JXB Advance Access originally published online on June 18, 2004
Journal of Experimental Botany 2004 55(403):1697-1706; doi:10.1093/jxb/erh191
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RESEARCH PAPER |
In vivo transport of folded EGFP by the 
pH/TAT-dependent pathway in chloroplasts of Arabidopsis thaliana
1Institut für Pflanzenphysiologie, Martin-Luther-Universität Halle-Wittenberg, Weinbergweg 10, D-06120 Halle (Saale), Germany
2Biozentrum der Universität, Martin-Luther-Universität Halle-Wittenberg, Weinbergweg 22, D-06120 Halle (Saale), Germany
* To whom correspondence should be addressed. Fax: +49 345 5527095. E-mail: marques{at}pflanzenphys.uni-halle.de
Received 25 February 2004; Accepted 4 May 2004
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Abstract |
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Among the protein translocation pathways of the thylakoid membrane in chloroplasts, the
pH/TAT pathway is unique in several aspects. In vitro transport assays with isolated chloroplasts or thylakoids have defined the trans-thylakoidal proton gradient as the sole requirement for effecting transport. From these studies, evidence has also accumulated indicating that, in contrast to the remaining protein transport pathways present in the thylakoid membrane, the pH/TAT pathway is able to mediate the transport of folded proteins. The present work has established a novel approach to demonstrate the transport of folded proteins by this pathway in vivo. For this purpose, Arabidopsis thaliana plants were stably transformed with gene constructs expressing enhanced green fluorescent protein (EGFP) alone or fused to the transit peptides of different chloroplast proteins under the control of the 35S CAMV promoter. The intracellular and intraorganellar distribution of EGFP in the resulting transformants showed that while all the chloroplast transit peptides efficiently mediated the transport of EGFP into plastids, only those specific for the pH/TAT pathway were able to direct the protein into the thylakoid lumen as well. This could be demonstrated both by fluorescence and immunoelectron microscopy. Analysis of isolated and fractionated chloroplasts using western blot and spectrofluorometric assays confirmed the presence of folded EGFP solely within the thylakoid lumen of these lines. These results strongly suggest that the protein adopts a folded state in the chloroplast stroma and thus, can only be translocated further into the chloroplast lumen by the pH/TAT pathway.
Key words:
Chloroplast, green fluorescent protein (GFP), pH/TAT pathway, protein transport, thylakoid membrane
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Introduction |
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Chloroplasts are structurally complex organelles, which are surrounded by an envelope consisting of two membranes that enclose the soluble stromal phase. In addition, an independent and highly organized membrane system is present, the thylakoids, which further defines a luminal space. Selective protein transport into each of these compartments poses a formidable task for the cell. This is further complicated by the fact that some chloroplast proteins are still encoded in the organelle, while the large majority is encoded in the cell nucleus. Among the latter, the proteins of the thylakoid lumen are of particular interest because they must be transported across both envelope membranes plus the thylakoid membrane during biogenesis. For this purpose, these proteins are synthesized in the cytosol as precursor molecules comprising bipartite transit peptides which carry two transport signals in tandem: an amino terminal envelope transport signal followed by a thylakoid targeting and translocating signal. Depending on the specificity of the thylakoid targeting signal, transport into the thylakoid lumen is known to take place through one of two distinct, protein-specific pathways (reviewed in Klösgen, 1997
; Dalbey and Robinson, 1999). One of these, denominated the Sec pathway by analogy to the bacterial general secretory pathway, depends on the presence of nucleoside triphosphates (NTP) and the chloroplast analogues to the bacterial SecYE membrane complex as well as to the stromal SecA ATPase. The second pathway, called the pH-dependent or twin arginine translocation (TAT) pathway, has several unique features. In higher plants, in vitro transport experiments with isolated chloroplasts have established the transthylakoidal proton gradient as the single energy source required for transport, depending neither on stromal proteins nor on NTPs (Mould and Robinson, 1991; Cline et al., 1992; Klösgen et al., 1992). Recent experiments with Chlamydomonas reinhardtii suggest that, in this organism, protein import of established pH/TAT substrates might even be independent of the pH, requiring in this case an unknown intracellular factor instead (Finazzi et al., 2003). Particularly unusual is the apparent capacity of the pH/TAT pathway to mediate transport of fully folded globular proteins across the membrane bilayer. The first indication for this property was observed after in vitro chloroplast import experiments showing that the 23 kDa subunit of the oxygen-evolving complex of photosystem II (PSII-P), a natural substrate of the pH/TAT pathway, assumes a folded conformation during its passage through the stromal space (Creighton et al., 1995). Supporting evidence was provided by the observation that bovine pancreatic trypsin inhibitor (BPTI) could be transported into thylakoids of isolated chloroplasts when fused to a pH-dependent pathway precursor, even if unfolding of BPTI was prevented by irreversible cross-linking (Clark and Theg, 1997). Similarly, dihydrofolate reductase could only be targeted in vitro to the thylakoid lumen by pH/TAT but not by Sec-type transit peptides when the folded state of the protein was stabilized by methotrexate (Hynds et al., 1998). Finally, it could be shown in such in vitro assays that EGFP (enhanced green fluorescent protein) can only be efficiently translocated across the thylakoid membrane by the pH/TAT pathway, presumably due to folding of the protein within the stroma (Marques et al., 2003).
Although an independent line of evidence for the transport of folded proteins by the TAT pathway has been provided by the analysis of the homologous bacterial pathway in vivo (reviewed in Palmer and Berks, 2003), no direct proof is available so far that clearly demonstrates the capacity of the thylakoidal pH/TAT pathway to translocate folded proteins similarly in vivo. In order to approach this question the authors have established a novel methodological approach based on the generation of Arabidopsis thaliana lines stably expressing protein chimeras consisting of transit peptides from different chloroplast proteins fused to EGFP. This is a variant of the green fluorescent protein (GFP) (Yang et al., 1996), which is widely used as a reporter protein due to its advantageous properties: it is small, it is not of plant origin nor displays any significant homology to plastid proteins, and it has no inherent toxicity to cellular processes (reviewed in Zimmer, 2002). Furthermore, as shown by several stroma-targeting experiments, GFP can be successfully translocated across the chloroplast envelope membrane both in vitro (Marques et al., 2003) and in vivo (Köhler et al., 1997). EGFP combines these characteristics with several improvements over the authentic GFP. These include a higher extinction coefficient (which is reflected by an increased sensitivity), reduced photobleaching, and elimination of a cryptic intron present in the original gene which is efficiently recognized in A. thaliana, thus leading to a defective protein product (Haseloff et al., 1997).
In the present study, constructs encoding protein chimeras have been used, which in a previous work were shown to be efficiently transported into isolated chloroplasts in in vitro assays (Marques et al., 2003). These protein chimeras consist of EGFP fusions to the transit peptides of either the 16 kDa (PSII-Q), 23 kDa (PSII-P), or 33 kDa (PSII-O) subunits of the oxygen-evolving system of PSII, plastocyanin (PC), or ferredoxin NADP(H) oxidoreductase (FNR). While the transit peptide of FNR has solely stroma-targeting properties, all other transit peptides are of bipartite nature, comprising a N-terminal envelope transport and a C-terminal thylakoid transport signal (Clausmeyer et al., 1993). These bipartite transit peptides are further grouped into targeting signals specific for the thylakoidal pH/TAT pathway (16 and 23) or the Sec pathway (33 and PC) (Klösgen, 1997).
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Materials
Restriction endonucleases and T4 DNA ligase were obtained from Roche, and plant growth reagents from Duchefa. All further reagents and chemicals were obtained from Merck or Sigma.
Generation of transgenic A. thaliana lines expressing the transit peptide/EGFP fusion proteins
Cassettes containing the transit peptide/EGFP chimeras described in Marques et al. (2003) were cloned into the binary vector pBIN 426 Bgl (Troidl, 1995) using EcoRV and SacI. The resulting constructs were transferred to Agrobacterium tumefaciens LBA 4404 using triparental conjugation. A. thaliana plants (ecotype Columbia 0) were subsequently transformed following the vacuum infiltration method (Bechtold et al., 1993). Seeds of transformed lines were surface-sterilized and selected on solid medium containing 1.5% sucrose supplemented with half MS nutrients and 50 µg ml–1 kanamycin. Plants were further grown in soil under short-day conditions (8 h light to 16 h dark). Segregation analysis and DNA gel blots were performed in the following progeny to assay for insertion number and homozygosity status. For subsequent microscopy and biochemical experiments, homozygous plants from at least two independent, single insertion lines from each construct were used.
Optical microscopy and image analysis
Fresh plant tissues were collected, mounted in water, and immediately used for microscopy. Plant fluorescence was monitored by epifluorescence microscopy using a Zeiss Axioscope 2 microscope (Carl Zeiss, Jena, Germany) equipped with a 50 W mercury lamp and visualized using either filter ‘13’ from Zeiss (Carl Zeiss, Jena, Germany) or the ‘Endow GFP’ filter from Chroma (Chroma Technology Corp., Rockingham, VT, USA). Fluorescence images were captured with a Spot 2E liquid-crystal display camera using the Spot software v3.5 (Spot Diagnostic Instruments, Burroughs, MI, USA). Confocal laser scanning microscopy was performed using a Zeiss confocal laser scanning microscope (Carl Zeiss, Jena, Germany) equipped with argon and HeNe lasers. Plant tissue autofluorescence was excited with the 633 nm line of the He/Ne laser, while EGFP fluorescence was induced with the 488 nm line of the argon laser. The fluorescence images were collected in red and green channels, respectively. Corresponding bright field images were collected through the differential interference contrast channel and detected using the transmission detector of the confocal microscope. Images were processed using the image software supplied by the microscope manufacturer (Carl Zeiss, Jena, Germany), contrast and brightness levels adjusted with Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA, USA), and, when necessary, two-dimensional deconvolution applied using Autodeblur (AutoQuant Imaging, Watervliet, NY, USA).
For fixation, plant tissues were cut in fragments of about 5 mm in length in a solution containing 3.7% (w/v) paraformaldehyde in PBS (135 mM NaCl, 3 mM KCl, 1.5 mM KH2PO4, 8 mM Na2HPO4). After 30 min at 4 °C the fixing solution was removed by repeated washings with PBS. Iodine and Rhodamine B staining were performed, as described by Nultsch (1994) and Augsten (1964), respectively.
Electron microscopy
Small pieces of leaves were fixed with 3% (w/v) paraformaldehyde/0.25% (v/v) glutaraldehyde in PBS, dehydrated by a graded series of ethanol, and embedded in LR-White (Polysciences, Warrington, PA, USA). Sections of 90 nm thickness were immunolabelled with a mouse monoclonal antibody directed against EGFP (Clontech, Palo Alto, CA, USA; Living Colors Monoclonal antibody (JL-8), batch 97875) that was diluted 1:500 in PBS containing 1% (w/v) acetylated bovine serum albumin and 0.1% (v/v) Tween 20. Subsequently, an anti-mouse-IgG antibody conjugated with 10 nm gold (Sigma, Taufkirchen, Germany) was used according to the supplier's instructions. After immunolabelling, sections were post-stained with uranyl acetate and lead citrate and observed using a Zeiss EM 900 electron microscope (Carl Zeiss, Jena, Germany).
Cell fractionation
Intact chloroplasts were isolated from A. thaliana according to Kunst (1998) with the following modifications: in the last phase of chloroplast purification, a two-step gradient was used consisting of an 80% Percoll cushion followed by a 50% Percoll step. For the preparation of thylakoid vesicles, chloroplasts were lysed by osmotic shock for 5 min on ice at 2 mg chlorophyll ml–1 in HM buffer (10 mM HEPES, pH 8.0, 5 mM MgCl2). Thylakoids and stromal fractions were separated after centrifugation for 5 min at 20 000 g. Thylakoids were washed twice in HM buffer and finally resuspended at 2 mg chlorophyll ml–1 in HM buffer. For further analysis, both the chloroplasts and the thylakoid fractions were solubilized in HMES (HM plus 10 mM EDTA and 1% SDS) at a chlorophyll concentration of 2 mg ml–1. For the direct comparison of stroma and thylakoid aliquots, a factor of 0.35 µg stromal protein µg–1 of thylakoid chlorophyll was employed. This was the highest stromal concentration µg–1 of chlorophyll reproducibly measured in wild-type A. thaliana plants.
Western blots
Proteins corresponding to 10 µg of thylakoid chlorophyll were separated by SDS–PAGE (Laemmli, 1970) and subsequently transferred to polyvinylidene fluoride membranes (PVDF, Millipore). For the immunological detection of EGFP a monoclonal antibody from Clontech (Living Colors Monoclonal antibody (JL-8), batch 97875) was used. The 33 kDa protein of PSII was decorated by a polyclonal antisera raised against the protein homologue from spinach. After immunolabelling the membranes were developed using alkaline phosphatase-coupled secondary antibodies according to Bauw et al. (1987) and Towbin et al. (1979).
Quantification of EGFP fluorescence
From each chloroplast fraction at least three independent aliquots were quantified in a Shimatzu spectrofluorophotometer RF-1502 (Shimatzu Europa, Duisburg, Germany). In order to neutralize the effect of plant pigment fluorescence, a calibration curve consisting of aliquots from untransformed A. thaliana chloroplast fractions that contained different concentrations of commercially available EGFP (Clontech) was generated as described in Remans et al. (1999).
Miscellaneous
All other methods followed the protocols of Sambrook et al. (1989).
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Results |
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The transit peptides of the 16 and 23 kDa subunits of the oxygen-evolving system of PSII mediate translocation of folded EGFP into the thylakoid lumen in vivo
Protein constructs consisting of EGFP fused to the transit peptides of FNR, PC, or either of the 16, 23, and 33 kDa subunits of the oxygen-evolving system of PSII have previously been shown to be efficiently imported in vitro into plant chloroplasts isolated from either pea or spinach (Marques et al., 2003
). However, only the constructs comprising a transit peptide specific for the thylakoidal pH/TAT pathway, namely those from the 16 and 23 kDa subunits of PSII, were also able to mediate transport of EGFP into the thylakoid lumen in these assays. Since EGFP is known to fold quickly and spontaneously (Patterson et al., 1997), these results have led to the formulation of the hypothesis that the transport specificity of EGFP for the pH/TAT pathway might be due to folding of the protein during its passage through the stromal space. In order to examine whether a similar transport specificity is observed in vivo, the authors have generated transgenic A. thaliana lines expressing EGFP alone or in combination with either of the transit peptides mentioned above under the control of the CAMV 35S promoter.
For the analysis of the expression and conformation of EGFP in these plants, this study has taken advantage of the fluorescence capacity of EGFP, which is restricted to the correctly folded polypeptide. Several plant tissues and cell types, including leaf mesophyll, epidermis, guard cells, and trichomes, were prepared and examined under UV light by both epifluorescence and confocal laser scanning microscopy. In all plant lines analysed, green fluorescence (absent in the wild-type controls) could be detected when using the appropriate GFP filters. Independently from the tissue analysed, the fluorescence pattern observed clearly correlates with the type of fusion protein expressed in the corresponding plant line. In plants expressing EGFP alone, a dim green fluorescence was observed not only in the cytoplasm but also in the nucleus, apparently a consequence of cryptic nuclear targeting sequences present in the protein sequence (reviewed in Ruitjer et al., 2003) (Fig. 1a). On the other hand, in all plants expressing chimeras carrying chloroplast-targeting transit peptides fluorescence was restricted to plastids (Fig. 1b–d). Among these, in plants expressing the FNR/EGFP chimera, a strong green fluorescence was observed in the chloroplasts from mesophyll tissue and guard cells that was almost homogeneously distributed within the stroma (Fig. 1b, e). In some of these chloroplasts, large non-fluorescent areas were frequently observed which turned out to be starch granules, as confirmed after staining with iodine (Fig. 1f). At higher magnification, smaller non-fluorescent areas could also be detected (Fig. 1b, e), which are assumed to consist of the grana stacks of thylakoid membranes as they co-localize with the autofluorescence of chlorophyll and exhibit green coloration under transmission microscopy (Fig. 1b). Furthermore, these structures stain with Rhodamine B (Fig. 1g), a membrane-specific dye frequently used to detect membrane compartments inside plastids (Bartels, 1955).
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Plant tissues expressing EGFP fusions with thylakoid-targeting transit peptides revealed two distinct fluorescent patterns, depending on the type of transit peptide present. Plastids from plants expressing either PC/EGFP or 33/EGFP, both substrates of the Sec pathway, showed an almost homogeneous green fluorescence (Fig. 1c, h), which was virtually indistinguishable from that observed within the FNR/EGFP lines. Since both chimeras were also not translocated into the thylakoid lumen in in vitro assays but were shown to accumulate quantitatively in the stroma after import into isolated chloroplasts (Marques et al., 2003
), this homogeneous green fluorescence has been interpreted to result from a similar stromal accumulation of EGFP in the transgenic plants. In contrast, in those plant lines expressing EGFP fused to either of the transit peptides for the pH/TAT pathway (16/EGFP or 23/EGFP), a distinct and complex fluorescence pattern was observed within the chloroplasts. Fluorescence was restricted to definite fluorescent areas whose shape and distribution pattern are characteristic of the structures previously identified as the grana stacks of thylakoid membranes (Fig. 1d, i). This identity is particularly evident after comparison with the pattern of chlorophyll autofluorescence in the chloroplasts of wild-type plants. (Fig. 1i, j). These observations suggest that in these plant lines EGFP is transported not only into the chloroplasts but is also further translocated into the thylakoid lumen.
Fluorescence of EGFP within the thylakoid lumen of mesophyll chloroplasts is light dependent
Interestingly, the intensity of EGFP fluorescence within the thylakoid lumen of chloroplasts from mesophyll cells in plants expressing the 16/EGFP and 23/EGFP constructs was shown to vary according to the time point of observation. While in the chloroplasts of guard cells bright green EGFP fluorescence could be observed during the entire light and dark phases, in mesophyll chloroplasts EGFP fluorescence could only be detected in the thylakoid lumen within a short time period, notably the first 3–4 h of the dark phase (Fig. 2a, b). Afterwards, EGFP apparently becomes retained in the stroma as the fluorescence in this compartment increases. The resulting chloroplast pattern becomes indistinguishable from that of plants expressing either the FNR/EGFP, PC/EGFP, or 33/EGFP constructs (Fig. 2c).
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Considering that EGFP suffers a reversible conformational change which leads to the loss of fluorescence emission if exposed to pH values lower than 5.8 (Patterson et al., 1997
; Haupts et al., 1998; Kneen et al., 1998), the absence of fluorescence during the light phase was interpreted as being a possible consequence of the acidic pH in the lumen of the photosynthetically active thylakoids (Kramer et al., 1999, 2003). In order to examine this possibility, an attempt was made to disrupt the trans-thylakoidal proton gradient, thus leading to an increase of both the pH and EGFP fluorescence within the lumen. Firstly, tissue sections were incubated with several ionophores. By contrast with intact living cells where no fluorescence could be detected, fluorescence associated with chloroplasts could be observed in cells with severe damage (absence of cytoplasm flow, membrane disruption). However, the poor tissue preservation of these damaged cells did not allow any definitive conclusions concerning the fluorescence localization within the chloroplasts (data not shown). These results were interpreted as reflecting the incapacity of the ionophores to reach the thylakoid membrane in intact cells through the natural cell barriers consisting of plasma membrane, cytoplasm, plastid envelopes, and stroma. As an alternative to the disruption of the trans-thylakoidal proton gradient, the experimenters therefore opted for the fixation of light-exposed mesophyll cells with paraformaldehyde prior to microscopy. While this treatment was expected to result in cross linking of proteins and thus damage to membrane integrity, it was equally expected to preserve the internal structure of cells and respective organelles. Indeed, after this treatment, EGFP fluorescence could be restored and clearly localized within the thylakoid lumen of 16/EGFP- and 23/EGFP-expressing plants (Fig. 2d). As seen after comparison of Fig. 2b–d, the resulting pattern was indistinguishable from that observed in the first 3–4 h after the onset of the dark period.
Immunogold labelling specifically detects EGFP within the thylakoid lumen of plants expressing the 16/EGFP and 23/EGFP constructs only
In order to examine the organellar localization of EGFP in plants expressing the different protein fusions in an independent approach, EGFP immunolocalization was performed using anti-EGFP antibodies. For this purpose, ultra-thin sections of the leaf mesophyll from the different plant lines were immunodecorated with gold particles after detection with anti-EGFP antibodies and analysed by electron microscopy. It turned out that the distribution pattern of EGFP as determined by this method was essentially identical to that obtained by fluorescence microscopy. In contrast to the wild-type controls, in all plant lines expressing EGFP fusions carrying a plastid transit peptide a specific labelling was observed in the chloroplasts (Fig. 3a). In order to obtain a representative sample of EGFP distribution within the chloroplasts of the different plant lines, a detailed counting of the gold particles in the different regions of 20 organelles was performed for each case. This analysis revealed a preferential accumulation of EGFP in the stroma of those lines expressing EGFP chimeras carrying the transit peptides of FNR, PC, or the 33 kDa subunit of PSII (Fig. 3b), thus confirming the results obtained by optical microscopy. Likewise, the specific transport of EGFP into the thylakoid system of those plants expressing either 16/EGFP or 23/EGFP could be confirmed by this approach. In both instances, approximately 60% of the gold particles were found within the thylakoid lumen, whereas in both the PC/EGFP and 33/EGFP lines labelling of this compartment was only in the range of 5–10% of all gold particles counted, a value close to that observed for the FNR/EGFP plants (Fig. 3b).
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In isolated chloroplast fractions, EGFP can only be detected within the thylakoid lumen if targeted to the
pH/TAT pathwayAs a third approach to determine the subcellular and suborganellar location of EGFP in the different transgenic lines, chloroplasts were isolated from these plants and fractionated into stroma and thylakoids. Subsequently, stoichiometric aliquots of total cell extract, chloroplasts, stroma, and thylakoids were assayed for EGFP content both by western blot analysis and spectrofluorometric fluorescence measurements. The results of the western blot analysis using anti-EGFP antibodies correspond well with the microscopy data (Fig. 4). While in plants expressing EGFP alone the protein is detected exclusively in the full cell extract, in all plants expressing chimeric EGFP constructs the protein is also found in the chloroplasts. Among those, FNR/EGFP, PC/EGFP, and 33/EGFP, EGFP accumulates exclusively in the stroma and is not detectable in significant amounts in the thylakoid fractions. Since the FNR/EGFP construct carries a strictly stroma-targeting transit peptide, the exclusive localization of EGFP in this compartment was expected in this case. However, the absence of EGFP from the thylakoid fractions of PC/EGFP- and 33/EGFP-expressing plants as well confirms that, when targeted to the Sec pathway, translocation of the protein across the thylakoid membrane does not take place. In contrast, when fused to the transit peptides of either the 16 kDa or 23 kDa subunits of the oxygen-evolving system of PSII, the majority of EGFP is detected in the thylakoid fractions (arrows in Fig. 4).
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Remarkably, a fraction of EGFP is always present in the stroma of 16/EGFP- and 23/EGFP-expressing plants (Fig. 4). Although identical in size to the original EGFP, this protein probably represents transport intermediates that were stopped in the stromal space due to the removal of their thylakoid targeting signals by an unknown proteolytic activity, able to remove unfolded segments from otherwise fully folded proteins. The alternative explanation, leakage of EGFP from the thylakoid during the fractionation procedure, can be excluded from the analysis of the 33 kDa protein which was detected in parallel (Fig. 4).
To quantify the amounts of folded EGFP in each chloroplast fraction, a spectrofluorometric analysis was performed using the same preparations used for the western blots. The results obtained essentially confirm the EGFP distribution pattern obtained with all other experimental approaches (Fig. 5). Particularly remarkable are the high levels of EGFP in the thylakoid lumen of plants expressing the 16/EGFP and 23/EGFP constructs. Interestingly, both in the immunological and spectrofluorometric measurements a higher amount of EGFP is found in the stroma of plants expressing the 23/EGFP than in those expressing the 16/EGFP chimera (Figs 4, 5). Since similar amounts of EGFP can be detected within the thylakoid vesicles of both lines, the higher levels of EGFP in the stroma from plants expressing the 23/EGFP construct most probably result from a limiting translocation by the pH/TAT pathway rather than a lower translocation efficiency of the 23/EGFP chimera in relation to the 16/EGFP one.
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The analysis of A. thaliana lines expressing chimeric proteins consisting of EGFP and various transit peptides of nuclear encoded chloroplast proteins has shown that EGFP is efficiently imported into chloroplasts by all transit peptides analysed. However, it can only be further transported into the thylakoids if the transit peptide is a
pH/TAT-specific thylakoid transport signal. If fused to a Sec-type transit peptide instead, the localization of EGFP is indistinguishable from that of plants expressing the stroma-targeted FNR/EGFP fusion, i.e. no significant amounts of EGFP can be detected in the thylakoid fractions either by microscopy or after cell fractionation. These data are in agreement with in vitro experimental evidence showing that transport of EGFP through the Sec pathway, if it happens at all, occurs rather inefficiently. PC/EGFP accumulates quantitatively in the stroma after import into chloroplasts isolated from either spinach or pea. Although 33/EGFP is further transported into the thylakoid lumen, this takes place only in intact spinach chloroplasts. However, even in this case, the majority of the protein accumulates in the stroma and only minute amounts are found in the thylakoid lumen (Marques et al., 2003).
From these observations the question arises of the factors responsible for the clear targeting preference of EGFP for the pH/TAT pathway. The answer might reside in the folding status of this protein. The polypeptide chain of EGFP, like wild-type GFP, spontaneously folds, originating a mature protein form comprising the fluorescent chromophore (Patterson et al., 1997). The folded protein adopts a relatively rigid structure and is particularly resistant to proteolysis (reviewed in Zimmer, 2002). As demonstrated by the stromal fluorescence in the FNR/EGFP, PC/EGFP, and 33/EGFP lines, a significant fraction of EGFP apparently folds in this compartment. Indeed, assays performed after in vitro import of EGFP into isolated chloroplasts have shown that stromal EGFP is remarkably stable against protease treatment, a feature characteristic of the folded protein (E Fan, personal communication). Since transport by the Sec pathway requires the passenger proteins in an unfolded form, transport of folded EGFP by this route appears unlikely. In contrast, transport of EGFP in a folded conformation into the thylakoid lumen by the pH/TAT pathway would be compatible with the data available from in vitro import assays in isolated chloroplasts (Marques et al., 2003) as well as from in vivo experiments in prokaryotic organisms using GFP as a passenger protein. Both in Escherichia coli and Synechocystis PCC6803, GFP was shown to be efficiently translocated by the bacterial TAT machinery across the cytoplasmic membrane into the periplasmic space when fused to the TAT-specific targeting signal from trimethylamine N-oxide (TMAO) reductase (TorA) (Santini et al., 2001; Thomas et al., 2001; Barret et al., 2003; Spence et al., 2003). However, upon fusion of GFP to the maltose binding protein, a substrate of the Sec pathway, only marginal amounts of GFP could be transported into the periplasm of E. coli. Further, the protein secreted by the Sec pathway did not emit fluorescence and was particularly prone to proteolytic degradation, suggesting a misfolded conformation (Feilmeier et al., 2000). These data thus suggest that even the residual amounts of GFP which are translocated by the bacterial Sec pathway across the cytoplasmic membrane are in an unfolded conformation. Although it cannot strictly be excluded that, similar to bacteria, minimal amounts of EGFP remain unfolded in the stroma and under this conformation are translocated into the thylakoid lumen by the Sec pathway, no evidence for such a transport has been found in the transgenic plants used in this study.
The targeting and subsequent translocation of EGFP into the thylakoid lumen has further led to a number of observations of significant biological relevance. Of particular interest is the absence of EGFP fluorescence during the light period in the thylakoid lumen of mesophyll chloroplasts from the 16/EGFP and 23/EGFP lines. Since fluorescence can be restored after disruption of the pH gradient by fixation with paraformaldehyde, its disappearance under physiological conditions most probably reflects the acidification of the lumen. According to this scenario, the emission of fluorescence at onset of the dark phase can be interpreted to result from the disappearance of the trans-thylakoidal pH and the consequent increase of pH within the thylakoid lumen. Most remarkable, however, is the redistribution of EGFP fluorescence after the first 3–4 h of the dark phase. While the lumenal fluorescence during this period might represent the degree of EGFP stability in this compartment, the increasing accumulation of the protein in the chloroplast stroma strongly suggests that, upon the disappearance of the pH across the thylakoid membrane, an essential requirement for the translocation of EGFP into the thylakoid lumen becomes lost. Whether this requirement consists of the trans-thylakoidal pH itself, as suggested by the mass of data obtained from in vitro import experiments with isolated chloroplasts, or whether it is based on an alternative factor, as recently postulated for Chlamydomonas reinhardtii (Finazzi et al., 2003) cannot be decided at this point. Nevertheless, the observations in this study are the first indications that, in higher plants, directly or indirectly, the trans-thylakoidal pH is an essential requirement for the pH/TAT pathway to effect in vivo protein translocation across the thylakoid membrane.
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Acknowledgements |
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This work was supported by the Deutsche Forschungsgemeinschaft (grants SFB 363 and KL862/1–1). S Platzer and G Kuhnert are acknowledged for their excellent technical assistance.
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References |
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