Different in Vitro and in Vivo Targeting Properties of the Transit Peptide of a Chloroplast Envelope Inner Membrane Protein

Marcio De Castro Silva-Filho,Marie-Christine Wieërs,Ulf-Ingo Flügge,François Chaumont,Marc Boutry

doi:10.1074/jbc.272.24.15264

Marcio De Castro Silva-Filho, Marie-Christine Wieërs + Show 3 more

Open Access

https://doi.org/10.1074/jbc.272.24.15264

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Abstract

The triose phosphate 3-phosphoglycerate phosphate translocator (TPT) is a chloroplast envelope inner membrane protein whose transit peptide has structural properties typical of a mitochondrial presequence. To study the TPT transit peptide in more detail, we constructed two chimeric genes encompassing the TPT transit peptide and either 5 or 23 amino-terminal residues of the mature TPT, both linked to the reporter chloramphenicol acetyltransferase (cat) gene. The precursors were synthesized in vitro and translocated to and processed in purified plant mitochondria. However, this import was not specific since both precursors were also imported into isolated chloroplasts. To extend this analysis in vivo, the chimeric genes were introduced into tobacco by genetic transformation. Analysis of CAT distribution in subcellular fractions of transgenic plants did not confirm the data obtained in vitro. With the construct retaining only 5 residues of the mature TPT, CAT was found in the cytosolic fraction. Extension of the TPT transit peptide to 23 residues of the mature TPT allowed specific import and processing of CAT into chloroplasts. These results indicate that, despite its unusual structure, the TPT transit peptide is able to target a passenger protein specifically into chloroplasts, provided that NH2-terminal residues of the mature TPT are still present. The discrepancy between the in vitro and in vivo data suggests that the translocation machinery is more stringent in the latter case and that sorting of proteins might not be addressed adequately by in vitro experiments.

Highlights

Mitochondria and chloroplasts have their own genetic information
The second construct, TPT23-chloramphenicol acetyltransferase (CAT), included 23 residues of the mature triose phosphate 3-phosphoglycerate-phosphate translocator (TPT), in case the amino-terminal part of the mature TPT was involved in targeting, as this had already been shown for the chlorophyll a/b-binding protein [25, 26]
TPT precursor, is imported in vitro into mitochondria isolated from yeast and plants, and this process has shown the typical features of specific protein import such as energy requirement and protease-sensitive binding of the precursor to the yeast MOM19 receptor [16]

Summary

EXPERIMENTAL PROCEDURES

Gene Construction—Standard procedures were used for DNA manipulations [17]. The constructs assembling the TPT transit peptide and CAT were made as follows. After polymerase chain reaction amplification, the fragments were digested with HindIII and cloned into the HindIII site of the polylinker region of SK(ϩ) Bluescript (Stratagene), resulting in the TPT5 and TPT23 plasmids, respectively. The HindIII site previously used for inserting targeting sequences [8, 18] is localized 75 nucleotides upstream of the CAT translation initiation codon. We engineered a new CAT gene provided with a HindIII site 10 nucleotides upstream of the CAT translation initiation codon. Signals were quantitated using an Image Master densitometer (Pharmacia Biotech Inc.)

RESULTS

Targeting of a Chloroplast Envelope Protein

DISCUSSION