energy poisons. At best,
we observe 40% inhibition. Although the explanation for this
observation is currently unclear, we have excluded several
trivial possibilities, i.e. that increasing the preincubation
period beforepulse labeling orthe concentrations of inhibitors
increases the proportion of pre-I1 and that mature radiola-
beled subunit I1arises solely from incorporation of radiolabel
into the carboxyl terminus of nascent, processed subunit I1
polypeptides.'
To test whether a A;,+ is required for insertion of pre-I1
into the inner membrane, labeled mitochondria were treated
with 0.1 M sodium carbonate, under conditions that convert

3. 10116 Biogenesis of Cytochrome c Oxidase Subunit 11
A. 1 2 3 4 5 6 7 8 9 1 0
B. S 1 2 3 4 5 6 7 8 9 1 0
I-
ATPase6 -
m-
FIG.2. Pre-11, the precursorform of cytochrome c oxidase
subunit 11, accumulates in the presence of energy poisons.
Mitochondrial translation products were synthesized during a 60-min
pulse in the presence of the drugs indicatedbelow. A, an aliquot from
each reaction was analyzed by SDS-PAGE and fluorography; B, a
secondaliquot was subjected to immunoprecipitationusing anti-
subunit I1 IgG adsorbed to protein A-Sepharose CL-4B beads, and
the recovered polypeptides were analyzed as above. Lanes I, control
(no addition);lanes 2, +ethanol (2% (v/v) final concentration); lanes
3, +oligomycin (30 pg/ml final concentration); lanes 4, +potassium
cyanide (1mM final concentration); lanes 5, +carbonyl cyanide m-
chlorophenylhydrazone (20 pM final concentration); lanes 6, +valin-
omycin (1.25 pg/ml final concentration); lanes 7, potassium cyanide
(1mM) + oligomycin (30 pg/ml); lanes 8, carbonyl cyanide m-chlo-
rophenylhydrazone (20 p ~ )+ oligomycin (30 pg/ml); lanes 9,valin-
omycin (1.25 pg/ml) + oligomycin (30 pg/ml); lanes IO, + aurintri-
carboxylic acid (0.5 mM final concentration). Lane S in E contains
total translation products from a control reaction. The positions of
subunits 1-111of cytochrome c oxidase (I,ZZ,ZZZ), the precursor form
of subunit 11 (pre-ZZ),apocytochrome b (b),subunit 6 of oligomycin-
sensitive ATPase (ATPase 6), and the Var 1polypeptides are indi-
cated.
mitochondria into open membrane sheets andrelease content
proteins and peripheral membrane proteins(24). As shown in
Table I, carbonate extraction of mitochondria from a control
reaction, lacking inhibitors, releases approximately two-thirds
of the totalprotein into thesoluble fraction. The presence of
CCCP and oligomycinhas no significanteffect on thisdistri-
bution. Incontrast, most of the radiolabeled polypeptides
synthesized in the presence or absence of inhibitors are lo-
catedin the membrane pellet, although the percentage of
membrane-associated trichloroacetic acid-precipitable radio-
activity is reduced slightly by the inclusion of CCCP and
oligomycin.
TABLEI
Distribution of total protein and radiolabeled mitochondrial gene
products after carbonate extraction of pulse-labeled mitochondria
Carbonate extraction of mitochondria pulse-labeled in the absence
or presence of CCCP and oligomycin was performed as described in
the legend of Fig. 3.
Total protein"
Trichloroaceticacid-
radioactivityb
precipitable
Control
CCCP + CCCP +
olieomvcin oliaomvcin
%
"
%
"
Na2C03pellet 34.4 f 4.1 31.7 f 1.6 95.0 f 0.8 90.3 f 1.9
Na2C03soluble 65.6 f 4.1 68.3 f 1.6 5.0 f 0.8 9.7 f 1.9
Mean f S.D. of three experiments.
Mean f S.D. of four experiments.
b -
II-
m.
ATPase6
1 2 3 4 5 6-. -
Pc - pre-lI
FIG.3. Carbonate extractionofpulse-labeled mitochondria.
Mitochondria were pulse-labeled during a control reaction or in the
presence of CCCP (20 pM) and oligomycin (30 pglml). After run-off
and washing, the organelles were resuspended in 0.1 M Na2C03,
incubatedon ice for 30 min, andthen separated into pellet and
supernatant fractions by centrifugation for 1h at 226,000 X g,, and
2 "C. To collect soluble proteins, supernatants were adjusted to 10%
trichloroacetic acid, incubated on ice for 15 min, and centrifuged (1
h, 5,000 X g,,,.,, 2 "C).Membranepellets and precipitated soluble
proteins were rinsed (24) and dissolved in 0.5% SDS. After aliquots
were taken for protein determination, the samples were diluted with
0.25 volumes of 5 X protein dissociation buffer (18)and analyzed by
SDS-PAGE and autoradiography. Lanes 1-3 are control mitochon-
dria, lanes 4-6 are mitochondria incubated in the presence of CCCP
and oligomycin. Lanes I and 4, unextractedmitochondria; lanes 2
and 5,Na2CO3-insoluble;lanes 3 and 6,Na~CO3-soluble.In bothcases,
15%of the totalrecovered pellet and supernatantmaterial was loaded
on thegel.
All of the translationproducts labeled in a controlreaction
except for the ribosomal Var 1polypeptide are refractory to
carbonateextraction (Fig. 3, lanes 2 and 3). This result is
expected, since these proteins are very hydrophobic integral
components of the inner membrane (27). The same proteins
are also refractory to carbonate extraction when synthesized
in the presence of CCCP and oligomycin (lunes 5 and 6).
Moreover,the majority of the pre-I1generated in thepresence
of CCCP and oligomycinis also resistant tocarbonate extrac-
tion (lunes5 and 6),indicating that insertion per se of pre-I1
into theinner membranedoes not require a A ~ H + .The partial
susceptibility of pre-I1 to carbonate extractionmay reflect the
existence of asubpopulation of precursor molecules which
does not integrate into the lipid bilayer. Alternatively, such
partitioning may result from a weakened interaction of pre-
I1 with the innermembrane.

4. Biogenesis of Cytochrome cOxidase Subunit II 10117
DISCUSSION
In thisreport, we show for the firsttime that mitochondrial
energy poisons can be used to inhibit processing of pre-11, the
precursor form of cytochromec oxidasesubunit 11,synthesized
in isolated yeast mitochondria. It is unlikely that pre-I1 ac-
cumulation results from nonspecific effects of the inhibitors
used, because accumulation can be achieved with several
different drug treatments, and because although a combina-
tion of KCN plus oligomycin effects pre-I1 accumulation,
neither drug is effective on its own. The most plausible
explanation for the observed accumulation of pre-I1 is that a
A;,+ is involved in the biogenesis of subunit 11.
Inhibition of pre-I1 processing is not observed when mito-
chondrial translation products aresynthesized in the presence
of oligomycin or cyanide alone, presumably because under
these two conditions a A;,+ can still be generated via respi-
ration or hydrolysis of matrix ATP, respectively. Weak pre-
I1 accumulation occurs when mitochondria are pulse-labeled
in the presence of CCCP. Since a combination of CCCP and
oligomycin enhances accumulation ofpre-11, it appears that
CCCP alone is insufficient to completely dissipate A;,+. This
is not surprising, because although CCCP renders the inner
membrane permeable to protons and therefore tends toabol-
ish A;H+, it simultaneously stimulates protonpumping by the
FIFo-ATPase(26).Like CCCP, valinomycin alone also effects
a slight accumulation of pre-11. This result may be explained
in one of two ways. On the one hand, if valinomycin com-
pletely abolishes A$, the component of A;,+ arising from the
separation of charged species across the inner membrane,
then a proton gradientwould be sufficient to support almost
normal levels of pre-I1 processing. Alternatively, if A$ is not
completely abolished by valinomycin, the weak accumulation
of pre-I1 would reflect the importance of A$ in normal proc-
essing. Further study is required to decide between the two
possibilities.
The initial interaction of nuclear-encoded precursor poly-
peptides with the inner mitochondrial membrane requires a
A;,+ (7, 8, 10). Therefore, an energized inner membrane is
apparently required for the biogenesis of polypeptides that
enter this bilayer from opposite sides. For nuclear-encoded
precursors, the membrane potential is thought to exert an
electrophoretic effect on positively charged domains of these
molecules (6, 7). However, from the results presented here, it
appears unlikely that theASH+plays an analogous role in the
insertion of mitochondrial gene products intothe inner mem-
brane; whereas a A;,+ is required for processing of pre-11, it
is not required for association of pre-I1 (or any of the other
hydrophobic mitochondrial translationproducts) with the
inner membrane as integralpolypeptides.
Why does subunit I1 biogenesis require a A;H+? Several
possible roles of the membrane potential in subunit I1biogen-
esis cancurrently be envisaged. First, an energized inner
membrane maybe required for proper (ie.processing-com-
patible) insertion of pre-I1 into theinner membrane. Second,
the existence of a ASH+ may promote folding of the nascent
pre-I1 polypeptide in a conformation that is optimal for pro-
teolytic processing. Evidence for the importance of polypep-
tide conformation in subunit I1 biogenesis comes from the
observation3that a point mutationwhich changes residue 109
of the mature subunitI1sequencefrom tryptophan to arginine
causes essentially complete inhibition of pre-I1 processing.
Although the effect of this mutation ismore severe than that
obtained by using energy poisons, replacement of the trypto-
V. L. Cameron and R. 0.Poyton, manuscript in preparation.
phan residue may exert a more deleterious effect on polypep-
tide foldingthan does a reduction in A;,+. Third, it is possible
that pre-I1 processing requires prior assembly of subunit I1
with a cytoplasmically synthesized protein (e.g. a nuclear-
encodedsubunit of holocytochromec oxidase)that is imported
into the inner membrane in a AbH+-dependentmanner. It has
been demonstrated previously that isolated yeast mitochon-
dria contain adepletable, endogenous pool of cytoplasmically
synthesized polypeptides (28) and that subunits I, 11,and I11
made in isolated mitochondria are capable of assembling with
an endogenous pool of the nuclear-encoded subunits of cyto-
chrome c oxidase (29).Therefore, it is conceivable that accu-
mulation of pre-I1 occurs, because in the absence of a normal
A;,+, a significant fraction of one or more of these nuclear-
encoded subunits accumulate(s)outside the innermembrane,
in afashion analogous to the“stage3” intermediate described
by Pfanner and Neupert(8)for the ADP/ATP carrier. Inthe
above scenario, the incomplete inhibition of pre-I1processing
observed in our experiments would reflect the preexistence in
the inner membrane of a small population of the putative
partner protein that is available for interaction with newly
synthesized subunit I1polypeptides. Finally, the enzyme that
catalyzes removal of the pre-I1presequence may depend on a
A;,+ for optimalactivity. Bytesting each of these possibilities,
it should be possible to define more rigorouslythe role of the
membrane potential in the biogenesis of mitochondrial gene
products and thusderive a better understanding of how het-
erooligomeric respiratory complexes are formed.
Acknowledgments-We thank our colleagues in the laboratory for
valuable discussions, and Tom Patterson for developing conditions
for storageof translation-competent mitochondria.
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