Experimental Cell Research 263, 107–117 (2001)doi:10.1006/excr.2000.5096, available online at http://www.idealibrary.com on

Oxidative Stress Inhibits the Mitochondrial Import of Preproteins andLeads to Their Degradation

Gary Wright,*,1,2 Kazutoyo Terada,*,1 Masato Yano,* Igor Sergeev,† and Masataka Mori*,3

*Department of Molecular Genetics, Kumamoto University School of Medicine, Honjo 2-2-1, Kumamoto 860-0811, Japan; and

†Department of Anatomy, Cell and Neurobiology, Marshall School of Medicine, Huntington, West Virginia 25704-9388

The mitochondrion depends upon the import of cy-tosolically synthesized preproteins for most of the pro-teins that comprise its structural elements and meta-bolic pathways. Here we have examined the influenceof redox conditions on mitochondrial preprotein im-port and processing by mammalian mitochondria.Paraquat pretreatment of isolated mitochondria in-hibited the subsequent import preornithine transcar-bamylase (pOTC) in vitro. In intact cells oxidizing con-ditions led to decreased levels of mature OTC andaccumulation of its preprotein. Implicating a mito-chondrial import lesion, the fluorescence of pOTC-GFP (a protein in which the presequence of pOTC wasfused to green fluorescent protein) transfected cellswas decreased by paraquat treatment while cytosolicwild-type GFP remained largely unaffected. The accu-mulation of preproteins was enhanced by proteasomeinhibitors. We observed that precursor proteins thatfailed to be imported, due to oxidizing conditions or anintrinsically slower import rate, are susceptible todegradation. Inhibition of the proteasome was alsofound to lead to higher levels of the translocase outermembrane protein 20 (Tom20) and to the perinuclearaccumulation of mitochondria. These studies indicatethat cellular redox conditions influence mitochondrialimport, which, in turn, affects mitochondrial proteinlevels. A role for the proteasome in this process and ingeneral mitochondrial function was also indicated.© 2001 Academic Press

Key Words: mitochondrial processing; proteasome;redox; oxygen radicals; perinuclear aggregation; pre-protein import.

1 Gary Wright and Kazutoyo Terada contributed equally to thiswork.

2 Present address: University of Maryland at Baltimore, Depart-ment of Biochemistry, School of Medicine, 108 North Green StreetBaltimore, MD 21201.

3 To whom correspondence and reprint request should be ad-dressed at Department of Molecular Genetics, Kumamoto UniversitySchool of Medicine, Honjo 2-2-1, Kumamoto 860-0811, Japan. Fax:

181-96-373-5140. E-mail: masa@gpo.kumamoto-u.ac.jp.

107

INTRODUCTION

The mitochondrion imports the majority of the pro-teins that comprise its metabolic pathways and struc-tural elements. Mitochondrial matrix proteins are usu-ally synthesized in the cytosol as precursor proteinscontaining an N-terminal presequence of between 20and 70 amino acids in length that is sufficient to directthem to the mitochondrial matrix. This presequence issubsequently removed in the matrix by a specific mi-tochondrial processing peptidase. Although demon-stration of the completed precursor proteins in thecytosol and their rapid conversion to the mitochondrialmature forms in intact cells indicates posttranslationalimport [1], a report suggesting cotranslational importof a chimeric precursor protein appeared recently [2].The translocation of cytosolic preproteins into the mi-tochondrion is a complex process which requires cyto-solic factors, a mitochondrial inner and outer mem-brane translocational machinery, and matrix proteinsto result in a properly localized and assembled mito-chondrial protein [3, 4]. The deletion of only five mito-chondrial proteins has been reported to lead to a non-viable phenotype in yeast [5]. That all five of theseproteins are involved in some aspect of mitochondrialprotein import indicates the critical nature of this cel-lular parameter. These observations coupled to theidentification of mutations in proteins involved in themitochondrial import machinery as responsible fordeafness dystonia syndrome [6] suggest a role for dys-functional mitochondrial import in pathologies.

While the mechanism and components of the importmachinery responsible for preprotein translocationacross the mitochondrial outer and inner membraneshave been extensively studied, especially in yeast, apaucity of information is available concerning factorswhich may affect this process under either physiologi-cal or pathological conditions. However, recent evi-dence has begun to challenge the view that mitochon-drial internalization of preproteins is a constitutiveunregulated process. For instance, in plant cells themitochondrial import of proteins has been shown to be

a dynamically regulated process that is influenced by

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108 WRIGHT ET AL.

the light–dark cycle, wounding, and developmentalcues [7, 8]. In mammalian cells, differences observed inthe import rates of skeletal muscle intermyofibrillarand subsarcolemmal mitochondrial subpopulationshave been suggested to underlie the different func-tional characteristics of these two populations of mito-chondria [9]. In addition, it has also been recentlyreported that contractile activity and thyroid hormonetreatments both specifically stimulate mitochondrialimport rates [10, 11]. Taken together these observa-tions point to a role for mitochondrial import rates inmodulating mitochondrial phenotype and function inresponse to changing metabolic demands.

The present studies are motivated by our previousobservations that oxidizing conditions lead to an inhi-bition of processing of mitochondrial manganese-de-pendent superoxide dismutase in the baculovirus in-sect cell expression system [12, 13]. Here we haveexamined the influence of oxidizing conditions on theimport and processing of several mitochondrial matrixproteins in mammalian cells. The data indicate thatoxygen radicals inhibit mitochondrial import, which inturn affects the levels of mature mitochondrial pro-teins. These studies further suggest an important rolefor proteasome activity in the degradation of nonim-ported precursor proteins, as well as in general mito-chondrial function and localization.

MATERIALS AND METHODS

Reagents, antibodies and plasmids. 5,59,6,69-Tetrachloro-1,13,39-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) was purchasedfrom Molecular Probes. Lactacystin, MG101, MG115, and protea-some inhibitor 1 were purchased from Calbiochem. Paraquat waspurchased from Sigma. Preparation of antisera against Tom20, OTC,and GFP was previously described [14, 15]. Mouse monoclonal anti-body against glyceraldehyde 3-phosphate dehydrogenase (GAPDH)was purchased from Chemicon. An antibody against rat acetoacetyl-CoA thiolase [16] was provided by T. Hashimoto (Shinshu Univer-sity, Japan). Mammalian expression plasmids for pOTC, GFP, andpOTC-GFP in pCAGGS or pCXN2 [17] and the pGEM-3Zf(1)-hpOTCwhich was used for a template for in vitro translation of pOTC havebeen previously characterized [14, 15].

Western blots. Cells were harvested with trypsin, washed twicewith PBS, pH 7.4, and sonicated in buffer containing 10 mM Tris zHCl (pH 7.4), 5 mM MgCl2, 100 mM PMSF, 1 mM antipain, and 1 mMeupeptin. Sonicates were cleared of debris via centrifugation, andupernatant protein concentrations were determined using Bio-Radrotein Micro Assay with bovine serum albumin as protein standard.DS–PAGE was followed by semidry electrotransfer of proteins toitrocellulose membranes. Proteins were immunoprobed and visual-

zed with ECL (Amersham Pharmacia Biotech) according to theanufacturer’s protocols.Cell culture and stable and transient transfectants. COS-7 andeLa cells were cultured in Dulbecco’s modified Eagle’s medium

upplemented with 10% fetal calf serum, at 37°C in a 5% CO2/95%air atmosphere. CHO cells were cultured in Ham’s medium F-12supplemented with 10% fetal calf serum, at 37°C in a 5%/95% CO2 toair atmosphere. Transient transfections were preformed with 2 mg ofexpression plasmids and 8 ml TransIt per 60-mm tissue culture dish

4 h incubation) following TransIt-LT1 polyamine transfection re-

gent protocols (Pan Vera). Cell tranfectional efficiencies were about%. To select for stable transformants, cells were cultured continu-usly in 0.4–0.8 mg/ml Geneticin (G-418 sulfate; GIBCO BRL) for upo a month. Once stable lines were obtained G418 treatment wasiscontinued for periods of up to 2 weeks without an appreciable lossf expressing cells (in the case of GFP). For hyperoxic culture condi-ions cells were placed in an airtight vacuum desiccator with entrynd exit ports. A mixture of 95% O2/5% CO2 was circulated through

the chamber for at least 10 min and the ports were sealed. Thehyperoxic atmosphere was replenished within 12 h.

Fluorescent imaging. pOTC-GFP stable expressing cells werefixed directly with 3% paraformaldehyde in PBS onto tissue cultureplates. Cells were viewed on an Olympus IX70 fluorescent micro-scope equipped with a GFP filter, and images of GFP fluorescencewere acquired using a C5810 color chilled 3 CCD video camerasystem (Hamamatsu Photonics, Japan). For quantitation of singlecell fluorescence, cells were grown on coverslips, fixed with 3% para-formaldehyde in PBS, and mounted on slides using slowfade mount-ing gel. Images were acquired with a Nikon Diaphot inverted micro-scope and quantitative analysis was performed using Image-1/FLanalysis software. Fields of view were randomly selected and fluo-rescent cells were manually traced so that nonfluorescent nucleiwere excluded from the analysis. Confocal images were obtainedwith a Zeiss LSM410 confocal laser scanning microscope, using a633/NA 1.4 objective.

In vitro import and membrane potential measurements. Mito-chondrial import experiments were performed as previously de-scribed [18, 19]. mRNA for human pOTC was sythesized by in vitrotranscription, and used for in vitro translation of precursor protein inthe rabbit reticulocyte system (Promega). Precursor protein waslabeled with Pro-mix (Amersham Pharmacia Biotech) containingL-[35S]methionine and L-[35S]cysteine. Mitochondria were isolated as

reviously described [20] and treated with paraquat in incubationuffer (50 ml) containing 200 mM sucrose, 10 mM Tris z Mops (pH.4), 5 mM Succinate z Tris, 1 mM Pi z Tris, and 0.01 mM EGTA z Tris.

The import reaction, containing 4 mL of the lysate (8.8–65 kBq) and00 mg mitochondria, was carried out 25°C in import buffer (50 mL)ontaining 230 mM mannitol, 50 mM KCl, 10 mM Mops z KOH (pH.4), 1 mM ATP, 0.01 mM ADP, 1 mM methionine, 1 mM succinate,mM MgCl2, 5 mM K3PO4, and 1% bovine serum albumin.Cell fractionation. To generate cytosolic-, mitochondrial-, and nu-

lear-enriched fractions cultured cells were homogenized in mito-hondrial isolation buffer (210 mM D-mannitol, 70 mM sucrose, 0.2

mM EGTA z HCl, pH 8.2, Hepes z KOH, pH 7.5, 100 mM PMSF, 1 mMantipain, 1 mM leupeptin, and 5 mg/mL bovine serum albumin). The

omogenates were centrifuged at 600g generating the nuclear pellet.he supernatant was centrifuged at 12,000g to generate the mito-

chondrial pellet and soluble cytosolic fractions.Other methods. Membrane potential measurements were per-

formed under similar conditions using modification of methods pre-viously described [21]. Paraquat was solubilized in H2O. Mitochon-

ria 250 mg were incubated with paraquat in 100 mL of incubationbuffer. Samples were centrifuged and resupended in 0.5 mL of im-port buffer containing 310 nM JC-1. Fluorescence was measured at590 nm using a Hitachi F3010 fluorescent spectrophotometer (exci-tation, 575 nm). ATP in COS-7 cells was measured using the somaticcell ATP assay kit (Sigma).

RESULTS

Effect of Oxidizing Conditions on the Import andProcessing of Precursor OTC by Mitochondriain Vitro

The preincubation of isolated rat liver mitochondria

with paraquat, a redox cycling compound that gener-

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109OXIDATIVE STRESS ON MITOCHONDRIAL PROTEIN IMPORT

ates superoxide radicals, was found to inhibit the rateof protein processing when mitochondria were subse-quently resuspended in import buffer and an in vitro-ynthesized precursor protein 35S-labeled pOTC was

FIG. 1. Paraquat preincubation of isolated rat liver mitochon-ria inhibits subsequent mitochondrial import but does not affectembrane potential. (A) 100 mg of purified mitochondria was incu-

ated in 50 ml of buffer A with or without 1 mM paraquat for 10 minn ice, and then pelleted, washed, and resuspended in import buffer.he import reaction was initiated with the addition of 35S-pOTC-

containing rabbit reticulocyte lysate (10 ml) and was stopped afterincubation for the indicated times at 25°C with the addition of 1 mMdinitrophenol. The mitochondrial suspensions were centrifuged andthe pellets were dissolved in SDS–PAGE sample buffer for electro-phoresis and fluorography analysis. p, pOTC; m, mature OTC. (B)Dose response of paraquat inhibition of mitochondrial import. Mito-chondria were preincubated in the indicated concentrations of para-quat, the import reactions were preformed for 20 min at 25°C, andthe mitochondria were reisolated and subjected to electrophoresisand fluorographic analysis. The amount of mature OTC was quan-titated by image plate analysis. The amount of OTC of untreatedmitochondria was set at 100%, for the three independent experi-ments and data are expressed as means 6SE (n 5 4–8). (C) The

embrane potential of isolated mitochondria treated with paraquatnder conditions similar to those described above was assessed usinghe membrane potential sensitive dye JC-1, as described under Ma-erials and Methods. DNP, dinitrophenol. Data are expressed as theean relative fluorescence intensity 6SE (n 5 4).

added (Fig. 1A). The import and processing rates of

paraquat-treated mitochondria were diminished asjudged from the lower amounts of mature OTC prod-uct. In addition, greater amounts of pOTC were asso-ciated with the paraquat-treated mitochondrial pellet.This may indicate an accumulation of pOTC on themitochondrial import receptors due to slower importrates. The sensitivity of in vitro mitochondrial process-ing inhibition was found to reach maximal at approx-imately 55% inhibition at paraquat concentrations.0.5 mM paraquat with a half-maximal effect between0.05 and 0.25 mM paraquat concentrations (Fig. 1B).Residual paraquat present during the import reaction(when pOTC is added) could in theory lead to directeffects such as aggregation of pOTC. Because mito-chondria were washed before they were resuspended inimport buffer, very little residual paraquat is expectedto be present during the import reaction. These lowlevels of paraquat are unlikely to have a direct effect onthe pOTC or cytosolic factors present in the rabbitreticulocyte. No evidence of aggregation of pOTC wasobserved when pOTC synthesized in rabbit reticulo-cyte was incubated in the presence of 1 mM or higherparaquat concentrations in conditions identical asthose described for the mitochondrial import reaction(data not shown).

Previous reports have indicated that with paraquattreatment in these conditions, mitochondrial mem-brane proteins, which have susceptible redox sensitivevincinal SH-groups, become oxidized but that mito-chondrial permeability transition is not induced andthere is no loss of membrane potential or integrity [22,23]. To confirm this, the membrane potential of mito-chondria treated with paraquat was evaluated withthe membrane potential sensitive dye JC-1 (Fig. 1C)and found unaffected in these experimental conditions.

Paraquat Inhibits Mitochondrial PreproteinProcessing in Intact Cells as Revealed byProteasome Inhibition

Increasing amounts of paraquat led to a decrease inthe amount of mature OTC in the absence of a corre-sponding increase in pOTC in a HeLa cell line stablyexpressing pOTC (Fig. 2A). Results were comparablewhen a CHO cell line that was stably expressing pOTCwas examined (data not shown). As a measure of cellviability, and because the synthesis and mitochondrialprocessing of pOTC are both dependent upon intracel-lular ATP [1], its levels were assessed and found to bedecreased only slightly by paraquat treatment (Fig.2B). This suggested that general toxicity, althoughprobably a component, may not explain the decreasedlevel of OTC.

The absence of detectable precursor protein accumu-lation led us to explore the possibility that pOTC was

being proteolytically degraded. We found that lactacys-

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110 WRIGHT ET AL.

tin, a specific inhibitor of the proteasome, causes theappearance of pOTC in control cells and a larger accu-mulation of the preprotein in paraquat-treated cells(Fig. 2C). In the presence of lactacystin, two fastermigrating polypeptides of 31 and 29 kDa (Fig. 2C, aand b) were detected in association with pOTC accu-mulation. These polypeptides are presumably degrada-tion intermediates of pOTC/OTC as they appear only in

FIG. 2. The effect of paraquat and lactacystin on the levels ofmitochondrial preproteins and mature proteins in cultured cells. (A)HeLa cells stably expressing pOTC were treated with the indicatedconcentrations of paraquat and harvested after 24 h. Cell extracts(10 mg protein) were subjected to for immunoblot analysis using annti-OTC rabbit polyclonal antiserum (1:1,000 dilution). Represen-ative results of four independent experiments are shown. (B) CHOells were treated with the indicated concentrations of paraquat andTP concentrations were measured as described under Materialsnd Methods. The data are expressed as means 6SE (n 5 4). (C)HO cells stably expressing pOTC were treated with combinations of.6 mM paraquat and lactacystin for 20 h. Both reagents wereolubilized in H2O. Cell extracts (15 mg protein) were subjected tommunoblot analysis using the anti-OTC anti-serum or rabbit anti-at acetoacetyl-CoA thiolase (T2) IgG (2 mg/ml). p and m, precursornd mature forms of proteins; a and b, putative degradation inter-ediates of pOTC.

pOTC-expressing cultures treated with proteasome in- s

hibitors. Accumulation of a preprotein in the presenceof lactacystin was also observed for acetoacetyl-CoAthiolase, an endogenous mitochondrial matrix proteinpresent in CHO cells (Fig. 2C). However, paraquat didnot further increase this precursor’s accumulation.

The effects of paraquat on the processing of an arti-ficial mitochondrial targeted GFP was evaluated in aCHO cell line stably expressing pOTC-GFP, a con-struct in which the presequence of pOTC was fused toGFP and that has been shown to be a useful probe forassessing mitochondrial import and processing of pro-teins in intact cells [14]. The results were similar tothose with the pOTC-expressing cells, in that paraquattreatment led to reduced levels of mature mitochon-drial protein (Fig. 3A). In contrast to the pOTC-ex-pressing cells, the appearance of precursor was ob-served in these cells when treated with paraquat evenin the absence of lactacystin. This may indicate thatthe proteasome is less efficient at recognizing or de-grading the artificial fusion protein. However, treat-ment of the these cells with lactacystin demonstratedthat, in fact, large amounts of pOTC-GFP are beingdegraded by the proteasome in these cells even underbasal conditions. Large amounts of pOTC-GFP weredetected in cells treated with lactacystin in both thepresence and the absence of paraquat (Fig. 3B). Theeffect of paraquat in reducing levels of mature GFPwas abolished when the proteasome was inactivated inthese cells.

In the case of pOTC-GFP, mitochondrial GFP fluo-rescence is dependent upon the entire protein process-ing apparatus of the mitochondrion, and can thus beconsidered the penultimate product of this process,while wild-type GFP displays no such dependency onmitochondrial processing for fluorescence. Cells weretransiently transfected with mammalian expressionvectors encoding either pOTC-GFP or cytosolic wild-

FIG. 3. The effect of paraquat on the levels of pOTC-GFP andmature GFP in pOTC-GFP stably expressing CHO cells in the pre-sense and absense of lactacystin. CHO cells were treated with theindicated concentrations of paraquat for 24 h in the absence (A) orpresence of 25 mM lactacystin (B). Cell extracts (10 mg protein) wereubjected to immunoblot analysis using an anti-GFP rabbit anti-

erum (1:1,000 dilution).

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111OXIDATIVE STRESS ON MITOCHONDRIAL PROTEIN IMPORT

type GFP and the effect of paraquat on cellular fluo-rescence was assessed as an indicator of pOTC-GFPprocessing in these cells. While higher concentrationsof paraquat tended to decrease wild-type GFP cell flu-orescence, the effects of paraquat on pOTC-GFP-trans-fected cells were far more severe, with no detectablefluorescence observed in cells treated with paraquatconcentrations .0.6 mM (Fig. 4A). Western blot anal-sis confirmed that paraquat treatment decreased ma-ure GFP protein levels in the pOTC-GFP-transfectedells (Fig. 4B). In these transiently transfected cells aarallel increase in pOTC-GFP was found to accom-any the decreases in mature GFP with paraquatreatment. In contrast to the stable expressing cellines, the effects of paraquat in reducing mature pro-ein levels in these transiently expressing cells wereimilar in both lactacystin-treated and untreated cells.

comparison of precursor protein in lactacystin-reated versus untreated cells indicated that a signifi-ant amount of pOTC-GFP was degraded in these cellsnder basal conditions (Fig. 3). It should be noted thathe expression levels of pOTC-GFP obtained in the

FIG. 4. The effect of paraquat on the mitochondrial processingand fluorescence of the fusion construct pOTC-GFP. (A) COS-7 cellsgrown on glass coverslips were transfected with either a pCAGGSexpression vector encoding pOTC-GFP fusion protein or wild-typeGFP (2 mg of DNA per 60-mm plate). After treatment with thendicated concentrations of paraquat for an additional 22 h, theuorescent intensity of individual cells (n 5 9–18) was measured, asescribed under Materials and Methods using an inverted fluores-ent microscope and image analysis software. The data are expresseds means of the arbitrary fluorescent units per cell. (B) COS-7 cellsere transfected with pOTC-GFP expression vector as describedbove and allowed to incubate for 12 h before treatment with thendicated concentrations of reagents for an additional 18 h. Proteinxtracts (3 mg/lane) underwent immunoblot analysis using anti-GFP

polyclonal antiserum.

ransiently transfected COS-7 cells was extremely high l

nd was estimated to be at least 15% of total cellularrotein. The high levels of pOTC-GFP apparently over-helm the proteasome and mitochondrial processing

apacity of the cells so that even in cells untreated withactacystin a large amount of precursor protein isresent. This is in marked contrast to the stable pOTC-FP-expressing CHO and pOTC-expressing HeLa cell

ines where the expression levels are much lower, mea-ured at 0.5–1.0% of total cellular protein (data nothown), respectively, and precursor protein was notetected except in the presence of lactacystin.

ffect of Hyperoxia on pOTC Processingin Intact Cells

The increased levels of superoxide generated by theitochondrial electron transport chain in conditions of

yperoxia is thought to be the primary factor in hyper-xia toxicity. For this reason it was of interest to ex-lore the potential effects of hyperoxia on mitochon-rial precursor processing. Hyperoxic cultureonditions (95% O2/5% CO2) was found to lead to a

modest but consistent reduction of approximately 20%of mature OTC in the stable expressing CHO cell line(Fig. 5B). This decline was prevented by the addition oflactacystin to the culture medium. In addition, accu-mulation of precursor OTC was only observed in hy-peroxia-cultured cells in which lactacystin was alsopresent, indicating import inhibition in hyperoxic con-ditions (Fig. 5A). The two faster migrating bands were

FIG. 5. The effect of hyperoxia on the processing of pOTC inCHO cells. (A) The stably expressing pOTC-CHO cell line was cul-tured in a hyperoxic (HYP) (95% O2/ 5% CO2) or air (22%O2/ 5%CO2)tmosphere, in the presence or absence of lactacystin (25 mM) for

24 h. Protein extracts (10 mg/lane) were analyzed by immunoblotanalysis followed by ECL chemiluminescent detection. In order todetect pOTC some overexposure of the mature band chemilumines-cent intensity was necessary. (B) The same blot as A with a lowerexposure time (4- versus 22-s exposure) of the ECL procedure inwhich the intensity of the signal for mature OTC is within the rangeof linearity. Macbas densitometric quantitative analysis of the ma-ture band was performed, with control air cultured levels set to 100%in each experimental set. The data are expressed as means % aircontrol 6SE (n 5 6–10): hyperoxia-treated, 81 6 6.9; air plus

actacytin-treated, 107 6 8.2; hyperoxia plus lactacystin, 104 6 13.2.

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112 WRIGHT ET AL.

again observed in this CHO cell line when lactacys-tin was present in the medium of hyperoxia-treatedcells.

Effects of Protease Inhibitors on Accumulationof Preproteins

Having observed the appearance of preproteins anddegradation intermediates in the presence of lactacys-tin, we confirmed the specificity of this effect to protea-some inhibition by testing other proteasome and non-proteasome inhibitors (Fig. 6). Accumulation of pOTCwas observed in the presence of proteasome inhibitorsMG115 and proteasome inhibitor 1 (PS1) as well aslactacystin. The two faster migrating polypeptides (aand b) were consistently observed in the presence oflactacystin, whereas the polypeptide b was not evidentin the presence of MG115 and proteasome inhibitor 1.In contrast, no accumulation of pOTC was observed inthe presence of MG101 (calpain inhibitor) or of themixture of antipain and leupeptin (inhibitors of severalserine and cysteine proteases) and E-64 (cysteine pro-tease inhibitor). Similar results were obtained for thefusion protein pOTC-GFP. These results suggest thatthe proteasome is responsible for the degradation ofpreproteins.

Characterization of the Nonprocessed pOTC andpOTC-GFP

Proteasomal degradation of proteins is generallypreceded by ubiquitination (covalent attachment of theubiqitin polypeptide) of the target protein. Thus inhi-

FIG. 6. The effect of proteasome inhibitors on accumulation ofpreproteins. CHO cells were treated for 16 h with 10 mM of MG101,MG115, lactacystin, proteasome inhibitor 1 (PS1), or a combinationof antipain (AP), leupeptin (LP), and E-64. Cell extracts (15 mgprotein) were subjected to immunoblot analysis using the anti-OTCantiserum (A) or anti-GFP antiserum (B).

bition of proteasome often leads to accumulation of r

higher molecular weight ubiquitin conjugates. A pro-tein ladder larger than the precursor protein was notdetected with the highly specific and robust anti-OTCpolyclonal antibody preparation. In the case of pOTC-GFP-expressing cells, one slower migrating proteinwas sometimes observed but it was not shifted enoughto represent a ubiquitin–pOTC-GFP conjugate (Figs.3B and 6B). We are not certain of the nature of thisprotein but it may represent a rarely expressed pOTC-GFP (i.e., alternate initiation or stop codon) that issusceptible to proteasome degradation.

One explanation for the detection of unprocessedprecursor proteins during proteasome inhibition bylactacystin, is that unprocessed precursor protein mayaccumulate due to the failure of its import into themitochondrion. Alternatively, a failure of the protein tobe processed in the mitochondrial matrix of the mito-chondrion by the processing peptidase after being im-ported could result in accumulation of precursor pro-tein. The sensitivity of pOTC-GFP in digitonin-permeabilized cells to proteinase K digestion suggeststhe extramitochondrial localization of this precursor(Fig. 7). It was also observed that precursor proteinwas associated exclusively with the pellet and was notfound in the cytosolic soluble fraction in the digitonin-treated cells.

To more fully explore the localization of the pOTC-GFP and pOTC in these CHO cell lines they werefractionated via differential centrifugation into nucle-ar-, mitochondrial-, and cytosolic-enriched fractions.pOTC and pOTC-GFP were found associated with boththe nuclear- and the mitochondrial-enriched fractionsin lactacystin-treated cells (Fig. 8A, panels a and b). Incontrast, in the pOTC-GFP stable cells not treated withlactacystin, mature GFP was highly enriched in themitochondrial fraction and was nearly absent from thenuclear fraction (Fig. 8A, panel c). Likewise in un-treated cell fractions Tom20, a component of the outermitochondrial membrane import receptor, was almost

FIG. 7. Sensitivity of pOTC-GFP to proteinase K digestion indi-cates extramitochondrial localization of the accumulated preprotein.Stable pOTC-GFP-expressing CHO cells, treated with paraquat (0.6mM) and lactacystin (25 mM), were permeabilized with digitonin.Cells were incubated on ice in a 0.1% digitonin–PBS solution with/without the presence of proteinase K (40 mg/ml) for 5 min. Pellet andoluble fractions were separated by centrifugation at 12,000g. West-rn blot analysis was performed with anti-GFP or anti-GAPDH

abbit polyclonal and mouse monoclonal antibody, respectively.

113OXIDATIVE STRESS ON MITOCHONDRIAL PROTEIN IMPORT

exclusively associated with the mitochondria-enrichedfraction as expected (Fig. 7A, panel d) while Tom20was found to be present in both the nuclear and mito-chondrial fractions in lactacystin-treated cells (Fig. 8A,panel e). A considerable amount of mature OTC andGFP was observed in the cytosolic fraction under allconditions, indicating some amount of mitochondrialbreakage during homogenization. GAPDH was, as ex-pected, found exclusively in the cytosolic fraction (Fig.8A, panel f).

A comparison between panels d and e suggested thatthe increase in Tom20 in the nuclear fraction repre-

FIG. 8. Cellular localization of accumulated pOTC and pOTC-GFP in stably expressing CHO cell lines. (A) CHO cells were frac-tionated into nuclear (N), mitochondrial (M), and cytosolic (C)-en-riched fractions as described under Materials and Methods. All cellswere treated with their respective reagents for 20 h. FractionatedpOTC-expressing cells treated with 0.6 mM paraquat and 25 mMlactacystin and probed with anti-OTC polyclonal antibody (a); frac-tionated pOTC-GFP expressing cells treated with 25 mM lactacytinand probed with anti-GFP polyclonal antibody (b); fractionated un-treated pOTC-GFP expressing cells probed with anti-GFP polyclonalantibody (c); fractionated untreated pOTC-GFP expressing cellsprobed with anti-hTom20 polyclonal antibody (d); fractionatedpOTC-GFP expressing cells treated with 25 mM lactacytin andprobed with anti-Tom20 polyclonal antibody (e); fractionated un-treated pOTC-GFP expressing cells probed with anti-GAPDH mousemonoclonal antibody (f). Protein was loaded stoiciometrically foreach fraction obtained. Using this fractionation procedure proteinratios were found to vary some between experiments and treatmentgroups but were ;1:1.5:4 for N, M, and C fractions, respectively, onaverage. (B) Whole cell lysates of HeLa and CHO cells treated withand without lactacystin (25 mM) for 24 h. Samples were analyzed bySDS–PAGE and Western blotting and probed with anti-Tom20 an-tibody. Results are representative of four independent experiments.

sented not just a shift in the localization of Tom20 from

the mitochondrial fraction, but also a gross increase inthe amount of Tom20 in the lactacystin-treated cells. Adirect comparison between panel d and e can be madebecause these samples were processed, run on Westernblots, and detected in parallel. The induction in thelevel of Tom20 was confirmed with whole-cell homoge-nates of lactacytin-treated cultures, the results ofwhich are shown (Fig. 8B). The observation thatTom20 levels increase when the proteasome is inhib-ited suggested a reason for the shift of mitochondriaproteins OTC, pOTC, pOTC-GFP, and GFP to the nu-clear fraction in lactacystin-treated cells because wehave previously shown that perinuclear accumulationof mitochondria results when Tom20 is overexpressed[14]. Perinuclear accumulation of mitochondria wouldoffer an explanation for the heavy mitochondrial pro-tein contamination observed in the nuclear-enrichedfraction of the lactacystin-treated cells.

Proteasome Inhibition Induces PerinuclearAccumulation of Mitochondria

Paraquat (0.6 mM) treatment of stable pOTC-GFP-expressing CHO cells did not appear to alter mitochon-drial morphology, although a decrease in GFP fluores-cence was evident after 20 h of treatment. Lactacystintreatment resulted in a relocation of mitochondria tothe perinuclear region (Figs. 9E and 9J). Treatmentwith lactacystin and paraquat in conjunction did notchange the mitochondrial distribution patterns fromthose seen with lactacystin treatment alone. Similarlyconfocal microscopy of COS-7 cells transiently trans-fected with the pOTC-GFP plasmid showed perinu-clear accumulation of rounded abnormal mitochondriain lactacystin-treated cells (Fig. 9J). In untreated cells,elongated mitochondria were dispersed throughout thecytoplasm (Fig. 9I). Similar effects were observed withother proteasome inhibitors, MG115 and proteasomeinhibitor 1 (data not shown).

DISCUSSION

In this article we have investigated the influence ofredox conditions on mitochondrial preprotein process-ing. In addition the role of the proteasome was as-sessed both in this process and in other general mito-chondrial parameters. Paraquat, a superoxide-generating molecule, is routinely used as a modelcompound to probe the effects of oxygen radicals. Ahallmark of pathologically oxidizing conditions, as oc-curs with paraquat intoxication, is dysfunctional mito-chondrial performance. Although poorly defined it isthought that the mitochondrion is a primary target ofparaquat and other oxidative stressors in the intactcell.

One form of mitochondrial damage that has been

114 WRIGHT ET AL.

FIG. 9. The effect of lactacytin and paraquat on mitochondrial localization in pOTC-GFP-expressing CHO cells. Cells were treated with

the indicated combinations of paraquat and/or lactacystin for 20 h, whereupon they were fixed with 4% paraformaldehyde in PBS and

115OXIDATIVE STRESS ON MITOCHONDRIAL PROTEIN IMPORT

extensively investigated in relation to oxidative stressis the mitochondrial permeability transition phenome-non. Mitochondrial permeability transition essentiallyinvolves the opening of the large permeability transi-tion pore (MTP), which leads to loss of membrane po-tential and the escape of metabolites from the mito-chondrion [24]. The MTP contains redox-activevincinal thiol groups that modulate the sensitivity ofthis channel to opening by inducing agents [25]. Para-quat treatment of isolated mitochondria has beenshown to lead to oxidation of these critical sulfhydrylgroups, which, in turn, induces mitochondrial perme-ability transition under conditions of high calcium andwith an inhibition of complex I of the electron transportchain [22]. This may be of relevance to mitochondrialprotein import because it has been suggested that theMTP is a dysfunctional opening of the mitochondrialimport pore [26–28].

The conditions employed in present experimentshave been shown to lead to oxidation of the redox-sensitive sulfhydryl groups of the MTP (or putativeprotein import pore) but not to the induction of perme-ability transition. The absence of any overt effects ongeneral mitochondrial integrity or membrane potentialby paraquat treatment was also confirmed using themembrane potential sensitive dye JC-1. In these con-ditions the mitochondrial import rate was inhibited byparaquat pretreatment of mitochondrial preparationsin the in vitro assay. In a similar vein, protein importby isolated plant mitochondria has been shown to besensitive to the redox status of sulfhydryl groups on theouter surface of the inner mitochondrial membrane[29]. Taken together, these in vitro results point to amodulation of protein import transduced through re-dox-sensitive SH-groups of the inner membrane mito-chondrial import machinery. Further studies will berequired to confirm this contention and to identify thecomponents of the translocase of the inner membranethat are redox-sensitive. It is of interest that two con-served cysteine residues are present in Tim17s fromSaccharomyces cerevisiae, Caenorhabditis elegans,Arabidopsis thaliana, Drosophila melanogaster, andHomo sapiens [30].

In contrast to the situation in vitro, the evaluation ofthe effect of oxidizing conditions on mitochondrial pro-cessing in the intact cell was complicated by the deg-radation of precursor protein. Only when the protea-some was inhibited with lactacystin, was a processinglesion unequivocally demonstrated in paraquat treated

fluorescent images were digitaly obtained as described under Materiacan be directly compared; however, phase contrast images were gainand (B) phae contrast images (same field) of control pOTC-GFP-exprtreated with 25 mM lactacystin; (G, H) cells treated with both lactacy

transfected cells, one untreated (I) and one treated (J) with lactacystin

cells. The apparently efficient degradation of pOTC,when import is inhibited, and of pOTC-GFP underbasal conditions, indicates two potential fates for cyto-solic precursor proteins; they are efficiently importedinto the mitochondrion or they undergo rapid degrada-tion. In liver cells pOTC has been reported to be clearedfrom the cytosol with a half-life of ;3 min followinginhibition of import with rhodamine123 [31], an obser-vation explained by efficient proteasomal degradationof the preprotein. The competition of mitochondrialimport and a degradation pathway for preproteins mayhave interesting implications for the regulation of pro-tein import under different conditions. The lowamounts or absence of pOTC coupled with the failure ofOTC levels to increase in lactacystin-treated cells sug-gests that the great majority of pOTC is rapidly andefficiently imported under normal conditions. In thecase of artificial pOTC-GFP, however, significant pro-teasomal degradation of this precursor protein wasfound to occur basally. We have previously shown thatthe import rate of pOTC-GFP is much slower than thatof the more efficiently imported pOTC [14, 15], proba-bly due to partial folding of the GFP portion of thefusion protein in the cytoplasm. The slower import rateof pOTC-GFP may result in the shift of a considerableamount of this precursor to the degradation pathway.The tendency of lactacystin to counter the effects ofimport inhibition of pOTC and pOTC-GFP by paraquatand hyperoxia may reflect the fact that by eliminatingdegradation, the available substrate for the import re-action is increased, thus compensating for the lowerimport rates. The lactacystin-induced increase in ma-ture product in the face of import inhibition furtherimplies that the accumulated precursor protein that isobserved is import-competent, in that it can eventuallybe imported and processed if its degradation in thecytosol is prevented. However, in cells where the pro-teasome is active, a decrease in import would be ex-pected to shift substrate to this irreversible degrada-tion pathway resulting in lower levels of mature (hencefunctional) mitochondrial protein which we observe asa result of paraquat or hyperoxia treatment. In thecase of transiently transfected COS-7 cells where theamount of precursor protein is greater than saturatinglevels, the mitochondrial import rate is substrate-inde-pendent, and hence no increase in mature protein isseen to result from proteasomal inhibition when theimport rate is lowered.

The most direct interpretation of the finding that a

nd Methods. GFP images were taken under identical conditions andnd light-adjusted to obtain maximum contrast. (A) GFP fluorescentng CHO cels. (C, D) Cells treated with 0.6 mM paraquat; (E, F) cellsand paraquat. (I, J) Confocal GFP fluorescent images of transiently

ls a- a

essistin

. The cell edge has been traced manualy for perspective.

116 WRIGHT ET AL.

number of specific proteasome inhibitors lead to theaccumulation of mitochondrial precursors is that theproteasome is responsible for their degradation. How-ever, several observations caution against directly as-cribing the degradation of the precursor proteins to theproteasome. Despite extensive experimental effort,which included but was not limited to pulse chaselabeling followed with immunoprecipitation of mito-chondrial precursors and coexpression of ubiquitinpathway components (K. Terada, unpublished observa-tions), we were unable to demonstrate ubiquitinatedpreproteins. Additionally, the appearance of degrada-tion products (bands a and b) is inconsistent with whatis known about proteasome degradation, suggestingthe involvement of other cellular proteases.

The mitochondrial phenotypic changes that mightresult in pathologically oxidizing conditions, as a con-sequence of import inhibition, are difficult to predictbecause so many of the mitochondrial components de-pend upon import and processing. Presumably, mito-chondrial proteins with short half-lives that occupycritical metabolic pathways would be expected to de-cline when import is inhibited and gradually lead todiffuse mitochondrial dysfunction. Oxygen radicals af-fect numerous cellular processes, and thus, it is un-clear whether inhibition of mitochondrial import is aprimary or a secondary factor in the mitochondrialdysfunction observed during pathologically oxidizingconditions. Because the proteasomal degradation ofproteins limits the accumulation of precursor protein,and effectively masks import defects, it is possible thatdeclines in import rates may underlie or contribute tomitochondrial dysfunction seen in a variety of patho-logical conditions.

One important implication of the present findings isthe possibility of a role of redox modulation of mito-chondrial import under physiological conditions. Theamount of superoxide produced by the electron trans-port chain is directly proportional to local oxygen ten-sion [32, 33] and large oxygen gradients exist in tissuesand cells [34, 35]. In this scenario a physiological mech-anism to modulate protein import may be usurped bypathologically oxidizing conditions that inappropri-ately signal the shutdown of mitochondrial import.

An unexpected finding that has arisen from thepresent studies is the apparent intimate relationshipbetween proteasome activity and mitochondrial func-tion. Three observations point to the importance ofproteasome activity to the mitochondrion: (1) mito-chondrial precursor protein accumulation; (2) Tom20accumulation; and (3) a dramatic redistribution of themitochondria to the perinuclear region of the cell asso-ciated with proteasome activity inhibition. The findingthat Tom20, a core component of the mitochondrialouter membrane protein import receptor, levels rise

when cells are treated with lactacystin suggests that

the proteasome may play a quality control role with themitochondrial outer membrane proteins which is sim-ilar to that described for the endoplasmic reticulum[36]. We suspect that turnover of Tom20 is somehowmediated by proteasome. The perinuclear accumula-tion of mitochondria that coincides with the elevationof Tom20 suggests that the two events may be related.We have previously observed a similar localization ofmitochondria when cells were made to overexpressTom20 [14, 15].

In summary, this report shows that oxidizing condi-tions lead to a mitochondrial-processing lesion thatresults in the posttranslational decrease of mitochon-drial proteins. We speculate that this inhibition of pre-protein processing may represent a mode of physiolog-ical regulation that allows the mitochondrion torespond to localized redox conditions. These studiesalso implicate the proteasome as having a role not onlyin preprotein degradation during import inhibition,but also in general mitochondrial function and local-ization. Future studies will focus upon the contributionof import inhibition, caused either by oxidizing condi-tions or by other factors, in the mitochondrial dysfunc-tion observed in many pathologies.

This work was supported by grants-in-aid (09276103 and10470034 to M.M.) from the Ministry of Education, Science, Sportsand Culture of Japan, and by a fellowship from the Japan Society forthe Promotion of Science (to G.W.).

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Received July 31, 2000Revised version received October 16, 2000Published online January 3, 2001