Chloroplast Protein Import

Abstract

Proteins have to be at least partially unfolded upon passage through the biological membranes. Previous studies with a dihydrofolate reductase fusion protein containing a chloroplast transit peptide showed that stabilization of the tertiary structure of the fusion protein by binding of a ligand, methotrexate, failed to block its translocation across the envelopes, suggesting that chloroplast envelopes have strong activity to unfold proteins [America, T., Hageman, J., Guéra, A., Rook, F., Archer, K., Keegstra, K. & Weisbeek, P. (1994) Plant Mol. Biol. 24, 283–294]. In the present study, we have analyzed in vitro translocation of a fusion protein consisting of the entire plastocyanin precursor and dihydrofolate reductase across the chloroplast envelope membranes and the thylakoid membrane. In the presence of methotrexate, the fusion protein was imported into the stroma but its translocation across the thylakoid membrane was blocked. The fusion protein that bound to the envelope became susceptible to digestion by thermolysin. These results suggest that, while the envelope membranes can unfold the methotrexate‐bound fusion protein to allow its passage, the thylakoid membrane cannot unfold the fusion protein that has re‐bound to methotrexate in the stroma.

A folded protein can be transported across the chloroplast envelope and thylakoid membranes.

“…Schnell and Blobel (1993) Endo et al (1994) found that a full-~l ength PC molecule was halted in its transfer across the thylakoid membrane by the presence of a DHFR-B. OE17-BPTI MTX C-terminal domain, this protein is transported via the so-called Sec-related pathway and likely traverses the membrane through a pore similar to the -I 20-60-A channel formed by Sec6lp (Hamman and Johnson, 1996;Hanein et al, 1996).…”

“…Endo et al (1994) showed that a C-terminal MTX-bound DHFR domain can halt the translocation of PC on the Sec-related thylakoid pathway. However, no arrested translocation intermediates have yet been generated in the ApH-dependent pathway.…”

A 1-Megadalton Translocation Complex Containing Tic20 and Tic21 Mediates Chloroplast Protein Import at the Inner Envelope Membrane

“…The resulting mRNA was translated in a wheat germ extract (Promega) at 258C for 2 h in the presence of [ 35 S]Met. A plasmid for the in vitro expression of pSSU-DHFR, which consists of the full-length precursor to SSU fused to the entire mouse dihydrofolate reductase, was constructed by inserting the pSSU coding sequence lacking the stop codon into pPC-DHFR/SP (Endo et al, 1994) at a PstI/BamHI site. The fusion protein pSSU-DHFR was synthesized using TNT SP6-coupled reticulocyte lysate system (Promega) at 308C for 90 min in the presence of [ 35 S]Met.…”

Import inhibition of poly(His) containing chloroplast precursor proteins by Ni2+ ions

“…Furthermore, the binding constant of methotrexate to DHFR is lower than that of Ni 2+ to (His)6. Eventually the unfolding capacity of the chloroplast envelope membrane [8,9] is able to discriminate between the two binding constants, thereby explaining why the import of (His)6-containing precursor protein is inhibited by Ni 24 , while DHFR fusion proteins are still imported in the presence of methotrexate. To further investigate this point we currently produce a precursor fusion protein with two C-terminal additions: a DHFR sequence followed by a hexahistidyl peptide.…”

“…On the one hand the import of a transit peptide-DHFR fusion protein is not blocked by the presence of methotrexate in higher plants, pointing to a strong unfolding activity associated with chloroplast envelope membranes [7,8]. Also the protease sensitivity of a chimeric precursor protein containing the ricin A chain, when bound to chloroplasts, points to an unfolding activity of the chloroplast envelope [9].…”