Insertion of the 34-kDa chloroplast protein import component, IAP34, into the chloroplast outer membrane is dependent on its intrinsic GTP-binding capacity

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.