Plant Molecular Biology 12:13-18 (1989) © Kluwer Academic Publishers, Dordrecht - Printed in the Netherlands
13
Chloroplast import characteristics of chimeric proteins
Thomas H. Lubben, 1, 3. Anthony A. Gatenby, 2, 3 Paul Ahlquist 2 and Kenneth Keegstra l
1Department of Botany and 2Institute for Molecular Virology and Department of Plant Pathology, University of Wisconsin, Madison, WI 53706, USA; 3present address: Central Research and Development Department, E402, E. L Du Pont de Nemours and Co., Wilmington, DE 19898, USA (*author for correspondence; Telephone (302) 695-9246) Received 1 July 1988; accepted in revised form 27 September 1988
Key words: precursor, BMV coat protein; Rubisco small subunit; protein targeting; transit peptide Abstract We have examined the import o f a series o f chimeric precursor proteins into chloroplasts. These fusion proteins contained the transit peptide, and various amounts o f the amino-terminal region of the mature peptide, from the small subunit of ribulose 1,5-bisphosphate carboxylase, linked to the coat protein o f brome mosaic virus. Chimeric genes were cloned into SP6 plasmids and in vitro transcription/translation was used to produce fusion proteins, which were examined in a quantitative in vitro import assay. A chimeric protein which contained only the transit peptide fused to the coat protein was imported into chloroplasts. A second chimeric precursor, which also contained a small portion of the mature peptide, was imported at nearly the same rate. A chimeric protein which contained the transit peptide and most o f the mature peptide fused to the coat protein was not imported. These results suggest that secondary or tertiary structural features of precursor proteins are important for protein import, and that the presence o f a transit peptide in a protein does not necessarily ensure import o f that protein into chloroplasts.
Introduction Chloroplasts contain many proteins that are encoded in the nucleus, synthesized on cytoplasmic ribosomes as higher molecular weight precursors and imported by a post-translation process (for reviews, see [3, 8]). Recent studies have demonstrated that the information required for the correct targeting of these proteins resides in an amino-terminal transit peptide. This has been shown in several instances where transit peptides from precursors to nuclearencoded chloroplast proteins were attached to various passenger proteins which are normally not imported into chloroplasts. The resulting chimeric proteins were imported into chloroplasts in vivo [6, 9,
11] and in vitro [5, 7, 10, 12]. However, many questions concerning the import o f foreign proteins remain to be answered. Because foreign passenger proteins are imported with lower efficiency than the molecules from which the transit peptides are derived [5-7, 9, 10, 12], it is possible that some targeting information resides outside the transit peptide. Another possibility is that certain proteins may contain particular primary sequences or secondary structural features which preclude their import into chloroplasts. To address these points, we have constructed fusion proteins which contain the transit peptide as well as portions o f the mature peptide from the precursor to the small subunit o f ribulose 1,5-bisphosphate carboxylase (Rubisco), fused
14
to the coat protein of brome mosaic virus (BMV).
Materials and methods
The sources of materials, plasmid isolation, in vitro preparation of radiolabeled precursor proteins, and quantitation of import have been described previously [7]. Plasmid pSP81/4 [7] encodes a chimeric Rubisco small-subunit precursor, which we designate TPSS, and which contains a soybean Rubisco small-subunit transit peptide and pea small-subunit mature peptide.
Construction of plasmids Plasmid pTPCP3A, which codes for the protein TPCP, was made from pGM22/3 and an 867 bp fragment of BMV cDNA coat protein gene from pB3PM1 [1]. pB3PM1 was cut with Sal I, bluntended with Klenow fragment, and cut with Eco RI. This (Klenow) blunt-ended Sal I/Eco R1867 bp fragment was ligated between a (Klenow) blunt-ended Sal I and Eco RI site in the polylinker of pUC18. Plasmid pGM22/3 encodes the transit peptide of soybean Rubisco small subunit and contains the Hind III/Sph I fragment of pSRS2.1 [2], cloned into the Hind III/Sph I sites of pGEM3. To produce plasmid pTPCP3A, the fragment which encoded the BMV coat protein was excised with Sph I and Eco RI from the pUC18 polylinker and ligated into Sph I/Eco RI-cut pGM22/3. Plasmid pTPSPCP codes for the protein TPSPCP, which contains the transit peptide and 14 amino acids (aa) of the mature peptide from TPSS, and the same region of the BMV coat protein present in TPCP. It was made by ligation of a (mung bean nuclease) blunt-ended Sau 3A/Eco RI fragment of pTPCP3A (encoding the BMV coat protein) into (mung bean nuclease) blunt-ended Xba I-cut and Eco RI-cut pSP26/4, pSP26/4 is a plasmid which encodes the transit peptide and the first 14 aa of TPSS, and contains the Hind I/Hinf III fragment of pSP81/4 [7], ligated into the Hind III site of pSP64. Plasmid pTPSSCP encodes the protein TPSSCP, which contains the transit peptide and 117 aa of 123
aa of the mature peptide from TPSS, and the same BMV coat protein region used in TPSPCP. It was made by ligation of the same blunt-ended Sau3A/Eco RI fragment, used in construction of pTPSPCP, and an Sph I/Fsp I fragment of pSP81/4, into Sph I/Eco RI-cut pGM22/3.
Import of proteins into chloroplasts Precursor proteins were prepared as described previously [4], except that proteins were labelled with either 35S-methionine or 3H-leucine. Import assays contained precursor proteins at various concentrations from 1.5 nM to 13 nM. Time course reactions were performed as described previously [7], except that reactions were stopped using an improved, more rapid procedure as follows. The entire reaction mixture was applied to the top of a step-gradient which consisted of 100/~1 of Wacker AR200 silicone oil (a gift of Wacker Silicones, Adrian, MI) on top of 100/~1 of 1 M perchloric acid, 2 mM EDTA in a 400/~1 microcentrifuge tube. This was centrifuged for 30 s at 9000 xg. Under these conditions, broken chloroplasts remained at the top of the silicone oil layer, whereas intact chloroplasts were pelleted into the perchloric acid phase. This method stopped time points quickly and reduced the likelihood of proteolysis occurring in subsequent steps. The protein pellets were resuspended in SDS sample buffer and analyzed by SDS-polyacrylamide gel electrophoresis.
Results
The composition of the various precursor proteins is diagrammed in Fig. 1. TPSS is a chimeric precursor to Rubisco small subunit, described previously [4], and was used as a positive control. TPCP contains the transit peptide of TPSS and the coat protein of BMV. TPSPCP contains the transit peptide and 14 aa of the mature peptide of TPSS and the coat protein of BMV. TPSSCP contains the transit peptide and 117 (of a total of 123) aa of the mature peptide of TPSS and the coat protein of BMV. Precursor proteins of the correct size were produced
15 TPSS- I TP [~ii~".":'~:;.] TPCP-l TP ~ f f P . , ~ . . ~ l ~SS TPSPCP -I TP F ~ , ~ x ~ . " - . P L ~ ~
I
0
I
I
100
I
I
200
I
I
500
I
I
400
size (Qmino Qcids) Fig. 1. Chimeric precursor proteins diagrammed to show their composition. Bars are drawn to scale. The label within the shaded bars show the origin of that segment. TP, the transit peptide of Rubisco small subunit precursor; SS, Rubisco mature peptide, CP, BMV coat protein.
by in vitro transcription/translation of genes cloned into plasmids containing an SP6 RNA polymerase promoter (Fig. 2).
Import of chimeric proteins When precursor proteins were incubated with intact chloroplasts, TPSS, T P C P and T P S P C P were imported and proteolytically processed. However, with T P C P the imported molecule was 17 kDa, 3 kDa
Fig. 2. Chimeric precursor proteins which were made by in vitro transcription/translation of cloned genes were analyzed by SDS gel electrophoresis and fluorography. Lane 1, TPSS; lane 2, TPCP; lane 3, TPSPCP; lane 4, TPSSCP. The band which is TPSSCP is indicated by a dash (-); the band marked with an asterisk (*) is the correct size to have resulted from initiation of translation at the methionine which is the first amino acid of Rubisco mature peptide. The migration of molecular weight markers is shown: ovalbumin, 43 kDa; ~t-chymotrypsin, 26 kDa; /3lactoglobulin, 18 kDa; lysozyme, 14 kDa.
less than expected for BMV coat protein (Fig. 3). To verify that this was not due to anomalous migration o f BMV coat protein, authentic BMV coat protein was produced by in vitro transcription/translation, and found to migrate at the expected size o f 20 kDa (data not shown). Therefore, although T P C P was imported into chloroplasts, it was not processed correctly. With TPSPCP, an imported protein of 22 kDa, the size expected if processing had occurred at the correct site, was observed. In addition, a 17 kDa protein, presumably the same protein as that found during import o f TPCP, also was present (Fig. 3). Apparently, the presence of the 14 aa portion of the Rubisco small-subunit mature peptide partially restored correct processing.
Import rates In our studies with these precursor proteins, we noticed that the import o f certain chimeric precursor proteins, such as T P C P and TPSPCP, proceeded at a slower initial rate, and for longer periods of time, compared to TPSS. This phenomenon was most evident if the import time course o f TPSS and T P S P C P was compared (Fig. 4). The fact that import continued for different periods of time with different precursors made it important that we compare the various precursors using initial rates, rather than extent of import. The relative import rates of the three chimeric precursor proteins were compared to the import rate o f TPSS by conducting time course studies with each precursor. Because it was not possible to obtain sufficient quantities o f precursors to perform these studies using saturating precursor concentrations, import assays were done with the various precursors over a range of subsaturating concentrations, from 1.5 nM to 13.0 nM (Fig. 4 and Table 1). The initial rates o f import for each precursor were plotted versus initial concentration. Straight lines were obtained over the concentration ranges tested. This confirmed that as expected for subsaturation precursor concentrations, rates were proportional to concentration. This linearity allowed us to compare the import rates of the different precursors. When both the 17 kDa and 22 kDa bands were included in the
16
Fig. 3. Precursor proteins were incubated with intact chloroplasts for various times, and import reactions stopped by centrifugation through AR200 silicone oil into perchloric acid. Protein pellets were dissolved in SDS sample buffer and portions of each analyzed by SDS-polyacrylamide gel electrophoresis. Fluorograms of the gels are shown. Times of incubation are shown in minutes under the gel lanes; samples which were treated with protease to remove externally bound proteins were run in lanes designated P. Panel A, TPSS; Panel B, TPSPCP; Panel C, TPCP; Panel D, TPSSCP. The migration of molecular weight markers is shown: (a) ovalbumin, 43 kDa; (b) ¢xchymotrypsin, 26 kDa; (c) B-lactoglobulin, 18 kDa; (d) lysozyme, 14 kDa.
recoverable as either intact precursor or imported proteins (data not shown). Thus, we conclude that the rates we observed were accurate measurements of the import rates of these precursors.
data for TPSPCP, both T P C P and T P S P C P were imported at the same rate. This rate was approximately 2.5°7o of that found for TPSS (Table 1). Import o f T P S S C P into chloroplasts could not be detected during a 30-min assay, within a limit of detection of 0.507o o f the TPSS rate (Fig. 4 and Table 1). In order to determine whether the import rates of TPCP, T P S P C P and T P S S C P were artificially low because of proteolysis, all fractions from import reactions were analyzed. For TPSS, TPCP, T P S P C P and TPSSCP, over 80070 o f the starting material was
Discussion
The results presented here provide an additional example o f the import of a foreign protein, BMV coat protein, into chloroplasts. However, compared to
oo
o
40 ¸
8 TPSPCP total
X
8 TPCP
no
20
4
o
Q.
E o~ ....1 (3 o
E
2 0
.
0
5
10
0
,
10
.
0
,
20
30
0
10
20
30
time of import (min.)
Fig. 4. Imported proteins were excised from the gel of Fig. 3 and quantitated by liquid scintillation. Initial precursor concentrations for the reactions shown were: TPSS, 2.7 nM; TPSPCP, 7.7 nM; and TPCP, 6.1 nM.
17 Table L Relative rates of import of TPSS, TPCP, TPSPCP and TPSSCP. Import reactions similar to those shown in Fig. 3 were done with each precursor at various initial concentrations from 1.5 nM to 13.0 nM. The initial rates of import for each precursor were compared at identical concentrations as described earlier in the Results section. For TPSSCP, the limit of detection was approximately 0.5% of the observed rate for TPSS import.
Precursor
Relative rate (%)
TPSS TPCP TPSPCP (total) TPSSCP
100 2.3 + 0.4 2.1 + 0.6 not detectable
T P S S , t h e rate o f i m p o r t o f either c h i m e r i c precurs o r w h i c h c o n t a i n e d t h e B M V c o a t proteins was poor. T h e p o o r i m p o r t rate o f T P C P p r o v i d e d a n o p p o r t u n i t y to investigate t h e h y p o t h e s i s t h a t targeting i n f o r m a t i o n , r e q u i r e d for efficient i m p o r t , m a y b e p r e s e n t in the m a t u r e p e p t i d e as well as t h e t r a n s i t p e p t i d e o f p r e c u r s o r proteins. O u r results i n d i c a t e t h a t t h e first 14 a a o f t h e m a t u r e p e p t i d e d o n o t sign i f i c a n t l y affect i m p o r t . I n c l u s i o n o f m o s t o f the m a t u r e p e p t i d e (117 o u t o f 123 aa) y i e l d e d a c h i m e r i c p r e c u r s o r ( T P S S C P ) which, despite t h e fact t h a t it c o n t a i n e d a t r a n s i t peptide, was n o t d e t e c t a b l y imported. T h e s e results c o n t r a s t with t h o s e o f W a s m a n n et al. [12], w h o o b s e r v e d t h a t the i n c l u s i o n o f 23 a a o f t h e m a t u r e p e p t i d e o f R u b i s c o small s u b u n i t greatly e n h a n c e d the extent o f in vitro i m p o r t o f a c h i m e r i c p r e c u r s o r which o t h e r w i s e c o n s i s t e d o f b a c t e r i a l neo m y c i n p h o s p h o t r a n s f e r a s e , as a p a s s e n g e r protein, a t t a c h e d to t h e t r a n s i t p e p t i d e o f R u b i s c o smalls u b u n i t precursor. T h e y suggested t h a t the a d d i t i o n al 23 a m i n o acids f u n c t i o n e d as a s p a c e r a n d p r o v i d ed a s e p a r a t i o n o f the t r a n s i t p e p t i d e f r o m the p a s senger protein, w h i c h t h e r e b y allowed the t r a n s i t p e p t i d e to a s s u m e its native c o n f o r m a t i o n . I f the h y p o t h e s i s o f W a s m a n n et al. [12] is correct, a n d the s e g m e n t o f the m a t u r e p e p t i d e f u n c t i o n s as a s p a c e r to keep the t r a n s i t p e p t i d e r e m o v e d f r o m t h e p a s senger protein, t h e n t h e 14 a a s e g m e n t in T P S P C P represents a n i n a d e q u a t e spacer. However, the 117 a a o f m a t u r e p e p t i d e p r e s e n t in T P S S C P s h o u l d be sufficiently l o n g to fulfill any s p a c e r requirements.
F u r t h e r m o r e , b e c a u s e T P S S C P c o n t a i n s o n l y sequences w h i c h were i m p o r t e d in T P S S o r T P C P , this suggests t h a t i m p o r t c o m p e t e n c e o f a p r o t e i n is n o t a s i m p l e f u n c t i o n o f p r i m a r y sequence, b u t it also i n f l u e n c e d b y p r o t e i n c o n f o r m a t i o n . O n e way this c o u l d o c c u r is if t h e p r e c u r s o r p r o t e i n folds in such a way t h a t the transit p e p t i d e is sterically h i n d e r e d a n d c a n n o t interact with the i m p o r t a p p a r a t u s . A n o t h e r p o s s i b i l i t y is t h a t i n t e r a c t i o n s between distal segments o f the c h i m e r i c p r o t e i n m a y i n h i b i t u n f o l d ing o r o t h e r c o n f o r m a t i o n a l changes which m a y be n e c e s s a r y for i m p o r t . W h a t e v e r the e x p l a n a t i o n , the results p r e s e n t e d here d e m o n s t r a t e t h a t t h e presence o f a transit p e p tide is n o t always sufficient to cause the i m p o r t o f a p r o t e i n into c h l o r o p l a s t . Moreover, it is likely t h a t s e c o n d a r y a n d t e r t i a r y s t r u c t u r a l features o f the entire p r e c u r s o r p r o t e i n are i m p o r t a n t for i m p o r t o f p r o t e i n s into chloroplasts.
References 1. Ahlquist P, Janda M: cDNA cloning and in vitro transcription of the complete Brome Mosaic Virus genome. Mol Cell Biol 4:2876-2882 (1984). 2. Berry-Lowe SL, McKnight TD, Shah DM, Meagher RB" The nucleotide sequence, expression, and evolution of one member of a multigene family encoding the small subunit of ribulose 1,5-bisphosphate carboxylase in soybean. J Mol Appl Genet 1:483-498 (1982). 3. Cashmore A, Szabo L, Timko M, Kausch A, van den Broeck G, Schreier P, Bohnert H, Herrera-Estrella L, Van Montagu M, Schell J: Import of polypeptides into chloroplasts. BioTechnology 3:803-808 (1985). 4. Cline K, Werner-Washburne M, Lubben T, Keegstra K: Precursor to two nuclear-encoded chloroplast proteins bind to the outer envelope membrane before being imported into chloroplasts. J Biol Chem 260:3691-3696 (1985). 5. Gatenby AA, Lubben TH, Ahlquist P, Keegstra K: Imported large subunits of ribulose bisphosphate carboxylase/oxygenase, but not imported ~-ATP synthase subunits, are assembled into holoenzyme in isolated chloroplasts. EMBO J 7:1307-1314 (1988). 6. Kuntz M, Simmons A, Schell J, Schreier PH" Targeting of proteins to chloroplasts in transgenic plants by fusion to mutated transit peptide. Mol Gen Genet 205:454-460 (1986). 7. Lubben TH, Keegstra K: Efficient in vitro import of a cytosolic heat shock protein into pea chloroplasts. Proc Natl Acad Sci USA 83: 5502-5506, 1986. 8. Schmidt GW, Mishkind ML: The transport of proteins into chloroplasts. Ann Rev Biochem 55:879-912 (1986).
18 9. Schreier P, Seftor EA, Schell J, Bohnert H: The use of nuclear-encoded sequences to direct the light-regulated synthesis and transport of a foreign protein into plant chloroplasts. EMBO J 4:25-32 (1985). 10. Smeekens S, van Steeg H, Bauede C, Bettenbroek H, Keegstra K, Weisbeek P: Import into chloroplasts of a yeast mitochondrial protein directed by ferredoxin and plastocyanin transit peptides. Plant Mol Biol 9:377-388 (1987). 11. van den Broeck G, Timko PM, Kausch AP, Cashmore AR,
Van Montagu M, Herrera-Estrella L: Targeting of a foreign protein to chloroplasts by fusion to the transit peptide from the small subunit of Ribulose 1,5-bisphosphate carboxylase. Nature (London) 313:358-363 (1985). 12. Wasmann CC, Reiss B, Barlett SG, Bohnert H J: The importance of the transit peptide and the transported protein for protein import into chloroplasts. Mol Gen Genet 205: 446-453 (1986).
13
Chloroplast import characteristics of chimeric proteins
Thomas H. Lubben, 1, 3. Anthony A. Gatenby, 2, 3 Paul Ahlquist 2 and Kenneth Keegstra l
1Department of Botany and 2Institute for Molecular Virology and Department of Plant Pathology, University of Wisconsin, Madison, WI 53706, USA; 3present address: Central Research and Development Department, E402, E. L Du Pont de Nemours and Co., Wilmington, DE 19898, USA (*author for correspondence; Telephone (302) 695-9246) Received 1 July 1988; accepted in revised form 27 September 1988
Key words: precursor, BMV coat protein; Rubisco small subunit; protein targeting; transit peptide Abstract We have examined the import o f a series o f chimeric precursor proteins into chloroplasts. These fusion proteins contained the transit peptide, and various amounts o f the amino-terminal region of the mature peptide, from the small subunit of ribulose 1,5-bisphosphate carboxylase, linked to the coat protein o f brome mosaic virus. Chimeric genes were cloned into SP6 plasmids and in vitro transcription/translation was used to produce fusion proteins, which were examined in a quantitative in vitro import assay. A chimeric protein which contained only the transit peptide fused to the coat protein was imported into chloroplasts. A second chimeric precursor, which also contained a small portion of the mature peptide, was imported at nearly the same rate. A chimeric protein which contained the transit peptide and most o f the mature peptide fused to the coat protein was not imported. These results suggest that secondary or tertiary structural features of precursor proteins are important for protein import, and that the presence o f a transit peptide in a protein does not necessarily ensure import o f that protein into chloroplasts.
Introduction Chloroplasts contain many proteins that are encoded in the nucleus, synthesized on cytoplasmic ribosomes as higher molecular weight precursors and imported by a post-translation process (for reviews, see [3, 8]). Recent studies have demonstrated that the information required for the correct targeting of these proteins resides in an amino-terminal transit peptide. This has been shown in several instances where transit peptides from precursors to nuclearencoded chloroplast proteins were attached to various passenger proteins which are normally not imported into chloroplasts. The resulting chimeric proteins were imported into chloroplasts in vivo [6, 9,
11] and in vitro [5, 7, 10, 12]. However, many questions concerning the import o f foreign proteins remain to be answered. Because foreign passenger proteins are imported with lower efficiency than the molecules from which the transit peptides are derived [5-7, 9, 10, 12], it is possible that some targeting information resides outside the transit peptide. Another possibility is that certain proteins may contain particular primary sequences or secondary structural features which preclude their import into chloroplasts. To address these points, we have constructed fusion proteins which contain the transit peptide as well as portions o f the mature peptide from the precursor to the small subunit o f ribulose 1,5-bisphosphate carboxylase (Rubisco), fused
14
to the coat protein of brome mosaic virus (BMV).
Materials and methods
The sources of materials, plasmid isolation, in vitro preparation of radiolabeled precursor proteins, and quantitation of import have been described previously [7]. Plasmid pSP81/4 [7] encodes a chimeric Rubisco small-subunit precursor, which we designate TPSS, and which contains a soybean Rubisco small-subunit transit peptide and pea small-subunit mature peptide.
Construction of plasmids Plasmid pTPCP3A, which codes for the protein TPCP, was made from pGM22/3 and an 867 bp fragment of BMV cDNA coat protein gene from pB3PM1 [1]. pB3PM1 was cut with Sal I, bluntended with Klenow fragment, and cut with Eco RI. This (Klenow) blunt-ended Sal I/Eco R1867 bp fragment was ligated between a (Klenow) blunt-ended Sal I and Eco RI site in the polylinker of pUC18. Plasmid pGM22/3 encodes the transit peptide of soybean Rubisco small subunit and contains the Hind III/Sph I fragment of pSRS2.1 [2], cloned into the Hind III/Sph I sites of pGEM3. To produce plasmid pTPCP3A, the fragment which encoded the BMV coat protein was excised with Sph I and Eco RI from the pUC18 polylinker and ligated into Sph I/Eco RI-cut pGM22/3. Plasmid pTPSPCP codes for the protein TPSPCP, which contains the transit peptide and 14 amino acids (aa) of the mature peptide from TPSS, and the same region of the BMV coat protein present in TPCP. It was made by ligation of a (mung bean nuclease) blunt-ended Sau 3A/Eco RI fragment of pTPCP3A (encoding the BMV coat protein) into (mung bean nuclease) blunt-ended Xba I-cut and Eco RI-cut pSP26/4, pSP26/4 is a plasmid which encodes the transit peptide and the first 14 aa of TPSS, and contains the Hind I/Hinf III fragment of pSP81/4 [7], ligated into the Hind III site of pSP64. Plasmid pTPSSCP encodes the protein TPSSCP, which contains the transit peptide and 117 aa of 123
aa of the mature peptide from TPSS, and the same BMV coat protein region used in TPSPCP. It was made by ligation of the same blunt-ended Sau3A/Eco RI fragment, used in construction of pTPSPCP, and an Sph I/Fsp I fragment of pSP81/4, into Sph I/Eco RI-cut pGM22/3.
Import of proteins into chloroplasts Precursor proteins were prepared as described previously [4], except that proteins were labelled with either 35S-methionine or 3H-leucine. Import assays contained precursor proteins at various concentrations from 1.5 nM to 13 nM. Time course reactions were performed as described previously [7], except that reactions were stopped using an improved, more rapid procedure as follows. The entire reaction mixture was applied to the top of a step-gradient which consisted of 100/~1 of Wacker AR200 silicone oil (a gift of Wacker Silicones, Adrian, MI) on top of 100/~1 of 1 M perchloric acid, 2 mM EDTA in a 400/~1 microcentrifuge tube. This was centrifuged for 30 s at 9000 xg. Under these conditions, broken chloroplasts remained at the top of the silicone oil layer, whereas intact chloroplasts were pelleted into the perchloric acid phase. This method stopped time points quickly and reduced the likelihood of proteolysis occurring in subsequent steps. The protein pellets were resuspended in SDS sample buffer and analyzed by SDS-polyacrylamide gel electrophoresis.
Results
The composition of the various precursor proteins is diagrammed in Fig. 1. TPSS is a chimeric precursor to Rubisco small subunit, described previously [4], and was used as a positive control. TPCP contains the transit peptide of TPSS and the coat protein of BMV. TPSPCP contains the transit peptide and 14 aa of the mature peptide of TPSS and the coat protein of BMV. TPSSCP contains the transit peptide and 117 (of a total of 123) aa of the mature peptide of TPSS and the coat protein of BMV. Precursor proteins of the correct size were produced
15 TPSS- I TP [~ii~".":'~:;.] TPCP-l TP ~ f f P . , ~ . . ~ l ~SS TPSPCP -I TP F ~ , ~ x ~ . " - . P L ~ ~
I
0
I
I
100
I
I
200
I
I
500
I
I
400
size (Qmino Qcids) Fig. 1. Chimeric precursor proteins diagrammed to show their composition. Bars are drawn to scale. The label within the shaded bars show the origin of that segment. TP, the transit peptide of Rubisco small subunit precursor; SS, Rubisco mature peptide, CP, BMV coat protein.
by in vitro transcription/translation of genes cloned into plasmids containing an SP6 RNA polymerase promoter (Fig. 2).
Import of chimeric proteins When precursor proteins were incubated with intact chloroplasts, TPSS, T P C P and T P S P C P were imported and proteolytically processed. However, with T P C P the imported molecule was 17 kDa, 3 kDa
Fig. 2. Chimeric precursor proteins which were made by in vitro transcription/translation of cloned genes were analyzed by SDS gel electrophoresis and fluorography. Lane 1, TPSS; lane 2, TPCP; lane 3, TPSPCP; lane 4, TPSSCP. The band which is TPSSCP is indicated by a dash (-); the band marked with an asterisk (*) is the correct size to have resulted from initiation of translation at the methionine which is the first amino acid of Rubisco mature peptide. The migration of molecular weight markers is shown: ovalbumin, 43 kDa; ~t-chymotrypsin, 26 kDa; /3lactoglobulin, 18 kDa; lysozyme, 14 kDa.
less than expected for BMV coat protein (Fig. 3). To verify that this was not due to anomalous migration o f BMV coat protein, authentic BMV coat protein was produced by in vitro transcription/translation, and found to migrate at the expected size o f 20 kDa (data not shown). Therefore, although T P C P was imported into chloroplasts, it was not processed correctly. With TPSPCP, an imported protein of 22 kDa, the size expected if processing had occurred at the correct site, was observed. In addition, a 17 kDa protein, presumably the same protein as that found during import o f TPCP, also was present (Fig. 3). Apparently, the presence of the 14 aa portion of the Rubisco small-subunit mature peptide partially restored correct processing.
Import rates In our studies with these precursor proteins, we noticed that the import o f certain chimeric precursor proteins, such as T P C P and TPSPCP, proceeded at a slower initial rate, and for longer periods of time, compared to TPSS. This phenomenon was most evident if the import time course o f TPSS and T P S P C P was compared (Fig. 4). The fact that import continued for different periods of time with different precursors made it important that we compare the various precursors using initial rates, rather than extent of import. The relative import rates of the three chimeric precursor proteins were compared to the import rate o f TPSS by conducting time course studies with each precursor. Because it was not possible to obtain sufficient quantities o f precursors to perform these studies using saturating precursor concentrations, import assays were done with the various precursors over a range of subsaturating concentrations, from 1.5 nM to 13.0 nM (Fig. 4 and Table 1). The initial rates o f import for each precursor were plotted versus initial concentration. Straight lines were obtained over the concentration ranges tested. This confirmed that as expected for subsaturation precursor concentrations, rates were proportional to concentration. This linearity allowed us to compare the import rates of the different precursors. When both the 17 kDa and 22 kDa bands were included in the
16
Fig. 3. Precursor proteins were incubated with intact chloroplasts for various times, and import reactions stopped by centrifugation through AR200 silicone oil into perchloric acid. Protein pellets were dissolved in SDS sample buffer and portions of each analyzed by SDS-polyacrylamide gel electrophoresis. Fluorograms of the gels are shown. Times of incubation are shown in minutes under the gel lanes; samples which were treated with protease to remove externally bound proteins were run in lanes designated P. Panel A, TPSS; Panel B, TPSPCP; Panel C, TPCP; Panel D, TPSSCP. The migration of molecular weight markers is shown: (a) ovalbumin, 43 kDa; (b) ¢xchymotrypsin, 26 kDa; (c) B-lactoglobulin, 18 kDa; (d) lysozyme, 14 kDa.
recoverable as either intact precursor or imported proteins (data not shown). Thus, we conclude that the rates we observed were accurate measurements of the import rates of these precursors.
data for TPSPCP, both T P C P and T P S P C P were imported at the same rate. This rate was approximately 2.5°7o of that found for TPSS (Table 1). Import o f T P S S C P into chloroplasts could not be detected during a 30-min assay, within a limit of detection of 0.507o o f the TPSS rate (Fig. 4 and Table 1). In order to determine whether the import rates of TPCP, T P S P C P and T P S S C P were artificially low because of proteolysis, all fractions from import reactions were analyzed. For TPSS, TPCP, T P S P C P and TPSSCP, over 80070 o f the starting material was
Discussion
The results presented here provide an additional example o f the import of a foreign protein, BMV coat protein, into chloroplasts. However, compared to
oo
o
40 ¸
8 TPSPCP total
X
8 TPCP
no
20
4
o
Q.
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E
2 0
.
0
5
10
0
,
10
.
0
,
20
30
0
10
20
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time of import (min.)
Fig. 4. Imported proteins were excised from the gel of Fig. 3 and quantitated by liquid scintillation. Initial precursor concentrations for the reactions shown were: TPSS, 2.7 nM; TPSPCP, 7.7 nM; and TPCP, 6.1 nM.
17 Table L Relative rates of import of TPSS, TPCP, TPSPCP and TPSSCP. Import reactions similar to those shown in Fig. 3 were done with each precursor at various initial concentrations from 1.5 nM to 13.0 nM. The initial rates of import for each precursor were compared at identical concentrations as described earlier in the Results section. For TPSSCP, the limit of detection was approximately 0.5% of the observed rate for TPSS import.
Precursor
Relative rate (%)
TPSS TPCP TPSPCP (total) TPSSCP
100 2.3 + 0.4 2.1 + 0.6 not detectable
T P S S , t h e rate o f i m p o r t o f either c h i m e r i c precurs o r w h i c h c o n t a i n e d t h e B M V c o a t proteins was poor. T h e p o o r i m p o r t rate o f T P C P p r o v i d e d a n o p p o r t u n i t y to investigate t h e h y p o t h e s i s t h a t targeting i n f o r m a t i o n , r e q u i r e d for efficient i m p o r t , m a y b e p r e s e n t in the m a t u r e p e p t i d e as well as t h e t r a n s i t p e p t i d e o f p r e c u r s o r proteins. O u r results i n d i c a t e t h a t t h e first 14 a a o f t h e m a t u r e p e p t i d e d o n o t sign i f i c a n t l y affect i m p o r t . I n c l u s i o n o f m o s t o f the m a t u r e p e p t i d e (117 o u t o f 123 aa) y i e l d e d a c h i m e r i c p r e c u r s o r ( T P S S C P ) which, despite t h e fact t h a t it c o n t a i n e d a t r a n s i t peptide, was n o t d e t e c t a b l y imported. T h e s e results c o n t r a s t with t h o s e o f W a s m a n n et al. [12], w h o o b s e r v e d t h a t the i n c l u s i o n o f 23 a a o f t h e m a t u r e p e p t i d e o f R u b i s c o small s u b u n i t greatly e n h a n c e d the extent o f in vitro i m p o r t o f a c h i m e r i c p r e c u r s o r which o t h e r w i s e c o n s i s t e d o f b a c t e r i a l neo m y c i n p h o s p h o t r a n s f e r a s e , as a p a s s e n g e r protein, a t t a c h e d to t h e t r a n s i t p e p t i d e o f R u b i s c o smalls u b u n i t precursor. T h e y suggested t h a t the a d d i t i o n al 23 a m i n o acids f u n c t i o n e d as a s p a c e r a n d p r o v i d ed a s e p a r a t i o n o f the t r a n s i t p e p t i d e f r o m the p a s senger protein, w h i c h t h e r e b y allowed the t r a n s i t p e p t i d e to a s s u m e its native c o n f o r m a t i o n . I f the h y p o t h e s i s o f W a s m a n n et al. [12] is correct, a n d the s e g m e n t o f the m a t u r e p e p t i d e f u n c t i o n s as a s p a c e r to keep the t r a n s i t p e p t i d e r e m o v e d f r o m t h e p a s senger protein, t h e n t h e 14 a a s e g m e n t in T P S P C P represents a n i n a d e q u a t e spacer. However, the 117 a a o f m a t u r e p e p t i d e p r e s e n t in T P S S C P s h o u l d be sufficiently l o n g to fulfill any s p a c e r requirements.
F u r t h e r m o r e , b e c a u s e T P S S C P c o n t a i n s o n l y sequences w h i c h were i m p o r t e d in T P S S o r T P C P , this suggests t h a t i m p o r t c o m p e t e n c e o f a p r o t e i n is n o t a s i m p l e f u n c t i o n o f p r i m a r y sequence, b u t it also i n f l u e n c e d b y p r o t e i n c o n f o r m a t i o n . O n e way this c o u l d o c c u r is if t h e p r e c u r s o r p r o t e i n folds in such a way t h a t the transit p e p t i d e is sterically h i n d e r e d a n d c a n n o t interact with the i m p o r t a p p a r a t u s . A n o t h e r p o s s i b i l i t y is t h a t i n t e r a c t i o n s between distal segments o f the c h i m e r i c p r o t e i n m a y i n h i b i t u n f o l d ing o r o t h e r c o n f o r m a t i o n a l changes which m a y be n e c e s s a r y for i m p o r t . W h a t e v e r the e x p l a n a t i o n , the results p r e s e n t e d here d e m o n s t r a t e t h a t t h e presence o f a transit p e p tide is n o t always sufficient to cause the i m p o r t o f a p r o t e i n into c h l o r o p l a s t . Moreover, it is likely t h a t s e c o n d a r y a n d t e r t i a r y s t r u c t u r a l features o f the entire p r e c u r s o r p r o t e i n are i m p o r t a n t for i m p o r t o f p r o t e i n s into chloroplasts.
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18 9. Schreier P, Seftor EA, Schell J, Bohnert H: The use of nuclear-encoded sequences to direct the light-regulated synthesis and transport of a foreign protein into plant chloroplasts. EMBO J 4:25-32 (1985). 10. Smeekens S, van Steeg H, Bauede C, Bettenbroek H, Keegstra K, Weisbeek P: Import into chloroplasts of a yeast mitochondrial protein directed by ferredoxin and plastocyanin transit peptides. Plant Mol Biol 9:377-388 (1987). 11. van den Broeck G, Timko PM, Kausch AP, Cashmore AR,
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