Proteomic approach to identify novel mitochondrial proteins in Arabidopsis.

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 O(2), 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 at http://www.gartenbau.uni-hannover.de/genetik/AMPP.

Purification and characterization of the preprotein translocase of the outer mitochondrial membrane from Arabidopsis. Identification of multiple forms of TOM20.

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.

The spliceosomal snRNP core complex of Trypanosoma brucei: cloning and functional analysis reveals seven Sm protein constituents.

Each of the trypanosome small nuclear ribonucleoproteins (snRNPs) U2, U4/U6, and U5, as well as the spliced leader (SL) RNP, contains a core of common proteins, which we have previously identified. This core is unusual because it is not recognized by anti-Sm Abs and it associates with an Sm-related sequence in the trypanosome small nuclear RNAs (snRNAs). Using peptide sequences derived from affinity-purified U2 snRNP proteins, we have cloned cDNAs for five common proteins of 8.5, 10, 12.5, 14, and 15 kDa of Trypanosoma brucei and identified them as Sm proteins SmF (8.5 kDa), -E (10 kDa), -D1 (12.5 kDa), -G (14 kDa), and -D2 (15 kDa), respectively. Furthermore, we found the trypanosome SmB (T. brucei) and SmD3 (Trypanosoma cruzi) homologues through database searches, thus completing a set of seven canonical Sm proteins. Sequence comparisons of the trypanosome proteins revealed several deviations in highly conserved positions from the Sm consensus motif. We have identified a network of specific heterodimeric and -trimeric Sm protein interactions in vitro. These results are summarized in a model of the trypanosome Sm core, which argues for a strong conservation of the Sm particle structure. The conservation extends also to the functional level, because at least one trypanosome Sm protein, SmG, was able to specifically complement a corresponding mutation in yeast.

SR protein-specific kinase 1 is highly expressed in testis and phosphorylates protamine 1.

Arginine/serine protein kinases constitute a novel class of enzymes that can modify arginine/serine (RS) dipeptide motifs. SR splicing factors that are essential for pre-mRNA splicing are among the best characterized proteins that contain RS domains. TwoSRprotein-specifickinases, SRPK1 and SRPK2, have been considered as highly specific for the phosphorylation of these proteins, thereby contributing to splicing regulation. However, despite the fact that SR proteins are more or less conserved among metazoa and have a rather ubiquitous tissue distribution we now demonstrate that SRPK1 is predominantly expressed in testis. In situ expression analysis on transverse sections of adult mouse testis shows that SRPK1 mRNA is abundant in all germinal cells but not in mature spermatozoa. RS kinase activity was found primarily in the cytosol and only minimal activity was detected in the nucleus. In a search for testis-specific substrates of SRPK1 we found that the enzyme phosphorylates human protamine 1 as well as a cytoplasmic pool of SR proteins present in the testis. Protamine 1 belongs to a family of small basic arginine-rich proteins that replace histones during the development of mature spermatozoa. The result of this progressive replacement is the formation of a highly compact chromatin structure devoid of any transcriptional activity. These findings indicate that SRPK1 may have a role not only in pre-mRNA splicing, but also in the condensation of sperm chromatin.

Unique composition of the preprotein translocase of the outer mitochondrial membrane from plants.

Transport of most nuclear encoded mitochondrial proteins into mitochondria is mediated by heteropolymeric translocases in the membranes of the organelles. The translocase of the outer mitochondrial membrane (TOM) was characterized in fungi, and it was shown that TOM from yeast comprises nine different subunits. This publication is the first report on the preparation of the TOM complex from plant mitochondria. The protein complex from potato was purified by (a) blue native polyacrylamide gel electrophoresis and (b) by immunoaffinity chromatography. On blue native gels, the potato TOM complex runs close to cytochrome c oxidase at 230 kDa and hence only comprises about half of the size of fungal TOM complexes. Analysis of the TOM complex from potato by SDS-polyacrylamide gel electrophoresis allows separation of seven different subunits of 70, 36, 23, 9, 8, 7, and 6 kDa. The 23-kDa protein is identical to the previously characterized potato TOM20 receptor, as shown by in vitro assembly of this protein into the 230-kDa complex, by immunoblotting and by direct protein sequencing. Partial amino acid sequence data of the other subunits allowed us to identify sequence similarity between the 36-kDa protein and fungal TOM40. Sequence analysis of cDNAs encoding the 7-kDa protein revealed significant sequence homology of this protein to TOM7 from yeast. However, potato TOM7 has a N-terminal extension, which is very rich in basic amino acids. Counterparts to the TOM22 and TOM37 proteins from yeast seem to be absent in the potato TOM complex, whereas an additional low molecular mass subunit occurs. Functional implications of these findings are discussed.

New insights into the co-evolution of cytochrome c reductase and the mitochondrial processing peptidase.

The mitochondrial processing peptidase (MPP) is a heterodimeric enzyme that forms part of the cytochrome c reductase complex from higher plants. Mitochondria from mammals and yeast contain two homologous enzymes: (i) an active MPP within the mitochondrial matrix and (ii) an inactive MPP within the cytochrome c reductase complex. To elucidate the evolution of MPP, the cytochrome c reductase complexes from lower plants were isolated and tested for processing activity. Mitochondria were prepared from the staghorn fern Platycerium bifurcatum, from the horsetail Equisetum arvense, and from the colorless algae Polytomella, and cytochrome c reductase complexes were purified by a micro-isolation procedure based on Blue-native polyacrylamide gel electrophoresis and electroelution. This is the first report on the subunit composition of a respiratory enzyme complex from a fern or a horsetail. The cytochrome c reductase complexes from P. bifurcatum and E. arvense are shown to efficiently process mitochondrial precursor proteins, whereas the enzyme complex from Polytomella lacks proteolytic activity. An evolutionary model is suggested that assumes a correlation between the presence of an active MPP within the cytochrome c reductase complex and the occurrence of chloroplasts.

The modular organization of multifunctional peptide synthetases.

Gramicidin S synthetase 2 from B. brevis was affinity labeled at its valine thiolation center with the thiol reagent N-[3H]ethylmaleimide. From a tryptic digest of the enzyme-inhibitor complex a radioactive fragment was isolated in pure form by two reversed-phase HPLC steps. It was identified by liquid-phase N-terminal sequencing in combination with electrospray mass spectrometry (ESI-MS) as a hexadecapeptide containing the thiolation motif LGG(H/D)S(L/I). By ESI-MS it was demonstrated that a 4'-phosphopantetheine cofactor was attached to this fragment at its reactive serine. These results are consistent with the "Multiple Carrier Model" of nonribosomal peptide biosynthesis. Site-specific mutagenesis has been performed in thiolation, elongation, and epimerization motifs of some of the modules of surfactin synthetase from B. subtilis to clarify the function of prominent conserved amino acid residues in the intermediate steps of peptide biosynthesis. The modular structure of multifunctional peptide synthetases is discussed.

Trypanosoma brucei gBP21. An arginine-rich mitochondrial protein that binds to guide RNA with high affinity.

RNA editing in Trypanosoma brucei is a mitochondrial RNA processing reaction that results in the insertion and deletion of uridylate residues into otherwise untranslatable mRNAs. The process is directed by guide RNAs which function to specify the edited sequence. RNA editing in vitro requires mitochondrial protein extracts and guide RNAs have been identified as part of high molecular weight ribonucleoprotein complexes. Within the complexes, the RNAs are in close contact with several mitochondrial proteins and here we describe the isolation and cloning of a gRNA-interacting polypeptide from Trypanosoma brucei. The protein was named gBP21 for guide RNA-binding protein of 21 kDa. gBP21 shows no homology to proteins in other organisms, it is arginine-rich and binds to gRNA molecules with a dissociation constant in the nanomolar range. The protein does not discriminate for differences in the primary structures of gRNAs and thus likely binds to higher order structural features common to all gRNA molecules. gBP21 binding does not perturb the overall structure of gRNAs but the gRNA/gBP21 ribonucleoprotein complex is more stable than naked guide RNAs. Although the protein is arginine-rich, the free amino acid or an arginine-rich peptide were not able to inhibit the association to the RNAs. In contrast, the gRNA-gBP21 complex formation was sensitive to potassium and ammonium cations, thus indicating a contribution of ionic contacts to the binding.

4-Dihydromethyltrisporate dehydrogenase from Mucor mucedo, an enzyme of the sexual hormone pathway: purification, and cloning of the corresponding gene.

We have purified the NADP-dependent 4-dihydromethyltrisporate dehydrogenase from the zygomycete Mucor mucedo. The enzyme is involved in the biosynthesis of trisporic acid, the sexual hormone of zygomycetes, which induces the first steps of zygophore development. Protein was obtained from the (-) mating type of M. mucedo after induction with trisporic acid, and purified by gel filtration and affinity chromatography steps. On SDS-PAGE a band with an apparent molecular mass of 33 kDa was ascribed to the enzyme. After transferring onto PVDF membranes the protein was digested with endoprotease Lys-C, and several peptides were sequenced. Oligonucleotides derived from protein sequence data were used for PCR amplification of genomic M. mucedo DNA. The PCR fragment was used as probe for isolation of the corresponding cDNA and complete genomic DNA clones. Comparison of protein and DNA sequence data showed that the cloned fragment corresponded to the purified protein. Search for similarity with protein sequences of the Swiss-Prot database revealed a relationship to enzymes belonging to the aldo/keto reductase superfamily. Southern-blot analysis of genomic DNA with the labelled cloned fragment detected a single-copy gene in both mating types of M. mucedo. PCR with genomic DNA from other zygomycetes gave rise to several fragments. Hybridization analysis with the cloned M. mucedo fragment showed that a fragment of similar length cross-hybridized in Blakeslea trispora (Choanephoraceae) as well as in Parasitella parasitica and Absidia glauca (Mucoraceae). The promoter region of the gene contains DNA elements with similarity to a cAMP-regulated gene of Dictyostelium discoideum.

bli-4, a gene that is rapidly induced by blue light, encodes a novel mitochondrial, short-chain alcohol dehydrogenase-like protein in Neurospora crassa.

Blue light plays an important role in developmental control throughout nature. The bli-4 gene of Neurospora crassa, together with bli-3, al-1 and al-2, is rapidly inducible by blue light. Induction leads to a ninety-fold increase in transcription rate over the dark control level, and the gene therefore appears to be of prime importance in the blue-light induction pathway of N. crassa. We describe the sequencing and analysis of bli-4 and the 38 kDa protein it encodes. We show that the protein is very rapidly imported into the mitochondria and exhibits high homology with the family of short-chain alcohol dehydrogenases.

The multiple carrier model of nonribosomal peptide biosynthesis at modular multienzymatic templates.

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.

New insights into the composition, molecular mass and stoichiometry of the protein complexes of plant mitochondria.

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.

Partial amino acid sequence of an L-amino acid oxidase from the cyanobacterium Synechococcus PCC6301, cloning and DNA sequence analysis of the aoxA gene.

A novel type of L-amino acid oxidase from Synechococcus PCC6301 was purified and subjected to amino acid sequence analysis. Since the N-terminus of the L-amino acid oxidase protein was not accessible for Edman degradation, the protein was partially hydrolysed and a contiguous sequence of 17 amino acid residues was obtained from an endogenous peptide fragment. Based on the partial peptide sequence two oligonucleotides were designed, which were used as probes in Southern hybridization experiments in order to identify the corresponding aoxA gene. The aoxA gene was isolated from a size-fractionated genomic library of Synechococcus PCC6301 and subsequently sequenced. From the nucleotide sequence (data base accession number Z48565) it can be deduced that the L-amino acid protein consists of 355 amino acid residues resulting in a molar mass of 39.2 kDa. The calculated isoelectric point of the protein is 9.81. The L-amino acid oxidase from Synechococcus PCC6301 shows low homologies to other flavin oxidases/dehydrogenases, especially amine oxidases, but no homologies to other so far sequenced L- or D-amino acid oxidases.

Peptide environment of the peptidyl transferase center from Escherichia coli 70 S ribosomes as determined by thermoaffinity labeling with dihydrospiramycin.

In an attempt to gain information about the peptidyl transferase center at the peptide level we cross-linked the spiramycin derivative dihydrospiramycin to its functional binding site in the 70 S ribosome of Escherichia coli. In this manner ribosomal proteins S12, S14, L17, L18, L27 and L35 were found specifically affinity-labeled. Proteolytic fragmentation of these proteins, separation by C18 reversed-phase high performance liquid chromatography of the peptide mixtures, and subsequent sequence analysis of labeled peptides revealed peptide regions at positions Ala1-Lys9 and Tyr116-Lys119 of S12, Leu47-Asp53 of protein S14, Ser6-Lys35 of protein L17, Ala57-Lys63 of protein L18, Ala5-Lys18 and Val66-Lys71 of protein L27, and Thr5-Lys11 of protein L35. This approach is a valuable tool to characterize the binding site of spiramycin as well as the peptidyl transferase center at the molecular level.

Protein-rRNA binding features and their structural and functional implications in ribosomes as determined by cross-linking studies.

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.

Cytochrome c reductase from potato does not comprise three core proteins but contains an additional low-molecular-mass subunit.

Analysis of cytochrome c reductase from potato by Tricine/SDS/PAGE reveals 10 bands representing 10 different subunits. In comparison to glycine/SDS/PAGE one additional small protein becomes visible, whereas the three large core proteins are not resolved. The identity of the subunits was determined by immunoblotting and direct sequence determination. Sequence data for the novel small component were used to derive oligonucleotides for probing a potato cDNA-library and isolating corresponding clones. The newly identified subunit is a 6.7-kDa protein, that exhibits significant sequence similarity to a 8.5-kDa subunit of cytochrome c reductase from yeast and the 6.5-kDa iron-sulfur-protein-binding factor from the equivalent enzyme complex from beef. Also the potato 6.7-kDa subunit can be dissociated from the cytochrome c reductase complex together with the iron-sulfur protein. To address the question of whether three or two core subunits occur simultaneously in monomeric cytochrome c reductase complexes from potato, a peptide-specific antibody was generated. The antiserum is capable of discriminating between the 55-kDa and 53-kDa core proteins, which can be separated by glycine/SDS/PAGE and which were previously found to be structurally related. Immunoprecipitations of isolated cytochrome c reductase from potato using this antibody revealed an enzyme complex containing only two core proteins. The simultaneous occurrence of only two core subunits was confirmed by a comparison of the molecular masses of cytochrome c reductase from potato and beef by blue-native-gel electrophoresis. Hence the cytochrome c reductase complexes from potato, beef and yeast have a very conserved subunit composition. The evolutionary implications of these findings are discussed.

The general mitochondrial processing peptidase from wheat is integrated into the cytochrome bc1-complex of the respiratory chain.

The bc1-complex (EC 1.10.2.2.) from Triticum aestivum L. was purified by cytochrome-c affinity chromatography and gel filtration using either etiolated seedlings or wheat-germ extract as starting material. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the isolated enzyme revealed ten bands, which were analysed by immunoblotting and direct amino-acid sequencing. The enzyme from wheat is the first bc1-complex that is reported to contain four core proteins (55.5, 55.0, 51.5 and 51.0 kDa). In addition, the wheat bc1-complex comprises cytochrome b (35 kDa), cytochrome c1 (33 kDa) the "Rieske" iron-sulphur protein (25 kDa) and three small subunits < 15 kDa. This composition differs from the one reported in fungi, mammals and potato. Partial sequence determination of the large subunits suggests that the 55.5- and 55.0-kDa-proteins represent the beta-subunit of the general mitochondrial processing peptidase, and the 51.5- and 51.0-kDa proteins the alpha-subunit of this enzyme. The bc1-complex from wheat efficiently processes mitochondrial precursor proteins as shown in an in-vitro processing assay. In control experiments the isolated bc1-complexes from potato, yeast, Neurospora and beef, all purified by the same isolation procedure, were also tested for processing activity. Only the protein complexes from plants contain the general mitochondrial processing peptidase. The composition of the wheat bc1-complex sheds new light on the co-evolution of the processing peptidase and the middle segment of the respiratory chain.

Primary structure, cell-free synthesis and mitochondrial targeting of the 8.2 kDa protein of cytochrome c reductase from potato.

Cytochrome c reductase from potato comprises ten subunits with apparent molecular sizes between 55 and < 10 kDa. The subunit with the highest electrophoretic mobility on SDS-polyacrylamide gels was isolated and analysed by cyclic Edman degradation. Mixtures of degenerative oligonucleotides were derived from the obtained sequence data and used for the isolation of corresponding cDNA clones. The clones encode a protein of 72 amino acids which exhibits significant sequence identity with a 9.5 kDa subunit of cytochrome c reductase from bovine and a 11 kDa subunit of the enzyme complex from yeast. Comparison between the deduced amino acid sequence of the open reading frame and the sequence of the mature protein reveals that only the initiator methionine is absent in the functional subunit. Hence the protein has a calculated molecular mass of 8.2 kDa. Transcripts of the potato 8.2 kDa protein were not translated in reticulocyte lysates but in vitro translation worked efficiently with wheat germ lysate. Import of the radiolabelled protein into isolated mitochondria from potato seems to depend on a potential across the inner membrane and confirms the absence of a cleavable mitochondrial presequence.

Analysis of the Bacillus subtilis bacteriophages SPP1 and SF6 gene 1 product: a protein involved in the initiation of headful packaging.

Gene 1 product (G1P) of the related Bacillus subtilis bacteriophages SPP1, SF6, and rho 15 is essential for DNA maturation and packaging. A DNA segment containing gene 1 of phage SF6 or rho 15 origin was cloned and sequenced. SF6 and rho 15 G1P (both with predicted molecular mass of 16.7 kDa) share 71% identity with G1P of SPP1. The G1P of all three phages contains three conserved segments (I, II, and III). Within segments I and II helix-turn-helix DNA binding and nucleotide binding motifs were predicted. G1P of both SPP1 and SF6 origin was purified. SPP1 G1P protein (20.7 kDa), purified from cells overexpressing the cloned gene, purifies together with another polypeptide, having a molecular mass of about 13 kDa. The 13-kDa polypeptide results from a translation start signal within gene 1, and hence was termed SPP1 G1P*. G1P of both SPP1 and SF6 binds specifically to a pac-containing DNA fragment, whereas G1P*, which lacks segment I, does not. Chimeric G1P proteins were obtained by domain swapping between gene 1 of SPP1 and SF6. The results presented here suggest that the G1P DNA binding motif lies in segment I and the major determinant for G1P::G1P interaction might lie in segment II. Segment III and the extended C-terminal part of SPP1 G1P are dispensable. The G1P::G2P interacting region remains uncharacterized.

Analysis of the puromycin binding site in the 70 S ribosome of Escherichia coli at the peptide level.

Photoinduced cross-linking of Escherichia coli 70 S ribosomes with [3H]puromycin has led to the labeling of ribosomal proteins S7, S14, S18, L18, and L29. Proteolytic fragmentation of these proteins and separation of the peptide mixtures by C18 reversed-phase high performance liquid chromatography resulted in six puromycin-labeled peptides which were applied to sequence analysis. The following peptides were found labeled: Pro1-Lys10 of S7, Ala28-Lys46 and Ala7-Lys11 of S14, Asp24-Lys29 of S18, Tyr64-Lys68 of L18, and Thr55-Lys60 of L29. For the first time the molecular environment of an antibiotic in the procaryotic ribosome is presented at the peptide level.