 instead of RR. Charged residues are in bold, and hydrophobic regions are underlined. The invariant GxGxxG motif, which occurs well into the mature protein sequence of PPO, is boxed. ♦, Experimentally determined cleavage site; ▾, cleavage site predicted by TargetP; ↓, TPP cleavage site predicted by LumenP. B, In silico analysis of C. reinhardtii presequences. TargetP cTP, Chloroplast-targeting peptide; RC, reliability coefficient (1 is high and 5 is low); LumenP prediction cutoff, LumenP score ≥ 0.47 and cleavage site score (CSscore) ≥ 6.8; lTPlen, the predicted luminal-targeting peptide length. Predicted lTPlen for ISP and LHCII are incorrect, as these precursors do not contain TPP cleavage sites. C, Cys residues in the precursor sequences of plant and C. reinhardtii FeCs. The I and II suffixes refer to the isoforms present in photosynthetic and nonphotosynthetic tissues, respectively, as described in the text. ▾, Cleavage sites predicted by TargetP.
Interestingly, the presequences of the C. reinhardtii PPO and FeC precursors show certain characteristics of chloroplast precursors that are targeted to the thylakoid lumen via the twin-Arg translocase (Tat) pathway. The Tat pathway is capable of translocating proteins across the thylakoid membrane in their folded state. Tat domains consist of two adjacent Arg residues followed by a hydrophobic residue two or three residues afterward (Tat motif), a central hydrophobic region (H-region) directly followed by a consensus thylakoid processing peptidase (TPP) cleavage site (AxA↓x; for review, see Robinson and Bolhuis, 2004). The C. reinhardtii PPO and FeC precursors both contain Tat motifs (Fig. 3A) and possible TPP cleavage sites, as found in most lumen-targeted proteins (Mayfield et al., 1989; von Heijne et al., 1989; Merchant et al., 1990). The program LumenP, a neural network predictor of lumen targeting in plants (Westerlund et al., 2003), produced very high scores for known lumenal proteins such as the Rieske iron-sulfur protein (ISP), the PSII oxygen-evolving enhancer proteins OEE2 and OEE3 (Mayfield et al., 1989; Finazzi et al., 2003 and refs. therein), and a putative ascorbate peroxidase (APX2), which is considered very likely to be lumenal because it is found in the lumen in Arabidopsis (NM_116970; Kieselbach et al., 2000; Peltier et al., 2002). Light-harvesting chlorophyll a/b-binding protein (LHCII) receives a low score because it does not contain lumen-targeting sequences (Lamppa, 1988). Although the C. reinhardtii PPO and FeC precursors yielded relatively high LumenP scores (Fig. 3B), the LumenP program returned TPP cleavage site positions well into the mature protein, in the case of the PPO precursor directly after a predicted transmembrane helix, and for the FeC precursor inside a moderately hydrophobic stretch (Fig. 3A). Moreover, the segment that typically comprises the H-region in valid Tat domains, directly following the twin Args, could not be unequivocally identified in the C. reinhardtii precursors. Therefore, although the presence of Tat signals in these proteins is striking, their functional validity is uncertain. However, it should be noted that all of the analyzed C. reinhardtii precursors that are known to be targeted to the lumen exhibit two separate twin Args in their presequences, which is also true for the FeC precursor and potentially also for the PPO precursor, depending on the position of the N terminus of the latter.
Another interesting feature of FeC presequences from photosynthetic eukaryotes, including that of C. reinhardtii, is the presence of several Cys residues (Fig. 3C). A survey of plant chloroplast-targeting peptides indicates that the occurrence of multiple Cys residues is uncommon (data not shown).
The predicted mature regions of the PPO and FeC were overexpressed in E. coli using the pQE30 expression vector, which introduces an N-terminal His tag. The recombinant proteins were highly expressed (Fig. 4) and invariably found in inclusion bodies (data not shown). These inclusion bodies were dissolved in 6 m guanidinium hydrochloride and the proteins were purified under denaturing conditions. On SDS-PAGE, purified, overexpressed PPO appeared as a single 59-kD band, whereas purified FeC was seen as a 47-kD monomer but also as a set of higher Mr bands even in the presence of 1% (w/v) SDS and 100 mm dithiothreitol, suggestive of a strong tendency of the protein to aggregate, at least under denaturing conditions. Purified PPO and FeC were used to raise antibodies in rabbits. The obtained antisera cross-reacted with their corresponding overexpressed proteins (data not shown) and recognized bands of the appropriate size in C. reinhardtii cell extracts (Fig. 5).
Expression and purification of cloned C. reinhardtii PPO and FeC. Coomassie Blue-stained SDS-PAGE of His-tagged C. reinhardtii PPO and FeC proteins in extracts of E. coli cells before (0) and after 4 h of induction (4), and after purification of the expressed proteins by N-affinity chromatography (Ni-NTA). Positions of molecular mass marker proteins are indicated.
Cell fractionation and immunoblot hybridization with antibodies raised against the overexpressed PPO and FeC proteins. In FeC immunoblots, a 36-kD contaminant of chloroplast origin was usually observed. TMBZ detected heme-containing proteins indicative of mitochondria (cyt c and cyt c1) and chloroplasts (cyt f). Antibody against Rubisco detected the two subunits of 50 and 15 kD only in cells and the chloroplast fraction. For each sample, 100 μg of total proteins were loaded onto the gel. W, Whole cells; C, chloroplasts; M, mitochondria; RBC, Rubisco.
To determine whether the products of the single genes encoding PPO and FeC in C. reinhardtii are targeted to specific organelles in the cell, chloroplasts and mitochondria were isolated from mixotrophically grown cells. The subcellular fractions were analyzed on SDS-PAGE, and the proteins were transferred to nitrocellulose for immunoblotting with specific antisera that were raised against the purified, overexpressed PPO and FeC. Coomassie Blue-stained SDS-PAGE gels revealed clearly different polypeptide patterns for the chloroplast and mitochondrial fractions (Fig. 5). In the chloroplast fraction, heme-mediated peroxidase activity staining detected cytochrome (cyt) f, as expected, but also low levels of a 12-kD c-type cyt that was attributed to contamination by mitochondrial cyt c (Fig. 5). In contrast, the mitochondrial fraction contains heme-staining bands at positions corresponding to cyt c and cyt c1, but this fraction contained no evidence for the presence of chloroplast cytochromes. In addition, an anti-Rubisco antibody detected high levels of the large and small Rubisco subunits in the chloroplast fraction. The mitochondrial fraction contained some cross-reacting bands, but none of those bands corresponded to the Rubisco large and small subunits. It can therefore be concluded that the chloroplast fraction might be slightly contaminated by mitochondria whereas the mitochondrial fraction is free of chloroplast contamination. Immunoblot analysis with the anti-PPO antibodies revealed in whole cells a single band of an apparent molecular mass of 59 kD. In the organellar fractions, this protein was detected exclusively in the chloroplast fraction (Fig. 5). Anti-FeC antibodies detected a major band of 47 kD in whole cells and in the chloroplast fraction. Several very faint bands were detected in the mitochondrial fraction, but none were detected at the position of FeC. The apparent molecular mass of the mature PPO was approximately 4 kD higher (59 kD) than predicted; since the overexpressed PPO exhibited the same apparent molecular mass, the observed electrophoretic mobility might be influenced by the high pI value of the PPO protein. The FeC band correlated with the mass calculated for the predicted mature protein. A 36-kD nonspecific band, especially visible in the FeC immunoblot, was caused by a common contaminant in antisera of preimmunized rabbits for C. reinhardtii proteins (data not shown). Altogether, these results indicate that in C. reinhardtii, both PPO and FeC are located exclusively in the chloroplasts.
To determine whether C. reinhardtii PPO and FeC proteins are associated with membranes, intact chloroplasts were fractionated into membrane and soluble fractions. Immunoblot analysis revealed that both PPO and FeC were located only in the membrane fraction (Fig. 6). Membranes were further subjected to high salt or alkaline treatment to examine the nature of the membrane association of the enzymes. PPO was not extracted by 1 m NaCl and was mostly resistant to extraction by alkaline treatments to a similar level as the LHC proteins, indicating that the protein is intrinsic to the membrane. No dissociation of FeC by either 1 m NaCl or alkaline treatment could be detected.
Distribution of PPO and FeC between chloroplast membrane and soluble fractions. Chloroplast membranes were treated either with high pH (20 mm Na2CO3, pH 11.0) or high salt (1 m NaCl) to determine the nature of membrane associations. An anti-LHC antibody was used as control for a set of proteins known to be confined to the membranes.