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Identification of Protein-Receptor Components Required for the Import of Prealdehyde Dehydrogenase into Rat Liver Mitochondria
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 323, No. 1, October 20, pp. 54–62, 1995
Identification of Protein-Receptor Components Required for the Import of Prealdehyde Dehydrogenase into Rat Liver Mitochondria1 Olga Pchelintseva,2 Youngmi Kim Pak,2 and Henry Weiner3,4 Department of Biochemistry, Purdue University, 1153 Biochemistry Building, West Lafayette, Indiana 47907-1153
Received May 24, 1995, and in revised form July 31, 1995
Mitochondrial aldehyde dehydrogenase is synthesized as a high-molecular-weight precursor in cytosol and transported into mitochondrial matrix space where it is processed to the mature enzyme. To identify components of the transport machinery on liver mitochondria, anti-idiotypic antibodies against the rabbit anti-prealdehyde dehydrogenase signal peptide antibodies were produced in chicken eggs and rabbit. Both anti-idiotypic antibodies inhibited the import of prealdehyde dehydrogenase (pALDH) into isolated rat liver mitochondria. The rabbit anti-idiotypic antibody could recognize by Western blotting five mitochondrial membrane proteins with apparent molecular weights of 66, 60, 42, 34, and 29 kDa. The anti-idiotypic antibodies were cross-linked to mitochondrial membrane proteins using sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3 *-dithiopropionate which is an iodinatable, heterofunctional, and photoreactive cross-linker. Mitochondrial proteins with apparent molecular weights of 66, 60, and 42 kDa were identified using the chicken antibody. The 66and 34-kDa proteins were cross-linked to the rabbit antibody as the major components and the 42-kDa protein as a minor one. Antibodies against the 60- and 42-kDa proteins, as well as Fab fragments, inhibited the import of pALDH, suggesting that these proteins are receptor/translocator components for pALDH import. q 1995 Academic Press, Inc. 1 This work was supported in part by Grant AA05812 from the National Institute on Alcohol Abuse and Alcoholism and the Indiana Elks Charities, Inc., Purdue University Cancer Center. This is Journal Paper No. 14739 from the Purdue University Agricultural Experiment Station. 2 Each author made an equal contribution. 3 H. Weiner was a recipient of Senior Scientist Award AA00028 from the National Institute on Alcohol Abuse and Alcoholism. 4 To whom correspondence should be addressed. Fax: (317) 4947897.
Key Words: mitochondria; receptor; import; aldehyde dehydrogenase; anti-idiotypic antibodies; cross-linking.
Proteins destined for mitochondria are encoded by nuclear genes and synthesized in cytoplasm as higher molecular weight precursors. Transport of the proteins from cytoplasm into mitochondria requires energy, one or more targeting signals on the transported protein, and a machinery that decodes these signals and moves the protein to its correct intramitochondrial location (1). The targeting signal in a precursor is called a signal peptide, presequence, or leader sequence (for review see 1–3). The precursor protein is processed by a protease in the matrix space to produce the mature protein (4, 5). We have been investigating the relationship between the structure of the leader sequence of aldehyde dehydrogenase and how it binds to mitochondria (6–8). The leader must ultimately bind to a membrane-spanning receptor/translocator complex so that it can be brought into the mitochondria. During the past few years many components of the complex have been identified from yeast and Neurospora crassa. Three recent review articles summarize what is known about the system (9– 11). Two highly protease-sensitive outer membrane proteins of approximate molecular weights 70 and 20 kDa, respectively, have been identified as an integral part of the import receptor. The larger protein has been termed MAS70 in Saccharomyces cerevisiae (12) or MOM72 in N. crassa (13) and the smaller one MOM19 in N. crassa (14) or MAS20 in S. cerevisiae (15). The two import receptors MOM19 and MOM72 were found in a high-molecular-weight complex in the mitochondrial outer membrane. In both organisms, the complex contained at least five additional proteins,
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0003-9861/95 $12.00 Copyright q 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
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termed MOM7, MOM8, MOM22, MOM30, and MOM38 (16–18). Four of these proteins, MOM7, MOM8, MOM30, and MOM38, are believed to be involved in the formation of the general insertion pore (GIP)5 that is responsible for the insertion of preproteins into the outer membrane and their translocation across this membrane (3). The recent studies with S. cerevisiae and N. crassa have identified a third receptor, a 22-kDa protein termed Mas22p in yeast and MOM22 in N. crassa (19, 20). MOM22, anchored in the outer membrane by a single transmembrane segment, is required for the transfer of preproteins from the receptor to the GIP. Antibodies directed against MOM22 did not interfere with the binding of preproteins to their receptors, but inhibited the import of all analyzed preproteins that use the mitochondrial receptor complex. In contrast, antibodies directed against the receptors MOM19 and MOM72 showed different inhibitory effects on various subclasses of preproteins. These data suggest that MOM22 is required for insertion of proteins into the GIP. The import pathways using both receptors MOM19 and MOM72 appear to converge at MOM22 (19). One subunit of the putative outer membrane channel was identified by cross-linking it to a precursor protein that spanned the protein import channel of both membranes (21). This import-site protein (ISP42) proved to be an integral outer membrane protein essential for cell viability (22). The N. crassa homolog, termed MOM38, was shown to be part of a complex that also contained one of the import receptor components (16). There is now strong evidence that the mitochondrial inner membrane has a protein import system distinct from that in the outer membrane (23–25). However, only a few inner membrane import components have been identified. One component is a 45-kDa protein from S. cerevisiae, ISP45, which is associated with the outer face of the inner membrane. ISP45 was identified as one of several proteins that could be cross-linked to a precursor blocked in transit across the mitochondrial inner membrane (26). The other inner membrane import component is the MP11 gene product MIM44. It was found to behave as a peripheral membrane protein firmly associated with the mitochondrial inner membrane. Using cross-linking, it was demonstrated that MIM44 is in close proximity to the precursor at an early stage of translocation across the inner membrane (27). 5 Abbreviations used: pALDH, prealdehyde dehydrogenase; mALDH, mature aldehyde dehydrogenase; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; SASD, sulfosuccinimidyl 2-(p-azido-salicylamide)ethyl-1,3*-dithiopropionate; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; GIP, general insertion pore; ISP, import-site protein.
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Mas6p, a 23-kDa protein with three or four potential transmembrane domains, is an integral membrane protein of the yeast mitochondrial inner membrane, which is essential for protein import. Depletion of Mas6p from yeast cells resulted in the accumulation of mitochondrial precursor proteins. Antibodies to Mas6p inhibited protein import into mitochondria, but only when the outer membrane was disrupted to allow the antibodies access to the inner membrane (28). In spite of all that is known about transport apparatus in lower eukaryotes, less is known about the import complex in higher organisms. Chloroplast components have been characterized (29, 30). A 42-kDa plant mitochondria outer membrane protein has been identified as being necessary for import (31). Synthetic peptides derived from presequences of precursor proteins were used to identify the putative signal-sequence receptor components for liver. These were either radiolabeled and cross-linked to mitochondrial proteins or used as ligands on an affinity chromatography column. Two proteins of 29 and 52 kDa were purified from rat liver mitochondria, and antibodies raised against them inhibited protein import (32). In this paper, we utilized anti-idiotypic antibodies mimicking the signal peptide of rat liver prealdehyde dehydrogenase (pALDH) for cross-linking to the protein components on the surface of rat and beef liver mitochondria. Anti-idiotypic antibodies were generated in chicken and rabbit. Rabbit anti-idiotypic antibodies were used to identify receptor proteins by immunoblotting, as well as by cross-linking. Three proteins, 42, 60, and 66 kDa, were identified and purified from liver mitochondria. Antibodies raised against these proteins inhibited import of pALDH into mitochondria. We propose that the 42-, 60-, and, possibly, 66-kDa proteins function as receptor/translocator components for pALDH import into liver mitochondria. EXPERIMENTAL PROCEDURES Animals. Male Wistar rats of about 200 g were used for mitochondria preparation. Beef livers were obtained from a local slaughterhouse. New Zealand white rabbits and laying hens were used for antibody preparation. Materials. [ 35S]Methionine (ú800 Ci/mmol) was from New England Nuclear. [ 125I]NaI (2.4 Ci/mmol) was from Amersham. IodoGen, SASD, and ImmunoPure Fab preparation kit were purchased from Pierce. Keyhole limpet hemocyanin and substrate system (nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate) for phosphatase-labeled antibodies were purchased from Sigma Co. CNBractivated Sepharose 4B was purchased from Pharmacia. Molecular weight markers for SDS–PAGE and alkaline phosphatase-coupled secondary antibodies were purchased from Bio-Rad. Preparation of anti-signal peptide antibody. The pALDH signal peptide was synthesized in the Purdue Medical Chemistry Department with a sequence of MLRAALSTARKGPRLSRLLSYA-CONH2 deduced from pALDH cDNA sequence (33) and used previously by Pak and Weiner (34). The synthesized signal peptide (5 mg) was
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conjugated to a keyhole limpet hemocyanin carrier protein (1 mg) as described (35). Antisera were prepared by injecting the signal peptide–hemocyanin conjugate into rabbits (36). The IgG from antisera was purified by ammonium sulfate fractionation and DEAE–cellulose chromatography (37). The specific antibodies against the signal peptide was purified from the antisera using the signal peptidebound Sepharose affinity column, which was prepared by attaching the signal peptide to CNBr-activated Sepharose 4B following the manufacturer’s instruction (Pharmacia). Antisera were first loaded on the affinity column with phosphate-buffered saline (PBS; 6 mM sodium phosphate, pH 7.2, 0.17 M NaCl, 3 mM KCl, 0.02% sodium azide) and the bound antibodies were eluted with 0.2 M glycine buffer, pH 2.8, containing 0.5 M NaCl. The peak fractions detected by optical density at 280 nm were pooled and the pH was adjusted to 7.2 with 1 M Tris. The purified antibodies were dialyzed against PBS and concentrated using an Amicon Centricon. The specificity of the antibodies was determined by Western blotting after 10% SDS–PAGE of crude pALDH or mALDH expressed in Escherichia coli. Expression and labeling of pALDH and mALDH in E. coli. pALDH and mALDH were expressed in E. coli (BL 21/pLysS) using the pT7-7 expression vector (42). The E. coli cells from a 10-ml culture were harvested and resuspended in 1 ml of buffer (0.05 M sodium phosphate, pH 7.2, 1 mM EDTA, 0.02% b-mercaptoethanol) and used for immunoblotting. Preparation of anti-idiotypic antibodies. The anti-idiotypic antibodies were raised in two laying hens by subcutaneous injection of the purified rabbit anti-signal peptide antibody (5 mg) in PBS with an equal volume of Freund’s complete adjuvant into five sites. After the initial three injections, one every other week, eggs were collected daily. The anti-signal peptide antibodies from individual egg yolks were subjected to 10% SDS–PAGE and immunoblotted. The egg yolks which showed a positive reaction were pooled and IgY was extracted (43). Anti-idiotypic antibodies from IgY fractions were purified by passing the extract through the anti-signal peptide antibody– Sepharose 4B affinity column. The eluted anti-idiotypic antibodies in glycine buffer were pooled, dialyzed against PBS, and concentrated to 1 mg/ml. Anti-idiotypic antibodies were also generated in rabbits by subcutaneous injection of the purified anti-signal peptide antibodies. The IgG from antisera was purified by caprylic acid precipitation and ammonium sulfate fractionation (37). The Fab fragments of IgG were prepared using ImmunoPure Fab preparation kit following the manufacturer’s instruction. Briefly, the anti-idiotypic IgG was digested with immobilized papain. The Fab fragments were purified from the Fc fragments by passing through a protein A column, dialyzed against PBS, and concentrated to 1 mg/ml. The molecular weight of anti-idiotypic antibody was determined by size-exclusion HPLC (TSK-GEL G3000-SW, 30 1 7.5 cm) to be 158 kDa. Inhibition of pALDH import by antibodies. Rat liver mitochondria were isolated by differential centrifugation (38). Mitochondria (75 mg) in the import assay mixture (39) were preincubated with the anti-idiotypic antibodies (8 mg protein each) for 60 min at 47C, washed twice, and resuspended in 12.5 ml of mitochondria isolation buffer (0.025 M Tris–Cl, pH 7.4, 0.25 M sucrose, 1 mM EDTA). As a control, mitochondria were preincubated with preimmune IgY or IgG (27 mg protein) or buffer. Pretreated mitochondria were incubated with in vitro translated [ 35S]pALDH at 307C for 30 min, treated with proteinase K (200 mg/ml final concentration) on ice for 15 min followed by PMSF addition (1 mM final concentration), and analyzed by 10% SDS–PAGE and fluorography (34, 39). Various amounts of the Fab fragments from rabbit anti-idiotypic antibodies also were preincubated with mitochondria in the import assay mixture, and pALDH import into mitochondria was performed as stated above. Cross-linking of anti-idiotypic antibody with mitochondrial proteins. SASD was iodinated with [ 125I]NaI using Iodo-Gen in 0.1 M sodium phosphate buffer, pH 7.4, and incubated with purified
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chicken or rabbit anti-idiotypic antibody (38 mg protein) at room temperature for 30 min in the dark. The unreacted 125I and SASD were removed by spinning through a 1-ml G-25 spun column (40). [ 125I]SASD conjugated to anti-idiotypic antibody was incubated in the dark with liver mitochondria (1.8 mg) in the import assay mixture at 07C for 30 min and then at room temperature for 30 min. The reaction mixture was irradiated with a hand-held long wavelength uv lamp (Mineralight UVS 11) for 10 min and then with a bright light (200 W) for 3 min. The mitochondria were isolated, washed, resuspended in 60 ml of the mitochondria isolation buffer, and divided into two parts. Each was dissolved in SDS sample buffer with and without 10% (v/v) b-mercaptoethanol, respectively, and analyzed on 7.5% SDS–PAGE. The gels were dried and autoradiographed. Purification of mitochondrial membrane proteins. Mitochondria were prepared from rat and beef liver by differential centrifugation (38, 41) and suspended in the mitochondria isolation buffer at a protein concentration of 7 mg/ml. All procedures were carried out at 47C. Mitochondria were separated into membranes and matrix fractions as described (44). The membrane pellet was resuspended in 220 mM mannitol containing 70 mM sucrose, 10 mM Hepes–KOH buffer, pH 7.6, 2 mM DTT, and 0.5 mM PMSF and stored at 0807C until use. For preparation of antibodies to the membrane proteins, the mitochondrial membrane fraction was fractionated by preparative 10% SDS–PAGE (1.5 1 130 1 120 mm) and the bands of 66-, 60-, and 42-kDa proteins were cut from the gel after staining with Coomassie brilliant blue. The proteins were eluted from pieces of gel by 20 mM Tris–Cl, pH 7.4, 0.5 mM PMSF buffer two times while stirred at room temperature for 1 h, concentrated using a Amicon Centricon, and used for immunizing the rabbits. Antibody titers were monitored by immunoblotting using mitochondrial membrane proteins resolved on 10% SDS–PAGE. IgG and Fab fragments were prepared by the methods described above. Various amounts of the IgG and Fab fragments from rabbit anti66-, -60-, and -42-kDa antibodies were preincubated with rat liver mitochondria, and pALDH import was performed as stated above. Other methods. The integrity of the mitochondria was routinely checked by measuring cytochrome c oxidase activity as a mitochondrial marker (45). Trypsin- or CCCP-treated mitochondria were prepared and mitochondria were treated with proteinase K as described previously (34). Yeast mitochondria were prepared from cells (XK251B) grown aerobically (46), then lysed by freezing and thawing three times to prepare a crude matrix homogenate containing the signal peptide peptidase. Protein concentration was determined by Bradford method (47). Antisera against mALDH were prepared in rabbits using purified rat liver mitochondria ALDH. Western blot analysis was performed according to standard methods (48). The proteins were immunodecorated with the suitable antibodies and the reactions were detected with the goat anti-rabbit antibodies conjugated to alkaline phosphatase (Bio-Rad) using the nitroblue tetrazolium/ 5-bromo-4-chloro-3-indolyl phosphate substrate system.
RESULTS
Specificity of anti-signal peptide antibody. Antisera raised against the chemically synthesized pALDH signal peptide recognized only pALDH, not mALDH, while both were recognized by antibodies produced against purified mALDH. Expressed pALDH was incubated with yeast mitochondria lysate which contained the signal peptide peptidase. Since yeast mitochondrial ALDH did not cross-react with anti-rat liver mitochondrial ALDH antibody (49), only expressed rat ALDH in the assay mixture could be detected by it. After incuba-
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FIG. 1. Specificity of the anti-signal peptide antibody determined by immunoblotting. pALDH and mALDH in pT7-7 were expressed in E. coli (BL21/pLysS) cells. Aliquots (5 ml) of cells from a 10-ml culture were analyzed by 10% SDS–PAGE and immunoblotting with anti-signal peptide antisera (aSP; A) and anti-rat liver mitochondrial ALDH antisera (aRM-ALDH; B). Lane 1, mALDH; lane 2, pALDH; lane 3, pALDH incubated with lysed yeast mitochondria which contained the signal peptide peptidase (57) for 2 h at 307C to cleave signal peptide from pALDH. The upper band represents precursor (pALDH) and lower band represents mature or processed mALDH.
tion about 50% of expressed pALDH was processed to mALDH. The processed mALDH was not recognized by anti-signal peptide antibody, verifying that anti-signal peptide antibody reacted with the signal peptide portion of pALDH (Fig. 1). A second set of antibodies were raised in chicken and in rabbit against the purified rabbit anti-signal peptide antibody to obtain the anti-idiotypic antibody that could recognize the peptide-binding site of the receptor/ translocator. The anti-idiotypic antibodies inhibited pALDH import into mitochondria. The inhibition of pALDH import by anti-idiotypic antibodies was investigated to determine if these interact with the import machinery. Preincubation of mitochondria with chicken, as well as rabbit, anti-idiotypic antibodies dramatically inhibited the import of pALDH, while incubation with excessive amounts of preimmune IgY or IgG caused slight inhibition of import (Fig. 2A). Similarly, Fab fragments of rabbit anti-idiotypic antibody completely inhibited pALDH import, while Fab-PI from preimmune IgG did not affect the import of pALDH (Fig. 2B). It appears from the data presented in Fig. 2 that the presence of IgG or the Fab fragments prevented binding of precur-
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sor protein to mitochondria. This could be expected to occur if these were interacting with components of the receptor/translocator. The fact that the Fab fragments were inhibitors implies that the inhibition was not just due to a large IgG molecule nonspecifically binding to the membrane. Five different mitochondrial proteins were recognized by rabbit anti-idiotypic antibody immunoblotting. Rat and beef liver mitochondrial membranes were resolved on SDS–PAGE and immunoblotted using the rabbit anti-idiotypic antibody. Five membrane proteins with apparent molecular weights of 66, 60, 42, 34, and 29 kDa were found when protein bands on the immunoblot were compared with those using preimmune IgG (Fig. 3). 66-, 60-, 42-, and 34-kDa mitochondrial proteins were cross-linked to anti-idiotypic antibody. Chicken antiidiotypic antibody was cross-linked to mitochondria using SASD to identify mitochondrial membrane proteins which may interact with the signal peptide. Radioactivity was found in the protein–antibody complex prior to reduction of the internal disulfide bond of SASD by b-mercaptoethanol. A radioactive protein complex of approximately 240 kDa was found (Fig. 4A). If the complex was reduced with b-mercaptoethanol, only the cross-linked mitochondrial protein would contain ra-
FIG. 2. Inhibition of pALDH import by anti-idiotypic antibody. (A) Rat liver mitochondria (75 mg protein) were preincubated with either chicken or rabbit anti-idiotypic antibodies (lane 2, 8 mg; lane 3, 16 mg) or with preimmune IgY or IgG (lane 1, 27 mg), as well as with PBS buffer (lane C) for 1 h at 47C. Pretreated mitochondria were incubated with reticulocyte lysate (15 ml) containing 35S-labeled pALDH in the import assay mixture for 30 min at 307C and were then treated with proteinase K (200 mg/ml). The mitochondria were reisolated by centrifugation and analyzed by 10% SDS–PAGE and fluorography. (B) The experiment was performed as described above with the following modifications. The mitochondria (75 mg protein) were preincubated with different amounts of Fab fragment from rabbit anti-idiotypic antibodies or from preimmune serum (Fab-PI) (lane 1, 3 mg; lane 2, 6 mg; lane 3, 13 mg), as well as with PBS buffer (lane C). 35S-labeled pALDH were added and the mixture was incubated for 30 min at 307C. The mitochondria were isolated by centrifugation and the pellet was analyzed by 10% SDS–PAGE and fluorography. The upper band (pALDH) represents binding; the lower band (mALDH) represents import.
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FIG. 3. Immunoblotting of rat liver mitochondrial membrane proteins using rabbit anti-idiotypic antibody. Rat and beef liver mitochondrial membranes were prepared as described (44). (A) Membrane proteins were separated on 10% SDS–PAGE and immunoblotted using rabbit anti-idiotypic antibody (IgG-aIT) or preimmune IgG (IgG-PI). Lane S, prestained protein molecular weight markers; lane 1, rat liver mitochondrial membranes; lane 2, beef liver mitochondrial membranes. (B) Coomassie blue-stained gel. Approximately 50 mg proteins was loaded in each lane.
mately 95 kDa did not involve mitochondrial proteins as it was a contaminant found in the cross-linking control experiment (Fig. 5, lane C). Inhibition of protein import by the antibodies raised against proteins identified by cross-linking. A number of proteins were identified by cross-linking as potential candidates of the translocator. Proteins of these subunit molecular weights (66, 60, and 42 kDa) were isolated from SDS–PAGE. The proteins were excised from the gels and injected into rabbits. It was possible to obtain sufficient quantities of the 66-kDa protein from rat to make antibodies. It was necessary to use beef liver mitochondria to obtain the 60- and 42-kDa proteins. The specificity of the antibodies raised to 66-, 60-, and 42-kDa SDS–PAGE fractions was verified by subjecting total mitochondrial membranes and matrix fraction as well as intact mitochondria to Western blot analysis. Antibodies against 66-, 60-, and 42-kDa proteins specifically detect corresponding protein when used to immunodecorate intact mitochondria and mitochondria membrane proteins (Fig. 6).
dioactivity. After reduction three major radioactive bands were found with apparent molecular weights of 66, 42, and 34 kDa (Fig. 4B). The same three protein bands were obtained by cross-linking anti-idiotypic antibody to CCCP-treated mitochondria (Fig. 4C). This was expected, since CCCP-treated mitochondria should still contain the protein translocator, even though import was inhibited by the uncoupler dissipating the membrane potential (3). Treating mitochondria with a low concentration of trypsin destroyed the outer membrane proteins and abolished precursor import (2). Therefore, the trypsintreated mitochondria should not possess the protein translocators which could bind to anti-idiotypic antibody. As expected, pALDH was not imported into trypsin-treated mitochondria as described previously (34) and no corresponding protein bands were found after cross-linking trypsin-treated mitochondria with antiidiotypic antibody (Fig. 4, lane T). After cross-linking, mitochondria were treated with proteinase K. This treatment destroyed the crosslinked components showing that the anti-idiotypic antibody–membrane protein complex was on the outside of the mitochondria (Fig. 4C). Rabbit anti-idiotypic antibody was used for the crosslinking to determine if the same molecular weight proteins found with chicken anti-idiotypic antibody could be identified. The major bands were the 66-, 60-, and 34-kDa proteins, while 42-kDa protein was a minor component, as shown in Fig. 5. The band at approxi-
FIG. 4. Cross-linking of chicken anti-idiotypic antibody to mitochondria. Rat liver mitochondria were treated with trypsin (T) or CCCP (C) (34). The chicken anti-idiotypic antibody was crosslinked to the treated (T or C) or untreated (U) mitochondria as described under Experimental Procedures. After reisolating, mitochondria were dissolved in SDS sample buffer with b-mercaptoethanol (B, /bME) or without b-mercaptoethanol (A, 0bME) and then analyzed by 7.5% SDS – PAGE and autoradiography. Mitochondria, treated with proteinase K after cross-linking, contained no radioactivity, as shown in C (K). Molecular weight markers are shown to the left of A.
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FIG. 5. Cross-linking of rabbit anti-idiotypic antibody to mitochondria. Rabbit anti-idiotypic antibody (lane 2) or preimmune IgG (lane 1) was cross-linked to either rat or beef liver mitochondria and analyzed by 10% SDS–PAGE under reducing conditions as described in the legend to Fig. 4. As a control, the SASD-linked anti-idiotypic antibodies were cross-linked without mitochondria incubation (lane C).
Cross-competition experiments were also performed to determine whether the anti-66, -60-, and -42-kDa protein antibodies were in fact recognizing the same components as the anti-idiotypic antibodies. Immunological cross-reactivity with mitochondrial membrane proteins of similar molecular weight has been detected between antibodies raised against SDS–PAGE purified proteins and anti-idiotypic antibodies (data not shown). The antibodies against the 66-, 60-, and 42-kDa mitochondria proteins and their corresponding Fab fragments were tested as potential inhibitors of protein import. Both anti-42-kDa and anti-60-kDa antibodies inhibited the import of pALDH into mitochondria, as shown in Fig. 7. The Fab fragments also caused the inhibition of import almost completely at a concentration of 0.88 mg/ml, while preimmune Fab had no effect at the same concentration. In contrast, neither antibodies against the 66-kDa protein nor the Fab fragments cause measurable inhibition of import to occur. The findings from import inhibition, immunoblotting, and ligand cross-linking lead us to conclude that the 60- and 42-kDa proteins are strong candidates for being components of the rat liver mitochondrial receptor/translocator complex. It remains to be investigated whether the 66-kDa protein functions as part of a receptor/translocator complex or may be involved in the import pathway of pALDH in some other manner. DISCUSSION
Several research groups identified receptor/translocator proteins on mitochondria from various species
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using different approachs as described in the Introduction. Anti-idiotypic antibodies have been used to probe for hormone and neurotransmitter receptors when the receptor proteins were unknown (50). A 30-kDa chloroplast envelope protein that participated in protein import was identified using immunoprecipitation with anti-idiotypic antibody (51). Since anti-idiotypic antibodies mimick the structure of the signal peptide, they should bind to the receptor. This chloroplast import receptor protein was purified and its primary structure was deduced from cDNA sequencing (52). The deduced molecular weight was 36 kDa instead of 30 kDa, and the protein showed a high homology to the phosphate translocator of chloroplast. Using a similar approach with anti-idiotypic antibody, the 32-kDa integral membrane protein of yeast mitochondria was identified (53) and its gene was cloned (54). p32 showed high amino acid identity (40%) with the mitochondrial phosphate translocator as did the chloroplast p36. However, some of the previous applications of this approach have led to identification of proteins that may not be directly implicated in targeting (55). It was not possible to identify unknown components of the receptor complex of rat liver mitochondria only on the basis of their relative molecular weights with recently identified components of the import apparatus in yeast and Neurospora. Although fundamental questions remain about the use of the antiidiotypic antibodies for identification of relatively low affinity receptors as might be involved in protein targeting, they could serve as an initial probe to identify
FIG. 6. Immunoblotting of mitochondrial membrane proteins using rabbit anti-66-, -60-, and -42-kDa protein antibodies. Rat and beef liver mitochondria were separated into membranes and matrix fractions as described (44). The mitochondrial protein fractions were separated by 10% SDS–PAGE, transferred to nitrocellulose, and immunodecorated with anti-66- (A), -60- (B), and -42-kDa protein IgG (C) at a dilution of 1:5000. Preimmune IgG was also employed. No stained bands were detected (not shown). Lane 1, intact mitochondria; lane 2, total mitochondrial membranes; lane 3, matrix fraction. Approximately 50 mg proteins was loaded in each lane. Molecular weight markers (lane C) were run with each.
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FIG. 7. Inhibition of pALDH import into mitochondria by anti-66, -60-, and -42-kDa Fab fragments. The Fab fragments were prepared following the manufacturer’s instruction (Pierce, ImmunoPure preparation kit). Mitochondria (7 mg/ml, final protein concentration) were preincubated with different concentrations of anti-66, -60-, and -42-kDa protein Fab fragments (lane 2, 0.2 mg/ml; lane 3, 0.4 mg/ml; lane 4, 0.88 mg/ml) or with Fab fragments prepared from a preimmune rabbit (lane 1, 0.88 mg/ml), as well as with PBS buffer (lane C). The mitochondria were reisolated by centrifugation, washed, and incubated with 35S-labeled pALDH in the import assay mixture as described in the legend to Fig. 2. Mitochondria were then analyzed for import of pALDH by 10% SDS–PAGE and fluorography. Bands of ALDH on the fluorogram were quantified by densitometry. In all cases, the values obtained with untreated mitochondria (lane C, PBS buffer) were set at 100%.
possible components of the receptor. Hence, we used anti-idiotypic antibodies to probe for the mitochondrial receptor/translocator proteins of rat liver. Both the chicken and rabbit anti-idiotypic antibodies mimicking the pALDH signal peptide inhibited the import of pALDH, implying that anti-idiotypic antibody reacted
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with the protein import machinery. When rat and beef membrane protein were immunoblotted using rabbit anti-idiotypic antibody, 66-, 60-, and 42-, 34-, and 29kDa proteins among others were identified. Chicken and rabbit anti-idiotypic antibodies were chemically cross-linked to mitochondrial membrane proteins and
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four components were labeled (66, 60, 42, and 34 kDa). Since cross-linking is dependent upon many experimental conditions such as ligand orientation and length of the cross-linker used (56), the proteins which were not cross-linked cannot be excluded as possible components of the receptor. Proteins (29-kDa and 52-kDa) from rat liver mitochondrial membranes were purified by Ono and Tuboi (44) using signal peptide affinity chromatography. The 29-kDa protein we identified by anti-idiotypic antibody immunoblotting is possibly the same component found by them. It is possible that the 29-kDa protein was not cross-linked because it is not fully exposed to the outer surface of mitochondria, but when solubilized it becomes immunoreactive to the anti-idiotypic antibodies. Our antibodies did not cross-react with a 52-kDa protein found by Ono and Tuboi (32). To further characterize protein components of a receptor their purification was necessary. The only available property of the import receptor components was their subunit molecular weights, for no convenient method was available to assay for them. To obtain the 66-, 60-, and 42-kDa proteins for antibody production, preparative SDS–PAGE was performed on rat and beef liver mitochondria. Fab fragments of the anti-60- and 42-kDa protein antibodies inhibited pALDH import into mitochondria. Import can be inhibited by prevention of precursor binding either to the membrane or to its translation across the membrane. From the data presented in Fig. 7 it appears that binding of precursor is diminished in the presence of Fab fragments. Their association with the translocator would be expected to affect the interaction of the precursor with the translocator just due to the steric presence of another large protein attached to the complex. We cannot state if the inhibition of import was related just to diminished binding or was also related to the antibody preventing the translocator from functioning. Since the 60- and 42-kDa proteins were identified by several different techniques, and antibodies against each inhibited the import of pALDH, we conclude that the 60- and 42kDa proteins were involved in the import of pALDH, possibly as components of the receptor/translocator. The 66-kDa protein was implicated to be involved, but since antibodies against it did not inhibit import we cannot suggest that it is part of the translocation apparatus. The 66-, 34-, and 29-kDa proteins, identified by cross-linking or Western blotting, may not interact directly with the pALDH signal peptide. However, each of them may form a complex with the 42- or 60 kDa proteins and this complex may function in the import process on the mitochondria. It will be necessary to isolate the import complex in order to determine which of the components are associated with it. Work is currently in progress to do this.
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REFERENCES 1. Hartl, F.-U., and Neupert, W. (1990) Science 247, 930–938. 2. Nicholson, D. W., and Neupert, W. (1989) in Protein Transfer and Organelle Biogenesis (Das, R. C., and Robbins, P. W., Eds.), pp. 637–746, Academic Press, San Diego. 3. Pfanner, N., Hartl, F.-U., and Neupert, W. (1988) Eur. J. Biochem. 175, 205–212. 4. Bo¨hni, P., Gasser, S., Leaver, C., and Schatz, G. (1980) in The Organization and Expression of the Mitochondrial Genome (Kroon, A. M., and Saccone, C., Eds.), pp. 424–433, Elsevier, Amsterdam. 5. Schneider, H., Arretz, M., Wachter, E., and Neupert, W. (1990) J. Biol. Chem. 265, 9881–9887. 6. Thornton, K., Wang, Y., Weiner, H., and Gorenstein, D. G. (1993) J. Biol. Chem. 268, 19906–19914. 7. Wang, Y., and Weiner, H. (1993) J. Biol. Chem. 268, 4759–4765. 8. Wang, Y., and Weiner, H. (1994) Biochemistry 33, 12860–12867. 9. Lithgow, T., Glick, B. S., and Schatz, G. (1995) TIBS 20, 98– 101. 10. Shore, G. C., McBride, M. M., Millar, D. G., Steenaart, N. A. E., and Nguyen, M. (1995) Eur. J. Biochem. 227, 9–18. 11. Ku¨brich, M., Dietmeier, K., and Pfanner, N. (1995) Curr. Genet. 27, 393–403. 12. Hines, V., Brandt, A., Griffity, G., Hotstmann, H., Bru¨ltsch, H., and Schatz, G. (1990) EMBO J. 9, 3191–3200. 13. So¨llner, T., Pfaller, R., Griffiths, G., Pfanner, N., and Neupert, W. (1990) Cell 62, 107–115. 14. So¨llner, T., Griffiths, G., Pfaller, R., Pfanner, N., and Neupert, W. (1989) Cell 59, 1061–1070. 15. Ramage, L., Junne, T., Hahne, K., Lithgow, T., and Schatz, G. (1993) EMBO J. 12, 4115–4123. 16. Kiebler, M., Pfaller, R., So¨llner, T., Griffiths, G., Horstmann, H., Pfanner, N., and Neupert, W. (1990) Nature 348, 610–616. 17. Meczko, M., Dietmeier, K. A., So¨llner, T., Segui-Real, B., Steger, H. F., Neupert, W., and Pfanner, N. (1992) FEBS Lett. 310, 265– 268. 18. So¨llner, T., Rassow, J., Wiedmann, M., Schossmann, J., Keil, P., Neupert, W., and Pfanner, N. (1992) Nature 355, 84–87. 19. Kiebler, M., Keil, P., Schneider, H., van der Klei, I. J., Pfanner, N., and Neupert, W. (1993) Cell 74, 483–492. 20. Lithgow, T., Junne, T., Suda, K., Gratzer, S., and Schatz, G. (1994) Proc. Natl. Acad. Sci. USA 91, 11973–11977. 21. Vestweber, D., Brunner, K., Baker, A., and Schatz, G. (1989) Nature 341, 205–209. 22. Baker, K. P., Schaniel, A., Vestweber, D., and Schatz, G. (1990) Nature 348, 605–609. 23. Pfanner, N., Rassow, J., van der Klei, I. J., and Neupert, W. (1992) Cell 68, 999–1002. 24. Horst, M., Jeno¨, P., Kronidou, N. G., Bolliger, L., Oppliger, W., Scherer, P., Manning-Krieg, U., Jascur, T., and Schatz, G. (1993) EMBO J. 12, 3035–3041. 25. Schatz, G. (1993) Protein Sci. 2, 141–146. 26. Scherer, P., Manning-Krieg, K., Jeno¨, P., Schatez, G., and Horst, M. (1992) Proc. Natl. Acad. Sci. USA 89, 11930–11934. 27. Blom, J., Ku¨brich, M., Rassow, J., Voos, W., Dekkler, P. J. T., Maarse, A., Meijer, M., and Pfanner, N. (1993) Mol. Cell. Biol. 13, 7354–7371. 28. Emtage, J. L., and Jensen, R. E. (1993) J. Cell Biol. 122, 1003– 1012.
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29. Schnell, D. J., Kessler, F., and Blobel, G. (1994) Science 266, 1007–1012. 30. Hirsch, S., Muckel, E., Heemeyer, F., von Heijne, G., and Soll, J. (1994) Science 266, 1989–1992. 31. Perryman, R. A., Mooney, B., and Harmey, M. A. (1995) Arch. Biochem. Biophys. 316, 659–664. 32. Ono, H., and Tuboi, S. (1991) Biochem. Biophys. Res. Commun. 180, 450–454. 33. Farre´s, J., Guan, K.-L., and Weiner, H. (1988) Biochem. Biophys. Res. Commun. 150, 1083–1087. 34. Pak, Y. K., and Weiner, H. (1990) J. Biol. Chem. 265, 14298– 14307. 35. Richardson, C. D., Berkovich, A., Rozenblatt, S., and Bellini, W. J. (1985) J. Virol. 54, 186–193. 36. Sakakibra, R., Horio, Y., Ishiguro, M., Kangawa, K., Matsuo, H., and Wada, H. (1987) Biochem. Biophys. Res. Commun. 148, 979– 988. 37. Hurn, B. A. L., and Chantler, S. M. (1980) Methods Enzymol. 70, 104–142. 38. Hajra, A. K., Burke, C. L., and Jones, C. L. (1979) J. Biol. Chem. 254, 10896–10900. 39. Wang, T. T. Y., Farre´s, J., and Weiner, H. (1989) Arch. Biochem. Biophys. 272, 440–449. 40. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, pp. 466–467, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 41. Woodfin, B. M., and Davis, L. E. (1986) J. Cell Biochem. 31, 271– 275. 42. Jeng, J. J., and Weiner, H. (1991) Arch. Biochem. Biophys. 289, 214–222.
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43. Song, C.-S., Yu, J.-H., Bai, D. H., Hester, P. Y., and Kim, K-H. (1985) J. Immunol. 135, 3354–3359. 44. Ono, H., and Tuboi, S. (1990) J. Biochem. 107, 840–845. 45. Storrie, E. B., and Madden, E. A. (1990) Methods Enzymol. 182, 214–215. 46. Daum, G., Bo¨hni, P. C., and Schatz, G. (1982) J. Biol. Chem. 257, 13028–13033. 47. Bradford, M. M. (1976) Anal. Biochem. 72, 248–254. 48. Wahl, G. M., Ono, E., Meinkath, J., Franco, R., and Barinaga, M. (1981) Methods for the Transfer of DNA, RNA and Protein in Nitrocellulose and Diazotized Paper Solid Supports, Schleicher & Schuell. 49. Weiner, H., McMichael, M., Lindahl-Hellstrom, E., and Tu, G. C. (1987) Alcohol Alcoholism Suppl. 1, 187–191. 50. Strosberg, A. D. (1989) Methods Enzymol. 178, 179–191. 51. Pain, D., Kanwar, Y. S., and Blobel, G. (1988) Nature 331, 232– 237. 52. Schnell, D. J., Blobel, G., and Pain, D. (1990) J. Cell Biol. 111, 1825–1838. 53. Pain, D., Murakami, H., and Blobel, G. (1990) Nature 347, 444– 449. 54. Murakami, H., Blobel, G., and Pain, D. (1990) Nature 347, 488– 491. 55. Savitz, A. J., and Meyer, D. I. (1990) Nature 346, 540–544. 56. Pilch, P. F., and Czech, M. P. (1984) in Membranes, Detergents, and Receptor Solubilization (Venter, J. C., and Harrison, L. C., Eds.), pp. 161–175, Liss, NY. 57. Witte, C., Jensen, R. E., Yaffe, M. P., and Schatz, G. (1988) EMBO J. 7, 1439–1447.
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Vol. 323, No. 1, October 20, pp. 54–62, 1995
Identification of Protein-Receptor Components Required for the Import of Prealdehyde Dehydrogenase into Rat Liver Mitochondria1 Olga Pchelintseva,2 Youngmi Kim Pak,2 and Henry Weiner3,4 Department of Biochemistry, Purdue University, 1153 Biochemistry Building, West Lafayette, Indiana 47907-1153
Received May 24, 1995, and in revised form July 31, 1995
Mitochondrial aldehyde dehydrogenase is synthesized as a high-molecular-weight precursor in cytosol and transported into mitochondrial matrix space where it is processed to the mature enzyme. To identify components of the transport machinery on liver mitochondria, anti-idiotypic antibodies against the rabbit anti-prealdehyde dehydrogenase signal peptide antibodies were produced in chicken eggs and rabbit. Both anti-idiotypic antibodies inhibited the import of prealdehyde dehydrogenase (pALDH) into isolated rat liver mitochondria. The rabbit anti-idiotypic antibody could recognize by Western blotting five mitochondrial membrane proteins with apparent molecular weights of 66, 60, 42, 34, and 29 kDa. The anti-idiotypic antibodies were cross-linked to mitochondrial membrane proteins using sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3 *-dithiopropionate which is an iodinatable, heterofunctional, and photoreactive cross-linker. Mitochondrial proteins with apparent molecular weights of 66, 60, and 42 kDa were identified using the chicken antibody. The 66and 34-kDa proteins were cross-linked to the rabbit antibody as the major components and the 42-kDa protein as a minor one. Antibodies against the 60- and 42-kDa proteins, as well as Fab fragments, inhibited the import of pALDH, suggesting that these proteins are receptor/translocator components for pALDH import. q 1995 Academic Press, Inc. 1 This work was supported in part by Grant AA05812 from the National Institute on Alcohol Abuse and Alcoholism and the Indiana Elks Charities, Inc., Purdue University Cancer Center. This is Journal Paper No. 14739 from the Purdue University Agricultural Experiment Station. 2 Each author made an equal contribution. 3 H. Weiner was a recipient of Senior Scientist Award AA00028 from the National Institute on Alcohol Abuse and Alcoholism. 4 To whom correspondence should be addressed. Fax: (317) 4947897.
Key Words: mitochondria; receptor; import; aldehyde dehydrogenase; anti-idiotypic antibodies; cross-linking.
Proteins destined for mitochondria are encoded by nuclear genes and synthesized in cytoplasm as higher molecular weight precursors. Transport of the proteins from cytoplasm into mitochondria requires energy, one or more targeting signals on the transported protein, and a machinery that decodes these signals and moves the protein to its correct intramitochondrial location (1). The targeting signal in a precursor is called a signal peptide, presequence, or leader sequence (for review see 1–3). The precursor protein is processed by a protease in the matrix space to produce the mature protein (4, 5). We have been investigating the relationship between the structure of the leader sequence of aldehyde dehydrogenase and how it binds to mitochondria (6–8). The leader must ultimately bind to a membrane-spanning receptor/translocator complex so that it can be brought into the mitochondria. During the past few years many components of the complex have been identified from yeast and Neurospora crassa. Three recent review articles summarize what is known about the system (9– 11). Two highly protease-sensitive outer membrane proteins of approximate molecular weights 70 and 20 kDa, respectively, have been identified as an integral part of the import receptor. The larger protein has been termed MAS70 in Saccharomyces cerevisiae (12) or MOM72 in N. crassa (13) and the smaller one MOM19 in N. crassa (14) or MAS20 in S. cerevisiae (15). The two import receptors MOM19 and MOM72 were found in a high-molecular-weight complex in the mitochondrial outer membrane. In both organisms, the complex contained at least five additional proteins,
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termed MOM7, MOM8, MOM22, MOM30, and MOM38 (16–18). Four of these proteins, MOM7, MOM8, MOM30, and MOM38, are believed to be involved in the formation of the general insertion pore (GIP)5 that is responsible for the insertion of preproteins into the outer membrane and their translocation across this membrane (3). The recent studies with S. cerevisiae and N. crassa have identified a third receptor, a 22-kDa protein termed Mas22p in yeast and MOM22 in N. crassa (19, 20). MOM22, anchored in the outer membrane by a single transmembrane segment, is required for the transfer of preproteins from the receptor to the GIP. Antibodies directed against MOM22 did not interfere with the binding of preproteins to their receptors, but inhibited the import of all analyzed preproteins that use the mitochondrial receptor complex. In contrast, antibodies directed against the receptors MOM19 and MOM72 showed different inhibitory effects on various subclasses of preproteins. These data suggest that MOM22 is required for insertion of proteins into the GIP. The import pathways using both receptors MOM19 and MOM72 appear to converge at MOM22 (19). One subunit of the putative outer membrane channel was identified by cross-linking it to a precursor protein that spanned the protein import channel of both membranes (21). This import-site protein (ISP42) proved to be an integral outer membrane protein essential for cell viability (22). The N. crassa homolog, termed MOM38, was shown to be part of a complex that also contained one of the import receptor components (16). There is now strong evidence that the mitochondrial inner membrane has a protein import system distinct from that in the outer membrane (23–25). However, only a few inner membrane import components have been identified. One component is a 45-kDa protein from S. cerevisiae, ISP45, which is associated with the outer face of the inner membrane. ISP45 was identified as one of several proteins that could be cross-linked to a precursor blocked in transit across the mitochondrial inner membrane (26). The other inner membrane import component is the MP11 gene product MIM44. It was found to behave as a peripheral membrane protein firmly associated with the mitochondrial inner membrane. Using cross-linking, it was demonstrated that MIM44 is in close proximity to the precursor at an early stage of translocation across the inner membrane (27). 5 Abbreviations used: pALDH, prealdehyde dehydrogenase; mALDH, mature aldehyde dehydrogenase; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; SASD, sulfosuccinimidyl 2-(p-azido-salicylamide)ethyl-1,3*-dithiopropionate; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; GIP, general insertion pore; ISP, import-site protein.
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Mas6p, a 23-kDa protein with three or four potential transmembrane domains, is an integral membrane protein of the yeast mitochondrial inner membrane, which is essential for protein import. Depletion of Mas6p from yeast cells resulted in the accumulation of mitochondrial precursor proteins. Antibodies to Mas6p inhibited protein import into mitochondria, but only when the outer membrane was disrupted to allow the antibodies access to the inner membrane (28). In spite of all that is known about transport apparatus in lower eukaryotes, less is known about the import complex in higher organisms. Chloroplast components have been characterized (29, 30). A 42-kDa plant mitochondria outer membrane protein has been identified as being necessary for import (31). Synthetic peptides derived from presequences of precursor proteins were used to identify the putative signal-sequence receptor components for liver. These were either radiolabeled and cross-linked to mitochondrial proteins or used as ligands on an affinity chromatography column. Two proteins of 29 and 52 kDa were purified from rat liver mitochondria, and antibodies raised against them inhibited protein import (32). In this paper, we utilized anti-idiotypic antibodies mimicking the signal peptide of rat liver prealdehyde dehydrogenase (pALDH) for cross-linking to the protein components on the surface of rat and beef liver mitochondria. Anti-idiotypic antibodies were generated in chicken and rabbit. Rabbit anti-idiotypic antibodies were used to identify receptor proteins by immunoblotting, as well as by cross-linking. Three proteins, 42, 60, and 66 kDa, were identified and purified from liver mitochondria. Antibodies raised against these proteins inhibited import of pALDH into mitochondria. We propose that the 42-, 60-, and, possibly, 66-kDa proteins function as receptor/translocator components for pALDH import into liver mitochondria. EXPERIMENTAL PROCEDURES Animals. Male Wistar rats of about 200 g were used for mitochondria preparation. Beef livers were obtained from a local slaughterhouse. New Zealand white rabbits and laying hens were used for antibody preparation. Materials. [ 35S]Methionine (ú800 Ci/mmol) was from New England Nuclear. [ 125I]NaI (2.4 Ci/mmol) was from Amersham. IodoGen, SASD, and ImmunoPure Fab preparation kit were purchased from Pierce. Keyhole limpet hemocyanin and substrate system (nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate) for phosphatase-labeled antibodies were purchased from Sigma Co. CNBractivated Sepharose 4B was purchased from Pharmacia. Molecular weight markers for SDS–PAGE and alkaline phosphatase-coupled secondary antibodies were purchased from Bio-Rad. Preparation of anti-signal peptide antibody. The pALDH signal peptide was synthesized in the Purdue Medical Chemistry Department with a sequence of MLRAALSTARKGPRLSRLLSYA-CONH2 deduced from pALDH cDNA sequence (33) and used previously by Pak and Weiner (34). The synthesized signal peptide (5 mg) was
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conjugated to a keyhole limpet hemocyanin carrier protein (1 mg) as described (35). Antisera were prepared by injecting the signal peptide–hemocyanin conjugate into rabbits (36). The IgG from antisera was purified by ammonium sulfate fractionation and DEAE–cellulose chromatography (37). The specific antibodies against the signal peptide was purified from the antisera using the signal peptidebound Sepharose affinity column, which was prepared by attaching the signal peptide to CNBr-activated Sepharose 4B following the manufacturer’s instruction (Pharmacia). Antisera were first loaded on the affinity column with phosphate-buffered saline (PBS; 6 mM sodium phosphate, pH 7.2, 0.17 M NaCl, 3 mM KCl, 0.02% sodium azide) and the bound antibodies were eluted with 0.2 M glycine buffer, pH 2.8, containing 0.5 M NaCl. The peak fractions detected by optical density at 280 nm were pooled and the pH was adjusted to 7.2 with 1 M Tris. The purified antibodies were dialyzed against PBS and concentrated using an Amicon Centricon. The specificity of the antibodies was determined by Western blotting after 10% SDS–PAGE of crude pALDH or mALDH expressed in Escherichia coli. Expression and labeling of pALDH and mALDH in E. coli. pALDH and mALDH were expressed in E. coli (BL 21/pLysS) using the pT7-7 expression vector (42). The E. coli cells from a 10-ml culture were harvested and resuspended in 1 ml of buffer (0.05 M sodium phosphate, pH 7.2, 1 mM EDTA, 0.02% b-mercaptoethanol) and used for immunoblotting. Preparation of anti-idiotypic antibodies. The anti-idiotypic antibodies were raised in two laying hens by subcutaneous injection of the purified rabbit anti-signal peptide antibody (5 mg) in PBS with an equal volume of Freund’s complete adjuvant into five sites. After the initial three injections, one every other week, eggs were collected daily. The anti-signal peptide antibodies from individual egg yolks were subjected to 10% SDS–PAGE and immunoblotted. The egg yolks which showed a positive reaction were pooled and IgY was extracted (43). Anti-idiotypic antibodies from IgY fractions were purified by passing the extract through the anti-signal peptide antibody– Sepharose 4B affinity column. The eluted anti-idiotypic antibodies in glycine buffer were pooled, dialyzed against PBS, and concentrated to 1 mg/ml. Anti-idiotypic antibodies were also generated in rabbits by subcutaneous injection of the purified anti-signal peptide antibodies. The IgG from antisera was purified by caprylic acid precipitation and ammonium sulfate fractionation (37). The Fab fragments of IgG were prepared using ImmunoPure Fab preparation kit following the manufacturer’s instruction. Briefly, the anti-idiotypic IgG was digested with immobilized papain. The Fab fragments were purified from the Fc fragments by passing through a protein A column, dialyzed against PBS, and concentrated to 1 mg/ml. The molecular weight of anti-idiotypic antibody was determined by size-exclusion HPLC (TSK-GEL G3000-SW, 30 1 7.5 cm) to be 158 kDa. Inhibition of pALDH import by antibodies. Rat liver mitochondria were isolated by differential centrifugation (38). Mitochondria (75 mg) in the import assay mixture (39) were preincubated with the anti-idiotypic antibodies (8 mg protein each) for 60 min at 47C, washed twice, and resuspended in 12.5 ml of mitochondria isolation buffer (0.025 M Tris–Cl, pH 7.4, 0.25 M sucrose, 1 mM EDTA). As a control, mitochondria were preincubated with preimmune IgY or IgG (27 mg protein) or buffer. Pretreated mitochondria were incubated with in vitro translated [ 35S]pALDH at 307C for 30 min, treated with proteinase K (200 mg/ml final concentration) on ice for 15 min followed by PMSF addition (1 mM final concentration), and analyzed by 10% SDS–PAGE and fluorography (34, 39). Various amounts of the Fab fragments from rabbit anti-idiotypic antibodies also were preincubated with mitochondria in the import assay mixture, and pALDH import into mitochondria was performed as stated above. Cross-linking of anti-idiotypic antibody with mitochondrial proteins. SASD was iodinated with [ 125I]NaI using Iodo-Gen in 0.1 M sodium phosphate buffer, pH 7.4, and incubated with purified
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chicken or rabbit anti-idiotypic antibody (38 mg protein) at room temperature for 30 min in the dark. The unreacted 125I and SASD were removed by spinning through a 1-ml G-25 spun column (40). [ 125I]SASD conjugated to anti-idiotypic antibody was incubated in the dark with liver mitochondria (1.8 mg) in the import assay mixture at 07C for 30 min and then at room temperature for 30 min. The reaction mixture was irradiated with a hand-held long wavelength uv lamp (Mineralight UVS 11) for 10 min and then with a bright light (200 W) for 3 min. The mitochondria were isolated, washed, resuspended in 60 ml of the mitochondria isolation buffer, and divided into two parts. Each was dissolved in SDS sample buffer with and without 10% (v/v) b-mercaptoethanol, respectively, and analyzed on 7.5% SDS–PAGE. The gels were dried and autoradiographed. Purification of mitochondrial membrane proteins. Mitochondria were prepared from rat and beef liver by differential centrifugation (38, 41) and suspended in the mitochondria isolation buffer at a protein concentration of 7 mg/ml. All procedures were carried out at 47C. Mitochondria were separated into membranes and matrix fractions as described (44). The membrane pellet was resuspended in 220 mM mannitol containing 70 mM sucrose, 10 mM Hepes–KOH buffer, pH 7.6, 2 mM DTT, and 0.5 mM PMSF and stored at 0807C until use. For preparation of antibodies to the membrane proteins, the mitochondrial membrane fraction was fractionated by preparative 10% SDS–PAGE (1.5 1 130 1 120 mm) and the bands of 66-, 60-, and 42-kDa proteins were cut from the gel after staining with Coomassie brilliant blue. The proteins were eluted from pieces of gel by 20 mM Tris–Cl, pH 7.4, 0.5 mM PMSF buffer two times while stirred at room temperature for 1 h, concentrated using a Amicon Centricon, and used for immunizing the rabbits. Antibody titers were monitored by immunoblotting using mitochondrial membrane proteins resolved on 10% SDS–PAGE. IgG and Fab fragments were prepared by the methods described above. Various amounts of the IgG and Fab fragments from rabbit anti66-, -60-, and -42-kDa antibodies were preincubated with rat liver mitochondria, and pALDH import was performed as stated above. Other methods. The integrity of the mitochondria was routinely checked by measuring cytochrome c oxidase activity as a mitochondrial marker (45). Trypsin- or CCCP-treated mitochondria were prepared and mitochondria were treated with proteinase K as described previously (34). Yeast mitochondria were prepared from cells (XK251B) grown aerobically (46), then lysed by freezing and thawing three times to prepare a crude matrix homogenate containing the signal peptide peptidase. Protein concentration was determined by Bradford method (47). Antisera against mALDH were prepared in rabbits using purified rat liver mitochondria ALDH. Western blot analysis was performed according to standard methods (48). The proteins were immunodecorated with the suitable antibodies and the reactions were detected with the goat anti-rabbit antibodies conjugated to alkaline phosphatase (Bio-Rad) using the nitroblue tetrazolium/ 5-bromo-4-chloro-3-indolyl phosphate substrate system.
RESULTS
Specificity of anti-signal peptide antibody. Antisera raised against the chemically synthesized pALDH signal peptide recognized only pALDH, not mALDH, while both were recognized by antibodies produced against purified mALDH. Expressed pALDH was incubated with yeast mitochondria lysate which contained the signal peptide peptidase. Since yeast mitochondrial ALDH did not cross-react with anti-rat liver mitochondrial ALDH antibody (49), only expressed rat ALDH in the assay mixture could be detected by it. After incuba-
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FIG. 1. Specificity of the anti-signal peptide antibody determined by immunoblotting. pALDH and mALDH in pT7-7 were expressed in E. coli (BL21/pLysS) cells. Aliquots (5 ml) of cells from a 10-ml culture were analyzed by 10% SDS–PAGE and immunoblotting with anti-signal peptide antisera (aSP; A) and anti-rat liver mitochondrial ALDH antisera (aRM-ALDH; B). Lane 1, mALDH; lane 2, pALDH; lane 3, pALDH incubated with lysed yeast mitochondria which contained the signal peptide peptidase (57) for 2 h at 307C to cleave signal peptide from pALDH. The upper band represents precursor (pALDH) and lower band represents mature or processed mALDH.
tion about 50% of expressed pALDH was processed to mALDH. The processed mALDH was not recognized by anti-signal peptide antibody, verifying that anti-signal peptide antibody reacted with the signal peptide portion of pALDH (Fig. 1). A second set of antibodies were raised in chicken and in rabbit against the purified rabbit anti-signal peptide antibody to obtain the anti-idiotypic antibody that could recognize the peptide-binding site of the receptor/ translocator. The anti-idiotypic antibodies inhibited pALDH import into mitochondria. The inhibition of pALDH import by anti-idiotypic antibodies was investigated to determine if these interact with the import machinery. Preincubation of mitochondria with chicken, as well as rabbit, anti-idiotypic antibodies dramatically inhibited the import of pALDH, while incubation with excessive amounts of preimmune IgY or IgG caused slight inhibition of import (Fig. 2A). Similarly, Fab fragments of rabbit anti-idiotypic antibody completely inhibited pALDH import, while Fab-PI from preimmune IgG did not affect the import of pALDH (Fig. 2B). It appears from the data presented in Fig. 2 that the presence of IgG or the Fab fragments prevented binding of precur-
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sor protein to mitochondria. This could be expected to occur if these were interacting with components of the receptor/translocator. The fact that the Fab fragments were inhibitors implies that the inhibition was not just due to a large IgG molecule nonspecifically binding to the membrane. Five different mitochondrial proteins were recognized by rabbit anti-idiotypic antibody immunoblotting. Rat and beef liver mitochondrial membranes were resolved on SDS–PAGE and immunoblotted using the rabbit anti-idiotypic antibody. Five membrane proteins with apparent molecular weights of 66, 60, 42, 34, and 29 kDa were found when protein bands on the immunoblot were compared with those using preimmune IgG (Fig. 3). 66-, 60-, 42-, and 34-kDa mitochondrial proteins were cross-linked to anti-idiotypic antibody. Chicken antiidiotypic antibody was cross-linked to mitochondria using SASD to identify mitochondrial membrane proteins which may interact with the signal peptide. Radioactivity was found in the protein–antibody complex prior to reduction of the internal disulfide bond of SASD by b-mercaptoethanol. A radioactive protein complex of approximately 240 kDa was found (Fig. 4A). If the complex was reduced with b-mercaptoethanol, only the cross-linked mitochondrial protein would contain ra-
FIG. 2. Inhibition of pALDH import by anti-idiotypic antibody. (A) Rat liver mitochondria (75 mg protein) were preincubated with either chicken or rabbit anti-idiotypic antibodies (lane 2, 8 mg; lane 3, 16 mg) or with preimmune IgY or IgG (lane 1, 27 mg), as well as with PBS buffer (lane C) for 1 h at 47C. Pretreated mitochondria were incubated with reticulocyte lysate (15 ml) containing 35S-labeled pALDH in the import assay mixture for 30 min at 307C and were then treated with proteinase K (200 mg/ml). The mitochondria were reisolated by centrifugation and analyzed by 10% SDS–PAGE and fluorography. (B) The experiment was performed as described above with the following modifications. The mitochondria (75 mg protein) were preincubated with different amounts of Fab fragment from rabbit anti-idiotypic antibodies or from preimmune serum (Fab-PI) (lane 1, 3 mg; lane 2, 6 mg; lane 3, 13 mg), as well as with PBS buffer (lane C). 35S-labeled pALDH were added and the mixture was incubated for 30 min at 307C. The mitochondria were isolated by centrifugation and the pellet was analyzed by 10% SDS–PAGE and fluorography. The upper band (pALDH) represents binding; the lower band (mALDH) represents import.
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FIG. 3. Immunoblotting of rat liver mitochondrial membrane proteins using rabbit anti-idiotypic antibody. Rat and beef liver mitochondrial membranes were prepared as described (44). (A) Membrane proteins were separated on 10% SDS–PAGE and immunoblotted using rabbit anti-idiotypic antibody (IgG-aIT) or preimmune IgG (IgG-PI). Lane S, prestained protein molecular weight markers; lane 1, rat liver mitochondrial membranes; lane 2, beef liver mitochondrial membranes. (B) Coomassie blue-stained gel. Approximately 50 mg proteins was loaded in each lane.
mately 95 kDa did not involve mitochondrial proteins as it was a contaminant found in the cross-linking control experiment (Fig. 5, lane C). Inhibition of protein import by the antibodies raised against proteins identified by cross-linking. A number of proteins were identified by cross-linking as potential candidates of the translocator. Proteins of these subunit molecular weights (66, 60, and 42 kDa) were isolated from SDS–PAGE. The proteins were excised from the gels and injected into rabbits. It was possible to obtain sufficient quantities of the 66-kDa protein from rat to make antibodies. It was necessary to use beef liver mitochondria to obtain the 60- and 42-kDa proteins. The specificity of the antibodies raised to 66-, 60-, and 42-kDa SDS–PAGE fractions was verified by subjecting total mitochondrial membranes and matrix fraction as well as intact mitochondria to Western blot analysis. Antibodies against 66-, 60-, and 42-kDa proteins specifically detect corresponding protein when used to immunodecorate intact mitochondria and mitochondria membrane proteins (Fig. 6).
dioactivity. After reduction three major radioactive bands were found with apparent molecular weights of 66, 42, and 34 kDa (Fig. 4B). The same three protein bands were obtained by cross-linking anti-idiotypic antibody to CCCP-treated mitochondria (Fig. 4C). This was expected, since CCCP-treated mitochondria should still contain the protein translocator, even though import was inhibited by the uncoupler dissipating the membrane potential (3). Treating mitochondria with a low concentration of trypsin destroyed the outer membrane proteins and abolished precursor import (2). Therefore, the trypsintreated mitochondria should not possess the protein translocators which could bind to anti-idiotypic antibody. As expected, pALDH was not imported into trypsin-treated mitochondria as described previously (34) and no corresponding protein bands were found after cross-linking trypsin-treated mitochondria with antiidiotypic antibody (Fig. 4, lane T). After cross-linking, mitochondria were treated with proteinase K. This treatment destroyed the crosslinked components showing that the anti-idiotypic antibody–membrane protein complex was on the outside of the mitochondria (Fig. 4C). Rabbit anti-idiotypic antibody was used for the crosslinking to determine if the same molecular weight proteins found with chicken anti-idiotypic antibody could be identified. The major bands were the 66-, 60-, and 34-kDa proteins, while 42-kDa protein was a minor component, as shown in Fig. 5. The band at approxi-
FIG. 4. Cross-linking of chicken anti-idiotypic antibody to mitochondria. Rat liver mitochondria were treated with trypsin (T) or CCCP (C) (34). The chicken anti-idiotypic antibody was crosslinked to the treated (T or C) or untreated (U) mitochondria as described under Experimental Procedures. After reisolating, mitochondria were dissolved in SDS sample buffer with b-mercaptoethanol (B, /bME) or without b-mercaptoethanol (A, 0bME) and then analyzed by 7.5% SDS – PAGE and autoradiography. Mitochondria, treated with proteinase K after cross-linking, contained no radioactivity, as shown in C (K). Molecular weight markers are shown to the left of A.
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FIG. 5. Cross-linking of rabbit anti-idiotypic antibody to mitochondria. Rabbit anti-idiotypic antibody (lane 2) or preimmune IgG (lane 1) was cross-linked to either rat or beef liver mitochondria and analyzed by 10% SDS–PAGE under reducing conditions as described in the legend to Fig. 4. As a control, the SASD-linked anti-idiotypic antibodies were cross-linked without mitochondria incubation (lane C).
Cross-competition experiments were also performed to determine whether the anti-66, -60-, and -42-kDa protein antibodies were in fact recognizing the same components as the anti-idiotypic antibodies. Immunological cross-reactivity with mitochondrial membrane proteins of similar molecular weight has been detected between antibodies raised against SDS–PAGE purified proteins and anti-idiotypic antibodies (data not shown). The antibodies against the 66-, 60-, and 42-kDa mitochondria proteins and their corresponding Fab fragments were tested as potential inhibitors of protein import. Both anti-42-kDa and anti-60-kDa antibodies inhibited the import of pALDH into mitochondria, as shown in Fig. 7. The Fab fragments also caused the inhibition of import almost completely at a concentration of 0.88 mg/ml, while preimmune Fab had no effect at the same concentration. In contrast, neither antibodies against the 66-kDa protein nor the Fab fragments cause measurable inhibition of import to occur. The findings from import inhibition, immunoblotting, and ligand cross-linking lead us to conclude that the 60- and 42-kDa proteins are strong candidates for being components of the rat liver mitochondrial receptor/translocator complex. It remains to be investigated whether the 66-kDa protein functions as part of a receptor/translocator complex or may be involved in the import pathway of pALDH in some other manner. DISCUSSION
Several research groups identified receptor/translocator proteins on mitochondria from various species
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using different approachs as described in the Introduction. Anti-idiotypic antibodies have been used to probe for hormone and neurotransmitter receptors when the receptor proteins were unknown (50). A 30-kDa chloroplast envelope protein that participated in protein import was identified using immunoprecipitation with anti-idiotypic antibody (51). Since anti-idiotypic antibodies mimick the structure of the signal peptide, they should bind to the receptor. This chloroplast import receptor protein was purified and its primary structure was deduced from cDNA sequencing (52). The deduced molecular weight was 36 kDa instead of 30 kDa, and the protein showed a high homology to the phosphate translocator of chloroplast. Using a similar approach with anti-idiotypic antibody, the 32-kDa integral membrane protein of yeast mitochondria was identified (53) and its gene was cloned (54). p32 showed high amino acid identity (40%) with the mitochondrial phosphate translocator as did the chloroplast p36. However, some of the previous applications of this approach have led to identification of proteins that may not be directly implicated in targeting (55). It was not possible to identify unknown components of the receptor complex of rat liver mitochondria only on the basis of their relative molecular weights with recently identified components of the import apparatus in yeast and Neurospora. Although fundamental questions remain about the use of the antiidiotypic antibodies for identification of relatively low affinity receptors as might be involved in protein targeting, they could serve as an initial probe to identify
FIG. 6. Immunoblotting of mitochondrial membrane proteins using rabbit anti-66-, -60-, and -42-kDa protein antibodies. Rat and beef liver mitochondria were separated into membranes and matrix fractions as described (44). The mitochondrial protein fractions were separated by 10% SDS–PAGE, transferred to nitrocellulose, and immunodecorated with anti-66- (A), -60- (B), and -42-kDa protein IgG (C) at a dilution of 1:5000. Preimmune IgG was also employed. No stained bands were detected (not shown). Lane 1, intact mitochondria; lane 2, total mitochondrial membranes; lane 3, matrix fraction. Approximately 50 mg proteins was loaded in each lane. Molecular weight markers (lane C) were run with each.
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FIG. 7. Inhibition of pALDH import into mitochondria by anti-66, -60-, and -42-kDa Fab fragments. The Fab fragments were prepared following the manufacturer’s instruction (Pierce, ImmunoPure preparation kit). Mitochondria (7 mg/ml, final protein concentration) were preincubated with different concentrations of anti-66, -60-, and -42-kDa protein Fab fragments (lane 2, 0.2 mg/ml; lane 3, 0.4 mg/ml; lane 4, 0.88 mg/ml) or with Fab fragments prepared from a preimmune rabbit (lane 1, 0.88 mg/ml), as well as with PBS buffer (lane C). The mitochondria were reisolated by centrifugation, washed, and incubated with 35S-labeled pALDH in the import assay mixture as described in the legend to Fig. 2. Mitochondria were then analyzed for import of pALDH by 10% SDS–PAGE and fluorography. Bands of ALDH on the fluorogram were quantified by densitometry. In all cases, the values obtained with untreated mitochondria (lane C, PBS buffer) were set at 100%.
possible components of the receptor. Hence, we used anti-idiotypic antibodies to probe for the mitochondrial receptor/translocator proteins of rat liver. Both the chicken and rabbit anti-idiotypic antibodies mimicking the pALDH signal peptide inhibited the import of pALDH, implying that anti-idiotypic antibody reacted
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with the protein import machinery. When rat and beef membrane protein were immunoblotted using rabbit anti-idiotypic antibody, 66-, 60-, and 42-, 34-, and 29kDa proteins among others were identified. Chicken and rabbit anti-idiotypic antibodies were chemically cross-linked to mitochondrial membrane proteins and
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four components were labeled (66, 60, 42, and 34 kDa). Since cross-linking is dependent upon many experimental conditions such as ligand orientation and length of the cross-linker used (56), the proteins which were not cross-linked cannot be excluded as possible components of the receptor. Proteins (29-kDa and 52-kDa) from rat liver mitochondrial membranes were purified by Ono and Tuboi (44) using signal peptide affinity chromatography. The 29-kDa protein we identified by anti-idiotypic antibody immunoblotting is possibly the same component found by them. It is possible that the 29-kDa protein was not cross-linked because it is not fully exposed to the outer surface of mitochondria, but when solubilized it becomes immunoreactive to the anti-idiotypic antibodies. Our antibodies did not cross-react with a 52-kDa protein found by Ono and Tuboi (32). To further characterize protein components of a receptor their purification was necessary. The only available property of the import receptor components was their subunit molecular weights, for no convenient method was available to assay for them. To obtain the 66-, 60-, and 42-kDa proteins for antibody production, preparative SDS–PAGE was performed on rat and beef liver mitochondria. Fab fragments of the anti-60- and 42-kDa protein antibodies inhibited pALDH import into mitochondria. Import can be inhibited by prevention of precursor binding either to the membrane or to its translation across the membrane. From the data presented in Fig. 7 it appears that binding of precursor is diminished in the presence of Fab fragments. Their association with the translocator would be expected to affect the interaction of the precursor with the translocator just due to the steric presence of another large protein attached to the complex. We cannot state if the inhibition of import was related just to diminished binding or was also related to the antibody preventing the translocator from functioning. Since the 60- and 42-kDa proteins were identified by several different techniques, and antibodies against each inhibited the import of pALDH, we conclude that the 60- and 42kDa proteins were involved in the import of pALDH, possibly as components of the receptor/translocator. The 66-kDa protein was implicated to be involved, but since antibodies against it did not inhibit import we cannot suggest that it is part of the translocation apparatus. The 66-, 34-, and 29-kDa proteins, identified by cross-linking or Western blotting, may not interact directly with the pALDH signal peptide. However, each of them may form a complex with the 42- or 60 kDa proteins and this complex may function in the import process on the mitochondria. It will be necessary to isolate the import complex in order to determine which of the components are associated with it. Work is currently in progress to do this.
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REFERENCES 1. Hartl, F.-U., and Neupert, W. (1990) Science 247, 930–938. 2. Nicholson, D. W., and Neupert, W. (1989) in Protein Transfer and Organelle Biogenesis (Das, R. C., and Robbins, P. W., Eds.), pp. 637–746, Academic Press, San Diego. 3. Pfanner, N., Hartl, F.-U., and Neupert, W. (1988) Eur. J. Biochem. 175, 205–212. 4. Bo¨hni, P., Gasser, S., Leaver, C., and Schatz, G. (1980) in The Organization and Expression of the Mitochondrial Genome (Kroon, A. M., and Saccone, C., Eds.), pp. 424–433, Elsevier, Amsterdam. 5. Schneider, H., Arretz, M., Wachter, E., and Neupert, W. (1990) J. Biol. Chem. 265, 9881–9887. 6. Thornton, K., Wang, Y., Weiner, H., and Gorenstein, D. G. (1993) J. Biol. Chem. 268, 19906–19914. 7. Wang, Y., and Weiner, H. (1993) J. Biol. Chem. 268, 4759–4765. 8. Wang, Y., and Weiner, H. (1994) Biochemistry 33, 12860–12867. 9. Lithgow, T., Glick, B. S., and Schatz, G. (1995) TIBS 20, 98– 101. 10. Shore, G. C., McBride, M. M., Millar, D. G., Steenaart, N. A. E., and Nguyen, M. (1995) Eur. J. Biochem. 227, 9–18. 11. Ku¨brich, M., Dietmeier, K., and Pfanner, N. (1995) Curr. Genet. 27, 393–403. 12. Hines, V., Brandt, A., Griffity, G., Hotstmann, H., Bru¨ltsch, H., and Schatz, G. (1990) EMBO J. 9, 3191–3200. 13. So¨llner, T., Pfaller, R., Griffiths, G., Pfanner, N., and Neupert, W. (1990) Cell 62, 107–115. 14. So¨llner, T., Griffiths, G., Pfaller, R., Pfanner, N., and Neupert, W. (1989) Cell 59, 1061–1070. 15. Ramage, L., Junne, T., Hahne, K., Lithgow, T., and Schatz, G. (1993) EMBO J. 12, 4115–4123. 16. Kiebler, M., Pfaller, R., So¨llner, T., Griffiths, G., Horstmann, H., Pfanner, N., and Neupert, W. (1990) Nature 348, 610–616. 17. Meczko, M., Dietmeier, K. A., So¨llner, T., Segui-Real, B., Steger, H. F., Neupert, W., and Pfanner, N. (1992) FEBS Lett. 310, 265– 268. 18. So¨llner, T., Rassow, J., Wiedmann, M., Schossmann, J., Keil, P., Neupert, W., and Pfanner, N. (1992) Nature 355, 84–87. 19. Kiebler, M., Keil, P., Schneider, H., van der Klei, I. J., Pfanner, N., and Neupert, W. (1993) Cell 74, 483–492. 20. Lithgow, T., Junne, T., Suda, K., Gratzer, S., and Schatz, G. (1994) Proc. Natl. Acad. Sci. USA 91, 11973–11977. 21. Vestweber, D., Brunner, K., Baker, A., and Schatz, G. (1989) Nature 341, 205–209. 22. Baker, K. P., Schaniel, A., Vestweber, D., and Schatz, G. (1990) Nature 348, 605–609. 23. Pfanner, N., Rassow, J., van der Klei, I. J., and Neupert, W. (1992) Cell 68, 999–1002. 24. Horst, M., Jeno¨, P., Kronidou, N. G., Bolliger, L., Oppliger, W., Scherer, P., Manning-Krieg, U., Jascur, T., and Schatz, G. (1993) EMBO J. 12, 3035–3041. 25. Schatz, G. (1993) Protein Sci. 2, 141–146. 26. Scherer, P., Manning-Krieg, K., Jeno¨, P., Schatez, G., and Horst, M. (1992) Proc. Natl. Acad. Sci. USA 89, 11930–11934. 27. Blom, J., Ku¨brich, M., Rassow, J., Voos, W., Dekkler, P. J. T., Maarse, A., Meijer, M., and Pfanner, N. (1993) Mol. Cell. Biol. 13, 7354–7371. 28. Emtage, J. L., and Jensen, R. E. (1993) J. Cell Biol. 122, 1003– 1012.
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29. Schnell, D. J., Kessler, F., and Blobel, G. (1994) Science 266, 1007–1012. 30. Hirsch, S., Muckel, E., Heemeyer, F., von Heijne, G., and Soll, J. (1994) Science 266, 1989–1992. 31. Perryman, R. A., Mooney, B., and Harmey, M. A. (1995) Arch. Biochem. Biophys. 316, 659–664. 32. Ono, H., and Tuboi, S. (1991) Biochem. Biophys. Res. Commun. 180, 450–454. 33. Farre´s, J., Guan, K.-L., and Weiner, H. (1988) Biochem. Biophys. Res. Commun. 150, 1083–1087. 34. Pak, Y. K., and Weiner, H. (1990) J. Biol. Chem. 265, 14298– 14307. 35. Richardson, C. D., Berkovich, A., Rozenblatt, S., and Bellini, W. J. (1985) J. Virol. 54, 186–193. 36. Sakakibra, R., Horio, Y., Ishiguro, M., Kangawa, K., Matsuo, H., and Wada, H. (1987) Biochem. Biophys. Res. Commun. 148, 979– 988. 37. Hurn, B. A. L., and Chantler, S. M. (1980) Methods Enzymol. 70, 104–142. 38. Hajra, A. K., Burke, C. L., and Jones, C. L. (1979) J. Biol. Chem. 254, 10896–10900. 39. Wang, T. T. Y., Farre´s, J., and Weiner, H. (1989) Arch. Biochem. Biophys. 272, 440–449. 40. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, pp. 466–467, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 41. Woodfin, B. M., and Davis, L. E. (1986) J. Cell Biochem. 31, 271– 275. 42. Jeng, J. J., and Weiner, H. (1991) Arch. Biochem. Biophys. 289, 214–222.
/ m4096$9108
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43. Song, C.-S., Yu, J.-H., Bai, D. H., Hester, P. Y., and Kim, K-H. (1985) J. Immunol. 135, 3354–3359. 44. Ono, H., and Tuboi, S. (1990) J. Biochem. 107, 840–845. 45. Storrie, E. B., and Madden, E. A. (1990) Methods Enzymol. 182, 214–215. 46. Daum, G., Bo¨hni, P. C., and Schatz, G. (1982) J. Biol. Chem. 257, 13028–13033. 47. Bradford, M. M. (1976) Anal. Biochem. 72, 248–254. 48. Wahl, G. M., Ono, E., Meinkath, J., Franco, R., and Barinaga, M. (1981) Methods for the Transfer of DNA, RNA and Protein in Nitrocellulose and Diazotized Paper Solid Supports, Schleicher & Schuell. 49. Weiner, H., McMichael, M., Lindahl-Hellstrom, E., and Tu, G. C. (1987) Alcohol Alcoholism Suppl. 1, 187–191. 50. Strosberg, A. D. (1989) Methods Enzymol. 178, 179–191. 51. Pain, D., Kanwar, Y. S., and Blobel, G. (1988) Nature 331, 232– 237. 52. Schnell, D. J., Blobel, G., and Pain, D. (1990) J. Cell Biol. 111, 1825–1838. 53. Pain, D., Murakami, H., and Blobel, G. (1990) Nature 347, 444– 449. 54. Murakami, H., Blobel, G., and Pain, D. (1990) Nature 347, 488– 491. 55. Savitz, A. J., and Meyer, D. I. (1990) Nature 346, 540–544. 56. Pilch, P. F., and Czech, M. P. (1984) in Membranes, Detergents, and Receptor Solubilization (Venter, J. C., and Harrison, L. C., Eds.), pp. 161–175, Liss, NY. 57. Witte, C., Jensen, R. E., Yaffe, M. P., and Schatz, G. (1988) EMBO J. 7, 1439–1447.
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