Inner envelope protein 32 is imported into chloroplasts by a novel pathway

Ahmed Nada 0 Jrgen Soll ) 0 0 Department of Biology I, Botany, University of Munich , Menzinger Str. 67, Munich 80638 , Germany - The 32 kDa chloroplast inner envelope protein (IEP32) is imported into the organelle in the absence of a cleavable Nterminal pre-sequence. The ten N-terminal amino acids form an essential portion of this targeting information as deduced from deletion mutants. Recognition and translocation of IEP32 is not catalysed by the general chloroplast outer envelope translocon subunits Toc159, Toc75III and Toc34, because IEP32 import is neither inhibited by proteolytic removal of Toc34 and Toc159 nor by inhibition of the Toc75 import channel by CuCl2 or spermine. Import of IEP32 only requires ATP Chloroplasts are organelles of endosymbiotic origin. During evolution most of the genes from the endosymbiont were transferred to the host nucleus. Today more than 95% of the chloroplast proteome is nuclear encoded. Pre-proteins are synthesized in the cytosol and post translationally imported into the organelle (Chen and Schnell, 1999; Keegstra and Froehlich, 1999; Bauer et al., 2001; Soll, 2002). In most cases pre-proteins are synthesized with an NH2-terminal presequence also called targeting signal. The pre-sequence is both necessary and sufficient for organellar targeting and translocation initiation. Upon import the pre-sequence is proteolytically removed by the stromal processing peptidase (Oblong and Lamppa, 1992) and the protein can attain its native conformation. Chloroplast pre-sequences seem to share little common sequence motifs or secondary structure (v. Heijne and Nishikawa, 1991). The NH2-proximal part is normally devoid of negatively charged amino acids and the central domain is rich in the hydroxylated amino acids serine and threonine. The C-proximal region can form a b -sheet structure, which includes the processing site. The pre-sequence is recognized at the chloroplast surface by receptors, which are integral subunits of the Toc-complex (translocon at the outer envelope of chloroplast) (Hirsch et al., 1994; Kessler et al., 1994; Schnell et al., 1994; Seedorf et al., 1995). The Toc complex has three distinct core subunits, the GTP-dependent Toc34 receptor (Hirsch et al., 1994; Kessler et al., 1994; Seedorf et al., 1995), a b -barrel type import channel Toc75 (Schnell et al., 1994; Tranel et al., 1995; Hinnah et al., 1997) and a GTP-dependent receptor and motor protein Toc159 (Hirsch et al., 1994; Kessler et al., 1994; Ma et al., 1996; Schleiff et al., 2003). Binding to and translocation across the concentrations of below 20 m M indicating that stromal chaperones are not involved in the process, but that IEP32 might be directly inserted from the intermembrane space into the inner envelope by a so far unidentified pathway. IEP32 may require the assistance of Tic22, an intermembrane space translocon subunit for import as indicated by the presence of a chemical crosslinked product between both polypeptides. Toc-complex is GTP dependent (Schleiff et al., 2003). Import across the inner envelope is facilitated by the Tic-complex and requires ATP in the stroma, most likely for the action of molecular chaperones (Flgge and Hinz, 1986; Schindler et al., 1987; Theg et al., 1989). The Tic complex is composed of several subunits: Tic110 and Tic20, which may form an import channel (Kouranov et al., 1998; Heins et al., 2002); Tic40 as a chaperone coordinating factor on the stromal site of the envelope (Stahl et al., 1999; Chou et al., 2003); Tic22 as an intermembrane space factor (Kouranov, 1998) and finally the redox proteins Tic62 and Tic55 (Caliebe et al., 1997; Kchler et al., 2002). ATP concentrations above 50 m M are generally required to complete import of a standard precursor protein into chloroplasts (Flgge and Hinz, 1986; Schindler et al., 1987; Theg et al., 1989). ATP hydrolysis by molecular chaperones such as HSP93 or HSP70 is thought to provide the driving force for the final membrane passage (Nielsen et al., 1997). Proteins of the outer envelope are generally targeted and inserted into the membrane by internal sequence information and they therefore do not contain a cleavable pre-sequence (Schleiff and Klsgen, 2001). No auxiliary factor or helper protein has been identified so far that facilitates insertion into the outer envelope. Indeed, in vitro insertion can occur spontaneously into protein-free liposomes. The only known exception is Toc75, which is synthesized with a cleavable presequence and which uses the Toc and the Tic translocon for import (Tranel and Keegstra, 1996). Several reports indicate that specialised import routes into chloroplasts might exist. A nuclear encoded heat-shockinduced protein of Chlamydomonas thylakoids did not contain a cleavable pre-sequence as deduced from sequence comparison. However no in vitro imports were conducted (Grimm et al., 1989). From in vivo studies using a GFP-fusion of the chloroplast inner envelope localized quinone oxido reductase (QORH) it was shown that internal sequence information was required for correct targeting and that neither N- or C-terminal transit peptides were required (Miras et al., 2002). Tic22 takes a different route again. Whereas the preprotein contains a cleavable pre-sequence and requires protease-sensitive receptors, its import needed only low concentrations of ATP, consistent with the idea that stromal chaperones are not involved in Tic22 import (Kouranov et al., 1999). In this paper we provide evidence for a distinct import pathway into the inner envelope of chloroplasts. The inner envelope protein IEP32, also named HP32, is targeted to chloroplasts independent of a cleavable pre-sequence and any protease-sensitive surface-exposed receptor protein. Import of IEP32 does not seem to require the Toc75 import channel as deduced from inhibitor studies. The involvement of stromal chaperones is also unlikely because ATP concentrations below 20 m M are sufficient for import. Materials and Methods Transcription and translation The coding region for IEP32 from pea (GenBank accession no. AY488758) was cloned into the vector PSP65 (Promega, Madison, USA) under the control of the SP6 promoter. Deletion mutants were constructed in the same vector using standard PCR protocols. All constructs were controlled by DNA sequencing. Transcription was performed in the presence of SP6 RNA polymerase and the resulting mRNA was translated in a reticulocyte lysate system (FlexiSystem, Promega, Madison, USA) in the presence of [35S]methionine (Waegemann and Soll, 1995) at 25 C for 45 minutes. The translation mixture was then centrifuged at 250,000 g for 10 minutes at 4 C and the post-ribosomal supernatant was used for all import studies. Chloroplast isolation and protein import Chloroplasts were isolated from leaves of 10- to 12-day-old pea plants (Pisum sativum, var. Golf) and purified through Percoll density gradients as described (Waegemann and Soll, 1991). A standard import reaction contai (...truncated)