Chloroplast Targeting Research Articles

Biophysical characterization of a transit peptide directing chloroplast protein import

We have investigated the biophysical properties of a 35 amino acid peptide representing the entire length of a chloroplastic targeting sequence. The peptide, termed gamma-tp, corresponds in sequence to the transit peptide of the gamma subunit of the chloroplast ATP synthase from Chlamydomonas reinhardtii. We found that gamma-tp blocks the import of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase into isolated pea chloroplasts (KI approximately 5 microM), suggesting that it interacts with higher plant plastids in a physiological manner. We also found the gamma-tp to have a high affinity for nonpolar environments, but not to cause a general disruption of membrane integrity. Hydrophobic moment analysis suggests that the gamma-tp can adopt an amphipathic beta structure. However, circular dichroism measurements indicate that the peptide is largely a random coil, in both the presence and absence of sodium laurylsulfate micelles. In the absence of a recognizable secondary structural targeting motif, we asked whether the presence of a transit peptide on a chloroplast protein increases the protein's overall affinity for nonpolar environments. Phase-partition experiments with Triton X-114 suggest that this is not the case. These results are discussed in relation to the mechanism of protein targeting to chloroplasts.

Chloroplast Targeting of Bacterial β-Glucuronidase with a Pea Transit Peptide in Transgenic Tobacco Plants

Chloroplast Targeting of Bacterial β-Glucuronidase with a Pea Transit Peptide in Transgenic Tobacco Plants

Nucleotide sequence of a cDNA encoding a tobacco thioredoxin

Thioredoxins are small (about 12 kDa) proteins which reduce disulphide bridges on target enzymes [6]. Protein or nucleic acid sequences have been established for different thioredoxins from vertebrates [7, 8, 15, 16], algae [3, 4, 12] and bacteria [6, 10]. The active site shows a highly conserved sequence -C-G-P-C-. Recently a larger consensus thioredoxin signature (AT)-x-W-C(AG)-(PH)-C has been defined in the PROSITE database [ 1 ]. It matches all thioredoxins but no other protein sequences. In higher plants a spinach f-type cDNA [9], an m-type cDNA [ 14] and an m-type protein [ 11 ] have been sequenced. The f-type has a signal peptide typical of chloroplast targeting. Both enzymes are encoded in the nucleus, but are localized and active in the chloroplast in their mature form. M-type activates NADP-malate dehydrogenase wild f-type activates enzymes of the photosynthetic carbon cycle. In addition, cytosolic thioredoxin activities have been characterized in spinach. The proteins have been purified but not yet sequenced [5]. We have isolated a complete or nearly complete clone from a cDNA library from Nicotiana tabacum cells cultivated in vitro. This 699 nucleotide insert hybridizes on northern blots with a 0.9 kb mRNA. It contains an ORF of 378 bp (Fig. 1). The 3' untranslated region is 242bp long, including 16bp of poly(A) ÷ tail. Two AT-rich regions could serve as polyadenylation signals. The ORF codes for a protein of 126 amino acids. At positions 43-49 it contains a sequence which perfectly matches the thioredoxin signature. In addition, the protein sequence shows 40-50 ~o homologies with Chlamydomonas Chl assumed to be a cytoplasmic protein [4] and vertebrate thioredoxins using FASTA [13]. In contrast, the homology with m and f spinach chloroplast thioredoxins is only 30~o. Figure2 presents a multiple alignment [2] with some of the published thioredoxin sequences sorted by decreasing homology with the tobacco sequence. Chlamydomonas [3, 4], rabbit [7], Anacystis [ 12], Corynebacterium [ 10] and m-type spinach 2 [ 11] sequences were obtained by protein sequencing. Human [16], rat [15], chicken [8], Escherichia coli [6], f-type [9] and m-type 1 [14] spinach sequences are derived from cDNA sequencing, showing the presence of a large signal peptide in the spinach thioredoxins. The tobacco sequence matches the mature protein sequences and has no signal peptide, suggesting that the protein encoded by the clone we have isolated is cytoplasmic.

Domain structure of mitochondrial and chloroplast targeting peptides.

Representative samples of mitochondrial and chloroplast targeting peptides have been analyzed in terms of amino acid composition, positional amino acid preferences and amphiphilic character. No highly conserved 'homology blocks' are found in either class of topogenic sequence. Mitochondrial-matrix-targeting peptides are composed of two domains with different amphiphilic properties. Arginine is frequently found either at position -10 or -2 relative to the cleavage site, suggesting that some targeting peptides may be cleaved twice in succession by two different matrix proteases. In stroma-targeting chloroplast transit peptides three distinct regions are evident: an uncharged amino-terminal domain, a central domain lacking acidic residues and a carboxy-terminal domain with the potential to form an amphiphilic beta-strand. Targeting peptides that route proteins to the mitochondrial intermembrane space or the lumen of chloroplast thylakoids have a mosaic design with an amino-terminal matrix- or stroma-targeting part attached to a carboxy-terminal extension that shares many characteristics with secretory signal peptides.

Chloroplast targeting ofneomycin phosphotransferase II with a pea transit peptide in electroporated barley mesophyll protoplasts

Routine methods for stable gene transfer to cereals are not yet available. To be able to study chloroplast targeting in barley (Hordeum vulgare L.) cells, the expression of recombinant genes was assayed in barley mesophyll protoplasts after electroporation of DNA. The CaMV 35S transcript promoter was attached to a chimeric gene consisting of a pea RuBisCo small subunit transit peptide coding sequence (tp) and the gene coding for neomycin phosphotransferase II (nptII). As a control, a construction with no transit peptide coding segment was used. 48 hours after electroporation, a fraction of the protoplasts was lysed and intact chloroplasts were isolated. Protoplasts electroporated with either of the gene constructions showed strong NPTII activity. However, enzyme activity was detected in chloroplasts only when thetp-nptII gene construction was used. Protease treatment of the chloroplasts confirms that the pea RuBisCo small subunit transit peptide is targeting the NPTII polypeptide into the chloroplasts, subsequent to the synthesis of the hybrid precursor in barley cells.