Vol. 180, No. 1, 1991 October 15, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 450-454
PURIFICATION OF 52 kDa PROTEIN: A PUTATIVE COMPONENT OF THE IMPORT MACHINERY FOR THE MITOCHONDRIAL PROTEIN-PRECURSOR IN RAT LIVER Hideyu Ono and Syozo Tuboi Department of Biochemistry, Yamagata University School of Medicine, Yamagata 990-23, JAPAN
Received September 6, 1991 Summary: A protein having a molecular mass of 52 kDa was purified to homogeneity from solubilized mitochondrial membrane proteins by affinity column chromatography using the s y n t h e t i c presequence of ornithine a m i n o t r a n s f e r a s e (OAT) as t h e ligand. This 52 kDa protein was s p e c i f i c a l l y bound to the a f f i n i t y column and e l u t e d with 1 mM OAT-presequence, indicating t h a t it recognized the presequence and bound to it specifically. Anti-52 kDa protein Feb fragments s p e c i f i c a l l y inhibited the import of OAT-precursor into mitochondria, showing that the 52 kDa protein plays an essential role in this process. These results suggest t h a t 52 k D a protein is a component of the import machinery of the mitochondrial p r o t e i n - p r e c u r s o r in the mitochondrial membrane, o 1991 ~cademic Press, lrnc.
The mitochondrial p r o t e i n - p r e c u r s o r s synthesized on cytoplasmic polysomes a r e t r a n s p o r t e d into mitochondria p o s t - t r a n s l a t i o n a l l y (1, 2}.
It has been shown
t h a t ATP and an inner membrane p o t e n t i a l (A~} a r e required as energy for this process (2).
Furthermore, t h e r e a r e also s e v e r a l reports on f a c t o r s t h a t a r e
e s s e n t i a l in the course of t r a n s l o c a t i o n of t h e mitochondrial proteins, and some of t h e s e f a c t o r s have been purified from the cytosol and the mitochondrial membrane (3-13). In the previous papers, we r e p o r t e d the purifications of a 28 kDa protein from rabbit r e t i c u l o c y t e l y s a t e (5) and a 29 kDa protein from r a t liver mitochondrial membranes (13} and showed t h a t these two proteins play essential roles in the t r a n s p o r t of mitochondrial proteins.
We purified t h e s e two proteins
on an a f f i n i t y column prepared with a chemically synthesized p e p t i d e composed of 54 amino acids containing the presequence of ornithine a m i n o t r a n s f e r a s e (OAT} a t its NH2-terminal as a ligand (5, 13).
Furthermore, we r e p o r t e d t h a t a 52 kDa
protein was co-purified with the 29 kDa protein from crude solubilized mitochondrial membrane proteins, although we did not d e t e r m i n e w h e t h e r this 52 kDa protein has any role in the import of mitochondrial proteins (13). In the p r e s e n t paper, we r e p o r t t h a t t h e 52 kDa protein was s p e c i f i c a l l y e l u t e d with the s y n t h e t i c OAT-presequence (composed of 34 amino acids) from an a f f i n i t y column and that anti-52 kDa protein antibody s p e c i f i c a l l y inhibited the 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
450
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
import of the OAT-precursor into mitochondria.
These findings i n d i c a t e that the
52 kDa protein is also an essential component of the import machinery of mitochondrial protein in the membrane.
MATERIALS AND METHODS SYNTHESIS OF PEPTIDES AND PREPARATION OF PEPTIDE-CONJUGATED AGAROSE: The two peptides used in this study, a 54 amino acids p e p t i d e (54-AA
peptide) and the OAT-presequence, were chemically synthesized by Applied Biesystems Japan Co.(Tokyo, Japan). The amino acid sequence of the 54-AA p e p t i d e is the same as t h a t from Met I to His 53 of the OAT-precursor r e p o r t e d by Mueckler and Pitot (14), with an additional C-terminal Cys 54 to f a c i l i t a t e chemical crosslinking, as described in our previous paper (13). The 52 kDa protein was purified from solubilized mitochondrial membrane p,'oteins with an a f f i n i t y column of t~-aminobt,tyl-agarose conjugated with 54-AA peptide by using m-maleimidobenzoyl-N-hydroxysuccinimide e s t e r (13). OTHER METHODS: Precursors of mitochondrial proteins were synthesized in a c e l l - f r e e translation system using t o t a l rat liver RNA and a n u c l e a s e - t r e a t e d r e t i c u l o c y t e l y s a t e as described previously (15). A f t e r the t r a n s l a t i o n r e a c t i o n , the r e a c t i o n mixture was c e n t r i f u g e d for 30 min at 320,000 xg to remove ribosomes and the s u p e r n a t a n t was used as a source of mitochondrial p r o t e i n precursors for import into mitochondria (15). S D S - p o l y a c r y l a m i d e slab gel e l e c t r o p h o r e s i s was performed by the method of Laemmli (16). Fab f r a g m e n t s w e r e p r e p a r e d from IgG by the method of Mage (17). Ornithine a m i n o t r a n s f e r a s e and porin were purified as described previously (18, 19) and antibodies against t h e s e enzymes hnd the 52 kDa protein were p r e p a r e d by immunizing r a b b i t s with t h e s e purified proteins as d e s c r i b e d previously (20). MATERIALS: [a SS ] Methionine C>800 Ci/mmol) was obtained from American Radiolabeled Chemicals, Inc., USA. ATP-agarose (A 9264) and t~-aminobutyla g a r o s e (A 6142) were purchased from Sigma Chemical Co.. Nikkol ( o c t a e t h y l e n e g l y c o l mono-n-dodecyl e t h e r ) was from Nikko Chemicals (Tokyo, JaPan) , and the silver staining kit was from Kanto Chemical Co. (Tokyo, Japan).
RESULTS AND D I S C U S S I O N PURIFICATION OF 52 kDa PROTEIN:
Crude o u t e r mitochondrial membranes and
a solubilized protein f r a c t i o n of mitochondrial membranes were p r e p a r e d as d e s c r i b e d previously (13).
The solubilized proteins (2,210 mg) were applied to an
A T P - a g a r o s e column and the unadsorbed fraction was applied to a 54-AA peptide c o n j u g a t e d agarose column as d e s c r i b e d previously (13).
The column was washed
with b u f f e r A (10 mM Hepes-KOH, 2 mM magnesium a c e t a t e , 2 mM GSH, 1 jag/ml leupeptin, 1 ~g/ml pepstatin, 1096 glycerol) (pH 7.6) containing 800 mM potassium a c e t a t e and 1~ Triton X-100, and then with buffer A c o n t a i n i n g 200 mM potassium a c e t a t e and 196 Nikkol.
The proteins bound to the column w e r e e l u t e d
with b u f f e r A containing 200 mM potassium a c e t a t e , 1~ Nikkol, and 1 mM OATpresequence.
Elution of the a f f i n i t y column with OAT-presequence yielded only
t h e 52 kDa protein (about 0.8 rag), indicating t h a t this protein r e c o g n i z e d at~d s p e c i f i c a l l y bound t o t h e
OAT-presequence (Fig. 1, lane 2).
The 29 kDa p r o t e i n
was eluted t o g e t h e r with 52 kDa protein with c h a o t r o p i c ion (Fig. 1, lane 1) (13), but it was not e l u t e d with the s y n t h e t i c OAT-presequence, suggesting t h a t 451
Vol. 180, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1
2
52 kDa
29kDa
,-
EP. PIG. 1. Electrophoretogram of the purified 52 kDa protein. A solubilized protein fraction prepared from mitochondrial outer membranes was partially purified on ATP-agarose. The unadsorbed fraction (2,000 rag) was applied to a 54-AA peptide conjugated agarose column (bed volume 3.3 ml), and adsorbed proteins were eluted with chaotropic ion (3 M MgCiz, lane 1) or 1 mM OAT-presequence (lane 2) as described in the text. Electrophoresis was performed on a 11~ polyacrylamide slab gel in the presence of 0 . 2 t SDS. Proteins were d e t e c t e d by silver staining. E.P., OAT presequence.
this 29 kDa protein recognizes both the OAT-presequence and a p a r t of the NH 2terminal amino acid sequence of the mature OAT contained in the ligand or t h a t it has a g r e a t e r a f f i n i t y to the presequence. These results strongly suggest t h a t the 52 kDa protein is p a r t of the import machinery in the mitochondrial membrane t o g e t h e r with the 29 kDa protein and o t h e r proteins t h a t have not y e t been identified.
To confirm this possibility, we
p r e p a r e d anti-52 kDa protein antibody by immunizing a rabbit with the purified 52 kDa protein, as described previously (19). EFFECT OF ANTI-52 kDa PROTEIN ANTIBODY ON THE IMPORT O F OATPRECURSOR INTO MITOCHONDRIA:
T r e a t m e n t of mitochondria with anti-52 kDa
protein Fab f r a g m e n t s strongly inhibited the import of the OAT-precursor (Fig. 2A).
This inhibition seemed to be caused by specific i n t e r a c t i o n of the
antigen with its antil~ody, because control Fab fragments prepared from nonimmune rabbit and anti-porin Fab fragments did not inhibit this i m p o r t - r e a c t i o n (Fig. 2A and B).
The p r e s e n t results suggest t h a t the 52 kDa p r o t e i n is an
e s s e n t i a l component of t h e import machinery for t h e mitochondrial p r o t e i n p r e c u r s o r in the mitochondrial o u t e r membrane. We have r e p o r t e d (5) t h a t a 28 kDa protein, a component of t h e t a r g e t i n g f a c t o r required for t h e t r a n s p o r t of mitochondrial p r o t e i n - p r e c u r s o r s to t h e mitochondrial surface, could be purified to homogeniety using t h e same a f f i n i t y 452
Vol. 180, No. 1, 1991
A lO0
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
o
B 100
~. 75
75
50.
~
~
25
i
25 0
i
o Concentrationof Fab (mg/rnl)
Concentrationof Fab (mg/ml)
FIG. 2. Effect of anti-52 kDa protein Fab fragments and anti-porin Fab fragments on the import of OAT-precursor. A) Effect of anti-52 kDa protein Fab fragments. Mitochondria (final concentration, 0.25 mg/ml) were incubated with anti-52 kDa protein Fab fragments (e) or control Fab fragments prepared from a non-immune rabbit (o) at the concentrations indicated in the Figure for 15 rain at 25°C and then for 90 rain at O°C. Then, each mitocbondrial fraction(100ul, 25 pg) was incubated with 100 pl of ceU-free translation products for 15 mir~at 30°C. The mitochondria were then reisolated and their imported OAT was analyzed (15). Bands of OAT on the fluorogram were quantified by densitometry. B) Effect of anti-porin Fab fragments. Experimental and analytical conditions were as described above, e, treated with anti-porin Fab fragments; o, treated with control Fab fragments.
column as t h a t used for purification of the 52 kDa protein, and that anti-28 kDa protein antibody inhibited the binding of the precursor to the mitochondria at 0°C as well as its import.
On the contrary, we found that anti-52 kDa protein
antibody had no e f f e c t on the binding of the OAT-precursor to the mitochondria at 0°C : on incubation with mitochondria at 0°C for 20 rain, the precursor bound to the mitochondria, as shown in Fig. 3, lane l, and this binding was not e f f e c t e d by p r e - t r e a t m e n t of the mitochondria with anti-52 kDa protein antibody (lanes 8
2
3
4
5
6
7
8
9
pOAT =--1
FIG. 3. Effects of anti-52 kDa protein Fab fragments and anti-porin Fab fragments on the binding of pOAT to mitochondria. Mitochondria (final concentration, 0.25 mg/ml) were treated with each antibody as described in the legend for Fig. 2. Then, the samples were incubated with 100 jal of cell-free translation products for 20 rain at 0°C. The mitochondria were then reisolated and OAT-precursor recovered from them was assayed by fluorography. Lane 1, untreated; lanes 2-4, treated with control Fab fragments at concentrations of 0.41 mg/ml, 0.83 mg/ml, and 1.7 mg/ml, respectively; lanes 5-7, treated with anti-porin Fab fragments at concentrations of 0.41 mg/ml, 0.83 mg/ml, and 1.7 mg/ml, respectively; lanes 8 and 9, treated with anti-52 kDa protein Fab fragments at concentrations of 0.83 mg/ml and 1.7 mg/ml, respectively. 453
VOI. 180, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and 9), control IgG (lanes 2-4) or anti-porin antibody (lanes 5-7).
We are now
carrying out immunochemical studies on the molecular events occurring during the binding of the precursor-protein to the mitochondria. As we reported (21) that the OAT-precursor specifically bound to the mitochondrial surface at 0*C and the OAT-precursor bound to the surface was efficiently imported into mitochondria, these results indicate that the 52 kDa protein may not have an essential role in the e a r l y binding step of the mitochondrial protein-precursors.
ACKNOWLEDGMENTS This work was supported in part by Grants-in-Aid for Scientific Research (0245454, 02770118) from the Ministry of Education, Science and Culture of Japan and by The Naito Foundation.
REFERENCES I.
2. 3. 4. 5.
6. 7. 8. 9. I0. II. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
Hay, R., BOhni, P., & Gasser, S. (1984) Biochim. Biophys. Acta 770, 65-87. Hartl, F,=U., Pfanner, N., Nicholson, D.W., & Neupert, W. (1989) Biochim. Biophys. Acta 988. 1-45. Deshaies, R.J., Koch, B.D., Werner-Washburne, M., Craig, E.A., & Schekman, R. (1988) Nature 332, 800-805. Murakami, H., Pain, D., & Blobel, G. (1988) J. Cell Biol. 107, 2051-2057. Ono, H. & Tuboi, S. (1990) Arch. Biochem. Biophys. 280, 299-304. Murakami, K. & Mori, M. (1990) EMBO J. 9, 3201-3208. S611ner, T., Griffiths, G., Pfaller, R., Pfanner, N., & Neupert, W. (1989) Cell 59, 1061-1070. SOllner, T., PfaUer, R., Griffiths, G. Pfanner, N., & Neupert, W. (1990) Cell 62, 107-115. Vestweberi D., Brunner, J., Baker, A., & Schatz, G. (1989) Nature 341, 205209. Hines, U., Brandt, A., Griffiths, G., Horstmann, H., Brtltsch, H., Schatz, G. (1990) EMBO J. 9, 3191-3200. Pain, D., Murakami, H., & Blobel, G. (1990) Nature 347, 444-449. Kiebler, M., Pfaller R., SOllner, T., Griffiths, G., Horstman, H., Pfanner, N., & Neupert, W. (1990) Nature, 348, 610-616. Ono, H. & Tuboi, S. (1990) J. Biochem. I07, 840-845. Mueckler, M.M. & Pitot, P.C. (1985) J. Biol. Chem. 260, 12993-12997. Ono, H., Yoshimura, N., Sato, M., & Tuboi, S. (1985) J. Biol. Chem. 260, 3402-3407. Laemmli, U.K. (1970) Nature 227, 680-685. Mage, M.G. (1981) Methods Enzymol. 73, 142-150. Peraino, C., Bunville, L.G. & Tahimisian, T.N. (1969) J. Biol. Chem. 244, 2241-2249. Ono, H. & Tuboi, S. (1987) Eur. J. Biochem. !68, 509-514. Ono, H., Ito, A., & Omura, T. (1982) J. Biochem. 91, 107-116. Ono, H. & Tuboi, S. (1986) Eur. J. Biochem. 155, 543-549.
454
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 450-454
PURIFICATION OF 52 kDa PROTEIN: A PUTATIVE COMPONENT OF THE IMPORT MACHINERY FOR THE MITOCHONDRIAL PROTEIN-PRECURSOR IN RAT LIVER Hideyu Ono and Syozo Tuboi Department of Biochemistry, Yamagata University School of Medicine, Yamagata 990-23, JAPAN
Received September 6, 1991 Summary: A protein having a molecular mass of 52 kDa was purified to homogeneity from solubilized mitochondrial membrane proteins by affinity column chromatography using the s y n t h e t i c presequence of ornithine a m i n o t r a n s f e r a s e (OAT) as t h e ligand. This 52 kDa protein was s p e c i f i c a l l y bound to the a f f i n i t y column and e l u t e d with 1 mM OAT-presequence, indicating t h a t it recognized the presequence and bound to it specifically. Anti-52 kDa protein Feb fragments s p e c i f i c a l l y inhibited the import of OAT-precursor into mitochondria, showing that the 52 kDa protein plays an essential role in this process. These results suggest t h a t 52 k D a protein is a component of the import machinery of the mitochondrial p r o t e i n - p r e c u r s o r in the mitochondrial membrane, o 1991 ~cademic Press, lrnc.
The mitochondrial p r o t e i n - p r e c u r s o r s synthesized on cytoplasmic polysomes a r e t r a n s p o r t e d into mitochondria p o s t - t r a n s l a t i o n a l l y (1, 2}.
It has been shown
t h a t ATP and an inner membrane p o t e n t i a l (A~} a r e required as energy for this process (2).
Furthermore, t h e r e a r e also s e v e r a l reports on f a c t o r s t h a t a r e
e s s e n t i a l in the course of t r a n s l o c a t i o n of t h e mitochondrial proteins, and some of t h e s e f a c t o r s have been purified from the cytosol and the mitochondrial membrane (3-13). In the previous papers, we r e p o r t e d the purifications of a 28 kDa protein from rabbit r e t i c u l o c y t e l y s a t e (5) and a 29 kDa protein from r a t liver mitochondrial membranes (13} and showed t h a t these two proteins play essential roles in the t r a n s p o r t of mitochondrial proteins.
We purified t h e s e two proteins
on an a f f i n i t y column prepared with a chemically synthesized p e p t i d e composed of 54 amino acids containing the presequence of ornithine a m i n o t r a n s f e r a s e (OAT} a t its NH2-terminal as a ligand (5, 13).
Furthermore, we r e p o r t e d t h a t a 52 kDa
protein was co-purified with the 29 kDa protein from crude solubilized mitochondrial membrane proteins, although we did not d e t e r m i n e w h e t h e r this 52 kDa protein has any role in the import of mitochondrial proteins (13). In the p r e s e n t paper, we r e p o r t t h a t t h e 52 kDa protein was s p e c i f i c a l l y e l u t e d with the s y n t h e t i c OAT-presequence (composed of 34 amino acids) from an a f f i n i t y column and that anti-52 kDa protein antibody s p e c i f i c a l l y inhibited the 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
450
Vol. 180, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
import of the OAT-precursor into mitochondria.
These findings i n d i c a t e that the
52 kDa protein is also an essential component of the import machinery of mitochondrial protein in the membrane.
MATERIALS AND METHODS SYNTHESIS OF PEPTIDES AND PREPARATION OF PEPTIDE-CONJUGATED AGAROSE: The two peptides used in this study, a 54 amino acids p e p t i d e (54-AA
peptide) and the OAT-presequence, were chemically synthesized by Applied Biesystems Japan Co.(Tokyo, Japan). The amino acid sequence of the 54-AA p e p t i d e is the same as t h a t from Met I to His 53 of the OAT-precursor r e p o r t e d by Mueckler and Pitot (14), with an additional C-terminal Cys 54 to f a c i l i t a t e chemical crosslinking, as described in our previous paper (13). The 52 kDa protein was purified from solubilized mitochondrial membrane p,'oteins with an a f f i n i t y column of t~-aminobt,tyl-agarose conjugated with 54-AA peptide by using m-maleimidobenzoyl-N-hydroxysuccinimide e s t e r (13). OTHER METHODS: Precursors of mitochondrial proteins were synthesized in a c e l l - f r e e translation system using t o t a l rat liver RNA and a n u c l e a s e - t r e a t e d r e t i c u l o c y t e l y s a t e as described previously (15). A f t e r the t r a n s l a t i o n r e a c t i o n , the r e a c t i o n mixture was c e n t r i f u g e d for 30 min at 320,000 xg to remove ribosomes and the s u p e r n a t a n t was used as a source of mitochondrial p r o t e i n precursors for import into mitochondria (15). S D S - p o l y a c r y l a m i d e slab gel e l e c t r o p h o r e s i s was performed by the method of Laemmli (16). Fab f r a g m e n t s w e r e p r e p a r e d from IgG by the method of Mage (17). Ornithine a m i n o t r a n s f e r a s e and porin were purified as described previously (18, 19) and antibodies against t h e s e enzymes hnd the 52 kDa protein were p r e p a r e d by immunizing r a b b i t s with t h e s e purified proteins as d e s c r i b e d previously (20). MATERIALS: [a SS ] Methionine C>800 Ci/mmol) was obtained from American Radiolabeled Chemicals, Inc., USA. ATP-agarose (A 9264) and t~-aminobutyla g a r o s e (A 6142) were purchased from Sigma Chemical Co.. Nikkol ( o c t a e t h y l e n e g l y c o l mono-n-dodecyl e t h e r ) was from Nikko Chemicals (Tokyo, JaPan) , and the silver staining kit was from Kanto Chemical Co. (Tokyo, Japan).
RESULTS AND D I S C U S S I O N PURIFICATION OF 52 kDa PROTEIN:
Crude o u t e r mitochondrial membranes and
a solubilized protein f r a c t i o n of mitochondrial membranes were p r e p a r e d as d e s c r i b e d previously (13).
The solubilized proteins (2,210 mg) were applied to an
A T P - a g a r o s e column and the unadsorbed fraction was applied to a 54-AA peptide c o n j u g a t e d agarose column as d e s c r i b e d previously (13).
The column was washed
with b u f f e r A (10 mM Hepes-KOH, 2 mM magnesium a c e t a t e , 2 mM GSH, 1 jag/ml leupeptin, 1 ~g/ml pepstatin, 1096 glycerol) (pH 7.6) containing 800 mM potassium a c e t a t e and 1~ Triton X-100, and then with buffer A c o n t a i n i n g 200 mM potassium a c e t a t e and 196 Nikkol.
The proteins bound to the column w e r e e l u t e d
with b u f f e r A containing 200 mM potassium a c e t a t e , 1~ Nikkol, and 1 mM OATpresequence.
Elution of the a f f i n i t y column with OAT-presequence yielded only
t h e 52 kDa protein (about 0.8 rag), indicating t h a t this protein r e c o g n i z e d at~d s p e c i f i c a l l y bound t o t h e
OAT-presequence (Fig. 1, lane 2).
The 29 kDa p r o t e i n
was eluted t o g e t h e r with 52 kDa protein with c h a o t r o p i c ion (Fig. 1, lane 1) (13), but it was not e l u t e d with the s y n t h e t i c OAT-presequence, suggesting t h a t 451
Vol. 180, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1
2
52 kDa
29kDa
,-
EP. PIG. 1. Electrophoretogram of the purified 52 kDa protein. A solubilized protein fraction prepared from mitochondrial outer membranes was partially purified on ATP-agarose. The unadsorbed fraction (2,000 rag) was applied to a 54-AA peptide conjugated agarose column (bed volume 3.3 ml), and adsorbed proteins were eluted with chaotropic ion (3 M MgCiz, lane 1) or 1 mM OAT-presequence (lane 2) as described in the text. Electrophoresis was performed on a 11~ polyacrylamide slab gel in the presence of 0 . 2 t SDS. Proteins were d e t e c t e d by silver staining. E.P., OAT presequence.
this 29 kDa protein recognizes both the OAT-presequence and a p a r t of the NH 2terminal amino acid sequence of the mature OAT contained in the ligand or t h a t it has a g r e a t e r a f f i n i t y to the presequence. These results strongly suggest t h a t the 52 kDa protein is p a r t of the import machinery in the mitochondrial membrane t o g e t h e r with the 29 kDa protein and o t h e r proteins t h a t have not y e t been identified.
To confirm this possibility, we
p r e p a r e d anti-52 kDa protein antibody by immunizing a rabbit with the purified 52 kDa protein, as described previously (19). EFFECT OF ANTI-52 kDa PROTEIN ANTIBODY ON THE IMPORT O F OATPRECURSOR INTO MITOCHONDRIA:
T r e a t m e n t of mitochondria with anti-52 kDa
protein Fab f r a g m e n t s strongly inhibited the import of the OAT-precursor (Fig. 2A).
This inhibition seemed to be caused by specific i n t e r a c t i o n of the
antigen with its antil~ody, because control Fab fragments prepared from nonimmune rabbit and anti-porin Fab fragments did not inhibit this i m p o r t - r e a c t i o n (Fig. 2A and B).
The p r e s e n t results suggest t h a t the 52 kDa p r o t e i n is an
e s s e n t i a l component of t h e import machinery for t h e mitochondrial p r o t e i n p r e c u r s o r in the mitochondrial o u t e r membrane. We have r e p o r t e d (5) t h a t a 28 kDa protein, a component of t h e t a r g e t i n g f a c t o r required for t h e t r a n s p o r t of mitochondrial p r o t e i n - p r e c u r s o r s to t h e mitochondrial surface, could be purified to homogeniety using t h e same a f f i n i t y 452
Vol. 180, No. 1, 1991
A lO0
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
o
B 100
~. 75
75
50.
~
~
25
i
25 0
i
o Concentrationof Fab (mg/rnl)
Concentrationof Fab (mg/ml)
FIG. 2. Effect of anti-52 kDa protein Fab fragments and anti-porin Fab fragments on the import of OAT-precursor. A) Effect of anti-52 kDa protein Fab fragments. Mitochondria (final concentration, 0.25 mg/ml) were incubated with anti-52 kDa protein Fab fragments (e) or control Fab fragments prepared from a non-immune rabbit (o) at the concentrations indicated in the Figure for 15 rain at 25°C and then for 90 rain at O°C. Then, each mitocbondrial fraction(100ul, 25 pg) was incubated with 100 pl of ceU-free translation products for 15 mir~at 30°C. The mitochondria were then reisolated and their imported OAT was analyzed (15). Bands of OAT on the fluorogram were quantified by densitometry. B) Effect of anti-porin Fab fragments. Experimental and analytical conditions were as described above, e, treated with anti-porin Fab fragments; o, treated with control Fab fragments.
column as t h a t used for purification of the 52 kDa protein, and that anti-28 kDa protein antibody inhibited the binding of the precursor to the mitochondria at 0°C as well as its import.
On the contrary, we found that anti-52 kDa protein
antibody had no e f f e c t on the binding of the OAT-precursor to the mitochondria at 0°C : on incubation with mitochondria at 0°C for 20 rain, the precursor bound to the mitochondria, as shown in Fig. 3, lane l, and this binding was not e f f e c t e d by p r e - t r e a t m e n t of the mitochondria with anti-52 kDa protein antibody (lanes 8
2
3
4
5
6
7
8
9
pOAT =--1
FIG. 3. Effects of anti-52 kDa protein Fab fragments and anti-porin Fab fragments on the binding of pOAT to mitochondria. Mitochondria (final concentration, 0.25 mg/ml) were treated with each antibody as described in the legend for Fig. 2. Then, the samples were incubated with 100 jal of cell-free translation products for 20 rain at 0°C. The mitochondria were then reisolated and OAT-precursor recovered from them was assayed by fluorography. Lane 1, untreated; lanes 2-4, treated with control Fab fragments at concentrations of 0.41 mg/ml, 0.83 mg/ml, and 1.7 mg/ml, respectively; lanes 5-7, treated with anti-porin Fab fragments at concentrations of 0.41 mg/ml, 0.83 mg/ml, and 1.7 mg/ml, respectively; lanes 8 and 9, treated with anti-52 kDa protein Fab fragments at concentrations of 0.83 mg/ml and 1.7 mg/ml, respectively. 453
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and 9), control IgG (lanes 2-4) or anti-porin antibody (lanes 5-7).
We are now
carrying out immunochemical studies on the molecular events occurring during the binding of the precursor-protein to the mitochondria. As we reported (21) that the OAT-precursor specifically bound to the mitochondrial surface at 0*C and the OAT-precursor bound to the surface was efficiently imported into mitochondria, these results indicate that the 52 kDa protein may not have an essential role in the e a r l y binding step of the mitochondrial protein-precursors.
ACKNOWLEDGMENTS This work was supported in part by Grants-in-Aid for Scientific Research (0245454, 02770118) from the Ministry of Education, Science and Culture of Japan and by The Naito Foundation.
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