W E JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and 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 Cytochrome c Oxidase Subunit 11, a Mitochondrial Gene Product*

(Received for publication, January 17, 1989)

George H. D. Clarkson and Robert 0. PoytonS From the Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, BouMer, Cokrado 80309-0347

Subunit I1 of yeast cytochrome c oxidase is synthe- sized on mitochondrial ribosomes as a precursor con- taining a transient NHa-terminal presequence and is inserted into the mitochondrial inner membrane from the matrix side. Using an optimized in vitro mitochon- drial translation system (McKee, E. E., and Poyton, R. 0. (1984) J. Biol. Chem. 259, 9320-9331), we have examined the requirement for an electrochemical po- tential (Ab,+) across the inner mitochondrial membrane during subunit I1 biogenesis. When mitochondrial gene products are synthesized under conditions that prevent formation of a normal Ai,+, accumulation of unproc- essed subunit I1 (pre-11) occurs. The majority of pre-I1 generated in this way is inserted into the lipid bilayer, as judged by resistance to extraction with 0.1 M Na2COs. Therefore, it appears that a Ab* is required for the normal biogenesis of subunit 11, and that the Ab,+ is required for a function other than the insertion of pre-I1 into the lipid bilayer of the inner membrane.

Most mitochondrial polypeptides are encoded by nuclear genes and are imported into mitochondria from the cytoplasm. However, a few are mitochondrial gene products and are synthesized on endogenous ribosomes (1). The majority of mitochondrially synthesized polypeptides are integral mem- brane components and are associated with nuclear gene prod- ucts in 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 pertaining to the import of cytoplasmically synthesized mitochondrial precursor polypeptides (reviewed in 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) and the involvement of nucleoside triphosphates in productive inter- action of precursors with the mitochondrial surface and in completion of precursor translocation (9-11). In contrast, 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 thus far undefined.

We have investigated the role of a A;,+ in the biogenesis

*This work was 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 accordance with 18 U.S.C. Section 1734 solely to 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-terminal presequence (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), and apparently 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 via two hydrophobic do- mains, with both termini protruding into the intermembrane space (19).

In this paper, we demonstrate that the precursor form of subunit I1 accumulates when mitochondrial translation prod- ucts are synthesized under conditions that prevent formation of a normal A;,+. Furthermore, we show that the majority 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 the Ai,+ is involved in the biogenesis of mitochondrial polypeptides that are synthesized on oppo- site sides of the inner membrane.

MATERIALS AND METHODS

Isolation of Mitochondria-The wild type Saccharomyces cereuisiae strain D273-10B (MATa, ATCC No. 24657) was grown 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 (3 mg/g wet weight cells), and the recovery 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 to pH 7.2 with KOH, and pelleted by centrifugation for 10 min at 13,000 X gmaX and 2 “2. Mitochondria that have been “rescued” in this way perform protein synthesis that is insensitive to the presence of ribonuclease A, a t rates greater than 90% of those exhibited by freshly isolated organ- elles. The results presented in this paper were obtained with such rescued mitochondria.

Conditions for in Vitro Mitochondrial 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 in ethanol 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) (50 mM) 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

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 labeled polypeptides 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 air dried.

Gel Electrophoresis-SDS-polyacrylamide slab gel electrophoresis was performed in a discontinuous buffer system (21). Resolving and stacking gels contained 16 and 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% Triton x-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 rocked with an equal volume of suspended beads for 2 h at room temperature. The beads were reisolated by centrifugation, washed three times with immunobuffer, once with distilled water, and immune complexes were 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 protein determination 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 contains a respiratory substrate, permitting synthesis of matrix ATP via oxidative phosphorylation, as well as an exogenous ATP-regenerating system. To investi- gate the role of a A;,+ in subunit I1 biogenesis, isolated mitochondria were suspended in PSM and pulse-labeled in the presence of various energy poisons. In these experiments, oligomycin was used to inhibit the FIFo-ATPase and 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 the rate and extent of protein synthesis. In contrast, 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 translation products 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 energy poisons 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 (10 pg of mitochondrial protein) were taken from each reaction, and the amount of trichloroacetic acid-precipitable radioactivity was determined. The maximum value obtained in 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 (20 p M 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 resolved by SDS-PAGE and visualized by fluorography (Fig. 2A). In the presence of oligomycin and cyanide (lane 7), oligomycin and CCCP (lane 8), 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 inhibitor of 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 (lane 5 ) or valinomycin (lane 6) alone, but not in mitochondria incubated with ethanol (lune Z), oligomycin (lane 3), or cya- nide (lane 4 ) . The identity of the extra band 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 that the extra band generated by these drug treatments is also recognized by anti-subunit I1 IgG. 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 it represents unprocessed pre-11.

Interestingly, we have never observed complete inhibition of pre-I1 processing 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 before pulse labeling or the concentrations of inhibitors increases the proportion of pre-I1 and that mature radiola- beled subunit I1 arises 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

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 precursor form 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 indicated below. A , an aliquot from each reaction was analyzed by SDS-PAGE and fluorography; B, a second aliquot was subjected to immunoprecipitation using 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 (1 mM final concentration); lanes 5, +carbonyl cyanide m- chlorophenylhydrazone (20 p M final concentration); lanes 6, +valin- omycin (1.25 pg/ml final concentration); lanes 7, potassium cyanide (1 mM) + 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 1-111 of 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 1 polypeptides are indi- cated.

mitochondria into open membrane sheets and release 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 total protein into the soluble fraction. The presence of CCCP and oligomycin has no significant effect on this distri- bution. In contrast, most of the radiolabeled polypeptides synthesized in the presence or absence of inhibitors are lo- cated in the membrane pellet, although the percentage of membrane-associated trichloroacetic acid-precipitable radio- activity is reduced slightly by the inclusion of CCCP and oligomycin.

TABLE I 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" Trichloroacetic acid-

radioactivityb precipitable

Control CCCP + CCCP + olieomvcin oliaomvcin

% "

% "

Na2C03 pellet 34.4 f 4.1 31.7 f 1.6 95.0 f 0.8 90.3 f 1.9 Na2C03 soluble 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. ATPase 6

1 2 3 4 5 6 -. -

P c - pre-lI

FIG. 3. Carbonate extraction of pulse-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, incubated on ice for 30 min, and then separated into pellet and supernatant fractions by centrifugation for 1 h 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). Membrane pellets 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, unextracted mitochondria; lanes 2 and 5, Na2CO3-insoluble; lanes 3 and 6, Na~CO3-soluble. In both cases, 15% of the total recovered pellet and supernatant material was loaded on the gel.

All of the translation products labeled in a control reaction except for the ribosomal Var 1 polypeptide are refractory to carbonate extraction (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-I1 generated in the presence of CCCP and oligomycin is also resistant to carbonate extrac- tion (lunes 5 and 6) , indicating that insertion per se of pre-I1 into the inner membrane does not require a A ~ H + . The partial susceptibility of pre-I1 to carbonate extraction may reflect the existence of a subpopulation 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 inner membrane.

Biogenesis of Cytochrome c Oxidase Subunit I I 10117

DISCUSSION

In this report, we show for the first time that mitochondrial energy poisons can be used to inhibit processing of pre-11, the precursor form of cytochrome c oxidase subunit 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 are synthesized 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 of pre-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 to abol- ish A;H+, it simultaneously stimulates proton pumping 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$, the component of A;,+ arising from the separation of charged species across the inner membrane, then a proton gradient would be sufficient to support almost normal levels of pre-I1 processing. Alternatively, if A$ 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 the ASH+ plays an analogous role in the insertion of mitochondrial gene products into the 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 translation products) with the inner membrane as integral polypeptides.

Why does subunit I1 biogenesis require a A;H+? Several possible roles of the membrane potential in subunit I1 biogen- esis can currently be envisaged. First, an energized inner membrane may be required for proper (ie. processing-com- patible) insertion of pre-I1 into the inner 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 observation3 that a point mutation which changes residue 109 of the mature subunit I1 sequence from tryptophan to arginine causes essentially complete inhibition of pre-I1 processing. Although the effect of this mutation is more 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 folding than 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- encoded subunit of holocytochrome c oxidase) that is imported into the inner membrane in a AbH+-dependent manner. It has been demonstrated previously that isolated yeast mitochon- dria contain a depletable, 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 inner membrane, in a fashion analogous to the “stage 3” intermediate described by Pfanner and Neupert (8) for the ADP/ATP carrier. In the above scenario, the incomplete inhibition of pre-I1 processing 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 I1 polypeptides. Finally, the enzyme that catalyzes removal of the pre-I1 presequence may depend on a A;,+ for optimal activity. By testing each of these possibilities, it should be possible to define more rigorously the role of the membrane potential in the biogenesis of mitochondrial gene products and thus derive 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 storage of translation-competent mitochondria.

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