A nuclear-coded chloroplastic inner envelope membrane protein uses a soluble sorting intermediate upon import into the organelle.

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.

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.

The Evolving Theory of Evolutionary Radiations.

Evolutionary radiations have intrigued biologists for more than 100 years, and our understanding of the patterns and processes associated with these radiations continues to grow and evolve. Recently it has been recognized that there are many different types of evolutionary radiation beyond the well-studied adaptive radiations. We focus here on multifarious types of evolutionary radiations, paying special attention to the abiotic factors that might trigger diversification in clades. We integrate concepts such as exaptation, species selection, coevolution, and the turnover-pulse hypothesis (TPH) into the theoretical framework of evolutionary radiations. We also discuss other phenomena that are related to, but distinct from, evolutionary radiations that have relevance for evolutionary biology.

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.

A receptor for protein import into potato mitochondria.

Five potential surface receptors for protein import into plant mitochondria were identified by gentle trypsin treatment of intact mitochondria from potato tubers and subsequent preparation of outer mitochondrial membranes. One of them, a 23 kDa protein, was purified to homogeneity and analysed by direct protein sequencing. Copy DNA clones encoding the corresponding polypeptide were isolated with labelled oligonucleotides derived from the amino acid data. The 23 kDa protein shares significant sequence similarity with protein import receptors from fungal mitochondria and contains one of their typical tetratricopeptide motifs. Its integration into the outer membrane is independent of protease accessible surface receptors and not accompanied by proteolytic processing. Monospecific antibodies against the 23 kDa protein significantly reduce import capacity of isolated mitochondria indicating that this component is indeed involved in the recognition or import of precursor proteins. As in fungi, immunological inhibition of protein import with IgGs against a single receptor is incomplete suggesting the existence of other receptors in the outer mitochondrial membrane of plant mitochondria.

Characterization of the interaction of plant transcription factors using a bacterial repressor protein.

Transcription initiation from a eukaryotic polymerase II promoter requires a functional interaction of regulatory transcriptional activators with at least one of the basal transcription factors binding in the vicinity of the TATA box. To characterize this type of interaction in vivo, we have inserted the bacterial Tet repressor-operator complex in nine different positions between an enhancer element (as-1) and the TATA box of the cauliflower mosaic virus (CaMV) 35S RNA promoter. A direct contact between the transcriptional activator ASF-1, which binds to as-1, and the transcriptional machinery should be affected by a repressor protein bound between them, as the spacing of only 34 base pairs (bp) between as-1 and the TATA box is too short to allow looping of the DNA around the repressor. In each construct, the distance of 34 bp was kept constant, while the position of the 19-bp tet operator relative to the TATA box differed by 2 bp. Thus, the position of the Tet repressor relative to the plant transcription factors was consecutively changed by 72 degrees, which allowed us to investigate whether repression depended on the stereospecific alignment of the repressor with the transcription factors. Binding of the Tet repressor to the operator blocked transcription only when the operator was inserted less tha 5 bp from the TATA box. In all other promoter derivatives, no inhibitory effect of the repressor was observed, which suggests that ASF-1 does not directly interact with the general transcription machinery.

The Tn10-encoded Tet repressor blocks early but not late steps of assembly of the RNA polymerase II initiation complex in vivo.

We have studied the effect of the Tn10-encoded Tet repressor on expression from 13 cauliflower mosaic virus (CaMV) 35S promoter derivatives that contain a tet operator sequence in various positions downstream of the TATAbox. When the operator sequence was inserted less than 33 bp away from the TATAbox (position +9 with respect to the transcription start site), the repressor interfered with transcription, whereas increasing the distance to 35 bp (position +11) abolished repression. This result indicates that initiation of transcription from the CaMV 35S promoter occurs in at least two different steps: (1) binding of transcription factors, involving sequences extending to position +9; this step can be inhibited by binding of the Tet repressor protein; and (2) initiation of transcription from this complex, which is not affected by the repressor protein. We suggest that the Tet repressor can be used to investigate whether transcription conditions in vitro truly reflect the in vivo situation.

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 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.

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 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.

Biochemical, molecular, and functional characterization of porin isoforms from potato mitochondria.

The mitochondrial outer membrane of eukaryotic cells contains a voltage-dependent anion channel termed porin. In the organisms studied so far only one type of porin has been identified at the protein level. Here we present a biochemical and molecular genetic analysis of two different porin polypeptides of M(r) 34,000 and 36,000 from the outer membranes of potato mitochondria (termed POM 34 and POM 36, respectively). N-terminal sequencing and the use of labeled oligonucleotide mixtures derived from these amino acid sequences allowed the isolation of cDNA clones encoding the 34- and 36-kDa proteins. They have similar steady state protein levels and share about 75% identical amino acids suggesting that they represent isoforms. In addition, a third cDNA clone coding for a slightly different isoform of the 36-kDa protein was characterized. The polypeptides encoded by the three cDNA clones share the highest degree of sequence identity with mitochondrial porins from fungi and mammals. Tentative models of the secondary structure of the 34- and 36-kDa proteins suggest the occurrence of a 16-stranded beta-barrel typical for bacterial and mitochondrial porins. Purification of the 34-kDa protein by hydroxyapatite chromatography allowed conductance measurements in artificial bilayers. The 34-kDa protein is a voltage-dependent, channel-forming component with single channel conductances of 3.5 and 2.0 nanosiemens in 1 M KCl. In spite of the striking functional similarities to mitochondrial porins from other organisms neither the 34- nor the 36-kDa proteins are able to complement the respiratory defect of a yeast por- mutant.

Molecular features, processing and import of the Rieske iron-sulfur protein from potato mitochondria.

The mitochondrial iron-sulfur protein (also termed Rieske iron-sulfur protein) of cytochrome c reductase was purified from potato tubers and identified with heterologous antibodies. The sequences of the N-terminus of this 25 kDa protein and of an internal peptide were determined to design oligonucleotide mixtures for screening a cDNA library. One class of cDNA clones containing an open reading frame of 265 amino acids was isolated. The encoded protein contains the peptide sequences of the 25 kDa protein and shares about 50% sequence identity with the Rieske iron-sulfur proteins from fungi and around 43% with those from mammals. In vitro transcription and translation of the cDNA reveals that the iron-sulfur protein is made as a larger precursor of 30 kDa which is processed by the cytochrome c reductase/processing peptidase complex from potato. The processing product obtained after in vitro processing has the same size as the mature protein imported into isolated mitochondria. The presequence, which targets the protein to the organelle, is 53 amino acids long and has molecular features different from those found in presequences of fungal iron-sulfur proteins, which are processed in two steps. Our results indicate that, unlike in yeast and Neurospora, the presequence of the iron-sulfur protein from potato is removed by a single processing enzyme in one step.