Jun 12, 1991 - a thylakoid transfer domain is not enough for routeing and proper processing. Only when the complete thylakoid lumen precursor plastocyanin ...
The EMBO Journal vol.10 no.10 pp.2765-2772, 1991
Protein targeting towards the thylakoid lumen of chloroplasts: proper localization of fusion proteins is only observed in vivo Douwe de Boer1'2, Hans Bakker, Alice Lever, Tjeerd Bouma, Elma Salentijn and Peter Weisbeek Department of Molecular Cell Biology and Institute of Molecular Biology, University of Utrecht, PO Box 80.056, 3508 TB Utrecht, The Netherlands
'Present address: Agrotechnological Research Institute (ATO), PO Box 17, 6700 AA Wageningen, The Netherlands 2Corresponding author Communicated by L.L.M.van Deenen
Routeing of fusion proteins to the thylakoid lumen of the chloroplast was compared in vitro and in vivo. The Escherichia coli protein ,B-lactamase was used as a passenger to study this intraorganellar sorting process. The first step, translocation of 3-lactamase into the chloroplast stroma, occurs properly both in vitro and in vivo and is dependent on the presence of a transit peptide in the protein construct. The second step, targeting towards the thylakoid lumen, is more complicated as was also observed previously when other passenger proteins were used. In vitro, the presence of a thylakoid transfer domain is not enough for routeing and proper processing. Only when the complete thylakoid lumen precursor plastocyanin was fused to ,B-lactamase was the fusion protein processed adequately, but routeing was still incomplete. However, in vivo, the information present in the thylakoid transfer domain was the only requirement for proper transport towards the thylakoid lumen. These data show that in vivo, the only requirement for targeting of passenger proteins towards the thylakoid lumen is the presence of a transit peptide and a thylakoid transfer domain. Furthermore, we demonstrate that the in vitro import system does not necessarily reflect the in vivo situation with respect to intraorganellar sorting. Key words: intraorganellar sorting/f-lactamase/plastocyanin/ protein transport/thylakoid lumen
Introduction Chloroplast proteins that are nuclear-encoded are synthesized in the cytoplasm, usually as higher molecular weight precursor proteins with an N-terminal transit peptide (for reviews see Keegstra et al., 1989; De Boer and Weisbeek, 1991). Cytosolic factors are sometimes (Waegemann et al., 1990) but not always (Pilon et al., 1990) required. They assist in the first stages of the import process, supposedly by preventing aggregation and by keeping the protein in an import-competent conformation. The precursor proteins bind to the chloroplast envelope most probably to a receptor protein (Cline et al., 1985; Pain et al., 1988; Schnell et al., 1990). Binding to the outer envelope membrane and also the actual translocation process require ATP (Olsen et al., Oxford University Press
1989; Theg et al., 1989). The transit peptide is removed by the stromal processing peptidase after translocation into the chloroplast (Robinson and Ellis, 1984). In the stroma, the imported proteins usually associate with a heat-shock chaperonin-60 complex (hsc-60), which assists in proper folding of the protein or assembly into enzyme complexes (Lubben et al., 1989). This default import pathway to the stroma may be followed by a routeing step towards one of the other chloroplast compartments, e.g. the thylakoid membrane or the thylakoid lumen. Routeing towards the thylakoid membrane is mainly studied with the light harvesting complex II protein. The protein is synthesized with a transit peptide, which is only required for translocation into the chloroplast (Lamppa, 1988; Van den Broeck et al., 1988; Hand et al., 1989). The thylakoid membrane targeting information is present in the mature part of the precursor protein (Viitanen et al., 1988; Kohorn and Tobin, 1989). Three membrane spanning domains are present in the mature protein and the most C-terminal one is essential for insertion (Karlin-Neumann et al., 1985; Kohorn and Tobin, 1989). Thylakoid lumen proteins like plastocyanin require a thylakoid transfer domain next to the transit peptide to enter the lumen (Smeekens et al., 1985, 1986). This routeing signal is not present in the mature part, but it is part of the cleavable extension of the precursor. Proteins that reside in the thylakoid lumen are routed to this compartment in a two step process (Smeekens et al., 1986; James et al., 1989; Ko and Cashmore, 1989). First, the transit peptide is removed after translocation into the chloroplast and an intermediate sized protein is found in the stroma. Next, the intermediate is translocated into the thylakoid lumen and processed to the mature size by the thylakoidal processing peptidase (Hageman et al., 1986; Kirwin et al., 1987, 1988). Although the thylakoid transfer domain was shown to be essential for routeing towards the thylakoid lumen, it is not always sufficient for thylakoid translocation. Passenger proteins fused to the targeting sequences of thylakoid lumen Table I. Fusion proteins used to study thylakoid lumen targeting
Targeting signal
Mature protein
Localization within chloroplast
Plastocyanin Plastocyanin Plastocyanin
DHFR ferredoxin SOD DHFR GO SSU
stroma stroma stroma
b
thylakoid lumen
d,
stroma
d d
33 kDa OEC 33 kDa OEC 33 kDa OEC
stroma/thylakoid
Reference a
c
e
lumen
OEC, SSU, GO, DHFR and SOD are the oxygen evolving complex, small subunit of Rubisco, glycolate oxidase, dihydrofolate reductase and superoxide dismutase, respectively. aHageman et al. (1990); bSmeekens et al. (1986); cSmeekens et al. (1987); dKo and Cashmore (1989); eMeadows et al. (1989).
2765
D.de Boer et al.
proteins were not routed to the thylakoid lumen in in vitro import experiments, apart from one exception (Table I). The proteins were arrested in the stroma as intermediate sized molecules. Only the targeting signal of the 33 kDa subunit of the oxygen evolving complex was able to mediate routeing of dehydrofolate reductase (DHFR) and a small amount of the small subunit of Rubisco (ssu) to the lumen (Ko and Cashmore, 1989). Alternatively, when the plastocyanin mature protein was fused to the transit peptide of the stromal protein ferredoxin and used in in vivo import studies, a small amount of plastocyanin was found in the thylakoid lumen (De Boer et al., 1988). These results together support the view that additional information for routeing may be present in the mature part of thylakoid lumen proteins. To study thylakoid lumen targeting in more detail, the mature part of the E. coli 3-lactamase protein was used as a passenger in a set of fusion proteins. The protein is a suitable passenger as it is normally translocated across membranes and because it has an enzymatic activity that can easily be assayed. The chloroplast import and routeing properties of the protein were studied both in vitro and in vivo.
was constructed to determine whether the 3-lactamase mature protein itself is capable of translocation across the chloroplast envelope. In vitro transcription of this plasmid and translation of the RNA in a wheatgerm system results in a 29.7 kDa protein. This protein, a fusion between mature ,3-lactamase and a few amino acids of the plastocyanin protein, is not imported into isolated pea chloroplasts (data not shown). In all other fusion proteins mature 3-lactamase is at least fused to a chloroplast transit peptide (Figure 1). The ferredoxin -f3-lactamase fusion protein (FDBLA) has a molecular weight of 34.1 kDa and contains the ferredoxin transit peptide and a few amino acids of the C-terminal end of mature ferredoxin in front of f-lactamase. These amino acids are present to ensure proper conformation around the processing site. The FDBLA fusion protein was shown to be converted into a processed form during a time course experiment with pea chloroplasts (Figure 2A). The apparent molecular weight of the processed form is close to that expected (29.2 kDa) for the normally processed fusion protein. Chloroplast fractionation experiments (Figure 2B) showed that this processed protein is localized in the stroma and that there is no interaction with the thylakoids. The other fusion proteins containing either a ferredoxin transit peptide or a transit peptide derived from plastocyanin are all imported normally into pea chloroplasts in vitro (see below). Import into chloroplasts of mature 3-lactamase therefore only requires the presence of a transit peptide. Three fusion proteins were constructed to determine whether the thylakoid transfer domain of plastocyanin is sufficient for translocation across the thylakoid membrane (Figure 1). The first, PCBLA, is a 35.7 kDa fusion protein
Results A thylakoid transfer domain is not sufficient for translocation of 3-lactamase into the thylakoid lumen in vitro
Several transcription plasmids coding for 3-lactamase fusion proteins were constructed to study routeing inside the chloroplast (Figure 1). One transcription plasmid, pTBLAI,
A
pTBLA 1
BLA
pTFB 2
FDBLA
pTPB 5
PCBLA
B-lactamase mature FD transit peptide M PC transit peptide E thylakoid transfer domain
[
=
FD or PC mature
E_ _
6 (11)PCBLA
pTPB 11-1
B
M
FUSION PROTEINS
PLASMIDS
t
pTFPB 2
FPBLA
pTPLB 51
pPCBLA
_
BLA
1 2 3 4 (1)(2)(3)(4)(5) accatggccgccgaggtcttgcGTCACCCAGAAACGCTGG m a a q v 1 r H P Q T L
FDBLA
v 1 2 3 4 (2)(3)(4)(5) agagtgactgcaatggccacatacaagCCAGAAACGCTGG r v t a m a t y k P Q T L
(1)(2)(3)(4)(5) v1 2 3 4 .i(11) PCBLA gccatggccgccgaggtcttgcGTCACCCAGAAACGCTGG
PCBLA
FPBLA
pPCBLA
a
m
a
a
q
v i
r
H
P
Q
T
L
90 91 92 93 94 95 96 97 98(2) (3) (4) (5) gctggtatggtaggaaaagttaccgttCCAGAAACGCTGG a
g
m
v
g
k
v
t
v
P
Q
T
L
Fig. 1. f-lactamase fusion proteins and transcription plasmids. (A) The different fusion proteins are schematically drawn with the separate domains of the targeting signals indicated. Processing sites are marked by arrows. The names for the corresponding transcription plasmids are also given. (B) The DNA and protein sequences around the fusion points with the 3-lactamase sequence are given. Nucleotides and amino acids derived from flactamase are capitalized. Numbers refer to the corresponding amino acids found in the ferredoxin or mature plastocyanin protein. Numbers between brackets refer to the corresponding amino acids in mature f-lactamase.
2766
Thylakoid lumen targeting of fusion proteins
with the targeting signal of plastocyanin and a few amino acids of the C-terminal end of the plastocyanin mature protein fused to mature 3-lactamase. The additional amino acids are present to ensure proper conformation around the second processing site. The second, b(( I)PCBLA is similar to PCBLA but has a deletion of 11 amino acids in the thylakoid transfer domain. The third, FPBLA, is also similar to PCBLA except that the plastocyanin transit peptide is exchanged for the ferredoxin transit peptide. All three fusion proteins were converted into processed forms during in vitro time course experiments, as is shown for PCBLA (Figure 2A). However, the apparent molecular weights of the processed products are comparable to intermediate sized proteins that have been processed at the first processing site only. Mature sized products processed at the second processing site were not found. The intermediate sized processed product was localized in the stroma, as is shown for PCBLA in the fractionation experiment in Figure 2B. Taken together these data show that in the case of ,B-lactamase, the thylakoid transfer domain of plastocyanin is not sufficient in vitro to direct a passenger protein to the thylakoid lumen. The presence or absence of a thylakoid transfer domain has no influence on the behaviour of the fusion protein inside a chloroplast. All three fusion proteins A
PCH L 1A
FDBLA
.
wg 0
1
_ -
p _m
2 5 5 10 20
_
_
~
w
c
t
1
2 5
55!
B LA
PCBLA
FDBLA
protease
0
wc
dp
~
t
f-
_
~
~
S
.
_
~
t
4-
Fig. 2. In vitro import and processing of the FDBLA and PCBLA fusion proteins. (A) Import time course of the FDBLA and PCBLA fusion proteins. The translation mixtures containing the fusion proteins were incubated with chloroplasts in the light. At the indicated time points, samples were taken and the import reactions were stopped. wg = wheat germ translation mixture before incubation with chloroplasts. p, i and m, are precursor, intermediate and mature proteins respectively. Molecular weight markers are in kDa. (B) Chloroplast localization of FDBLA and PCBLA after import. Translation mixtures were incubated with chloroplasts for 10 min in the light. c, s and t, are total chloroplast, stroma and thylakoids respectively. wg, as indicated above. Protease treatment is indicated. Stroma and thylakoid fractions are isolated from protease treated chloroplasts. Molecular weight markers as above. (C) In vitro processing of FDBLA and PCBLA with stromal (s) and thylakoidal (t) extracts. Translation products were incubated with the extracts for 1 h at 27°C. p, i and m, as indicated above.
tested were only processed at the first processing site and the intermediate sized protein was not routed within the chloroplast towards its proper location. The PCBLA fusion protein is processed at both processing sites in in vitro processing experiments The inability of PCBLA to be routed to the thylakoid lumen could be explained by assuming that the second processing site is not available for processing because it is shielded by the rest of the protein. Therefore we determined whether the PCBLA fusion protein could be processed in vitro by crude extracts of the stromal and the thylakoidal processing peptidases (Figure 2C). The FDBLA fusion protein was used as a control. This protein could only be processed by the stromal extract and not by the thylakoidal extract. The apparent molecular weight of the stromal processing product was identical to the one found in the import experiments. The absence of processing of FDBLA by the thylakoidal extract was expected because there is no second processing site present, but it also indicates that the stromal processing peptidase (SPP) was not active in the thylakoidal extract. The PCBLA fusion protein was processed by the stromal extract into the intermediate sized product that was also
observed during the import experiments (Figure 2C). The thylakoidal extract was able to convert the fusion protein into a smaller product with an apparent molecular weight comparable to the expected molecular weight for a mature sized protein (29.5 kDa). These in vitro processing experiments confirm the nature of the products found during the import experiments. In in vitro import experiments, FDBLA and PCBLA were only processed by the SPP. However, the thylakoidal processing peptidase (TPP) is able to process the second site in vitro. This indicates that the absence of processing at the second processing site and improper routeing during the in vitro import experiment is probably not caused by a conformation of the protein that is incompatible with processing. Proper processing at the second processing site and routeing to the thylakoids occurs in vitro when the complete precursor of plastocyanin is present in front of mature 3-lactamase A transcription plasmid (pTPLB51) with the coding sequence for the complete plastocyanin precursor in front of that for the f-lactamase mature protein was constructed (Figure 1). This plasmid was made to determine whether information in mature plastocyanin was necessary for targeting to the thylakoid lumen of a passenger protein. Plasmid pTPLB51 codes for a fusion protein (pPCBLA) of 426 amino acids with a molecular weight of 45.3 kDa. The expected molecular weights after processing at the first and the second processing site are 41.2 and 39.0 kDa, respectively. Figure 3 shows an import time course and a chloroplast fractionation with this fusion protein. The time course (Figure 3A) shows that the precursor is converted via an intermediate sized protein into a mature sized product. The time course data of Figure 3A are complicated because two different events lower the molecular weight of the precursor protein. First, the precursor band is shifted because of the presence of high amounts of the large subunit of Rubisco which runs at the same apparent molecular weight. Second, the precursor is reduced in molecular weight due to
2767
D.de Boer et al.
processing at the first and the second processing sites. The intermediate sized protein fractionates into the stroma fraction and the mature sized product in the thylakoid fractions (Figure 3B). The processed mature sized product is not stable and the maximum amount inside the chloroplast is reached after 5 min. At higher time points smaller products appear, which are probably degradation products of the mature sized protein. The products of mature size and the degradation products are not protected against protease treatment of the thylakoids from the outside and therefore are not present in the thylakoid lumen. Probably only the plastocyanin portion of the fusion protein crossed the membrane, still attached to the 3-lactamase in the membrane. Table II summarizes the in vitro import experiments. From these data it is obvious that the presence of mature plastocyanin in addition to the two-domain targeting signal in the fusion proteins results in processing at the second domain and interaction with the thylakoid membrane. However, despite the presence of the complete plastocyanin precursor, ,B-lactamase is not translocated into the thylakoid lumen completely in vitro. This again shows that the requirements for routeing inside the chloroplast are more difficult to define than translocation into the chloroplast.
Plants with the highest expression of RNA were analysed for expression of the fusion proteins. The enzymatic activity of f3-lactamase was used, since the activity was not lost in the various fusion proteins. There is almost no background activity for 3-lactamase present in plants. PADAC, a purplecoloured substrate analogue for 3-lactamase, which is normally used to detect 3-lactamase activity in prokaryotes (Kobayashi et al., 1988), was incubated with total protein isolated from the transgenic plants. When a 3-lactamase fusion protein was present, a colour change to yellow was observed. Figure 5 shows that for all different constructs the enzymatic activity in the transgenic plants was high enough to be easily detected within 10 min. However, the level of the enzymatic activity was variable. This was due to differences in the expression level of the protein, but also to differences of 3-lactamase activity depending on the fusion protein (data not shown). to the thylakoid lumen in vivo when a thylakoid transfer domain is present next to the transit peptide Chloroplasts were isolated from the six different transgenic plants and (-lactamase activity was measured. When information for a transit peptide was present in the construct used
fl-lactamase is properly routed
,3-lactamase fusion proteins are expressed in transgenic tobacco plants Since routeing studies turned out to be difficult to interpret in vitro, we decided to use the same constructs to study routeing in vivo, namely in transgenic plants. The coding sequences for all six 3-lactamase fusion proteins were cloned behind the cauliflower mosaic virus 35S promoter in the binary plant transformation vector pBINlg as outlined in Figure 4. Tobacco plants were transformed with these constructs (see Materials and methods) and several kanamycin resistant plants per construct were analysed for expression of RNA. Although the constructs did not contain plant-like polyadenylation signals, RNA could be detected (data not shown).
Table II. Localization of 3-lactamase fusion proteins after in vitro import into pea chloroplasts Fusion protein Chloroplast fraction Stroma Thylakoid membrane Thylakoid lumen BLA FDBLA b( 1 )PCBLA PCBLA FPBLA pPCBLA
mature mature
-
-
-
intermediate intermediate intermediate mature
~~T-DNA
i
mcs
A
{:::=f vHE=Ie~ ',-,I" ~
B
~
97
-
66 2 -
H '
p
42.7
35S-promotor --W
pBBLA7, pBFB 24 pBPB 12
-
pBPB53 wq
0
1
2.5 5 10 20 protease:
wg
c
c
t
pBFPB64
+
+
pBPLB83
Fig. 3. In vitro import of the pPCBLA fusion protein. (A) Import time course of the pPCBLA fusion protein. The translation mixture containing the fusion protein was incubated with chloroplasts in the light. At the indicated time points samples were taken and the import reaction was stopped. wg = wheatgerm translation mixture before incubation with chloroplasts. Molecular weight markers are in kDa. (B) Chloroplast localization of pPCBLA after import. The translation mixture was incubated for 7 min in the light. Experimental procedures and abbreviations are as indicated in Figure 2. Molecular weight markers as above. 2768
I
I
fusion gene
plasmid 31 (
(pBIN19)
~
fusion protein BLA FDBLA
PCBLA ,(1 1)PCBLA FPBLA pPCBLA
Fig. 4. Constructs used for expression of t3-lactamase fusion proteins in transgenic tobacco plants. The T-DNA region of the plant transformation vector pBIN19 is schematically shown. The different coding regions for the ,B-lactamase fusion proteins are clones behind the cauliflower mosaic virus 35S promoter in the EcoRI site of the multiple cloning site (mcs) of pBIN19. Details of the cloning are described in Materials and methods. The names of the constructed plasmids are given together with the fusion protein they encode. Arrows indicate the direction of transcription. NPT, H and E, are neomycin phosphotranspherase gene, HindIlI and EcoRI respectively.
Thylakoid lumen targeting of fusion proteins
for transformation, 3-lactamase activity was found inside the chloroplasts as was also found in vitro. Almost all activity present in a total cell lysate was detected in the chloroplast, showing efficient targeting (not shown). No enzymatic activity was localized in the chloroplast when a transit peptide was absent from the fusion protein, although activity was clearly present in a total cell lysate. Chloroplasts containing enzymatic activity were fractionated and the different fractions were assayed. Figure 6 shows a histogram of the activities per fraction relative to the total activity present in the chloroplasts. The FDBLA fusion protein is found mainly in the stromal fraction, whereas -20 % is associated with the thylakoids. The amount of 3-lactamase that is found associated with the thylakoids could be the result of fractionation artefacts but, alternatively, it is possible that the f-lactamase protein itself interacts with the thylakoid membrane. Most of the activity detected in the thylakoid fraction could be removed by protease treatment of the intact thylakoids. A similar fractionation result is observed for the ( I1 )PCBLA fusion protein, although the absolute amounts of enzymatic activity are lower than with FDBLA. Both fusion proteins contain a transit peptide and therefore are imported by the default pathway, but they lack an intact thylakoid transfer domain required for further routeing to the thylakoid lumen. In contrast to the results in vitro, the fusion proteins PCBLA and FPBLA, both containing an intact plastocyanin thylakoid transfer domain next to the transit peptide, are localized mainly in the thylakoids in vivo. Most of the enzymatic activity is protease resistant and fractionates into the thylakoid lumen. When the thylakoid lumen fraction is treated with protease, almost all enzymatic activity disappears. This clearly shows that in vivo, a passenger protein is routed to the thylakoid lumen when the thylakoid transfer domain is present next to the transit peptide. It is likely that the large amount ( - 30%) of protein found in the stromal fractions is the result of fractionation artefacts, since thylakoids isolated from intact tobacco plants tend to be more labile than thylakoids isolated from developing pea plants. We observed previously that a similar amount of the thylakoid lumen contents is released into the stromal fraction during fractionation of chloroplasts isolated from transgenic tomato and tobacco plants (D.de Boer, unpublished observations). Western blotting with antibodies against f-lactamase showed that the ,B-lactamase protein in the stromal fraction is of mature size (not shown), indicating that this protein is indeed probably released from the thylakoid lumen during fractionation. The fusion protein pPCBLA, with the complete plastocyanin precursor in front of f-lactamase, also fractionates in the thylakoids, but most of it is retained in the membranes. Although in vitro the protein was only found in the thylakoid membrane, - 38 % was found in the thylakoid lumen fraction in vivo. To exclude the possibility that the observed differences in localization in vitro and in vivo only resulted from the difference in the detection method, we also determined the enzymatic activities of the different sub-fractions of pea chloroplasts after in vitro import of the different constructs. The results were identical to the results that were obtained with autoradiography (not shown); none of the constructs was able to target 3-lactamase to the thylakoid lumen in vitro. Table III shows that there are distinct differences between in vitro and in vivo import results with respect to routeing of passenger proteins within the chloroplast. Although in both
systems the results of import into chloroplasts are comparable, the in vitro system does not reflect the in vivo situation with respect to proper routeing of passenger proteins.
Discussion We have analysed, both in vitro and in vivo, the import of proteins into chloroplasts and routeing towards the thylakoid lumen. Our results show that routeing within the chloroplast is more complicated than import into this organelle. Import
Cl) . 1.00
control
-
,8(1 1 )PCBLA
c
o r-
pPCBLA FDBLA
2:,
m
BLA
o
S
0.80
PCBLA FPBLA
0.60 6
4
2
0
12
10
8
14
time (min) fusion proteins in different of the Fig. 5. The 3-lactamase activity transgenic plants. Total protein was isolated from leaf tissue of transgenic tobacco plants transformed with the constructs described in Figure 4. The enzymatic activity of the 3-lactamase fusion proteins was determined by incubating comparable amounts of total protein with 500 AM PADAC (Calbiochem) in 30 mM KPO4, pH 7.5 at room temperature in a volume of 1 ml. The turnover of the substrate PADAC (Kobayashi et al.. 1988) was measured spectrophotometrically at 570 nm for the time indicated. The initial optical density was arbitrarily put to 1.0. Stock solutions of PADAC were made as described by Kobayashi et al. (1988). Total protein of transgenic plants was isolated as described previously for tomato plants (De Boer et al., 1988). Total protein isolated from transgenic tobacco plants transformed with pBINl9 was used as a control. FDBLA
b (11)PCBLA 100
001 _ 79.2
0:50 2
-
ae
20.8
o S
C
0
T
TM
TL
C
S
T
TM
TL
1930a00
100
53.8
°
3B50
° 350
S
T
TM
TL
50.3
3.
307
C
3 °
25.8
-
FPBLA 100
PCBLA 1
pPCBLA
74.5
'a
C
S
T
TM
TL
88.8 '-
100
U
51.0
*s
37.6
0
C
S
T
TM
TL
Fig. 6. Localization of 3-lactamase activity inside the chloroplast. Chloroplasts from transgenic tobacco plants transformed with the different 3-lactamase constructs were isolated and 3-lactamase activity was measured as outlined in Figure 5. Chloroplasts of the different plants were also fractionated and the activity per fraction was determined. The activity of the various fractions is given as a percentage of the total activity found in the chloroplast. Numbers above the bars refer to the percentage of that fraction. Chloroplast isolations and fractionations are performed as outlined for pea chloroplasts in Materials and methods. C, S, T, TM and TL are total chloroplast, stroma, total thylakoid. thylakoid membrane and thylakoid lumen respectively.
2769
D.de Boer et al. Table III. Comparison of in vitro and in vivo localization of B-lactamase fusion proteins Fusion protein
Localization of fusion protein In vitro In vivo
BLA FDBLA b(1 1)PCBLA PCBLA FPBLA pPCBLA
chloroplast stroma chloroplast stroma chloroplast stroma chloroplast stroma thylakoid membranea
cytoplasm chloroplast stroma chloroplast stroma thylakoid lumen thylakoid lumen thylakoid membrane/lumen
aAn intermediate sized processing product was found in the stroma
into chloroplasts of passenger proteins only requires a transit peptide, but the requirements for routeing are more difficult to define. The in vivo analyses showed that information in the thylakoid transfer domain was sufficient for routeing. However, in vitro, the presence of this domain was not enough for transport of 3-lactamase to the lumen, and it was suggested that additional information was present in the mature protein. All our fusion proteins were imported into chloroplasts when a transit peptide was present. A similar behaviour was reported previously for 30 different fusion proteins containing a transit peptide; most studies concerned an in vitro situation (for an overview see de Boer and Weisbeek, 1991). The presence of a transit peptide therefore is the most important requirement for the entry of a protein into the chloroplast. The in vivo import studies with the ,B-lactamase fusion proteins show that proper routeing is achieved when the thylakoid transfer domain of plastocyanin is present next to the transit peptide. Destroying the thylakoid transfer domain, as is the case with fusion protein b(1 1)PCBLA, abolishes routeing. Further, the presence of the mature part of plastocyanin is not required. In fact, when the mature part was present, less ,B-lactamase was found in the lumen and a considerable amount was found to be arrested in the thylakoid membrane. The presence of sufficient information in the thylakoid transfer domain for routeing of passenger proteins was suggested by the proper routeing in vitro of the 33 kDa subunit-DHFR fusion protein (Ko and Cashmore, 1989; Meadows et al., 1989). The absence of proper routeing in vitro of all the other fusion proteins, however, made this conclusion questionable. The observed absence of proper routeing of other fusion proteins was probably not caused by the lack of information in the different constructs, but by the in vitro import system. The difference between the in vitro and in vivo situations was clearly observed in this study with the ,B-lactamase fusion proteins. Since the same constructs were used both in vitro and in vivo, the same amount of routeing information was present in both systems. The fusion proteins PCBLA and FPBLA were routed towards the thylakoid lumen in vivo. However, in vitro they were arrested in the stroma, processed at the first processing site only. This shows that the in vitro system does not reflect the in vivo situation, implying that pea chloroplasts used in vitro are not suitable for studying the natural routeing within the organelle. Chloroplasts that are used to study protein transport in vitro are isolated from pea seedlings several days after germina-
2770
tion. It is possible that the developmental stage of the pea plants is not optimal for proper routeing. Similar observations were made for routeing of the LHCP-I protein towards the thylakoid membrane (Chitnis et al., 1986, 1987, 1988). We cannot exclude the possibility that species differences account for the observed dissimilarities. Authentic thylakoid lumen proteins like plastocyanin and subunits of the oxygen evolving complex are properly routed in vitro (Smeekens et al., 1986; James et al., 1989). It is possible that the presence of the mature protein stimulates translocation across the thylakoids in vitro, maybe by binding stromal factors that keep the proteins unfolded. For reasons unknown, one fusion protein, the 33 kDa subunit-DHFR fusion protein, is also properly targeted to the lumen in vitro (Ko and Cashmore, 1989; Meadows et al., 1989), whereas a fusion of the DHFR protein with the plastocyanin targeting signal did not result in thylakoid membrane translocation
(Hageman et al., 1990). When the complete plastocyanin precursor was present in the f3-lactamase fusion protein, the in vitro and in vivo routeing behaviour differed from that of the fusion proteins PCBLA and FPBLA. In vitro, this extended fusion protein was properly processed at both processing sites and was routed to the thylakoid membrane. Since the active site of the TPP is located at the luminal side of the thylakoid membrane (Kirwin et al., 1988), we expect that at least part of the mature sized fusion protein was translocated over the membrane. Although this result seems to suggest that essential information for routeing is present in the mature part of the plastocyanin protein, the in vivo transport studies with PCBLA and FPBLA show that this is unlikely. We assume that the routeing information that is present in the thylakoid transfer domain is presented more naturally when the mature plastocyanin protein is present. Hypothetically this could be achieved when a stromal factor binds to mature plastocyanin and keeps the protein in a partially unfolded conformation. The presence of the mature plastocyanin protein, however, also results in arrest of the fusion protein in the thylakoid membrane, both in vitro and in vivo. Although the exact cause is not known, it is possible that the energy that is generated for translocation across the thylakoid membrane is not sufficient to translocate this large fusion protein. Taken together, these results show that the presence of the complete precursor in the fusion does not optimize routeing in vivo, but gives rise to additional problems, probably relating to the greater length of the fusion. Previously we have shown that in transgenic tomato plants in vivo, a fusion protein containing the ferredoxin transit peptide and mature plastocyanin was localized in the thylakoid lumen, although the amount present was low (de Boer et al., 1988). We explained these results by assuming that some information for routeing is present in the mature part of plastocyanin. The results presented here show that information in the mature protein is not essential for routeing, but they do not exlude the presence of additional nonessential information. When this previously described FDPC fusion protein was expressed in transgenic tobacco plants, we did not find any protein product, although the RNA was present and could be translated in vitro (D.de Boer, unpublished results). In both transgenic tomato and tobacco plants, plastocyanin was expected to be directed to the stroma by the ferredoxin transit peptide. The absence of plastocyanin in this compartment in vivo can be explained if a high
Thylakoid lumen targeting of fusion proteins
of plastocyanin in this compartment is assumed. The small amount that is found in the thylakoid lumen of transgenic tomato plants might be the combined result of aspecific transport and a high stability in the lumen. Two major conclusions can be drawn from the results presented here. First, the in vitro import system with pea chloroplasts does not properly reflect the in vivo situation with respect to routeing within the organelle. Second, information present in the two-domain targeting signal of thylakoid lumen proteins is sufficient for routeing of passenger proteins to the thylakoid lumen in vivo. turnover
Materials and methods Cloning and sequencing procedures The Ecoli strain PC2495 (Van der Plas et al., 1989) was used for both cloning and sequencing procedures. Enzymes were obtained from New England Biolabs, Boehringer Mannheim and BRL. Plasmid preparations, restriction enzyme digestions, DNA electrophoresis, isolation of DNA fragments from low melting point agarose, Klenow treatment, SI nuclease treatment, ligations, transformation of bacterial cells and DNA sequencing were performed as previously described (Maniatis et al., 1982; Smeekens et
al., 1986).
Construction of transcription plasmids coding for 3-lactamase fusion proteins The mature part of the ,B-lactamase coding sequence was isolated from the plasmid pTG2, a pBR322 analogue (Kadonaga et al., 1984). pTG2 was first digested with HaeII and the 3' overhang was removed by treatment with Klenow polymerase. The coding sequences for the signal sequence and the mature protein were separated by digestion with BstEII. The 1876 bp fragment containing the sequence of the mature ,B-lactamase was isolated after electrophoresis in low melting point agarose. The fragment was cloned into the plasmid pSPFD22 (Smeekens et al., 1986) from which the mature ferredoxin sequence had been removed after BstEII and SmaI digestion, resulting in the plasmid pTKBI. The ferredoxin transit peptide coding sequence was fused in frame with the mature 3-lactamase sequence by digestion of pTKBI with BstEII, removal of the sticky ends by SI nuclease treatment and subsequent ligation. The resulting in vitro transcription plasmid with the coding sequence of the ferredoxin ,B-lactamase fusion protein was called pTFB2. A construct with the coding sequence of the two domain plastocyanin targeting signal fused in frame in front of the mature ,B-lactamase coding sequence was made as follows. pTKBI was digested with BstEl 1, the 5' overhang was filled in with Klenow and the ferredoxin transit peptide coding sequence was removed after HindIII digestion. The plastocyanin sequence was isolated from pSPPC74 (Smeekens et al., 1986) after AvaI digestion, S1 nuclease treatment and HindIll digestion and cloned into the vector fragment of pTKBI, resulting in the transcription plasmid pTPB5. A transcription plasmid (pTBLA1) coding for the f-lactamase protein without a targeting signal was derived from pTPB5 by isolating the coding sequence for mature 3-lactamase as an NcoI -EcoRI restriction fragment and inserting this fragment in the vector part of the NcoI-EcoRI digested plasmid pTPAP14 [the pTPAP14 plasmid is similar to pSPPC74 except that the NcoI site at the beginning of the mature protein has been removed and a new NcoI site is put in front of the precursor (de Boer, in preparation)]. A transcription plasmid with the complete coding sequence of the plastocyanin precursor in front of mature ,B-lactamase (pTPLB5 1) was constructed as follows. The plasmid pTKBI was digested with BstEII and treated with SI nuclease to remove the sticky ends. The fragment with the mature coding sequence of 13-lactamase was isolated after digestion with EcoRI. This fragment was cloned into the vector portion, with the coding sequence of the plastocyanin precursor, of the plasmid pPAP14 digested with HpaI and EcoRI. Two additional transcription plasmids were made, one containing the coding sequence for the two domain targeting signals of plastocyanin with a deletion of 11 amino acids in the second domain fused to the mature coding sequence of ,B-lactamase (pTPBb I l) and one with the coding sequences of the ferredoxin transit peptide and the plastocyanin thylakoid transfer domain in front of mature ,B-lactamase (pTFPB2). Plasmid pTPB6,1 - was made by cloning the NcoI-EcoRI fragment, with the mature 3-lactamase coding sequence, of pSPB5 into the NcoI-EcoRI digested vector fragment of plasmid pPCdel54-64 (Hageman et al., 1990). The plasmid pTFPB2 was similarly made but pFD-40PC (Hageman et al., 1990) was used instead of pPCdel54-64.
Isolation of chloroplasts and import experiments Pea chloroplasts were isolated from pea seedlings by Percoll centrifugation exactly as described by Smeekens et al. (1986). In vitro transcription of the plasmids with SP6 polymerase and translation of the RNA in a wheatgerm lysate were according to Smeekens et al. (1986). Either [3H]leucine (130 Ci/mmol, Amersham) or [35S]methionine (1300 Ci/mmol, Amersham) was used during the translations. Import reactions, reisolation of chloroplasts and quantifications were performed as described previously (Smeekens et al., 1986). Import reactions were stopped by diluting the samples at 0°C in a buffer containing the ionophore nigericin (Cline et al., 1985), to a final nigericin concentration of 0.2 AM. Stromal and thylakoidal extracts were made as described previously (Hageman et al., 1986). T-DNA plasmid construction and plant transformation The cauliflower mosaic virus 35S promoter was isolated as an EcoRI-HindL fragment from a 35S-CAT plasmid (Morelli et al., 1985). The fusion genes were isolated as HindIII-EcoRI fragments from the different transcription plasmids described above. The HindIlI sites of the different fusion genes were fused with the HindIII site of the promoter fragment and the resulting EcoRI fragments were cloned into the EcoRI site of the plant transformation plasmid pBINl9. Plasmids with an orientation of the EcoRI fragment as indicated in Figure 4 were used for plant transtormation. The different plasmids were conjugated into Agrobacterium tumefaciens LBA4404 containing the modified Ti plasmid pAL4404 (Hoekema et al., 1983) in a triparental mating event using the helper plasmid pRK2013 (Lam et al., 1985). The different constructs were subsequently introduced into the Nicotiana tabacum var. Petit Havana (SRI) using the leaf disc transformation method (Horsch et al., 1985). Regenerated plants were selected for kanamycin resistance.
Acknowledgements We thank Dr J.Tommassen for helpful suggestions, Dr M.Agterberg for making plasmid pTG2 available to us, Dr J.Hageman for his gift of the plasmids pFD-40PC and pPCdelS4-64, and Drs W.Bitter and B.Scheres for critically reading the manuscript. This work was supported in part by the Netherlands Organization for Advancement of Pure Science
(SON/NWO).
References Chitnis,P.R., Harel,E., Kohorn,B.D., Tobin,E.M. and Thomber,J.P. (1986) J. Cell Biol., 102, 982-988. Chitnis,P.R., Nechushtai,R. and Thomber,J.P. (1987) Plant Mol. Biol., 10, 3-11. Chitnis,P.R., Morishige,D.T., Nechushtai,R. and Thornber,J.P. (1988) Plant Mol. Biol., 11, 95-107. Cline,K., Werner-Washburne,M., Gubben,T.H. and Keegstra,K. (1985) J. Biol. Chem., 260, 3691-3696. de Boer,A.D. and Weisbeek,P.J. (1991) Biochim. Biophys. Acta, in press. de Boer,D., Cremers,F., Teertstra,R., Smits,L., Hille,J., Smeekens,S. and Weisbeek,P. (1988) EMBO J., 7, 2631-2636. Hageman,J., Robinson,C., Smeekens,S. and Weisbeek,P. (1986) Nature, 324, 567-569. Hageman,J., Baecke,C., Ebskamp,M., Pilon,R., Smeekens,S. and Weisbeek,P. (1990) Plant Cell, 2, 479-494. Hand,J.M., Szabo,L.J., Vasconcelos,A.C. and Cashmore,A.R. (1989) EMBOJ., 8, 3195-3206. Hoekema,A., Hirsch,P.R., Hooykaas,P.J.J. and Schilperoort,R.A. (1983) Nature, 303, 179-180. Horsch,R.B., Fry,J.E., Hoffman,N.L., Eichholtz,D., Rogers,S.G. and Frayley,R.T. (1985) Science, 227, 1229-1232. James,H.E., Bartling,D., Musgrove,J.E., Kirwin,P.M. and Herrmann,R.G. (1989) J. Biol. Chem., 264, 19573-19576. Kadonaga,J.T., Gautier,A.E., Straus,D.R., Charles,A.D., Edge,M.D. and Knowles,J.R. (1984) J. Biol. Chem., 259, 2149-2154. Karlin-Neumann,G.A., Kohorn,B.D., Thomber,J.P. and Tobin,E.M. (1985) J. Mol. Appl. Genet., 3, 45-61. Keegstra,K., Olsen,L.J. and Theg,S.M. (1989) Annu. Rev. Plant Phvsiol. Plant Mol. Biol., 40, 471-502. Kirwin,P.M., Elderfield,P.D. and Robinson,C. (1987) J. Biol. Chem., 262, 16386- 16390. Kirwin,P.M., Elderfield,P.D., Williams,R.S. and Robinson,C. (1988) J. Biol. Chem., 263, 18128-18132. Ko,K. and Cashmore,A.R. (1989) EMBO J., 8, 3187-3194.
2771
D.de Boer et al. Kobayashi,S., Susumu,A., Shoryo,H. and Takashi,S. (1988) Antimicrob. Agents Chemother., 32, 1040-1045. Kohorn,B.D. and Tobin,E.M. (1989) Plant Cell, 1, 159-166. Lam,S.T., Lam,B.S. and Strobel,G. (1985) Plasmid, 13, 200-204. Lamppa,G.K. (1988) J. Biol. Chem., 263, 14996-14999. Lubben,T.H., Donaldson,G.K., Viitanen,P.V. and Gatenby,A.A. (1989) Plant Cell, 1, 1223-1230. Maniatis,T., Fritsch,E.F. and Sambrook,J. (1982) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Meadows,J.W., Shackleton,J.B., Hulford,A. and Robinson,C. (1989) FEBS Lett., 253, 244-246. Morelli,G., Nagy,F., Frayley,R.T., Rogers,S.G. and Chua,N.-H. (1985) Nature, 315, 200-204. Olsen,L.J., Theg,S.M., Selman,B.R. and Keegstra,K. (1989) J. Biol. Chem., 264, 6724-6729. Pain,D., Kanwar,Y.S. and Blobel,G. (1988) Nature, 331, 232-237. Pilon,M., de Boer,A.D., Knols,S.L., Koppelman,M.H.G.M., Van der Graaf,R.M., De Kruijff,B. and Weisbeek,P.J. (1990) J. Biol. Chem., 265, 3358-3361. Robinson,C. and Ellis,R.J. (1984) Eur. J. Biochem., 142, 337-342. Schnell,D.J., Blobel,G. and Pain,D. (1990) J. Cell Biol., 111, 1825-1838. Smeekens,S., De Groot,M., Van Binsbergen,J. and Weisbeek,P. (1985) Nature, 317, 456-458. Smeekens,S., Bauerle,C., Hageman,J., Keegstra,K. and Weisbeek,P. (1986) Cell, 46, 365-376. Smeekens,S., Van Steeg,H., Bauerle,C., Bettenbroek,H. and Keegstra,K. (1987) Plant Mol. Biol., 9, 377-388. Theg,S.M., Bauerle,C., Olsen,L.J., Selman,B.R. and Keegstra,K. (1989) J. Biol. Chem., 264, 6730-6736. Van den Broeck,G., Van Houtven,A., Van Montagu,M. and HerreraEstrella,L. (1988) Plant Sci., 58, 171-176. Van der Plas,J., Bovy,A., Kruyt,F., De Vrieze,G., Dassen,E. and Weisbeek,P. (1989) Mol. Microbiol., 3, 275-284. Viitanen,P.V., Doran,E.R. and Dunsmuir,P. (1988) J. Biol. Chem., 263, 15000-15007. Waegemann,K., Paulsen,H. and Soll,J. (1990) FEBS Lett., 261, 89-92. Received on March 19, 1991; revised on June 12, 1991
2772
