- Email: [email protected]
[17] Analysis of mitochondrial protein import pathway in Saccharomyces cerevisiae with translocation intermediates
[17]
MITOCHONDRIAL PROTEIN IMPORT PATHWAY
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5. E l u t e the p r o t e i n s b o u n d to t h e b e a d s by e i t h e r i n c u b a t i n g the b e a d s in s o l u b i l i z a t i o n b u f f e r i n c l u d i n g 0.4 M i m i d a z o l e buffer, p H 7.4, or by a d d i n g 5 0 / , 1 2 x s a m p l e b u f f e r ~s a n d h e a t i n g for 5 min at 95 °. A n a l y z e the s a m p l e by S D S - P A G E .
[17] Analysis of Mitochondrial Protein Import Pathway in Saccharomyces cerevisiae with Translocation Intermediates By
DOUGLAS
M. CYR,
CHRISTIAN
UNGERMANN,
and
WALTER
NEUPERT
Introduction T h e c h a r a c t e r i z a t i o n of p r o t e i n t r a n s l o c a t i o n i n t e r m e d i a t e s has p r o v i d e d m a n y o f the s e m i n a l o b s e r v a t i o n s in the c h a r a c t e r i z a t i o n of the m i t o c h o n d r i a l p r o t e i n i m p o r t p a t h w a y . S t u d y o f i m p o r t i n t e r m e d i a t e s has h e l p e d define the e n e r g y r e q u i r e m e n t s for p r o t e i n i m p o r t , 1'2 the c o n f o r m a t i o n a l state of p r e c u r s o r p r o t e i n s d u r i n g t r a n s l o c a t i o n , 3 5 the c o m p o n e n t s of the i m p o r t m a c h i n e r y , T. S611ncr, J. Rassow. M. Wiedmann. J. Schlossmann, P. Kiel, W. Neupert, and N. Pfanner, Nature 355, 84 (1992). it T. S611ner, J. Rassow. and N. Pfanner, Methods Cell Biol. 34, 345 (1991).
METIIODS IN ENZYMOI OGY, VOI.. 260
CopFdght ~': 1995 by Academic Press, Inc All rights of reproduction in any foun reserved
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IMPORTOF PROTEINSAND RNA INTOMITOCHONDRIA
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G e n e r a t i o n of T r a n s l o c a t i o n I n t e r m e d i a t e s t h a t S p a n M e m b r a n e s of Isolated Mitoehondria
In vitro Synthesis of Mitochondrial Precursor Proteins Expression plasmids for mitochondrial precursor proteins are generated by cloning the gene of interest into a plasmid such as p G E M 4 (Promega Biochemicals) in which it is put under the control of the SP6 promoter. Purified plasmid D N A is used for in vitro transcription with SP6 RNA polymerase in the presence of NTPs and 7 m G p p G at 37 ° for 1 hr. ~2m R N A produced in this fashion is precipitated with LiC1, resuspended in RNasefree H20 and stored at 80 °. To synthesize 35S-labeled protein, rabbit reticulocyte lysate 13 (Promega Biochemicals) is programmed with m R N A and incubated for 60 rain at 30 ° in the presence of [35S]methionine (specific activity 1000 Ci/mM). For optimal synthesis of different precursor proteins, the concentrations of mRNA, magnesium ion (1 to 3 raM) and potassium ion (100 to 200 raM) must be determined. Lysates containing translated proteins are aliquoted, quick frozen in liquid nitrogen, stored at - 8 0 ° and thawed just prior to use,
Protein Import into Isolated Mitochondria Mitochondria are isolated from Saccharomyces cerevisiae strain D27310B according to the published protocol, 14 suspended in SEM buffer (sucrose 250 raM, E D T A 1 raM, and MOPS, pH 7.2) at 10 mg/ml, quick frozen in liquid N2, and stored at - 8 0 ° until use. Mitochondria (250/.tg/ml) are incubated with 35S-labeled precursor protein (1 to 10% reticulocyte lysate) in 100 to 1000/,1 of import buffer [500 mM sorbitol, 50 mM HEPES, pH 7.2, 80 mM KC1, 10 mM magnesium acetate, 2 mM potassium phosphate, 2 mM ATP, 2 mM N A D H , and fatty acid-free bovine serum albumin (BSA) (Sigma Chemical; 0.01 to 3%)] at 25 °. BSA is added to increase the import efficiency of different precursors, and the level required needs to be determined in each case. Import reactions are carried out in 1.5-ml microcentrifuge tubes, and reactions can be started by addition of either reticulocyte lysate or mitochondria with mixing. The incubation time (1 to 20 rain) depends on the kinetics for import of the precursor tested. After the import incubation, reaction mixtures are cooled on ice and mitochondria are reisolated by centrifugation at 12,000g for 10 rain in a refrigerated centrifuge 12j. Sambrook, E. F. Frilsch, and T. Maniatis, "Molecular Cloning. A Laboratory Manual." Cold Spring Harbor Laboratory Press, New York, 1989. ~3 H. R. B. Pelham and R. J. Jackson, Eur. J. Biochern. 67, 247 (1976). t4 G. Daum, S. Gasser, and G. Schatz, J. Biol. Chem. 257, 13,(/75 (1982).
[ 17]
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at 4 °. Protein translocation into mitochondria is typically monitored by determining the extent to which the higher molecular weight precursor (p-) form is converted to the lower molecular weight mature (m-) form. To demonstrate that the processed precursor protein has been translocated across the mitochondrial membranes, samples are treated with protease prior to reisolation of mitochondria. For the protease treatment, reaction mixtures are split into equal portions and the BSA concentration is adjusted to 0.5c}. Proteinase K (PK; 50/xg/ml) is added to one portion and incubated on ice for 30 min, while the other is mock treated. The protease treatment is quenched by addition of phenylmethylsulfonyl fluoride (PMSF, I mM) and an additional 5 min incubation on ice. Following this, mitochondria are reisolated and analyzed by SDS-polyacrylamide gel electrophoresis 15 ( S D S - P A G E ) and fluorography of dried gels that were treated with sodium salicylate. 16 Quantitation of autoradiographs is carried out by laser densitometry.17 Incubation at L o w Temperature to Accumulate Translocation Intermediates
One of the first protein translocation intermediates identified ~was generated by performing protein import reactions at low temperature. When import reactions were carried out at 8 °, the F~-ATPase/3 subunit precursor was only imported to a stage where its presequence was proteolytically processed, but the majority of the polypeptide remained outside of the mitochondria where it could be digested by protease. This result demonstrated that at low temperatures import intermediates accumulate in a conformation where they span both mitochondrial membranes. This interpretation led to the conclusion that protein translocation occurred at sites of close contact between the inner and outer mitochondrial membranes. To generate this type of import intermediate, 35S-labeled precursor protein is added to reaction mixtures containing isolated mitochondria preincubated at 8 ° and incubated for 15 rain. Precursor processing and translocation are then analyzed. Under these import conditions, precursors to cytochrome c~ and fusion proteins between dihydrofolate reductase ( D H F R ) and mitochondrial targeting signals, in addition to the F r A T P a s e /3 subunit, are observed to accumulate as processed preproteins that are not completely translocated into the matrix. To demonstrate the authenticity of such membrane spanning translocation intermediates, two controls are required. First, precursor protein should be incubated with deenergized r~ U. K. Laemmli, Nature 227, 680 (1970). t" J. P. Chamberlain, A n a l Biochem. 98, 132 (1979). L7D. M. Cyr and M. G. Douglas, ,/. Biol. Chem. 266, 21,700 (1991).
244
IMPORT OF PROTEINS AND R N A
INTO MITOCHONDRIA
[17]
mitochondria to exclude the possibility that presequence cleavage is being catalyzed by matrix-processing protease (MPP) that leaked from damaged mitochondria. Organelles treated with the proton ionophore CCCP (25/zM) are sufficient for this purpose. Second, it must be demonstrated that on raising the temperature from 8 to 25 ° in a second incubation, the translocation block is removed and the import intermediate is now efficiently translocated from the membrane into the matrix.
Reversible Arrest of Protein Import Generation of protein translocation intermediates using fusion proteins constructed with a passenger protein that can fold prior to import have been useful in the study of mitochondrial protein import. 3 5,7 A widely used passenger domain is murine DHFR. This protein is not normally targeted to mitochondria, but can be efficiently translocated into the matrix if it is fused to a mitochondrial targeting signal, l~ The D H F R domain on such constructs folds to the native state after synthesis. Its folded conformation is stabilized on binding of ligands such as the folate analog methotrexate. The stabilization of the folded state prevents the translocation of the D H F R domain across the outer mitochondrial membrane. 3,5 This observation led to the conclusion that proteins must assume an unfolded conformation in order to traverse the mitochondrial membranes. 3 In another study, different length amino-terminal regions of the cytochrome-b2 precursor were fused to D H F R , and translocation intermediates that spanned both mitochondrial membranes were generated by blocking their import with methotrexate. 5 Characterization of the ability of MPP to cleave the presequence from these different-length translocation intermediates demonstrated that it takes approximately 50 amino acids to span both membranes of mitochondria and that polypeptides are likely to assume an extended conformation during transit into the matrix. ~ To construct fusion proteins between murine D H F R and different amino-terminal regions of a mitochondrial precursor protein, standard cloning techniques are utilized. 5'~2A DNA fragment containing the open reading frame for murine D H F R 3 is subcloned into the polylinker of p G E M 4 ~ (Promega Biochemicals). PCR fragments encoding the presequence and different-length regions of adjacent mature portions of the precursor of choice are produced and fused in frame to the D H F R encoding DNA. 5 m R N A generated for such constructs is then translated in reticulocyte lysate as described earlier. To generate an arrested translocation intermediate that spans both ~s E. C. Hurt, B. Pesold-Hurt, and G. Schatz, F E B S Lett. 178, 306 (1984).
[17]
M I T O C H O N D R I A L PROTEIN IMPORT PATHWAY
245
mitochondrial membranes, translation lysates are added to import cocktails containing methotrexate at a concentration of 5 nM or 1 /zM and preincubated for 10 min on ice prior to addition of isolated mitochondria and shift to 25 °. The low concentration of methotrexate is used when further import of the precursor will be accomplished in a second reaction, whereas the high concentration is used to assure a complete block in translocation. In some cases, the region of mitochondrial preprotein fused to D H F R interferes with its folding after synthesis, especially if it is hydrophobic, and the fusion proteins aggregate in the translation lysate. This decreases the import efficiency of the precursor protein and prevents methotrexate from arresting its import. The folded state of the D H F R domain can be tested by determining if it is resistant to protease digestion. The folded D H F R domain on chimeric precursor proteins is resistant to digestion by PK (5 p,g/ml) when treated on ice for 30 rain, whereas unfolded D H F R is completely digested by the same treatment. 5 If incomplete folding of the D H F R domain is determined to be a problem, it can be overcome partly by lowering the temperature of the translation reaction to 25 ° and including methotrexate (1 /zM) cotranslationally to increase the folding efficiency of the precursor protein. If misfolding remains a problem, then the aggregates can be removed by spinning the reticulocyte lysates at 50,000g at 4 ° for 130 rain just prior to use. This centrifugation step reduces the quantity of ~SS-labeled precursor in the translation lysate by up to 80%, but the majority of preprotein remaining can usually bind methotrexate and have its import arrested. To demonstrate that the import-arrested preprotein found in ;association with mitochondria is an authentic translocation intermediate, its further import into mitochondria after removal of methotrexate can be accomplished. An example of such an experiment is demonstrated in Fig. 1. pSu9(1-86)-DHFR, which contains the first 86 amino acids of the FoATPase subunit 9 precursor 1~ fused to DHFR, was incubated in the presence and absence of methotrexate (5 nM). In the absence of ligand it was efficiently translocated into the matrix and processed twice to tile n7 form. In the presence of methotrexate, however, its import was arrested prior to insertion of the MPP cleavage site into the matrix and it accumulated with mitochondria in the precursor p form. When mitochondria that have accumulated the p form of Su9(1-86)-DHFR were isolated and washed to remove methotrexate and then incubated in a second import reaction, the p form was efficiently translocated into the matrix and converted to the m form. During the wash step, some of the p form of the import intermediate dissociated from the membrane (lanes 2 versus 3). Although this reduces the signal, this is typically not problematic because what remains bound is ~') A. V i e b r o c k , A. Perz, and W. Sebald, E M B O .I. 1, 565 (1982).
246
I M P O R T OF P R O T E I N S A N D R N A
1 [
Inc
1st -
Mtx
2 +
~NTO M I T O C H O N D R I A
[17]
3 4 [ 2nd [ +
-
i m
~
FIG. 1. Reversible arrest of protein translocation through stabilization of folded domains, pSu9(1-86)-DHFR (30,000 cpm/100/,d) was incubated with isolated yeast mitochondria (250 b~g/ml) at 25° for 5 min in standard import cocktail supplemented with 0.01% fatty acid-free BSA. The preprotein was preincubated in the reaction buffer for 10 rain on ice in the presence or absence of methotrexate (1 /xM). Import reactions were then started by addition of mitochondria. The sample analyzed in lane 1 was incubated in a 100-/,l reaction volume and then analyzed directly for imporl. For lanes 2 4, a single 400-/*L reaction was used for the initial import incubation (Inc). After the first import reaction, the reaction mixture was placed on ice and 3 aliquots of 100/xL were removed. One aliquot was analyzed immediately (lane 2). Mitochondria for the other reactions were reisolated by centrifugation and the supernalants were discarded. The mitochondrial pellets were rinsed with 1000/xl of standard import buffer (lane 4) or import buffer that was supplemented with methotrexate (1 /xM; lane 3). Samples were placed back in the centrifuge and spun at 10,000g to pellet the mitochondria, The wash buffer was then carefully removed and the mitochondria were resuspended in 100/*1 of import buffer with or without methotrexate and incubated for 20 rain at 25°. Import was analyzed by SDS-PAGE and fluorography. Precursor (p), intermediate (i), and mature (m) forms of Su9(I-86)-DHFR present in reaction mixtures.
t r a n s l o c a t e d i n t o t h e m a t r i x w i t h h i g h efficiency. F u r t h e r t r a n s l o c a t i o n in t h e s e c o n d i n c u b a t i o n o n l y o c c u r s if m e t h o t r e x a t e is r e m o v e d a n d is c o m p l e t e a f t e r a b o u t 20 rain of i n c u b a t i o n .
Cross-Linking
of Import Intermediates
to I m p o r t A p p a r a t u s
T h e first m e m b r a n e - b o u n d c o m p o n e n t of t h e i m p o r t a p p a r a t u s in y e a s t was identified by a cross-linking approach utilizing translocation intermediates. v O t h e r c o m p o n e n t s h a v e b e e n i d e n t i f i e d t h r o u g h t e s t i n g t h e e f f e c t of a n t i b o d i e s on t h e i m p o r t of p r e c u r s o r p r o t e i n s s'lu2°'21 a n d by g e n e t i c m e a n s . 02223 T h e a v a i l a b i l i t y of m o n o s p e c i f i c a n t i b o d i e s to d i f f e r e n t c o m p o nents of the import apparatus and cross-linking techniques have provided 2, p. E. Scherer, U. C. Manning-Krieg, P. Jen6. G. Schatz, and M. Horst, Proc. Natl. Acad. Sci. USA 89, 11,930 (1992). ~ T. S611ner, G. Griffiths, R. Pfaller, N. Planner, and W. Neupert, Cell S9, 1061 (1989). 22 A. C. Maarsc. J. Blom, L. A. Grivell, and M. Meyer, E M B O J. 11, 3619 (1992). 2;~j. L. T. Emtage and R. E. Jensen..1. Cell Biol. 122, 1(}03 (/993).
[ 17]
MI'IOCHONDRIALPROTEIN IMPORTPATHWAY
247
new powerful tools to analyze the stage of import where different translocation intermediates have become arrested. I° For instance, when import of Su9(1-86)-DHFR is blocked by methotrexate, the p form can be crosslinked with disuccinimidyl suberate (DSS: Pierce Chemicals) to several components of the import apparatus (Fig. 2). DSS is a homofunctional, m e m b r a n e - p e r m e a b l e , and amine-specific cross-linker. After performing import reactions to accumulate translocation intermediates, reaction mixtures are placed on ice and DSS (200/xM) is added. Cross-linking reactions are incubated for 30 min at 4 ° and then quenched by the addition of 10 m M Tris-HC1, p H 7.2, and further incubation of 4 ° for 30 min. Mitochondria are then reisolated and cross-linked adducts identified by determining
2
3
...... mt-Hsp70
~
- MIM44
........... ~ ' M I M | 7
MTX
+
-
+
CCCP
+
-
-
FJc;. 2. Cross-linking of protein translocation intermediates to components of the import apparatus, pSu9(1-86)-DHFR (5.0 x 10~ cpm in 10/xL of reticulocyte lysate) was incubated with isolated mitochondria (250/zg/ml) for 5 rain at 25 °. W h e r e indicated 25/xM CCCP and I /xM methotrexate were present in reaction mixtures. Mcthotrexate treatment of precursor was as described in the legend to Fig. 1. After the import reaction, samples were placed on ice and treated with DSS (200 tzM) as described in the text. The bands marked denote crosslinked adducts that were specitically immunoprecipitated with antisera against the indicated components: mtHsp70: mitochondrial Hsp70. MIM44: mitochondrial inner m e m b r a n e protein of 44 k D a in size and MIMI7: mitochondral inner m e m b r a n e protein of 17 kI)a. Shown is an overnight exposure of an X-ray lilm to the dried gel that was treated witb the ftuorophore sodium salicylate)"
248
IMPORT OF PROTEINSAND RNA ~NTOMITOCHONDRIA
[17]
the mobility shift of the 35S-labeled precursor protein on S D S - P A G E gels (Fig. 2). Note that mitochondria are not reisolated prior to addition of DSS because, at times, this treatment reduces the efficiency of the crosslinking reaction. Unbound precursor cross-linked to itself does not sediment with mitochondria and is removed from samples when mitochondria are pelleted. The identities of the proteins in the cross-linked adducts are determined by coimmunoprecipitation with antibodies against different components of the import apparatus by standard procedures. 24 Typically only 0.1 to 2% of the translocation intermediate is cross-linked to a particular component of the import apparatus. The efficiency of cross-linking depends on the proximity of the lysines in the translocation intermediate to those in the import component to which it is cross-linked. Due to the low efficiency of the cross-linking, high levels of 35S-labeled precursor (1 to 5.0 x 105 cpm) should be used in import reactions where cross-linked products will later be analyzed. When high levels of radioactive precursor are used. overnight exposures of X-ray films yield a detectable signal. Polyclonal sera is sufficient for the coimmunoprecipitation of cross-linked products. The sera should be titrated so that the quantity used is capable of quantitatively immunoprecipitating the protein of interest from the sample of mitochondria being analyzed. ~° We typically use 25/xl of a high titer sera per 50/xg of mitochondria analyzed. An example of such an analysis is shown in Fig. 2. These cross-linking data suggest that the amino terminus of the methotrexate-arrested form of pSu9(1-86)-DHFR was translocated to a position where it was in close proximity to the components of the inner membrane import apparatus MIM44 and MIM17 before its import was arrested by methotrexate (Fig. 2). pSu9(I-86)-DHFR could also be cross-linked to mitochondrial heatshock protein 70 (mtHsp70), demonstrating that this import intermediate was inserted through the inner membrane with its presequence exposed to the mitochondrial matrix. Control experiments should always be included to demonstrate that the cross-linked products detected are specific. For the methotrexate arrested form of pSu(1-86)-DHFR, this was accomplished by comparing cross-linked adducts formed when this precursor was incubated with deenergized mitochondria or in the absence of ligand (Fig. 2). Thus, tools are now available to pinpoint the step at which the import of different translocation intermediates has become arrested during import into mitochondria. 24E. Harlow and D. Lane, "Antibodies: A Laboratory Manual." Cold Spring Harbor Laboratory Press, New York (1988).
[17]
MITOCHONDRIAL PROTEIN IMPORT PATHWAY
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Assay for Sliding of Polypeptides in Import C h a n n e l s Analysis of p S u 9 - D H P R fusion proteins that extend different lengths of polypeptide into the mitochondrial matrix when their import is arrested by methotrexate has revealed that protein translocation across both mitochondrial membranes is a completely reversible process. 2s Import intermediates that exposed less than 10 amino acids in the matrix after the presequence was processed were observed to move in a retrograde direction and diffuse out of the import apparatus. Import intermediates that exposed more than 20 amino acids in the matrix after import arrest were firmly associated with mitochondria (mt). This resulted from the ATP--dependent binding of mtHsp70 to the length of polypeptide present in the matrix. Interference with the binding of mtHsp70 through depletion of matrix A T P caused the release of the translocation intermediate from mitochondria. This suggested that the import channels of mitochondria constitute a passive pore that only weakly interacts with precursor proteins when compared to mtHsp70. :5 These studies have provided the basis for an assay to study the sliding of polypeptides in the protein translocation channels of mitochondria and the factors that confer unidirectionally on the import process (Fig. 3). For this type of assay, pSu9(1-86)-DHFR was treated with methotrexate (1/xM) and incubated with mitochondria (250/xg/ml) in a 400-/xl reaction mixture that contained 0.01% BSA at 25 ° for 5 min. It is important to determine the time of incubation when import is complete and the majority of precursor is associated with mitochondria for this type of assay. After the initial incubation period, three 1004,1 aliquots of the reaction mixture are removed and placed in new tubes at 25 °. One tube is then placed on ice for later analysis. The other tubes are treated with either additional A T P (1 mM) and N A D H (2 mM) to maintain the energy status of the mitochondria or apyrase (40 units/ml) and oligomycin (20 /xM) to deplete the matrix of ATP and interfere with mtHsp70 function. 26 After further incubation for 5 min, reactions are stopped by placing the tubes on ice. Reaction mixtures are then split. A fraction of the reaction is treated with PK and the other receives no treatment. Mitochondria are then reisolated. The supernatant for the sample treated with PK is discarded. The supernatant from the nontreated sample is carefully removed so that the pellet is not disturbed, and transferred to another tube. Acetone is then added to a final concentration of 80% and samples are incubated on ice for 5 min to allow the precipitation of 35S-labeled precursor proteins present in the supernatant. 25 C. U n g c r m a n n , W. Neupert. and D. M. Cyr. Science, 266, 1250 (1994). "" D. M, Cyr, R. A. Stuart, and W. Neupert, .1. B i o t Chetn. 268, 23,751 (1993).
250
IMPORT OF PROTEINS AND R N A
1
2
I 1st
Ine
3 [
I x r o MITOCHONDRIA
4
I
2nd +
Mtx
-
+
+
ATP
+
+
+
~
~
Pellet
~
~
Pellet + Proteinase K
P i
~
P i m
~
i
[17]
Supernatant
FIG. 3. Import intermediates that accumulate with mitochondria arc translocated reversibly out of mitochondria if the matrix is depleted of ATP. Import conditions were as described in the legend to Fig. 1. When present, methotrexatc was 1 /xM. Experiments were carried out in a two-step sequence. In the first step, ~sS-labeled pSug(1-86)-DHFR fusion protein translated in reticulocyte lysate was incubated with mitochondria, as indicated below, for 5 min at 25 ° to allow import intermediates to accumulate (lanes 1 and 2). In lane 1. import was carried out in a 200-/xL reaction mixture and samples were analyzed as described in the text. For the other lanes, import reactions were carried out in a 40ll-p,L reaction volume. After the first incubation, the reaction mixture was placed on ice and 100-/zl aliquots were removed. One was analyzed immediately (lane 2), whereas the others were subjected to a second incubation. One reaction was mock treated (lane 3): to the other apyrase (40 units/ml) and oligomycin (20/xM) were added to deplete the matrix of ATP (lane 4). These reactions were then shifted back to 25 ° and incubated for an additional 10 min. Following this incubation, samples were analyzed as described above. Treatment of mitochondrial pellets with PK (50/,g/ml), and the analysis of the pellet and supernatants of reaction mixtures were as described in the text. Precursor (p), intermediate (i), and mature (m) forms of Su9(I-86)DHFR present in reaction mixtures.
M a t e r i a l i n s o l u b l e in 8 0 % a c e t o n e is t h e n s e d i m e n t e d b y c e n t r i f u g a t i o n a t 1 0 , 0 0 0 g f o r 10 m i n . T h e m i t o c h o n d r i a l p e l l e t s a n d a c e t o n e p r e c i p i t a t e s f r o m the supernatant fraction are then analyzed by SDS-PAGE. Analysis of the data from such an import experiment reveals that during t h e first i n c u b a t i o n p e r i o d m e t h o t r e x a t e - t r e a t e d p S u 9 ( l - 8 6 ) - D H F R is i m ported to a conformation where the tightly folded DHFR domain was
[ 1 7]
MITO(?HONDRIAL PROTE1N IMPORT PATHWAY
251
firmly apposed to the mitochondrial outer membrane and was resistant to digestion by PK.=: pSu9(1-86)-DHFR remains in this position during the second incubation period if mitochondria are fully energized (Fig. 3, lane 2 versus 3). However, when matrix A T P levels are reduced, pSug(1-86)D H F R moves in a retrograde direction out of the import channel and becomes sensitive to protease digestion (Fig. 3, lane 3 versus 4). In fact, about 50% of the precursor diffused all the way out of mitochondria and was found in the supernatant of reaction mixtures (Fig. 3, lane 3 versus 4). In the analysis of the supernatants of reaction mixtures for dissociation precursor proteins from mitochondria, it is important for the majority of the precursor to bind to the mitochondria during the initial reacl:ion. If this does not occur efficiently, a high background is observed. In these', instances, unbound precursor can be removed from the reactions by reisolating the mitochondria and resuspending them in new import buffer prior to the second incubation. In some preparations of mitochondria, MPP that has leaked from the matrix converts the p form released from milochondria to the ,n form (Fig. 3, lane 4). If this activity becomes problematic, E D T A in excess of the magnesium ion present in the import buffer can be added to inhibit soluble MPP. This treatment does not alter the activity of MPP in the matrix because E D T A cannot cross the membranes of mitochondria. Short-length translocation intermediates are optimal for use in the analysis of the sliding of polypeptides in mitochondrial membranes. Translocation intermediates that expose more than 60 amino acids in the matrix only slide backward on ATP depletion and become protease sensitive, but do not fall out of the import channel. This may result from interaction with additional chaperones or partial folding of the longer length precursor after its entrance into the matrix.
Concluding Remarks The future analysis of membrane-spanning translocation intermediates in studies on mitochondrial protein import should help identify functions of newly identified components of the import apparatus. It will be especially useful to accumulate translocation intermediates in mitochondria isolated from yeast strains harboring conditional mutations in specific import components and then ask if further import is defective when the mutant phenotype is induced. Critical information on the function of newly identified components of the import apparatus can also be obtained through determining where translocation intermediates accumulate in the import pathway when a specific component has been inactivated prior to the import incuba~7 E. Schwarz, T. Seyner, B. Guiard, and W. Neupcrt. E M B O Y. 12, 2295 (1993).
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IMPORT OF PROTEINS AND R N A INTO MITOCHONDRIA
[18]
tion. Both of these types of experiments will help delineate the stage of import at which the particular component is required. The analysis of translocation intermediates will also help determine the sequential series of interactions that occurs between precursor proteins and import components during protein import into the matrix. Determination of these reactions will provide an estimate of the affinity of different import components for the presequence and mature regions of precursor proteins. This information will provide a starting point for the reconstitution of protein translocation reactions with purified components. Acknowledgments D. M. C. is supported by a long-term fellowship from the Human Frontier Science Program Organization (HFSPO). C. U. is supported by a predoctoral fellowship from Boehringerlngelheim. W. N. is supported by grants from the Deutsche Forschungsgemeinschaft (SFB 184, Teilprojekt B12), the Fond der Chemischen Industrie and the HSFPO. The critical reading of the manuscript by Margaret Scully is greatly appreciated.
[18] P u r i f i e d a n d P r o t e i n - L o a d e d M i t o c h o n d r i a l O u t e r M e m b r a n e V e s i c l e s for F u n c t i o n a l A n a l y s i s of Preprotein Transport By ANDREAS MAYER, ARNOLD DRIESSEN, WALTER NEUPERT, a n d ROLAND LILL
Introduction The translocation of proteins into and across biological membranes is a complex, multistep process that requires the coordinated interplay of many integral membrane components and of soluble factors from both sides of the membrane (for a review see articles in Neupert and LilP). The functional analysis of the translocation process depends on the availability of defined biochemical in vitro systems. Many important mechanistic questions can only be answered conclusively, if purified biochemicals are utilized. This applies especially for protein translocation into and across the doublemembrane system of mitochondria. Detailed mechanistic dissection of the translocation processes across the individual membranes is often hampered by the fact that usually the preproteins pass simultaneously across the W. Neupert and R. Lill, "Membrane Biogenesis and Protein Targeting." Elsevier Sciencc Publishers, Amsterdam, 1992.
METHODS IN ENZYMOLOGY.VOI,. 260
Copyrighl~k~1~)95by AcademicPress,Inc. All rightsof reproduclionin any formrestored.
MITOCHONDRIAL PROTEIN IMPORT PATHWAY
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5. E l u t e the p r o t e i n s b o u n d to t h e b e a d s by e i t h e r i n c u b a t i n g the b e a d s in s o l u b i l i z a t i o n b u f f e r i n c l u d i n g 0.4 M i m i d a z o l e buffer, p H 7.4, or by a d d i n g 5 0 / , 1 2 x s a m p l e b u f f e r ~s a n d h e a t i n g for 5 min at 95 °. A n a l y z e the s a m p l e by S D S - P A G E .
[17] Analysis of Mitochondrial Protein Import Pathway in Saccharomyces cerevisiae with Translocation Intermediates By
DOUGLAS
M. CYR,
CHRISTIAN
UNGERMANN,
and
WALTER
NEUPERT
Introduction T h e c h a r a c t e r i z a t i o n of p r o t e i n t r a n s l o c a t i o n i n t e r m e d i a t e s has p r o v i d e d m a n y o f the s e m i n a l o b s e r v a t i o n s in the c h a r a c t e r i z a t i o n of the m i t o c h o n d r i a l p r o t e i n i m p o r t p a t h w a y . S t u d y o f i m p o r t i n t e r m e d i a t e s has h e l p e d define the e n e r g y r e q u i r e m e n t s for p r o t e i n i m p o r t , 1'2 the c o n f o r m a t i o n a l state of p r e c u r s o r p r o t e i n s d u r i n g t r a n s l o c a t i o n , 3 5 the c o m p o n e n t s of the i m p o r t m a c h i n e r y ,
METIIODS IN ENZYMOI OGY, VOI.. 260
CopFdght ~': 1995 by Academic Press, Inc All rights of reproduction in any foun reserved
242
IMPORTOF PROTEINSAND RNA INTOMITOCHONDRIA
[171
G e n e r a t i o n of T r a n s l o c a t i o n I n t e r m e d i a t e s t h a t S p a n M e m b r a n e s of Isolated Mitoehondria
In vitro Synthesis of Mitochondrial Precursor Proteins Expression plasmids for mitochondrial precursor proteins are generated by cloning the gene of interest into a plasmid such as p G E M 4 (Promega Biochemicals) in which it is put under the control of the SP6 promoter. Purified plasmid D N A is used for in vitro transcription with SP6 RNA polymerase in the presence of NTPs and 7 m G p p G at 37 ° for 1 hr. ~2m R N A produced in this fashion is precipitated with LiC1, resuspended in RNasefree H20 and stored at 80 °. To synthesize 35S-labeled protein, rabbit reticulocyte lysate 13 (Promega Biochemicals) is programmed with m R N A and incubated for 60 rain at 30 ° in the presence of [35S]methionine (specific activity 1000 Ci/mM). For optimal synthesis of different precursor proteins, the concentrations of mRNA, magnesium ion (1 to 3 raM) and potassium ion (100 to 200 raM) must be determined. Lysates containing translated proteins are aliquoted, quick frozen in liquid nitrogen, stored at - 8 0 ° and thawed just prior to use,
Protein Import into Isolated Mitochondria Mitochondria are isolated from Saccharomyces cerevisiae strain D27310B according to the published protocol, 14 suspended in SEM buffer (sucrose 250 raM, E D T A 1 raM, and MOPS, pH 7.2) at 10 mg/ml, quick frozen in liquid N2, and stored at - 8 0 ° until use. Mitochondria (250/.tg/ml) are incubated with 35S-labeled precursor protein (1 to 10% reticulocyte lysate) in 100 to 1000/,1 of import buffer [500 mM sorbitol, 50 mM HEPES, pH 7.2, 80 mM KC1, 10 mM magnesium acetate, 2 mM potassium phosphate, 2 mM ATP, 2 mM N A D H , and fatty acid-free bovine serum albumin (BSA) (Sigma Chemical; 0.01 to 3%)] at 25 °. BSA is added to increase the import efficiency of different precursors, and the level required needs to be determined in each case. Import reactions are carried out in 1.5-ml microcentrifuge tubes, and reactions can be started by addition of either reticulocyte lysate or mitochondria with mixing. The incubation time (1 to 20 rain) depends on the kinetics for import of the precursor tested. After the import incubation, reaction mixtures are cooled on ice and mitochondria are reisolated by centrifugation at 12,000g for 10 rain in a refrigerated centrifuge 12j. Sambrook, E. F. Frilsch, and T. Maniatis, "Molecular Cloning. A Laboratory Manual." Cold Spring Harbor Laboratory Press, New York, 1989. ~3 H. R. B. Pelham and R. J. Jackson, Eur. J. Biochern. 67, 247 (1976). t4 G. Daum, S. Gasser, and G. Schatz, J. Biol. Chem. 257, 13,(/75 (1982).
[ 17]
MrI'OCHONDRIAL PROTEIN IMPORT PATI IWAY
243
at 4 °. Protein translocation into mitochondria is typically monitored by determining the extent to which the higher molecular weight precursor (p-) form is converted to the lower molecular weight mature (m-) form. To demonstrate that the processed precursor protein has been translocated across the mitochondrial membranes, samples are treated with protease prior to reisolation of mitochondria. For the protease treatment, reaction mixtures are split into equal portions and the BSA concentration is adjusted to 0.5c}. Proteinase K (PK; 50/xg/ml) is added to one portion and incubated on ice for 30 min, while the other is mock treated. The protease treatment is quenched by addition of phenylmethylsulfonyl fluoride (PMSF, I mM) and an additional 5 min incubation on ice. Following this, mitochondria are reisolated and analyzed by SDS-polyacrylamide gel electrophoresis 15 ( S D S - P A G E ) and fluorography of dried gels that were treated with sodium salicylate. 16 Quantitation of autoradiographs is carried out by laser densitometry.17 Incubation at L o w Temperature to Accumulate Translocation Intermediates
One of the first protein translocation intermediates identified ~was generated by performing protein import reactions at low temperature. When import reactions were carried out at 8 °, the F~-ATPase/3 subunit precursor was only imported to a stage where its presequence was proteolytically processed, but the majority of the polypeptide remained outside of the mitochondria where it could be digested by protease. This result demonstrated that at low temperatures import intermediates accumulate in a conformation where they span both mitochondrial membranes. This interpretation led to the conclusion that protein translocation occurred at sites of close contact between the inner and outer mitochondrial membranes. To generate this type of import intermediate, 35S-labeled precursor protein is added to reaction mixtures containing isolated mitochondria preincubated at 8 ° and incubated for 15 rain. Precursor processing and translocation are then analyzed. Under these import conditions, precursors to cytochrome c~ and fusion proteins between dihydrofolate reductase ( D H F R ) and mitochondrial targeting signals, in addition to the F r A T P a s e /3 subunit, are observed to accumulate as processed preproteins that are not completely translocated into the matrix. To demonstrate the authenticity of such membrane spanning translocation intermediates, two controls are required. First, precursor protein should be incubated with deenergized r~ U. K. Laemmli, Nature 227, 680 (1970). t" J. P. Chamberlain, A n a l Biochem. 98, 132 (1979). L7D. M. Cyr and M. G. Douglas, ,/. Biol. Chem. 266, 21,700 (1991).
244
IMPORT OF PROTEINS AND R N A
INTO MITOCHONDRIA
[17]
mitochondria to exclude the possibility that presequence cleavage is being catalyzed by matrix-processing protease (MPP) that leaked from damaged mitochondria. Organelles treated with the proton ionophore CCCP (25/zM) are sufficient for this purpose. Second, it must be demonstrated that on raising the temperature from 8 to 25 ° in a second incubation, the translocation block is removed and the import intermediate is now efficiently translocated from the membrane into the matrix.
Reversible Arrest of Protein Import Generation of protein translocation intermediates using fusion proteins constructed with a passenger protein that can fold prior to import have been useful in the study of mitochondrial protein import. 3 5,7 A widely used passenger domain is murine DHFR. This protein is not normally targeted to mitochondria, but can be efficiently translocated into the matrix if it is fused to a mitochondrial targeting signal, l~ The D H F R domain on such constructs folds to the native state after synthesis. Its folded conformation is stabilized on binding of ligands such as the folate analog methotrexate. The stabilization of the folded state prevents the translocation of the D H F R domain across the outer mitochondrial membrane. 3,5 This observation led to the conclusion that proteins must assume an unfolded conformation in order to traverse the mitochondrial membranes. 3 In another study, different length amino-terminal regions of the cytochrome-b2 precursor were fused to D H F R , and translocation intermediates that spanned both mitochondrial membranes were generated by blocking their import with methotrexate. 5 Characterization of the ability of MPP to cleave the presequence from these different-length translocation intermediates demonstrated that it takes approximately 50 amino acids to span both membranes of mitochondria and that polypeptides are likely to assume an extended conformation during transit into the matrix. ~ To construct fusion proteins between murine D H F R and different amino-terminal regions of a mitochondrial precursor protein, standard cloning techniques are utilized. 5'~2A DNA fragment containing the open reading frame for murine D H F R 3 is subcloned into the polylinker of p G E M 4 ~ (Promega Biochemicals). PCR fragments encoding the presequence and different-length regions of adjacent mature portions of the precursor of choice are produced and fused in frame to the D H F R encoding DNA. 5 m R N A generated for such constructs is then translated in reticulocyte lysate as described earlier. To generate an arrested translocation intermediate that spans both ~s E. C. Hurt, B. Pesold-Hurt, and G. Schatz, F E B S Lett. 178, 306 (1984).
[17]
M I T O C H O N D R I A L PROTEIN IMPORT PATHWAY
245
mitochondrial membranes, translation lysates are added to import cocktails containing methotrexate at a concentration of 5 nM or 1 /zM and preincubated for 10 min on ice prior to addition of isolated mitochondria and shift to 25 °. The low concentration of methotrexate is used when further import of the precursor will be accomplished in a second reaction, whereas the high concentration is used to assure a complete block in translocation. In some cases, the region of mitochondrial preprotein fused to D H F R interferes with its folding after synthesis, especially if it is hydrophobic, and the fusion proteins aggregate in the translation lysate. This decreases the import efficiency of the precursor protein and prevents methotrexate from arresting its import. The folded state of the D H F R domain can be tested by determining if it is resistant to protease digestion. The folded D H F R domain on chimeric precursor proteins is resistant to digestion by PK (5 p,g/ml) when treated on ice for 30 rain, whereas unfolded D H F R is completely digested by the same treatment. 5 If incomplete folding of the D H F R domain is determined to be a problem, it can be overcome partly by lowering the temperature of the translation reaction to 25 ° and including methotrexate (1 /zM) cotranslationally to increase the folding efficiency of the precursor protein. If misfolding remains a problem, then the aggregates can be removed by spinning the reticulocyte lysates at 50,000g at 4 ° for 130 rain just prior to use. This centrifugation step reduces the quantity of ~SS-labeled precursor in the translation lysate by up to 80%, but the majority of preprotein remaining can usually bind methotrexate and have its import arrested. To demonstrate that the import-arrested preprotein found in ;association with mitochondria is an authentic translocation intermediate, its further import into mitochondria after removal of methotrexate can be accomplished. An example of such an experiment is demonstrated in Fig. 1. pSu9(1-86)-DHFR, which contains the first 86 amino acids of the FoATPase subunit 9 precursor 1~ fused to DHFR, was incubated in the presence and absence of methotrexate (5 nM). In the absence of ligand it was efficiently translocated into the matrix and processed twice to tile n7 form. In the presence of methotrexate, however, its import was arrested prior to insertion of the MPP cleavage site into the matrix and it accumulated with mitochondria in the precursor p form. When mitochondria that have accumulated the p form of Su9(1-86)-DHFR were isolated and washed to remove methotrexate and then incubated in a second import reaction, the p form was efficiently translocated into the matrix and converted to the m form. During the wash step, some of the p form of the import intermediate dissociated from the membrane (lanes 2 versus 3). Although this reduces the signal, this is typically not problematic because what remains bound is ~') A. V i e b r o c k , A. Perz, and W. Sebald, E M B O .I. 1, 565 (1982).
246
I M P O R T OF P R O T E I N S A N D R N A
1 [
Inc
1st -
Mtx
2 +
~NTO M I T O C H O N D R I A
[17]
3 4 [ 2nd [ +
-
i m
~
FIG. 1. Reversible arrest of protein translocation through stabilization of folded domains, pSu9(1-86)-DHFR (30,000 cpm/100/,d) was incubated with isolated yeast mitochondria (250 b~g/ml) at 25° for 5 min in standard import cocktail supplemented with 0.01% fatty acid-free BSA. The preprotein was preincubated in the reaction buffer for 10 rain on ice in the presence or absence of methotrexate (1 /xM). Import reactions were then started by addition of mitochondria. The sample analyzed in lane 1 was incubated in a 100-/,l reaction volume and then analyzed directly for imporl. For lanes 2 4, a single 400-/*L reaction was used for the initial import incubation (Inc). After the first import reaction, the reaction mixture was placed on ice and 3 aliquots of 100/xL were removed. One aliquot was analyzed immediately (lane 2). Mitochondria for the other reactions were reisolated by centrifugation and the supernalants were discarded. The mitochondrial pellets were rinsed with 1000/xl of standard import buffer (lane 4) or import buffer that was supplemented with methotrexate (1 /xM; lane 3). Samples were placed back in the centrifuge and spun at 10,000g to pellet the mitochondria, The wash buffer was then carefully removed and the mitochondria were resuspended in 100/*1 of import buffer with or without methotrexate and incubated for 20 rain at 25°. Import was analyzed by SDS-PAGE and fluorography. Precursor (p), intermediate (i), and mature (m) forms of Su9(I-86)-DHFR present in reaction mixtures.
t r a n s l o c a t e d i n t o t h e m a t r i x w i t h h i g h efficiency. F u r t h e r t r a n s l o c a t i o n in t h e s e c o n d i n c u b a t i o n o n l y o c c u r s if m e t h o t r e x a t e is r e m o v e d a n d is c o m p l e t e a f t e r a b o u t 20 rain of i n c u b a t i o n .
Cross-Linking
of Import Intermediates
to I m p o r t A p p a r a t u s
T h e first m e m b r a n e - b o u n d c o m p o n e n t of t h e i m p o r t a p p a r a t u s in y e a s t was identified by a cross-linking approach utilizing translocation intermediates. v O t h e r c o m p o n e n t s h a v e b e e n i d e n t i f i e d t h r o u g h t e s t i n g t h e e f f e c t of a n t i b o d i e s on t h e i m p o r t of p r e c u r s o r p r o t e i n s s'lu2°'21 a n d by g e n e t i c m e a n s . 02223 T h e a v a i l a b i l i t y of m o n o s p e c i f i c a n t i b o d i e s to d i f f e r e n t c o m p o nents of the import apparatus and cross-linking techniques have provided 2, p. E. Scherer, U. C. Manning-Krieg, P. Jen6. G. Schatz, and M. Horst, Proc. Natl. Acad. Sci. USA 89, 11,930 (1992). ~ T. S611ner, G. Griffiths, R. Pfaller, N. Planner, and W. Neupert, Cell S9, 1061 (1989). 22 A. C. Maarsc. J. Blom, L. A. Grivell, and M. Meyer, E M B O J. 11, 3619 (1992). 2;~j. L. T. Emtage and R. E. Jensen..1. Cell Biol. 122, 1(}03 (/993).
[ 17]
MI'IOCHONDRIALPROTEIN IMPORTPATHWAY
247
new powerful tools to analyze the stage of import where different translocation intermediates have become arrested. I° For instance, when import of Su9(1-86)-DHFR is blocked by methotrexate, the p form can be crosslinked with disuccinimidyl suberate (DSS: Pierce Chemicals) to several components of the import apparatus (Fig. 2). DSS is a homofunctional, m e m b r a n e - p e r m e a b l e , and amine-specific cross-linker. After performing import reactions to accumulate translocation intermediates, reaction mixtures are placed on ice and DSS (200/xM) is added. Cross-linking reactions are incubated for 30 min at 4 ° and then quenched by the addition of 10 m M Tris-HC1, p H 7.2, and further incubation of 4 ° for 30 min. Mitochondria are then reisolated and cross-linked adducts identified by determining
2
3
...... mt-Hsp70
~
- MIM44
........... ~ ' M I M | 7
MTX
+
-
+
CCCP
+
-
-
FJc;. 2. Cross-linking of protein translocation intermediates to components of the import apparatus, pSu9(1-86)-DHFR (5.0 x 10~ cpm in 10/xL of reticulocyte lysate) was incubated with isolated mitochondria (250/zg/ml) for 5 rain at 25 °. W h e r e indicated 25/xM CCCP and I /xM methotrexate were present in reaction mixtures. Mcthotrexate treatment of precursor was as described in the legend to Fig. 1. After the import reaction, samples were placed on ice and treated with DSS (200 tzM) as described in the text. The bands marked denote crosslinked adducts that were specitically immunoprecipitated with antisera against the indicated components: mtHsp70: mitochondrial Hsp70. MIM44: mitochondrial inner m e m b r a n e protein of 44 k D a in size and MIMI7: mitochondral inner m e m b r a n e protein of 17 kI)a. Shown is an overnight exposure of an X-ray lilm to the dried gel that was treated witb the ftuorophore sodium salicylate)"
248
IMPORT OF PROTEINSAND RNA ~NTOMITOCHONDRIA
[17]
the mobility shift of the 35S-labeled precursor protein on S D S - P A G E gels (Fig. 2). Note that mitochondria are not reisolated prior to addition of DSS because, at times, this treatment reduces the efficiency of the crosslinking reaction. Unbound precursor cross-linked to itself does not sediment with mitochondria and is removed from samples when mitochondria are pelleted. The identities of the proteins in the cross-linked adducts are determined by coimmunoprecipitation with antibodies against different components of the import apparatus by standard procedures. 24 Typically only 0.1 to 2% of the translocation intermediate is cross-linked to a particular component of the import apparatus. The efficiency of cross-linking depends on the proximity of the lysines in the translocation intermediate to those in the import component to which it is cross-linked. Due to the low efficiency of the cross-linking, high levels of 35S-labeled precursor (1 to 5.0 x 105 cpm) should be used in import reactions where cross-linked products will later be analyzed. When high levels of radioactive precursor are used. overnight exposures of X-ray films yield a detectable signal. Polyclonal sera is sufficient for the coimmunoprecipitation of cross-linked products. The sera should be titrated so that the quantity used is capable of quantitatively immunoprecipitating the protein of interest from the sample of mitochondria being analyzed. ~° We typically use 25/xl of a high titer sera per 50/xg of mitochondria analyzed. An example of such an analysis is shown in Fig. 2. These cross-linking data suggest that the amino terminus of the methotrexate-arrested form of pSu9(1-86)-DHFR was translocated to a position where it was in close proximity to the components of the inner membrane import apparatus MIM44 and MIM17 before its import was arrested by methotrexate (Fig. 2). pSu9(I-86)-DHFR could also be cross-linked to mitochondrial heatshock protein 70 (mtHsp70), demonstrating that this import intermediate was inserted through the inner membrane with its presequence exposed to the mitochondrial matrix. Control experiments should always be included to demonstrate that the cross-linked products detected are specific. For the methotrexate arrested form of pSu(1-86)-DHFR, this was accomplished by comparing cross-linked adducts formed when this precursor was incubated with deenergized mitochondria or in the absence of ligand (Fig. 2). Thus, tools are now available to pinpoint the step at which the import of different translocation intermediates has become arrested during import into mitochondria. 24E. Harlow and D. Lane, "Antibodies: A Laboratory Manual." Cold Spring Harbor Laboratory Press, New York (1988).
[17]
MITOCHONDRIAL PROTEIN IMPORT PATHWAY
249
Assay for Sliding of Polypeptides in Import C h a n n e l s Analysis of p S u 9 - D H P R fusion proteins that extend different lengths of polypeptide into the mitochondrial matrix when their import is arrested by methotrexate has revealed that protein translocation across both mitochondrial membranes is a completely reversible process. 2s Import intermediates that exposed less than 10 amino acids in the matrix after the presequence was processed were observed to move in a retrograde direction and diffuse out of the import apparatus. Import intermediates that exposed more than 20 amino acids in the matrix after import arrest were firmly associated with mitochondria (mt). This resulted from the ATP--dependent binding of mtHsp70 to the length of polypeptide present in the matrix. Interference with the binding of mtHsp70 through depletion of matrix A T P caused the release of the translocation intermediate from mitochondria. This suggested that the import channels of mitochondria constitute a passive pore that only weakly interacts with precursor proteins when compared to mtHsp70. :5 These studies have provided the basis for an assay to study the sliding of polypeptides in the protein translocation channels of mitochondria and the factors that confer unidirectionally on the import process (Fig. 3). For this type of assay, pSu9(1-86)-DHFR was treated with methotrexate (1/xM) and incubated with mitochondria (250/xg/ml) in a 400-/xl reaction mixture that contained 0.01% BSA at 25 ° for 5 min. It is important to determine the time of incubation when import is complete and the majority of precursor is associated with mitochondria for this type of assay. After the initial incubation period, three 1004,1 aliquots of the reaction mixture are removed and placed in new tubes at 25 °. One tube is then placed on ice for later analysis. The other tubes are treated with either additional A T P (1 mM) and N A D H (2 mM) to maintain the energy status of the mitochondria or apyrase (40 units/ml) and oligomycin (20 /xM) to deplete the matrix of ATP and interfere with mtHsp70 function. 26 After further incubation for 5 min, reactions are stopped by placing the tubes on ice. Reaction mixtures are then split. A fraction of the reaction is treated with PK and the other receives no treatment. Mitochondria are then reisolated. The supernatant for the sample treated with PK is discarded. The supernatant from the nontreated sample is carefully removed so that the pellet is not disturbed, and transferred to another tube. Acetone is then added to a final concentration of 80% and samples are incubated on ice for 5 min to allow the precipitation of 35S-labeled precursor proteins present in the supernatant. 25 C. U n g c r m a n n , W. Neupert. and D. M. Cyr. Science, 266, 1250 (1994). "" D. M, Cyr, R. A. Stuart, and W. Neupert, .1. B i o t Chetn. 268, 23,751 (1993).
250
IMPORT OF PROTEINS AND R N A
1
2
I 1st
Ine
3 [
I x r o MITOCHONDRIA
4
I
2nd +
Mtx
-
+
+
ATP
+
+
+
~
~
Pellet
~
~
Pellet + Proteinase K
P i
~
P i m
~
i
[17]
Supernatant
FIG. 3. Import intermediates that accumulate with mitochondria arc translocated reversibly out of mitochondria if the matrix is depleted of ATP. Import conditions were as described in the legend to Fig. 1. When present, methotrexatc was 1 /xM. Experiments were carried out in a two-step sequence. In the first step, ~sS-labeled pSug(1-86)-DHFR fusion protein translated in reticulocyte lysate was incubated with mitochondria, as indicated below, for 5 min at 25 ° to allow import intermediates to accumulate (lanes 1 and 2). In lane 1. import was carried out in a 200-/xL reaction mixture and samples were analyzed as described in the text. For the other lanes, import reactions were carried out in a 40ll-p,L reaction volume. After the first incubation, the reaction mixture was placed on ice and 100-/zl aliquots were removed. One was analyzed immediately (lane 2), whereas the others were subjected to a second incubation. One reaction was mock treated (lane 3): to the other apyrase (40 units/ml) and oligomycin (20/xM) were added to deplete the matrix of ATP (lane 4). These reactions were then shifted back to 25 ° and incubated for an additional 10 min. Following this incubation, samples were analyzed as described above. Treatment of mitochondrial pellets with PK (50/,g/ml), and the analysis of the pellet and supernatants of reaction mixtures were as described in the text. Precursor (p), intermediate (i), and mature (m) forms of Su9(I-86)DHFR present in reaction mixtures.
M a t e r i a l i n s o l u b l e in 8 0 % a c e t o n e is t h e n s e d i m e n t e d b y c e n t r i f u g a t i o n a t 1 0 , 0 0 0 g f o r 10 m i n . T h e m i t o c h o n d r i a l p e l l e t s a n d a c e t o n e p r e c i p i t a t e s f r o m the supernatant fraction are then analyzed by SDS-PAGE. Analysis of the data from such an import experiment reveals that during t h e first i n c u b a t i o n p e r i o d m e t h o t r e x a t e - t r e a t e d p S u 9 ( l - 8 6 ) - D H F R is i m ported to a conformation where the tightly folded DHFR domain was
[ 1 7]
MITO(?HONDRIAL PROTE1N IMPORT PATHWAY
251
firmly apposed to the mitochondrial outer membrane and was resistant to digestion by PK.=: pSu9(1-86)-DHFR remains in this position during the second incubation period if mitochondria are fully energized (Fig. 3, lane 2 versus 3). However, when matrix A T P levels are reduced, pSug(1-86)D H F R moves in a retrograde direction out of the import channel and becomes sensitive to protease digestion (Fig. 3, lane 3 versus 4). In fact, about 50% of the precursor diffused all the way out of mitochondria and was found in the supernatant of reaction mixtures (Fig. 3, lane 3 versus 4). In the analysis of the supernatants of reaction mixtures for dissociation precursor proteins from mitochondria, it is important for the majority of the precursor to bind to the mitochondria during the initial reacl:ion. If this does not occur efficiently, a high background is observed. In these', instances, unbound precursor can be removed from the reactions by reisolating the mitochondria and resuspending them in new import buffer prior to the second incubation. In some preparations of mitochondria, MPP that has leaked from the matrix converts the p form released from milochondria to the ,n form (Fig. 3, lane 4). If this activity becomes problematic, E D T A in excess of the magnesium ion present in the import buffer can be added to inhibit soluble MPP. This treatment does not alter the activity of MPP in the matrix because E D T A cannot cross the membranes of mitochondria. Short-length translocation intermediates are optimal for use in the analysis of the sliding of polypeptides in mitochondrial membranes. Translocation intermediates that expose more than 60 amino acids in the matrix only slide backward on ATP depletion and become protease sensitive, but do not fall out of the import channel. This may result from interaction with additional chaperones or partial folding of the longer length precursor after its entrance into the matrix.
Concluding Remarks The future analysis of membrane-spanning translocation intermediates in studies on mitochondrial protein import should help identify functions of newly identified components of the import apparatus. It will be especially useful to accumulate translocation intermediates in mitochondria isolated from yeast strains harboring conditional mutations in specific import components and then ask if further import is defective when the mutant phenotype is induced. Critical information on the function of newly identified components of the import apparatus can also be obtained through determining where translocation intermediates accumulate in the import pathway when a specific component has been inactivated prior to the import incuba~7 E. Schwarz, T. Seyner, B. Guiard, and W. Neupcrt. E M B O Y. 12, 2295 (1993).
252
IMPORT OF PROTEINS AND R N A INTO MITOCHONDRIA
[18]
tion. Both of these types of experiments will help delineate the stage of import at which the particular component is required. The analysis of translocation intermediates will also help determine the sequential series of interactions that occurs between precursor proteins and import components during protein import into the matrix. Determination of these reactions will provide an estimate of the affinity of different import components for the presequence and mature regions of precursor proteins. This information will provide a starting point for the reconstitution of protein translocation reactions with purified components. Acknowledgments D. M. C. is supported by a long-term fellowship from the Human Frontier Science Program Organization (HFSPO). C. U. is supported by a predoctoral fellowship from Boehringerlngelheim. W. N. is supported by grants from the Deutsche Forschungsgemeinschaft (SFB 184, Teilprojekt B12), the Fond der Chemischen Industrie and the HSFPO. The critical reading of the manuscript by Margaret Scully is greatly appreciated.
[18] P u r i f i e d a n d P r o t e i n - L o a d e d M i t o c h o n d r i a l O u t e r M e m b r a n e V e s i c l e s for F u n c t i o n a l A n a l y s i s of Preprotein Transport By ANDREAS MAYER, ARNOLD DRIESSEN, WALTER NEUPERT, a n d ROLAND LILL
Introduction The translocation of proteins into and across biological membranes is a complex, multistep process that requires the coordinated interplay of many integral membrane components and of soluble factors from both sides of the membrane (for a review see articles in Neupert and LilP). The functional analysis of the translocation process depends on the availability of defined biochemical in vitro systems. Many important mechanistic questions can only be answered conclusively, if purified biochemicals are utilized. This applies especially for protein translocation into and across the doublemembrane system of mitochondria. Detailed mechanistic dissection of the translocation processes across the individual membranes is often hampered by the fact that usually the preproteins pass simultaneously across the W. Neupert and R. Lill, "Membrane Biogenesis and Protein Targeting." Elsevier Sciencc Publishers, Amsterdam, 1992.
METHODS IN ENZYMOLOGY.VOI,. 260
Copyrighl~k~1~)95by AcademicPress,Inc. All rightsof reproduclionin any formrestored.