Gramicidin S synthetase 1 and 2 were affinity-labeled at their thiolation centers either by thioesterification with the amino acid substrate or by specific alkylation with the thiol reagent N-ethylmaleimide in combination with a substrate protection technique. The labeled proteins were digested either chemically by cyanogen bromide or by proteases. An efficient multistep high pressure liquid chromatography methodology was developed and used to isolate the active site peptide fragments of all five thiolation centers of gramicidin S synthetase in pure form. The structures of these fragments are investigated by N-terminal sequencing, mass spectrometry, and amino acid analysis. Each of the active site peptide fragments contains the consensus motif LGG(H/D)S(L/I), which is specific for thioester formation in nonribosomal peptide biosynthesis. It was demonstrated that a 4'-phosphopantetheine cofactor is attached to the central serine of the thiolation motif in each amino acid-activating module of the gramicidin S synthetase multienzyme system forming the thioester binding sites for the amino acid substrates and catalyzing the elongation process. Our data are strong support for a "multiple carrier model" of nonribosomal peptide biosynthesis at multifunctional templates, which is discussed in detail.

An Arabidopsis mitochondrial proteome project was started for a comprehensive investigation of mitochondrial functions in plants. Mitochondria were prepared from Arabidopsis stems and leaves or from Arabidopsis suspension cell cultures, and the purity of the generated fractions was tested by the resolution of organellar protein complexes applying two-dimensional blue-native/N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine (Tricine) sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Arabidopsis mitochondrial proteome was analyzed by two-dimensional isoelectric focusing/ Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 650 different proteins in a pI range of pH 3 to 10 were separated on single gels. Solubilization conditions, pH gradients for isoelectric focusing, and gel staining procedures were varied, and the number of separable proteins increased to about 800. Fifty-two protein spots were identified by immunoblotting, direct protein sequencing, and mass spectrometry. The characterized proteins cooperate in various processes, such as respiration, citric acid cycle, amino acid and nucleotide metabolism, protection against O2, mitochondrial assembly, molecular transport, and protein biosynthesis. More than 20% of the identified proteins were not described previously for plant mitochondria, indicating novel mitochondrial functions. The map of the Arabidopsis mitochondrial proteome should be useful for the analysis of knockout mutants concerning nuclear-encoded mitochondrial genes. Considerations of the total complexity of the Arabidopsis mitochondrial proteome are discussed. The data from this investigation will be made available athttp://www.gartenbau.uni-hannover.de/genetik/AMPP.

Recently a powerful electrophoresis method for the native preparation and characterization of the respiratory protein complexes of mitochondria from fungi and mammals has been developed, which employs Coomassie dyes to introduce charge shifts on proteins (Schägger and von Jagow (1991) Anal. Biochem. 199, 223-231). The procedure, which is called 'blue native-polyacrylamide gel electrophoresis' (BN-PAGE), was modified and introduced for the analysis of mitochondria from higher plants. BN-PAGE of mitochondrial protein from potato allows the separation of nine distinct protein complexes between 100 and 1000 kDa and reveals novel results for their composition, molecular mass and stoichiometry. For the first time soluble mitochondrial protein complexes, like the HSP60 complex (750 kDa) and a complex of 200 kDa, which includes a formate dehydrogenase, are analysed by BN-PAGE. Complex I from potato (1000 kDa) is about 100 kDa larger than the corresponding enzyme from beef and can be resolved into more than 30 different subunits on a second gel dimension. The F1F0 ATP synthase (580 kDa) and the cytochrome c oxidase (160 kDa) from potato seem to contain more subunits than hitherto reported. Direct sequencing of subunits revealed that the F1 part of the F1F0 ATP synthase lacks the oligomycin sensitivity conferring protein (OSCP), which was reported to be present in F1 parts of dicotyledonous plants, but contains the ATPase inhibitory protein. N-terminal sequences of 16 mitochondrial proteins were obtained, several of which are presented for the first time from a plant source. BN-PAGE allows the preparation of mitochondrial protein complexes from gram amounts of plant tissue, as the procedure only requires milligram amounts of organelles. This potential of BN-PAGE is demonstrated by the separation and characterization of the mitochondrial enzyme complexes from Arabidopsis thaliana. Further analysis of organellar protein complexes by BN-PAGE will allow the generation of 'protein maps' from different tissues and developmental stages or from mutant plants.

During tiatin of conjugative transfer of DNA containing the transfer origin (onTiT) of the promiscuous plasmid RP4, the proteins TraI, TraJ, and Trai interact and assemble a specialized nucleoprotein complex (the relaxosome) at onT. The structure can be visualized on electron micrographs. Site-and strand-specific nicking at the transfer origin in vitro is dependent on the proteins TraIl and TraJ and on Nicking and relaxosome formation require supercoiled DNA.Thus, a complicated structure involving multiple plasmidspecified proteins and a defined region of DNA must be formed at the transfer origin to prepare the plasmid for generating the single strand to be transferred.Bacterial conjugation mediates horizontal gene transfer between a wide variety of organisms (1, 2). The general model for initiation of conjugative DNA synthesis proposes that cleavage at the nic site within the transfer origin (oriT) allows creation of a single strand by subsequent strand displacement through rolling-circle-type replication. A prerequisite for the initial nicking reaction is the formation of a specialized nucleoprotein structure at oriT, the relaxosome. For the promiscuous IncPa plasmid RP4, it has been shown that genes specifying proteins which exert high-precision interactions with oriT sequences directly flank the transfer origin (3). The traJ gene encodes an oriT-recognizing protein essential for initiation of transfer DNA replication (3, 4). Genetic and biochemical data indicate that the product of the traf gene is an additional component of the relaxosome (3, 5).However, definite proof that the gene products specified by the relaxase operon (4) MATERIALS AND METHODS Bacterial Strains, Plasmids, and Media. Escherichia coli strain SCS1 (6) was used as host for plasmids. Recombinant plasmids pJF166uA2 (3), pMS2260 (7), pGZ192OA114 (8), pJF145n (3), and pWP392n (7) have been described. Expression systems used were the 17410/T7 gene 10 S/D vector pT7-7 obtained from Stanley Tabor (Harvard Medical School) and the Ptac/lacIQ vector pJF119HE (9). Cells were grown under the conditions described (5). When appropriate, antibiotics were used at the following concentrations: chloramphenicol, 10 /Lg/ml; ampicillin (sodium salt), 100 .g/ml.DNA Techniques. Standard molecular cloning techniques were performed as described (10).Proteins. TraJ protein was purified as described (4). A purification procedure for TraH protein (3) will be published elsewhere. Antiserum to the TraI protein was raised in rabbits by using a TraL/Tral hybrid polypeptide as immunogen (5).Immunoblot Assay. Proteins were electrophoresed in SDS/ 15% (wt/vol) polyacrylamide gels and transferred electrophoretically to a nitrocellulose membrane (12 V/cm, 90 min; ref. 11), followed by reaction with anti-TraL/Tral serum (1:500, 2 hr) and dichlorotriazinylaminofluorescein-conjugated goat anti-rabbit IgGs (Jackson ImmunoResearch; 1:50, 45 min).In Vitro Reconstitution of Relaxosomes. Under standard conditions, mixtures of Tra proteins and supercoiled plasmid DNA (0.7 ;...

Respiratory oxidative phosphorylation represents a central functionality in plant metabolism, but the subunit composition of the respiratory complexes in plants is still being defined. Most notably, complex II (succinate dehydrogenase) and complex IV (cytochrome c oxidase) are the least defined in plant mitochondria. Using Arabidopsis mitochondrial samples and 2D Blue-native/SDS-PAGE, we have separated complex II and IV from each other and displayed their individual subunits for analysis by tandem mass spectrometry and Edman sequencing. Complex II can be discretely separated from other complexes on Blue-native gels and consists of eight protein bands. It contains the four classical SDH subunits as well as four subunits unknown in mitochondria from other eukaryotes. Five of these proteins have previously been identified, while three are newly identified in this study. Complex IV consists of 9-10 protein bands, however, it is more diffuse in Blue-native gels and co-migrates in part with the translocase of the outer membrane (TOM) complex. Differential analysis of TOM and complex IV reveals that complex IV probably contains eight subunits with similarity to known complex IV subunits from other eukaryotes and a further six putative subunits which all represent proteins of unknown function in Arabidopsis . Comparison of the Arabidopsis data with Blue-native/SDS-PAGE separation of potato and bean mitochondria confirmed the protein band complexity of these two respiratory complexes in plants. Two-dimensional Blue-native/Blue-native PAGE, using digitonin followed by dodecylmaltoside in successive dimensions, separated a diffusely staining complex containing both TOM and complex IV. This suggests that the very similar mass of these complexes will likely prevent high purity separations based on size. The documented roles of several of the putative complex IV subunits in hypoxia response and ozone stress, and similarity between new complex II subunits and recently identified plant specific subunits of complex I, suggest novel biological insights can be gained from respiratory complex composition analysis.

The translocase of the outer mitochondrial membrane (TOM) complex is a preprotein translocase that mediates transport of nuclear-encoded mitochondrial proteins across the outer mitochondrial membrane. Here we report the purification of this protein complex from Arabidopsis. On blue-native gels the Arabidopsis TOM complex runs at 230 kD and can be dissected into subunits of 34, 23, 21, 8, 7, and 6 kD. The identity of four subunits could be determined by immunoblotting and/or direct protein sequencing. The 21-and the 23-kD subunits exhibit significant sequence homology to the TOM20 preprotein receptor from other organisms. Analysis by two-dimensional isoelectric focusing/Tricine sodium dodecyl sulfide-polyacrylamide gel electrophoresis revealed the presence of further forms for Arabidopsis TOM20. All TOM20 proteins comprise a large cytoplasmically exposed hydrophilic domain, which is degraded upon trypsination of intact mitochondria. Clones encoding four different forms of Arabidopsis TOM20 were identified and sequenced. The deduced amino acid sequences are rather conserved in the N-terminal half and in the very C-terminal part, but include a highly variable glycine-rich region close to the C terminus. Implications on the function of plant TOM complexes are discussed. Based on peptide and nucleic acid sequence data, the primary structure for Arabidopsis TOM40 is presented.Prerequisites for protein transport into mitochondria are targeting information of the proteins to be transported and a mitochondrial "protein import apparatus" that decodes the targeting information and mediates translocation of proteins across the organellar membranes (for review, see Neupert, 1997;Mori and Terada 1998; Braun and Schmitz, 1999;Voos et al., 1999). The targeting information often is localized on N-terminal extensions, termed presequences, which are removed within the organelles by processing peptidases (Braun and Schmitz, 1998). Central components of the protein import apparatus are translocase complexes: the preprotein translocase of the outer mitochondrial membrane (the so-called TOM complex) and the two preprotein translocases of the inner mitochondrial membrane (called TIM complexes). These translocases were first described for the fungi yeast and Neurospora crassa (for review, see Meisinger et al

The major mitochondrial processing activity removing presequences from nuclear encoded precursor proteins is present in the soluble fraction of fungal and mammalian mitochondria. We found that in potato, this activity resides in the inner mitochondrial membrane. Surprisingly, the proteolytic activity co‐purifies with cytochrome c reductase, a protein complex of the respiratory chain. The purified complex is bifunctional, as it has the ability to transfer electrons from ubiquinol to cytochrome c and to cleave off the presequences of mitochondrial precursor proteins. In contrast to the nine subunit fungal complex, cytochrome c reductase from potato comprises 10 polypeptides. Protein sequencing of peptides from individual subunits and analysis of corresponding cDNA clones reveals that subunit III of cytochrome c reductase (51 kDa) represents the general mitochondrial processing peptidase.

We have investigated protein‐rRNA cross‐links formed in 30S and 50S ribosomal subunits of Escherichia coli and Bacillus stearothermophilus at the molecular level using UV and 2‐iminothiolane as cross‐linking agents. We identified amino acids cross‐linked to rRNA for 13 ribosomal proteins from these organisms, namely derived from S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29 and L36. Several other peptide stretches cross‐linked to rRNA have been sequenced in which no direct cross‐linked amino acid could be detected. The cross‐linked amino acids are positioned within loop domains carrying RNA binding features such as conserved basic and aromatic residues. One of the cross‐linked peptides in ribosomal protein S3 shows a common primary sequence motif–the KH motif–directly involved in interaction with rRNA, and the cross‐linked amino acid in ribosomal protein L36 lies within the zinc finger‐like motif of this protein. The cross‐linked amino acids in ribosomal proteins S17 and L6 prove the proposed RNA interacting site derived from three‐dimensional models. A comparison of our structural data with mutations in ribosomal proteins that lead to antibiotic resistance, and with those from protein‐antibiotic cross‐linking experiments, reveals functional implications for ribosomal proteins that interact with rRNA.