Plant chloroplasts originated from an endosymbiotic event by which an ancestor of contemporary cyanobacteria was engulfed by an early eukaryotic cell and then transformed into an organelle. Oxygenic photosynthesis is the specific feature of cyanobacteria and chloroplasts, and the photosynthetic machinery resides in an internal membrane system, the thylakoids. The origin and genesis of thylakoid membranes, which are essential for oxygenic photosynthesis, are still an enigma. Vipp1 (vesicle-inducing protein in plastids 1) is a protein located in both the inner envelope and the thylakoids of Pisum sativum and Arabidopsis thaliana. In Arabidopsis disruption of the VIPP1 gene severely affects the plant's ability to form properly structured thylakoids and as a consequence to carry out photosynthesis. In contrast, Vipp1 in Synechocystis appears to be located exclusively in the plasma membrane. Yet, as in higher plants, disruption of the VIPP1 gene locus leads to the complete loss of thylakoid formation. So far VIPP1 genes are found only in organisms carrying out oxygenic photosynthesis. They share sequence homology with a subunit encoded by the bacterial phage shock operon (PspA) but differ from PspA by a C-terminal extension of about 30 amino acids. In two cyanobacteria, Synechocystis and Anabaena, both a VIPP1 and a pspA gene are present, and phylogenetic analysis indicates that VIPP1 originated from a gene duplication of the latter and thereafter acquired its new function. It also appears that the C-terminal extension that discriminates VIPP1 proteins from PspA is important for its function in thylakoid formation.

Pre‐protein translocation into chloroplasts is accomplished by two distinct translocation machineries in the outer and inner envelope, respectively. We have isolated the translocon at the inner envelope membrane (Tic complex) by blue‐native PAGE and describe a new Tic subunit, Tic62. Tic62, together with Tic110 and Tic55, forms a core translocation unit. The N‐terminus of Tic62 shows strong homologies to NAD(H) dehydrogenases in eukaryotes and to Ycf39‐like proteins present in cyanobacteria and non‐green algae. The stromal‐facing C‐terminus of Tic62 contains a novel, repetitive module that interacts with a ferredoxin‐NAD(P)+ oxidoreductase. Ferredoxin‐NAD(P)+ oxidoreductase catalyses the final electron transfer of oxygenic photosynthesis from ferredoxin to NAD(P). Substrates that interfere with either NAD binding, such as deamino‐NAD, or influence the ratio of NAD(P)/NAD(P)H, such as ruthenium hexamine trichloride, modulate the import characteristics of leaf‐specific ferredoxin‐NAD(P)+ oxidoreductase isologues differently. We conclude that the Tic complex can regulate protein import into chloroplasts by sensing and reacting to the redox state of the organelle.

Transport of precursor proteins across the chloroplastic envelope membranes requires the interaction of protein translocons localized in both the outer and inner envelope membranes. Analysis by blue native gel electrophoresis revealed that the translocon of the inner envelope membranes consisted of at least six proteins with molecular weights of 36, 45, 52, 60, 100 and 110 kDa, respectively. Tic110 and ClpC, identified as components of the protein import apparatus of the inner envelope membrane, were prominent constituents of this complex. The amino acid sequence of the 52 kDa protein, deduced from the cDNA, contains a predicted Rieske-type iron-sulfur cluster and a mononuclear iron-binding site. Diethylpyrocarbonate, a Rieske-type protein-modifying reagent, inhibits the translocation of precursor protein across the inner envelope membrane, whereas binding of the precursor to the outer envelope membrane is still possible. In another independent experimental approach, the 52 kDa protein could be copurified with a trapped precursor protein in association with the chloroplast protein translocon subunits Toc86, Toc75, Toc34 and Tic110. Together, these results strongly suggest that the 52 kDa protein, named Tic55 due to its calculated molecular weight, is a member of the chloroplastic inner envelope protein translocon.

Chloroplasts import post-translationally most of their constituent polypeptides via two distinct translocon units located in the outer and inner envelope. The protein import channel of the translocon of the outer envelope of chloroplasts, Toc75, is the most abundant protein in that membrane. We identify a novel Toc75 homologous protein, atToc75-V, a prominent protein that is clearly localized in the chloroplastic outer envelope. Phylogenetic analysis indicates that Toc75-V is more closely related to its prokaryotic ancestors than to Toc75 from plants. The presence of a second translocation channel suggests that alternative, previously unrecognized import routes into chloroplasts might exist.

The protein import translocon at the inner envelope of chloroplasts (Tic complex) is a heteroligomeric multisubunit complex. Here, we describe Tic40 from pea as a new component of this complex. Tic40 from pea is a homologue of a protein described earlier from Brassica napus as Cim/Com44 or the Toc36 subunit of the translocon at the outer envelope of chloroplasts, respectively (Wu, C., Seibert, F. S., and Ko, K. During endocytobiosis, chloroplasts and mitochondria have relinquished most of their genes to the host nucleus (1). Hence today, these organelles import the vast majority of their constituent proteins in a post-translational event from the cytoplasm. Nuclear-encoded chloroplast precursor proteins are imported to chloroplastidic subcompartments by preprotein translocases, which are located both in the outer and inner envelope membranes of the organelle. The translocon at the outer membrane of chloroplasts (Toc 1 complex) and the translocon inner membrane of chloroplasts (Tic complex) act cooperatively during the import process. According to the unified nomenclature (2), single subunits are named according to their calculated molecular size and their affiliation with either the chloroplastic outer or inner envelope membrane. The core subunits of the Toc complex from pea are Toc160, Toc75, and Toc34 (for review see Refs. 3-6). Toc160 is a putative precursor receptor and succeeds Toc86, which represents only a proteolytic C-terminal fragment of Toc160 (7). Toc75 forms the aqueous translocation pore, which is used by precursor proteins (8, 9). The function of Toc34 is less clear, but in the light of its GTP-binding properties, it could regulate precursor recognition (10 -12) or translocation. Brassica napus bnToc36, formerly named chloroplast inner membrane, chloroplast outer membrane protein of 44 kDa (Cim/Com44) or Bce44B (13, 14), might be another import relevant protein. Conflicting results do not allow the clear assignment of the protein to either the inner or the outer envelope membrane, reflected by its name Cim/ Com44 (13,14). Finally the protein was named Toc36 (2, 15) without additional experimentation or elucidation of its role in import. Two Hsp70 homologues associated either with the cytosolic site, i.e. Com70 (16) or the intermembrane face of the outer envelope membrane (17, 18), seem also to be involved in the translocation process.Subunits identified in the Tic complex include Tic110, Tic55, Tic22, and Tic20, however, their role in translocation is less well defined than for the Toc components. Tic110 might be involved in recruiting stromal Hsp100 or chaperonine 60 to the import site (10, 20, 21), whereas Tic110 domains exposed to the envelope intermembrane space could form translocation contact sites (22, 23). Tic55, containing a Rieske-type iron-sulfur motif, might act as a redox sensor to regulate import capacity of chloroplasts (24). Tic22 is peripherally associated with the intermembrane face of the inner envelope. Tic20 is an integral subunit of the Tic complex and is suggested to form pa...

The preprotein translocation at the inner envelope membrane of chloroplasts so far involves ®ve proteins: Tic110, Tic55, Tic40, Tic22 and Tic20. The molecular function of these proteins has not yet been established. Here, we demonstrate that Tic110 constitutes a central part of the preprotein translocation pore. Dependent on the presence of intact Tic110, radiolabelled preprotein speci®cally interacts with isolated inner envelope vesicles as well as with puri®ed, recombinant Tic110 reconstituted into liposomes. Circular dichroism analysis reveals that Tic110 consists mainly of b-sheets, a structure typically found in pore proteins. In planar lipid bilayers, recombinant Tic110 forms a cation-selective high-conductance channel with a calculated inner pore opening of 1.7 nm. Puri®ed transit peptide causes strong¯ickering and a voltage-dependent block of the channel. Moreover, at the inner envelope membrane, a peptide-sensitive channel is described that shows properties basically identical to the channel formed by recombinant Tic110. We conclude that Tic110 has a distinct preprotein binding site and functions as a preprotein translocation pore at the inner envelope membrane.

The chloroplastic inner envelope protein of 110 kD (IEP110) is part of the protein import machinery in the pea. Different hybrid proteins were constructed to assess the import and sorting pathway of IEP110. The IEP110 precursor (pIEP110) uses the general import pathway into chloroplasts, as shown by the mutual exchange of presequences with the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase (pSSU). Sorting information to the chloroplastic inner envelope is contained in an NH2-proximal part of mature IEP110 (110N). The NH2-terminus serves to anchor the protein into the membrane. Large COOH-terminal portions of this protein (80–90 kD) are exposed to the intermembrane space in situ. Successful sorting and integration of IEP110 and the derived constructs into the inner envelope are demonstrated by the inaccessability of processed mature protein to the protease thermolysin but accessibility to trypsin, i.e., the imported protein is exposed to the intermembrane space. A hybrid protein consisting of the transit sequence of SSU, the NH2-proximal part of mature IEP110, and mature SSU (tpSSU-110N-mSSU) is completely imported into the chloroplast stroma, from which it can be recovered as soluble, terminally processed 110NmSSU. The soluble 110N-mSSU then enters a reexport pathway, which results not only in the insertion of 110N-mSSU into the inner envelope membrane, but also in the extrusion of large portions of the protein into the intermembrane space. We conclude that chloroplasts possess a protein reexport machinery for IEPs in which soluble stromal components interact with a membrane-localized translocation machinery.

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