Petr Hajek and David M. Bedwell$. From the Department of ..... joined portions of cytochrome b2 and F,P (Becker et al., 1992). When rabbit RL was added to the ...
THEJOURNAL OF BIOWCICAL CHEMISTRY
Vol. 269,No. 10,Issue of March 11,pp. 7192-7200, 1994 Printed in U.S.A.
0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of the Mitochondrial Binding and Import Properties of Purified Yeast F1-ATPasep Subunit Precursor IMPORT REQUIRES EXTERNAL ATP* (Received for publication, September 29, 1993, and in revised form, December 21, 1993)
Petr Hajek and David M. Bedwell$ From the Department of Microbiology, The University of Alabama at Birmingham, Birmingham, Alabama35294
To better understand the early events of the mitochon- MOM22, along with the proteins MOM38, MOM30, MOMS, drial protein import process, we purified the precursorand MOM7, which are components of the general insertion pore of the F1-ATPaseP subunit (pre-FIP) and examinedits (Kiebler et al., 1990, 1993; Pfaller et al.,1988). Yeast homologs import into isolated mitochondria. Import of purified of many of these proteins have also been identified (Moczko et urea-denatured pre-FIP did not require cytosolic facal., 1992). In particular, the yeast proteinsMas70 and ISP42, tors. However, the period of productive import was propreviously shown to be involved in mitochondrial protein imlonged by the additionof reticulocyte lysate, suggestingport (Hines et al., 1990; Baker et al., 19901, appear to be hothatcytosolicfactorssuchasmolecularchaperones mologs of MOM72 and MOM38, respectively. Another low M , were acting to extend the period of import competence yeast outer membrane protein, ISP6, has been shown to diof pre-FIP. Purified pre-FIP bound extensively to bothrectly interact with ISP42 (Kassenbrock et al., 1993). Finally, cardiolipin-containingliposomesandtointactmitochondria, indicating that a direct interaction between the Mas6 proteinof yeast recently became the firstcomponent mitochondrial precursors and the mitochondrial outer of the inner membraneprotein import machinery to be identimembrane surface can occur. The ability to chase this fied (Emtage and Jensen,1993). Although recent studies have surface-bound pre-FIP into mitochondria suggests thatshown that the machineriesinvolved in translocating proteins precursors bound to the mitochondrial surface can be across each mitochondrial membrane can function independently of one another, they appear to normally interact in a maintained in an import competent conformation. Fidynamic fashion t o achieve simultaneous translocation across nally,ourdefinedmitochondrialimportsystemwas used to characterize the ATP requirements of pre-FIP both membranes (Segui-Real et al., 1993). Protein transport across the endoplasmic reticulum memimport in the absenceof cytosol. We found a strong requirement for ATP on both sides of the mitochondrial brane appears t o proceed by both co- and post-translational inner membrane, suggesting that one ormore previ- mechanisms. In thepost-translational pathway,molecular ously undetected mitochondrial proteins outside the chaperones inmaintainnewly synthesized precursors in a loosely ner membrane utilize ATP to promote efficient pre-FIP folded import-competentconformation prior to membrane import. translocation. In contrast,a ribonucleoprotein complex termed signal recognition particle has been shown to facilitate the co-translational movement of secretory proteins across the enet a l . , 1990). Other The compartmental organizationof eukaryotic cells requires doplasmic reticulummembrane(Poritz that specific and efficient machineries exist to transport pro- studies suggest that co- and post-translational pathways for teins from their siteof synthesis across oneor more membranes protein transportinto mitochondria may also exist. Recently, it to their final subcellular destination. Although the details of was suggestedthat bulk mitochondrial protein importproceeds these processes may vary, their common biophysical require- primarily by a co-translational mechanism in vivo (Fujiki and ments have led to a conservation of many general details of Verner, 1993). Presently, the specific cytosolic factors (if any) process are undefined. In contrast, the protein transport acrossbiological membranes. For proteins to required to mediate this be importedinto mitochondria, theymust be translocated molecular chaperones Hsp7O (Murakami et al., 1988) andYDJl across both the inner and outer membranes to reach the mito- (Caplan et al., 1992) appear to stimulate post-translational chondrial matrix. Most proteins destined for import into mito- mitochondrial protein import by maintaining precursors in a chondria are synthesized with an NH2-terminal targeting sig- loosely folded, import competent conformation prior to memnal having the general features of an amphipathic helix (von brane translocation. The importance of such an unfolded conHeijne, 1986; b i s e and Schatz, 1988; Bedwell et al., 1989). formation is supported by the observation that urea denaturof many precursors. In other cases, These targeting signals are thought to initiate mitochondrial ation stimulates the import import by a direct interaction with import receptors on the however, urea denaturation alone does not appear to be SUEmitochondrial surface. In Neurospora crassa, outer membrane cient to promote efficient mitochondrial import in uitro. For proteins involved in protein translocation include the protein example, the import of purified, urea-denatured pre-ornithine import receptors MOM19 and MOM72 andthe associated carbamoyltransferase has been shown t o require a reticulocyte factor, which lysate (RL)l factor called presequence binding appears t o bind specifically to thepresequence of pre-ornithine * The costs of publication of this article were defrayed in part by the carbamoyltransferase(Murakamiand Mori, 1990). Whether be hereby marked payment of page charges. This article must therefore “advertisement”inaccordancewith 18 U.S.C. Section 1734 solely to The abbreviations used are: RL, reticulocyte lysate;PMSF, phenylindicate this fact. methylsulfonylfluoride;MOPS, 4-morpholinepropanesulfonic acid; $ To whom correspondence should be addressed: Dept. of Microbiology, Bevill Biomedical Research Bldg., Rm. 432, The University of Ala- BSA, bovine serumalbumin;PAGE, polyacrylamidegel electrophoresis; bama at Birmingham, UAB Station, Birmingham, AL 35294-2170. “el.: CL, cardiolipin; PG,l-palmitoyl-2-oleoyl-sn-glycero-3~phospho-~ac-~lglycero1)l. 205-934-6593;Fax:205-975-5479.
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Mitochondrial Import of Purified Pre-Flp
7193
using yeast strain D273-10B essentially a s described (Glaserand Cumsky, 1990). The typical0.1-ml import reaction contained 10 nm MOPS, pH 7.2,2 mg/ml BSA (fatty acid-free), 250 m sucrose, 1m~ ATP, 10 m succinate, 86 m KOAc, 0.9 m MgOAc, 1 m dithiothreitol, 25 m creatine phosphate, 4 mg/ml creatine phosphokinase, and 0.5 mg/ml mitochondria. For ATP depletion experiments, bothATP and the ATP regenerating system were omitted, and the reaction mixture was pretreated with 1 unit/ml apyrase for 10 min on ice. The import reaction was started by the addition of 2.5 pl of purified urea-denatured 3sSlabeled pre-F1pa t 25 "C, resulting in a final urea concentration of 0.2 M. The reaction was stopped with 0.9 ml of ice-cold SEM buffer (250 m sucrose, 1 m EDTA, 10 nm MOPS, pH 7.2) containing 1pg/ml valinomycin. Parallel samples were treated with 50 pg/ml trypsin for 15 min on ice, then stopped by the additionof 500 pg/ml trypsin inhibitor. The samples were analyzed by SDS-PAGE and autoradiography,followed by quantitation using a PhosphorImager (Molecular Dynamics). Preparation of Liposomes-Multilamellar phospholipid liposomes were prepared by the method of Bangham (Bangham et al., 1965). Bovine heart phosphatidylcholine, bovine heartphosphatidylethanolamine, bovine heart cardiolipin (CL), and synthetic l-palmitoyl-2oleoyl-sn-glycero-3[phospho-rac-(l-glycerol~l (PG)wereallpurchased from AvantiPolarLipids.Phospholipidfilms (4 pmol)wereresuspended in 1 ml of binding buffer (86 nm KOAc, 0.9 m MgOAc, 10 m MOPS pH 7.2) by vortexing for 2 min. Liposomes were washed three times with binding buffer containing 1mg/ml BSA by centrifugation at 16,000 x g for 20 min. When liposomes were prepared for the import assays, the binding buffer was supplemented with 250 mM sucrose. If not indicated, the typical(15%CL) liposomes were composed of 45 mol % phosphatidylcholine, 30 mol % phosphatidylethanolamine, 15 mol % CL, and 10 mol % PG. Binding Assays-Binding assays were initiated by the addition of 2.5 pl of urea-denatured pre-F,P to the suspensionof liposomes pre-equiliMATERLALSAND METHODS brated at25 "C in binding buffer containing 1mg/ml BSA. After 20 min, Construction of Pre-F,P ExpressionPlasmid-A 1.6-kilobase HindIII binding was terminatedby a 10-fold dilution in ice-cold binding buffer. The liposomes were immediately re-isolated by centrifugation at 16,000 fragment containing the intact ATP2 gene (Bedwell et al., 1987) was x g for 20 min a t 4 "C and washed with 0.5 ml of binding buffer consubcloned into the HindIII site of M13mp18, yielding the construct DBM13-13. Site-directed mutagenesis was carried out using the oligo- taining 1 mg/ml BSA. The washed liposome pellet was resuspended in nucleotide 5'-CAAAAATAAAAAAATCATATGGmTGCC-3'to create a SDS boiling buffer, and pre-F,P was analyzed as described for mitochondrial protein import assays. The pre-FIP bound to liposomes was unique NdeI restriction site (underlined) which overlaps the translation expressed as total pelletable pre-FIP correctedfor nonspecific aggregainitiation codon of theATP2 gene. The HindIII fragment containing the NdeI site was subcloned into the T7 promoter expression vector PET-3a tion (in the absence of liposomes, 4 % of input). To assay binding to intact mitochondria, the binding reaction was carried out in protein (Studieret al., 1990).The pET3aIATP2 constructwasdesignated import buffer supplemented with 1 pg/ml valinomycin. pDB288. Expression and Purification of Pre-F,eEscherichia coli strain BL2UDE3) transformed with pDB288 was grown in minimal medium RESULTS containing 25 pg/ml ampicillin at 37 "C. When the culture density Expression and Purification of P r e - F l e P r e - F I P was overreached 0.6A- unitsfml, pre-F,p expression was inducedby the addiproduced in E. coli strain BL21(DE3) using the T7 RNA potion of 0.4 m isopropyl-1-thio-p-D-galactopyranoside. 5 min later 0.5 lymerase expression system (Studier et al., 1990). Using this mCi of [35Slmethionine/cysteine(DuPont NEN) was added1to ml of the culture, and cultivation was continuedfor 45 min a t 37 "C. Cells were system, pre-FIP was synthesized in large quantities (20-50 collected by centrifugation a t 16,000 x g for 2 min at 4 "C and washed mg/liter of culture) in both minimal and rich media. The rewith 1 ml of 10 m Tris-HC1, 1 nm EDTA, pH 7.5 (TE). The cell pellet combinant proteinwas found to accumulate in inclusion bodies, was resuspended in0.1 ml of lysis buffer containing1mg/ml lysozyme, which aided in its purification. Following cell lysis, the inclu50 nm EDTA, 1m PMSF, and 10m Tris-HC1, pH 7.5. Cells were lysed sion bodies were recovered by centrifugation and purified from by six freezelthaw cycles. Then 0.9 ml of 10 mM Tris-HC1, pH 7.5, 1 nm contaminating E. coli proteins by successive washes in buffers EDTA, 100 m NaCl (TEN) buffer containing 10 m MgCI2, 10 pg/ml DNase I, 1 mg/ml deoxycholate, and 1 m PMSF was added to the containing deoxycholate and Triton X-100 (see "Materials and lysate. After 10 min of incubation on ice, the lysate was centrifugeda t Methods"). The final pellet was solubilized in 8 M urea andwas 16,000 x g for 10 min at 4 "C. The pellet was treated with 1 ml of TEN found to contain highly purified pre-FIP (Fig. 1).35S-Labeled buffer containing 1%Triton X-100, 1 m PMSF for 1 h at 4 "C, centripre-FIP was alsoprepared using this system, and the resulting fuged at 16,000 x g for 10 min at 4 "C, and washed with 1 ml of TE buffer containing 1 nm PMSF. Finally, the pellet was solubilized in 50 radiolabeled denatured precursor was suitable for use in in pl of urea buffer (8 M urea, 20 m "is, 1 mM EDTA, 1 m PMSF), pH vitro import studies. 7.5, for 1 h a t 4 "C, and insoluble debris wasremoved by centrifugation Import of Purified Pre-Flpinto Isolated Mitochondria-The a t 16,000 X g for 20 min at 4 "C. The supernatant containing purified urea-solubilized 35S-labeled pre-FIP was found to import effiurea-denatured pre-F,P was stored in aliquotsat -80 "C. ciently into isolated yeast mitochondria in the absence of any In Vitro Synthesis o f P r e - F , e T h e DNA template for in vitro coupled transcriptiodtranslation of pre-F,p was pDB95, which containeda 1.6- cytosolic proteins (Fig. 2A). Import reached a plateau within 15-20 min, when 30% of the precursor was imported and prokilobase HindIII fragment encoding the ATP2 gene (Bedwell et al., cessed to the mature form. These results indicate that dena1987) cloned intotheHindIII site of plasmidpSP64.The coupled transcriptiodtranslation reaction contained 57% rabbit reticulocytely- tured pre-FIB does not require cytosolic factors to undergo misate, 25 m Tris-HC1, pH 7.5, 1.6 m magnesium acetate, 0.4 nm sper- tochondrial import. However, to exclude the possibility that midine, 0.4 m dithiothreitol, 0.5 mM each ofATP, CTP, GTP, and UTP, cytosolic factors that could facilitate pre-FIP import had co20 p~ amino acids (minus methionine), 0.6 mCi/ml [35S]methionine/ cysteine, 800 units/&RNasin, 40 pg/ml template DNA, and 400 purified with either the radiolabeled pre-Flp or the isolated units/ml SP6 RNA polymerase. The reaction was incubated for 2 h at mitochondria, both precursor and mitochondria were purified more extensively. To further purify pre-FIP, the urea-solubi30 "C and then stored frozen a t -80 "C until use. In Vitro Mitochondrial ProteinImport-Mitochondria were prepared lized precursor preparation was resolved by SDS-PAGE and
presequence binding factorrepresents anothermolecular chaperone or some other factor, such as a soluble receptor, remains to be determined. In contrast, cytosolic factors were not found to be required for the import of purified urea-denatured preadrenodoxin (Iwahashi et al., 1992) or a purified urea-denatured chimeric protein (Becker et al.,1992). These resultsdemonstrate that individualprecursor proteinsappeartohave differing cytosolic requirements for their import into mitochondria. These differences may correspond to unique properties associated with each protein. To better understand the early events in the mitochondrial protein import pathway, we have purified the precursor of the Fl-ATPase P subunit (pre-FIP) and characterized the specific conditions required for its import into isolated mitochondria. Import of urea-denatured pre-FIPdid not requirecytosolic factors, but was increased 2-fold by the addition of RL. Purified pre-Flp bound extensively to liposomes and to intact mitochondria, demonstrating that a direct interaction between mitochondrial precursors and the mitochondrial outer membrane can occur. Finally, our defined mitochondrial import system was used to characterize theATP requirements of pre-FIP import in the absence of cytosol. Our results indicated a strong requirement for ATP on both sides of the mitochondrial inner membrane, suggesting thatone or more previously undetected mitochondrial factors outside the inner membraneutilize ATP to promote efficient pre-F1P import.
Mitochondrial Import of Purified Pre-F,P
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FIG.1. Purification of pre-F,/3 overproducedin E. coli. Purification steps were analyzed by 8% SDS-PAGE: lane I , total cell homogenate: lane 2, 16,000 x g pellet; lane 3 , Triton X-100-washed pellet; lane 4 , Tris-EDTA-washed pellet: lane 5 , urea-solubilized pellet. The relative mobility of molecular mass standards are indicatedon the left in kDa.
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electroeluted from gel slices. To remove peripherally associated 10.0 proteins, mitochondria were subjected toa salt wash with0.25 M KCI. Both the rate of import and the total yield of imported / protein using gel-purified pre-FIP and salt-washed mitochondria were similar to the results shown in Fig. 2A (data not shown), excluding the possibility that contaminating proteins e facilitated the importof pre-F,P. Thus, urea-denatured pre-FIP 8 5.0 E does not requirecytosolic factors for import. These findings are consistent with results reported previously for purified adrenodoxin (Iwahashi et al., 1992) anda purified fusion protein t h a t 2.5 joined portions of cytochrome b2 and F,P (Becker et al., 1992). When rabbit RL was added to the import reaction, the yield of imported F,P was increased (Fig. 2 A ) . Under these condi0.0 tions, 70-80% of added precursor was routinely imported and d i 2 3 4 5 processed within 30 min. The RL stimulation does not appear to result from a general increase in protein concentration beTime (min) cause BSA alone did not elicit this effect. RL has been reported to contain a number of factors that stimulate mitochondrial protein import. Someof these factors, such as Hsp70, have been C reported to stimulate mitochondrial protein import by helping to maintain precursors in a n import-competent conformation. To better understand the mechanismof RL stimulation of preF,P import, we examined in greater detail the effect of RL on the initial kineticsof pre-FIP import (Fig.2B ). We found t h a t 7500 c the initial rateof pre-F,P import was identical in the presence a or absence of RL, indicating that RL does not stimulate import 8 5000 by increasing the absolute number of pre-F$ molecules undergoing import during the initialstages of the reaction. Rather, E the data indicate that the RL-mediated stimulation of pre-FIP 2500 import is caused primarily by a n extension of the total time period during which import occurs. This result is consistent with a stimulation by molecular chaperones, which would act to 0 prolong the window during which extramitochondrial pre-FIP 0 5 IO 15 20 is maintained ina loosely folded conformation compatible with mitochondrial import. Time (min) To exclude the possibility that RL exerts its stimulatoryefFIG. 2. Time course of pre-F,P importinto isolated mitochonfect by extendingtheimportcompetence of mitochondria dria.A, pre-F,P import was camed out in the presence of 1 4 6 RL SI00 rather than the import competence of pre-FIP, an import reac- (closed circles) or a corresponding concentration of BSA(open circles). B, tion was initiated with urea-denatured precursor in the abtime course of purified pre-F,P import during the first 5 min in the sence of RL. After import began to level off, a n additional ali- presence of 14% RL SlOO (closed circles) or a corresponding concentraquot of pre-F,Pwasadded(Fig. 2C). The rate of import tion of BSA (open circles). C, sequential addition of purified pre-F,P during mitochondrial import. The time course of pre-F,P import initifollowing precursor re-addition was almost identical to the ini- ated a t time 0 is indicated by the open circles. After the reaction had tial rate of pre-Flp import, indicating that the mitochondria proceeded for 10 min, it was divided into two aliquots, and 30 s later a remained capable of importing unfolded pre-FIP with similar fresh aliquot of urea-denatured pre-F,P was addedto one tube (closed kinetics throughout thefirst 20 min of import in the absence of circles). RL. These results confirm that RL factors stimulate pre-FIP
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v)
-B
v
I
Mitochondrial Import ofPre-F,P Purified
7195
assay, pre-FIP did not bind to theliposomes. This indicates that RL factor(s) interact with either the precursor or liposomes and prevents binding of the precursor protein. This effect is not simply due to high protein concentrations, since pre-FIP binding to CL-containing liposomes was not prevented by similar amounts of BSA. This finding is not consistent with the results of binding studies carried out with other precursors, since it +RL-translated pre-F, p has been shown that several RL-translated precursors are able to bind CL-containingliposomes (Ou et al., 1988).Therefore, we "0-Purified pre-F,S, BSA directly tested RL-translated pre-FIP(0.7% final RL concentration in the binding reaction)for its ability tobind CL-containing liposomes. Little or no binding wasobserved (
