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

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 NH 2 -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.

Signals Required for the Import and Processing of the Alternative Oxidase into Mitochondria

“…The transit sequence of the triose-phosphate translocator, which has the predicted ability to form an amphiphilic ␣-helix, has been shown to contain mitochondrial-targeting ability (40,41). We have carried out this study with the authentic "passenger" protein in an attempt to avoid any differential import with passenger proteins (23, 40 -42).…”

Differential usage of two in-frame translational start codons regulates subcellular localization ofArabidopsis thalianaTHI1

“…Although some mistargeting has been reported so far (Franzén et al, 1990;Hurt et al, 1986;Silva-Filho, 1999;Whelan et al, 1990), this is because of heterologous expression systems or unusual targeting sequences. In homologous systems mistargeting seems not to occur in vivo (Silva-Filho et al, 1997); therefore, dual targeting to mitochondria and chloroplasts is apparently not related to mistargeting but to cell requirement (Soll and Tien, 1998). An interesting report has shed light on the protein import specificity of both organelles.…”

Mutation of the TGD1 Chloroplast Envelope Protein Affects Phosphatidate Metabolism inArabidopsis

“…In contrast with our in vitro import assay system, chloroplasts might have difficulty importing and subsequently inserting a multispanning membrane protein into the chloroplastic envelope membrane. Indeed, discrepancies between in vitro and in vivo import systems have been reported previously (Silva-Filho et al, 1997). Likewise, attempts to employ in vitro import assay systems to investigate membrane proteins with multiple transmembrane domains, such as chloroplastic metabolite translocators, have proven to be very problematic and challenging (Schunemann et al, 1993;Fischer et al, 1994;Weber et al, 1995;Flü gge, 1998).…”