Insertion of the 34-kDa Chloroplast Protein Import Component, IAP34, into the Chloroplast Outer Membrane Is Dependent on Its Intrinsic GTP-binding Capacity

Dadong Chen,Danny J Schnell

doi:10.1074/jbc.272.10.6614

Abstract

IAP34 is a 34-kDa component of the outer membrane complex that mediates the initial stages of protein import into chloroplasts (Seedorf, M., Waegemann, K., and Soll, J. (1995) Plant J. 7, 401-411; Kessler, F., Blobel, G., Patel, H. A., and Schnell, D. J. (1994) Science 266, 1035-1039). We have investigated the targeting and insertion of IAP34 at the outer envelope membrane. The analyses of IAP34 deletion mutants and hybrid proteins (consisting of regions of IAP34 fused to the soluble IgG-binding domain of staphylococcal protein A) suggest that the transmembrane domain and C-terminal tail of IAP34 contain information essential but not sufficient for targeting to the outer membrane. Treatment of chloroplasts with exogenous proteases does not affect IAP34 insertion, indicating that targeting does not require surface-exposed receptors at the envelope. GTP or GDP is required for maximal integration of IAP34 into the outer membrane. The GTP/GDP requirement is attributed to the intrinsic GTP binding activity of IAP34 because GTP/GDP binding-deficient mutants are defective in outer membrane insertion. On the basis of these observations, we propose that IAP34 is targeted to the chloroplast by a C-terminal signal and efficiently integrated into the outer membrane by conformation-induced insertion upon GTP/GDP binding.

Highlights

The import of nuclear-encoded preproteins into chloroplasts is mediated by the coordinate interaction of protein conducting machineries in the outer and inner membranes of the chloroplast envelope (3)
Seedorf et al (1) previously had shown that the addition of ATP to an in vitro insertion assay containing isolated chloroplasts stimulated the association of IAP34 with the outer envelope membrane, whereas we had observed no effect of exogenous ATP on insertion (2)
We wished to reexamine the energetics of IAP34 insertion with precautions to eliminate any free nucleoside triphosphates (NTPs) from the in vitro transla

Summary

EXPERIMENTAL PROCEDURES

Chloroplast Isolation and Subfractionation—Intact chloroplasts were isolated from 10 –14-day-old pea seedlings The fragments were each inserted into the NcoI/XhoI sites of pET21b-protA to generate pET21b-protA-IAP34(237–310) and pET21b-protAIAP34(237–287), and they were used for coupled transcription and translation reactions. The chloroplasts were reisolated through 40% Percoll silica gel (Pharmacia Biotech Inc.) and lysed under hypotonic conditions (14), and the total membrane fraction was recovered by centrifugation at 30,000 ϫ g for 30 min. For assay of the insertion of E. coli synthesized substrates, the purified inclusion body fractions from extracts of induced E. coli cultures were dissolved in 6 M urea, 50 mM Tris-HCl, pH 7.5 to a concentration of 0.2 mg of protein/ml and diluted 10-fold with import buffer at room temperature. Antibody detection in immunoblots was performed using a chemiluminescence detection system (DuPont NEN)

RESULTS

Targeting of Proteins to Chloroplast Outer Envelope Membrane

DISCUSSION