Lisa Heins

The chloroplastic protein import machinery contains a Rieske‐type iron–sulfur cluster and a mononuclear iron‐binding protein

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 co‐purified 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.

Vipp1 deletion mutant of Synechocystis: A connection between bacterial phage shock and thylakoid biogenesis?

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 plants 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.

The preprotein conducting channel at the inner envelope membrane of plastids

The preprotein translocation at the inner envelope membrane of chloroplasts so far involves five 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 specifically interacts with isolated inner envelope vesicles as well as with purified, recombinant Tic110 reconstituted into liposomes. Circular dichroism analysis reveals that Tic110 consists mainly of β‐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. Purified transit peptide causes strong flickering 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.

Tic40, a New “Old” Subunit of the Chloroplast Protein Import Translocon

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. (1994)J. Biol. Chem. 269, 32264–32271; Ko, K., Budd, D., Wu, C., Seibert, F., Kourtz, L., and Ko, Z. W. (1995)J. Biol. Chem. 270, 28601–28608; Pang, P., Meathrel, K., and Ko, K. (1997) J. Biol. Chem. 272, 25623–25627). Tic40 can be covalently connected to Tic110 by the formation of a disulfide bridge under oxidizing conditions, indicating its close physical proximity to an established translocon component. The Tic40 protein is synthesized in the cytosol as a precursor with an N-terminal cleavable chloroplast targeting signal and imported into the organelle via the general import pathway. Immunoblotting and immunogold-labeling studies exclusively confine Tic40 to the chloroplastic inner envelope, in which it is anchored by a single putative transmembrane span.

The protein translocation apparatus of chloroplast envelopes

The evolution of the chloroplast from a photosynthetic prokaryote has resulted in the displacement of most of the prokaryote genes to the nucleus of the host eukaryote. Accordingly, the new organism has evolved targeting and translocation mechanisms on the organellar membranes for nuclear-encoded proteins. In plastids, the protein-import machinery is distinct from that of other organelles, in both composition and mechanism. Recently, proteins homologous to several subunits of the chloroplast import machinery were identified in the cyanobacterium Synechocystis PCC6803. It appears that parts of the protein-import machinery of chloroplasts are derived from ancient transport systems in cyanobacteria. These observations open up new avenues for elucidating the origin of the chloroplast membranes and functional properties of the protein-import machinery.

Chloroplast biogenesis: Mixing the prokaryotic and the eukaryotic?

Chloroplast biogenesis requires the translocation of proteins across the outer and inner envelopes. The membrane components of this transport machinery completely differ from those of other organelles, but recently homologues of some of the components have been detected in prokaryotes.

Structural and kinetic properties of adenylyl sulfate reductase from Catharanthus roseus cell cultures.

A cDNA encoding a plant-type APS reductase was isolated from an axenic cell suspension culture of Catharanthus roseus (Genbank/EMBL-databank accession number U63784). The open reading frame of 1392 bp (termed par) encoded for a protein (Mr=51394) consisting of a N-terminal transit peptide, a PAPS reductase-like core and a C-terminal extension with homology to the thioredoxin-like domain of protein disulfide isomerase. The APS reductase precursor was imported into pea chloroplasts in vitro and processed to give a mature protein of approximately 45 kDa. The homologous protein from pea chloroplast stroma was detected using anti:par polyclonal antibodies. To investigate the catalytical function of the different domains deleted par proteins were purified. ParDelta1 lacking the transit sequence liberated sulfite from APS (Km 2.5+/-0.23 microM) in vitro with glutathione (Km 3+/-0.64 mM) as reductant (Vmax 2.6+/-0.14 U mg-1, molecular activity 126 min-1). ParDelta2 lacking the transit sequence and C-terminal domain had to be reconstituted with exogenous thioredoxin as reductant (Km 15. 3+/-1.27 microM, Vmax 0.6+/-0.014 U mg-1). Glutaredoxin, GSH or DTT were ineffective substitutes. ParDelta1 (35.4%) and parDelta2 (21. 8%) both exhibited insulin reductase activity comparable to thioredoxin (100%). Protein disulfide isomerase activity was observed for parDelta1.

The Import and Sorting of Protein into Chloroplasts

Publisher Summary This chapter outlines the import and sorting of protein into chloroplasts. The characteristic organelles of plant cells are plastids. All plastids evolve from undifferentiated, semiautonomous proplastids. They contain their own genome but it comprises only a subset of the genetic information necessary for the biogenesis of diverse plastids. Depending on the tissue and developmental stage, plastids have different functions such as the synthesis of carotenoids in chromoplasts, which mainly reside in petals and fruits. The cytosol should not be imagined as an open space. Since presequences lack a strict consensus sequence, a specific modification might help to increase the fidelity of the chloroplast signal. Molecular chaperones such as members of the Hsp70 family have been shown to maintain preproteins in an unfolded, import-competent conformation after synthesis at cytosolic ribosomes. Hetero-oligomeric protein complexes at both the outer and the inner envelope membrane facilitate the joint translocation of preproteins containing a cleavable presequence. In the second study, dihydrofolate reductase was found to be efficiently transported across the thylakoid membrane by the Tat system after binding a folate analog in the active site. The targeting of thylakoid membrane proteins has also been studied with some enthusiasm and two further pathways have been identified for these hydrophobic proteins following their import into the plastid.