Toc64, a new component of the protein translocon of chloroplasts

Abstract

A subunit of the preprotein translocon of the outer envelope of chloroplasts (Toc complex) of 64 kD is described, Toc64. Toc64 copurifies on sucrose density gradients with the isolated Toc complex. Furthermore, it can be cross-linked in intact chloroplasts to a high molecular weight complex containing both Toc and Tic subunits and a precursor protein. The 0 A cross-linker CuCl(2) yields the reversible formation of disulfide bridge(s) between Toc64 and the established Toc complex subunits in purified outer envelope membranes. Toc64 contains three tetratricopeptide repeat motifs that are exposed at the chloroplast cytosol interface. We propose that Toc64 functions early in preprotein translocation, maybe as a docking protein for cytosolic cofactors of the protein import into chloroplasts.

Figure 1

Polypeptide composition of isolated pea chloroplast outer envelope (OE) membranes and the purified Toc complex. A silverstained SDS-PAGE is shown.

Figure 2

Sequence analysis of Toc64. (a) Protein sequence of pea Toc64 as deduced from the cDNA clone. The determined amino acid sequences are underlined. These sequence data are available from GenBank/EMBL/DDBJ under accession no. AF179282. (b) Sequence comparison of pea (Pis) Toc64 with a Toc64 homologue from Arabidopsis thaliana (Ara; sequence data available from EMBL/GenBank/DDBJ under accession no. H36863), amidases (amid) from Arabidopsis thaliana (sequence data available from EMBL/GenBank/DDBJ under accession no. AC000106), Rhodococcus rhodochrous (Rho; sequence data available from EMBL/GenBank/DDBJ under accession no. D16207), and Synechocystis sp. (Syn; sequence data available from EMBL/GenBank/DDBJ under accession no. D90907), Aquifex aeolicus glutamyl-tRNA^Gln amidotransferase subunit A (Aqu Glu-AdT; sequence data available from EMBL/GenBank/DDBJ under accession no. AE000680), and Pseudomonas syringae indoleacetamide hydrolase (Pse IAH; sequence data available from EMBL/GenBank/DDBJ under accession no. U04358) using the ClustalW 1.7 and BOXSHADE programs. The active residues of the amidases are indicated by asterisks. The arrow shows the amino acid exchange of Toc64. (c) Sequence alignment of TPR motifs of different polypeptides: pea Toc64, its homologue from Arabidopsis thaliana, hop from Rattus norvegicus (Rat; sequence data available from EMBL/GenBank/DDBJ under accession no. CAA75351) and its plant homologue Sti1 from Glycine max (Gly; sequence data available from EMBL/GenBank/DDBJ under accession no. S56658), human hTom34p (Hum; sequence data available from EMBL/GenBank/DDBJ under accession no. U58970), Podospora anserina (Pod) Tom70 (sequence data available from EMBL/GenBank/DDBJ under accession no. Y14750), and serine/threonine protein phosphatase 5 (PP5) from Rattus norvegicus (sequence data available from EMBL/GenBank/DDBJ under accession no. P53042) using the ClustalW 1.7 and BOXSHADE programs. (d) Hydropathy analysis of Toc64 according to Kyte and Doolittle 1982 using a window size of 19 amino acids.

Figure 3

Toc64 is an integral protein of the outer envelope of pea chloroplasts. (a) Immunoblot analysis of the different subcompartments using αToc64 antiserum. Proteins were separated on a 12.5% (wt/vol) acrylamide containing running gel (lanes 1–4). Total soluble chloroplast proteins were immunodecorated with αToc64 (lanes 5 and 6) or with antiserum against chaperonin 60 (lanes 7 and 8) before or after centrifugation. Proteins were separated on a 15% (wt/vol) acrylamide containing running gel at 35 mA for 5 h. (b) Purified outer envelope membranes were treated with 1 mM NaCl, 0.5 M Na₂CO₃, or 4 M urea as indicated and separated into soluble (S) and insoluble (P) protein fractions. An immunoblot is shown.

Figure 4

Immunoblot analysis of the purified Toc complex or isolated outer envelope membranes using the antisera indicated. The position of Toc160 and its typical proteolytical fragments is indicated. Identical samples were separated in distinct SDS-PAGE lanes and analyzed with antisera against Toc160, or Toc75, Toc64, Toc34, and OEP16, respectively.

Figure 5

Toc64 can be cross-linked to other Toc complex subunits. (a) Outer envelope membranes (40 μg protein) were either not treated or treated with 1 mM CuCl₂ or 1 mM DSP. (b) High molecular weight cross-links form in a concentration-dependent manner in the presence of CuCl₂. Cross-linking was performed for 15 min using the concentrations indicated. Cross-linked products were separated by SDS-PAGE and visualized by staining with Coomassie brilliant blue. Numbers 1 and 3 label identical bands as in a. (c) Prominent cross-linked products (bands 1–4 in a) were cut out of the gel, divided into equal aliquots, and separated by a second SDS-PAGE under reducing conditions. Proteins were analyzed either by silverstaining (left panel) or immunoblotting using the antisera indicated (right panel).

Figure 6

Toc64 is recovered in a precursor protein containing membrane complex. Intact pea chloroplasts were incubated with ³⁵S-labeled preSSU at 4°C and 100 μM ATP for 5 min. Chloroplasts were recovered from the incubation mixture, washed, and subjected to cross-linking in the absence or presence of 1 mM CuCl₂. Organelles were lysed, the membrane fractions extracted with 4 M urea, and envelope membranes enriched by centrifugation on a sucrose cushion. Envelope membranes were incubated with or without 20 mM DTT, washed, solubilized in SDS, and used for coimmunoprecipitation by αToc64 or preimmune serum as indicated. Lanes 1–4 contain 3% of the reaction mixture that was used in the immunoprecipitations presented in lanes 1′–4′. The immunoprecipitates were further analyzed by SDS-PAGE followed by immunoblotting using the antisera indicated or for the presence of [³⁵S]preSSU by autoradiography.

Figure 7

Topological arrangement of Toc64 in the outer envelope. (a) Insertion of ³⁵S-labeled Toc64 translation product (TL) either into pea chloroplasts (Chl) or isolated outer envelope membranes (OE). After insertion, organelles or membranes were either not treated or treated with the protease thermolysin as indicated. TL, 10% of translation product added to an insertion assay. A fluorogram is shown. (b) Intact chloroplasts or purified outer envelope were either not treated or treated with the protease thermolysin as indicated. Chloroplasts were treated at pH 11 as described for Fig. 6. Proteins were separated by SDS-PAGE and analyzed by immunoblotting using αToc64. Lane 3 contains a mixture of chloroplasts and envelope, which were combined after completion of proteolysis. (c) The topology of Toc64 was analyzed in right-side-out outer envelope membrane vesicles before or after treatment with thermolysin as indicated. Lanes marked IB contain 3 μg of recombinant Toc64, Toc64ΔTPR, and Toc64TPR protein. Thermolysin-treated membranes were separated into a supernatant (S) and a membrane fraction. The membranes were further treated with 0.5 M Na₂CO₃ and fractionated into a soluble (S) and insoluble (P) protein fractions as indicated. The immunoblots were incubated with total Toc64 serum (left panel) or serum that recognized the NH₂-terminal portion of Toc64 (αToc64ΔTPR, middle panel) or the COOH-terminal part of Toc64 (αToc64TPR, right panel). (d) Schematic representation of Toc64. The localization of distinct protein domains is indicated in the upper lane. The middle lane shows the beginning of the soluble 16-kD fragment as determined by sequencing. The lower lane indicates the position and size of truncated Toc64 proteins, which were heterologously expressed and used to affinity-isolate the different antibody populations.