Clp protease complexes from photosynthetic and non-photosynthetic plastids and mitochondria of plants, their predicted three-dimensional structures, and functional implications

Tetradecameric Clp protease core complexes in non-photosynthetic plastids of roots, flower petals, and in chloroplasts of leaves of Arabidopsis thaliana were purified based on native mass and isoelectric point and identified by mass spectrometry. The stoichiometry between the subunits was determined. The protease complex consisted of one to three copies of five different serine-type protease Clp proteins (ClpP1,3-6) and four non-proteolytic ClpR proteins (ClpR1-4). Three-dimensional homology modeling showed that the ClpP/R proteins fit well together in a tetradecameric complex and also indicated unique contributions for each protein. Lateral exit gates for proteolysis products are proposed. In addition, ClpS1,2, unique to land plants, tightly interacted with this core complex, with one copy of each per complex. The three-dimensional modeling show that they do fit well on the axial sites of the ClpPR cores. In contrast to plastids, plant mitochondria contained a single approximately 320-kDa homo-tetradecameric ClpP2 complex, without association of ClpR or ClpS proteins. It is surprising that the Clp core composition appears identical in all three plastid types, despite the remarkable differences in plastid proteome composition. This suggests that regulation of plastid proteolysis by the Clp machinery is not through differential regulation of ClpP/R/S gene expression, but rather through substrate recognition mechanisms and regulated interaction of chaperone-like molecules (ClpS1,2 and others) to the ClpP/R core.     

Multistep Processing of an Insertion Sequence in an Essential Subunit of the Chloroplast ClpP Complex

In Chlamydomonas reinhardtii, the clpP1 chloroplast gene encoding one of the catalytic subunits of the ClpP protease complex contains a large in-frame insertion sequence (IS1). Based on the Escherichia coli ClpP structure, IS1 is predicted to protrude at the apical surface of the complex, likely influencing the interaction of the catalytic core with ClpC/HSP100 chaperones. Immunoblotting with an anti-ClpP1 antibody detected two immunoreactive forms of ClpP1: ClpP1_H (59 kDa) and ClpP1_L (25 kDa). It has been proposed that IS1 is a new type of protein intron (different from inteins). By studying transformants harboring mutations at the predicted borders of IS1 and tags at the C terminus of ClpP1 (tandem affinity purification tag, His tag, Strep·Tag) or within the IS1 sequence (3-hemagglutinin tag), we show that IS1 is not a protein intron and that ClpP1_L results from endoproteolytic cleavage inside IS1. Processing sites have been identified in the middle of IS1 and near its C terminus. The sites can be mutated without abolishing processing.Clp proteases are self-compartmentalized serine proteases present in most eubacteria and, as a consequence of endosymbiotic events, in the mitochondrion and chloroplast of eukaryotes. In Escherichia coli, the organism in which they have been best characterized, Clp proteases associate a homo-oligomeric peptidase (ClpP) and a chaperone (ClpA or ClpX) that belongs to the Clp/HSP100 family, itself part of the large group of AAA⁺ ATPases (1–4). ClpP is composed of 14 identical subunits arranged in two heptameric rings related by central symmetry. They form a barrel-like structure with the 14 active sites facing an inner proteolytic chamber (5). ClpP alone is able to degrade only small peptides (6), and the recognition and unfolding of protein substrates are carried out by the Clp/HSP100 chaperone. The chaperone docks on the apical surfaces of ClpP and uses ATP hydrolysis to unfold and feed substrates through the ClpP axial pore into the proteolytic chamber (7–10).In chloroplasts, ClpP is present as a hetero-oligomer associating up to eight different types of subunit. This is the result of a gene diversification process that has begun in cyanobacteria and continues in the chloroplast of land plants. Not only has the number of clpP genes grown, but clpR genes have appeared that carry mutations in at least one residue of the catalytic triad and are thus presumed catalytically inactive. In the green alga Chlamydomonas reinhardtii, three clpP genes (clpP1, CLPP4, and CLPP5) and five clpR genes (CLPR1–CLPR4 and CLPR6) code for the subunits of the chloroplast ClpP complex (11). An additional CLPP2 gene codes for the homo-oligomeric mitochondrial ClpP.ClpP1 is the only subunit that is encoded in the chloroplast and probably the best conserved. In C. reinhardtii, clpP1 contains a large insertion sequence (IS1)3 translated in-frame with the conserved N- and C-terminal regions. This results in a protein about twice as large (∼59 kDa) as in other organisms. Chlamydomonas ClpP1 can be divided into two sequence domains, SD1 and SD2 (the latter containing the catalytic residues), corresponding to the conserved sequence, and one insertion sequence, IS1 (12). In C. reinhardtii, antisera raised against the entire open reading frame (ORF) recognize two products of clpP1 in Western blot: ClpP1_H (59 kDa) and ClpP1_L (21 kDa) (13). As the clpP1 mRNA does not undergo splicing (12), it has been proposed that IS1 could be a protein intron. Protein introns such as inteins (14) are defined as in-frame intervening sequences that disrupt a host gene and are post-translationally excised by a self-catalytic mechanism. In the case of clpP1, ClpP1_H would be the precursor protein and ClpP1_L the spliced form. However, IS1 lacks the sequence motifs characteristic of inteins. In addition, both ClpP1_L and ClpP1_H are stable, and both associate in the 540-kDa ClpP complex (11). Thus, if IS1 were a protein intron, it would be an unusual type. In the related species Chlamydomonas eugametos, clpP1 contains, in addition to IS1, another insertion sequence (IS2) displaying most of the sequence features of inteins. Indeed, IS2 can be induced to self-splice in E. coli by changing a single residue (15).In this study, we show that IS1 is not a protein intron and that ClpP1_L is the product of a complex proteolytic maturation of ClpP1_H. We have found similar insertion sequences in the clpP1 genes of other green algae from the group Chlorophyceae. Green algae accumulate such insertion sequences in many of their chloroplast genes, probably as a result of a high frequency of genome rearrangements.     

Detection of an IS2-encoded 46-kilodalton protein capable of binding terminal repeats of IS2.

The genome of the transposable element IS2 contains five open reading frames that are capable of encoding proteins greater than 50 amino acids; however, only one IS2 protein of 14 kDa had been detected. By replacing the major IS2 promoter located in the right terminal repeat of IS2 with the T7 promoter to express IS2 genes, we have detected another IS2 protein of 46 kDa. This 46-kDa protein was designated InsAB'. Analyses of the InsAB' sequence revealed motifs that are characteristic of transposases of other transposable elements. InsAB' has the ability to bind both terminal repeat sequences of IS2. It was shown to bind a 27-bp sequence (5'-GTTAAGTGATAACAGATGTCTGGAAAT-3', positions 1316 to 1290 by our numbering system [16 to 42 by the previous numbering system]) located at the inner end of the right terminal repeat and a 31-bp sequence (5'-TTATTTAAGTGATATTGGTTGTCTGGAGATT-3', positions 46 to 16 [1286 to 1316]), including the last 27 bp of the inner end and the adjacent 4 bp of the left terminal repeat of IS2. This result suggests that InsAB' is a transposase of IS2. Since there is no open reading frame capable of encoding a 46-kDa protein in the entire IS2 genome, this 46-kDa protein is probably produced by a translational frameshifting mechanism.     

The purification of the Chlamydomonas reinhardtii chloroplast ClpP complex: additional subunits and structural features

The ClpP peptidase is a major constituent of the proteolytic machinery of bacteria and organelles. The chloroplast ClpP complex is unusual, in that it associates a large number of subunits, one of which (ClpP1) is encoded in the chloroplast, the others in the nucleus. The complexity of these large hetero-oligomeric complexes has been a major difficulty in their overproduction and biochemical characterization. In this paper, we describe the purification of native chloroplast ClpP complex from the green alga Chlamydomonas reinhardtii, using a strain that carries the Strep-tag II at the C-terminus of the ClpP1 subunit. Similar to land plants, the algal complex comprises active and inactive subunits (3 ClpP and 5 ClpR, respectively). Evidence is presented that a sub-complex can be produced by dissociation, comprising ClpP1 and ClpR1, 2, 3 and 4, similar to the ClpR-ring described in land plants. Our Chlamydomonas ClpP preparation also contains two ClpT subunits, ClpT3 and ClpT4, which like the land plant ClpT1 and ClpT2 show 2 Clp-N domains. ClpTs are believed to function in substrate binding and/or assembly of the two heptameric rings. Phylogenetic analysis indicates that ClpT subunits have appeared independently in Chlorophycean algae, in land plants and in dispersed cyanobacterial genomes. Negative staining electron microscopy shows that the Chlamydomonas complex retains the barrel-like shape of homo-oligomeric ClpPs, with 4 additional peripheral masses that we speculate represent either the additional IS1 domain of ClpP1 (a feature unique to algae) or ClpTs or extensions of ClpR subunits.     

Characterization of the dihydrolipoamide acetyltransferase of the mitochondrial pyruvate dehydrogenase complex from potato and comparisons with similar enzymes in diverse plant species.

The pyruvate dehydrogenase complex (mPDC) from potato (Solanum tuberosum cv. Romano) can be disassociated in 1 M NaCl and 0.1 M glycine into a large dihydrolipoamide acetyltransferase (E2) complex and smaller pyruvate dehydrogenase (E1) and dihydrolipoamide dehydrogenase (E3) complexes. The E2 complex consists of 55 and 78-kDa polypeptides which are reversibly radiolabelled to a similar degree in the intact mPDC by [2-14C]pyruvate. Affinity-purified antibodies against the 55-kDa protein do not cross-react with the 78-kDa protein and the two proteins show different peptide patterns following partial proteolysis. The 78 and 55-kDa proteins are present in approximately equal abundance in the E2 complex and incorporate a similar amount of [14C] on incubation with [2-14C]pyruvate. Native mPDC and the E2 complex have sedimentation coefficients of 50S and 30S, respectively. Titration of electro-eluted polypeptides against the intact mPDC and E2 complex revealed that each mg of mPDC contains 0.4 mg of E1, 0.4 mg of E2 and 0.2 mg of E3. Labelling of partially purified mPDC from potato, pea, cauliflower, maize and barley, with [2-14C]pyruvate, suggest that a 78-kDa acetylatable protein is only found in the dicotyledonous species, while all plant species tested contained a smaller 52-60 kDa acetylatable protein.     

Subunit stoichiometry, evolution, and functional implications of an asymmetric plant plastid ClpP/R protease complex in Arabidopsis

The caseinolytic protease (Clp) protease system has been expanded in plant plastids compared with its prokaryotic progenitors. The plastid Clp core protease consists of five different proteolytic ClpP proteins and four different noncatalytic ClpR proteins, with each present in one or more copies and organized in two heptameric rings. We determined the exact subunit composition and stoichiometry for the intact core and each ring. The chloroplast ClpP/R protease was affinity purified from clpr4 and clpp3 Arabidopsis thaliana null mutants complemented with C-terminal StrepII-tagged versions of CLPR4 and CLPP3, respectively. The subunit stoichiometry was determined by mass spectrometry-based absolute quantification using stable isotope-labeled proteotypic peptides generated from a synthetic gene. One heptameric ring contained ClpP3,4,5,6 in a 1:2:3:1 ratio. The other ring contained ClpP1 and ClpR1,2,3,4 in a 3:1:1:1:1 ratio, resulting in only three catalytic sites. These ClpP1/R1-4 proteins are most closely related to the two subunits of the cyanobacterial P3/R complex and the identical P:R ratio suggests conserved adaptation. Furthermore, the plant-specific C-terminal extensions of the ClpP/R subunits were not proteolytically removed upon assembly, suggesting a regulatory role in Clp chaperone interaction. These results will now allow testing ClpP/R structure-function relationships using rationale design. The quantification workflow we have designed is applicable to other protein complexes.     

Identification of a 350-kDa ClpP protease complex with 10 different Clp isoforms in chloroplasts of Arabidopsis thaliana

A 350-kDa ClpP protease complex with 10 different subunits was identified in chloroplast of Arabidopsis thaliana, using Blue-Native gel electrophoresis, followed by matrix-assisted laser desorption ionization time-of-flight and nano-electrospray tandem mass spectrometry. The complex was copurified with the thylakoid membranes, and all identified Clp subunits show chloroplast targeting signals, supporting that this complex is indeed localized in the chloroplast. The complex contains chloroplast-encoded pClpP and six nuclear-encoded proteins nCpP1-6, as well as two unassigned Clp homologues (nClpP7, nClpP8). An additional Clp protein was identified in this complex; it does not belong to any of the known Clp genes families and is here assigned ClpS1. Expression and accumulation of several of these Clp proteins have never been shown earlier. Sequence and phylogenetic tree analysis suggests that nClpP5, nClpP2, and nClpP8 are not catalytically active and form a new group of Clp higher plant proteins, orthologous to the cyanobacterial ClpR protein, and are renamed ClpR1, -2, and -3, respectively. We speculate that ClpR1, -2, and -3 are part of the heptameric rings, whereas ClpS1 is a regulatory subunit positioned at the axial opening of the ClpP/R core. Several truncations and errors in intron and exon prediction of the annotated Clp genes were corrected using mass spectrometry data and by matching genomic sequences with cDNA sequences. This strategy will be widely applicable for the much needed verification of protein prediction from genomic sequence. The extreme complexity of the chloroplast Clp complex is discussed.     

A 20-kilodalton protein is required for efficient production of the Bacillus thuringiensis subsp. israelensis 27-kilodalton crystal protein in Escherichia coli.

The 27-kilodalton (kDa) mosquitocidal protein gene from Bacillus thuringiensis subsp. israelensis has been cloned as a 10-kilobase (kb) HindIII fragment from plasmid DNA; efficient expression in Escherichia coli KM1 depends on a region of DNA located approximately 4 kb upstream (K. McLean and H. R. Whiteley, J. Bacteriol. 169:1017-1023, 1987). We have cloned the upstream DNA region and show that it contains a complete open reading frame (ORF) encoding a protein with a molecular mass of 19,584 Da. Sequencing of adjacent stretches of DNA revealed two partial ORFs: one has 55.2% identity in an overlap of 319 amino acids to the putative transposase of IS231 of B. thuringiensis subsp. thuringiensis, and the other, a 78-codon partial ORF, may be the carboxyl terminus of the 67-kDa protein previously observed in maxicells of strain KM1. A 0.8-kb fragment containing only the 20-kDa protein gene greatly enhanced the expression of the 27-kDa protein in E. coli. The introduction of nonsense codons into the 20-kDa protein gene ORF abolished this effect, indicating that the gene product, not the mRNA or DNA, is required for the enhancement. The effect of the 20-kDa protein gene on various fusions of lacZ to the 27-kDa protein gene suggests that the 20-kDa protein acts after the initiation of translation of the 27-kDa protein gene.     

Characterization of a novel third member of the human cytomegalovirus glycoprotein H-glycoprotein L complex.

A prerequisite for understanding the molecular function of the human cytomegalovirus (HCMV) gH (UL75)-gL (UL115) complex is a detailed knowledge of the structure of this complex in its functional form, as it is present in mature virions. The gH protein is known to be a component of a 240-kDa envelope complex designated as gCIII (D. R. Gretch, B. Kari, L. Rasmussen, R. C. Gehrz, and M. F. Stinski, J. Virol. 62:875-881, 1988). However, the exact composition of the gCIII complex remains unknown. In this report, we attempted reconstitution of the gCIII complex by coexpression of gH and gL in the baculovirus expression system. Formation of recombinant gH-gL complexes of approximately 115 kDa was demonstrated; however, no higher-molecular-mass (approximately 240-kDa) recombinant gH-gL complexes were detected, suggesting that the presence of gH and gL alone is not sufficient for reconstitution of the gCIII complex. To identify other mammalian and/or HCMV factors which may be necessary for gCIII formation, immunoprecipitates of gH and gL from HCMV-infected fibroblasts and purified HCMV virions were examined. This analysis did reveal a number of coprecipitating proteins which associate either transiently or integrally with gH and gL. One coprecipitating protein of 145 kDa was shown to be an integral component of gCIII, along with gH and gL. Characterization of the 145-kDa protein demonstrates that it is structurally and antigenically unrelated to gH and gL and that it appears to be virally encoded. Together, these data indicate that the 145-kDa protein is a third novel component of the mature HCMV gH-gL complex.     

Subunit composition of photosystem I and identification of center X as a [4Fe-4S] iron-sulfur cluster

A photosystem I (PS-I) preparation from barley (Hordeum vulgare L.) containing the reaction center protein P700-chlorophyll a-protein 1 (CP1) and smaller polypeptides with apparent molecular masses of 18, 16, 14, 9.5, 9, 4, and 1.5 kDa has been analyzed with respect to subunit stoichiometry. CP1 contains two homologous subunits with approximate masses of 82 kDa. CP1 and the smaller polypeptides were isolated, and the amino acid composition of each component and of the PS-I preparation was determined. Based on the amino acid composition data and the determined ability of each isolated polypeptide to bind Coomassie Brilliant Blue, the PS-I complex is shown to contain 1 mol of each of the homologous 82-kDa polypeptides as well as 1 mol of the 18-, 16-, 9.5-, and 9-kDa polypeptides for each mol of P700. The total polypeptide mass of the PS-I complex is 209 kDa excluding tryptophan and approximately 220 kDa including tryptophan. The two 82-kDa subunits present/P700 provide cysteine residues for binding only one Fe-S center. In conjunction with the earlier reported binding of four iron and four acid-labile sulfides to CP1/P700 (H?j, P. B., Svendsen, I., Scheller, H. V., and M?ller, B. L. (1987) J. Biol. Chem. 262, 12676-12684), this demonstrates the center X is a [4Fe-4S] cluster and eliminates the possibility of center X being composed of two [2Fe-2S] clusters.     

Clp protease complexes and their diversity in chloroplasts

The Clp proteases represent a large, ancient ATP-dependent protease family which in higher plants is known to be located in chloroplasts. The soluble, presumably multisubunit, enzyme of the organelle stroma is of dual genetic origin. It consists of a nuclear-encoded, regulatory subunit ClpC, which is an ATPase, and a plastid-encoded proteolytic subunit ClpP, which is a serine protease. An additional, nuclear-encoded proteolytic subunit resembling ClpP has been recently reported from tomato (Schaller and Ryan, 1995 plant gene Register 95–00). We demonstrate that in both tomato Lycopersicon esculentum Mill. and Arabidopsis thaliana, (L.) Heynh. the nuclear-encoded ClpP (nClpP) is made as a precursor molecule that can be imported into isolated intact chloroplasts of spinach (Spinacia oleracea L.) and processed in two or three steps, respectively, to the size of the authentic protein. Furthermore, both gel electrophoresis under non-denaturing conditions and size-exclusion chromatography verified that the three proteins can form distinct heteromeric supramolecular complexes of approximately 860, 1380 and 1700 kDa (probably also of 600 kDa) molecular mass. The size ranges of the former two are reminiscent of those of Clp complexes described from Escherichia coli. In addition, various complexes between 160 and 560 kDa are detectable with the individual components. Both the processing “intermediates” and the mature nClpP are found in assembled form. Received: 11 March 1998 / Accepted: 8 July 1998     

Extrinsic proteins of photosystem II: an intermediate member of PsbQ protein family in red algal PS II.

The oxygen-evolving photosystem II (PS II) complex of red algae contains four extrinsic proteins of 12 kDa, 20 kDa, 33 kDa and cyt c-550, among which the 20 kDa protein is unique in that it is not found in other organisms. We cloned the gene for the 20-kDa protein from a red alga Cyanidium caldarium. The gene consists of a leader sequence which can be divided into two parts: one for transfer across the plastid envelope and the other for transfer into thylakoid lumen, indicating that the gene is encoded by the nuclear genome. The sequence of the mature 20-kDa protein has low but significant homology with the extrinsic 17-kDa (PsbQ) protein of PS II from green algae Volvox Carteri and Chlamydomonas reinhardtii, as well as the PsbQ protein of higher plants and PsbQ-like protein from cyanobacteria. Cross-reconstitution experiments with combinations of the extrinsic proteins and PS IIs from the red alga Cy. caldarium and green alga Ch. reinhardtii showed that the extrinsic 20-kDa protein was functional in place of the green algal 17-kDa protein on binding to the green algal PS II and restoration of oxygen evolution. From these results, we conclude that the 20-kDa protein is the ancestral form of the extrinsic 17-kDa protein in green algal and higher plant PS IIs. This provides an important clue to the evolution of the oxygen-evolving complex from prokaryotic cyanobacteria to eukaryotic higher plants. The gene coding for the extrinsic 20-kDa protein was named psbQ' (prime).     

Purification and subunit characterization of the rat liver endocytic hyaluronan receptor

The endocytic hyaluronan (HA) receptor of liver sinusoidal endothelial cells (LECs) is responsible for the clearance of HA and other glycosaminoglycans from the circulation in mammals. We report here for the first time the purification of this liver HA receptor. Using lectin and immuno-affinity chromatography, two HA receptor species were purified from detergent-solubilized membranes prepared from purified rat LECs. In nonreducing SDS-polyacrylamide gel electrophoresis (PAGE), these two proteins migrated at 175- and approximately 300 kDa corresponding to the two species previously identified by photoaffinity labeling of live cells as the HA receptor (Yannariello-Brown, J., Frost, S. J., and Weigel, P. H. (1992) J. Biol. Chem. 267, 20451-20456). These two proteins co-purify in a molar ratio of 2:1 (175:300), and both proteins are active, able to bind HA after SDS-PAGE, electrotransfer, and renaturation. After reduction, the 175-kDa protein migrates as a approximately 185-kDa protein and is not able to bind HA. The 300-kDa HA receptor is a complex of three disulfide-bonded subunits that migrate in reducing SDS-PAGE at approximately 260, 230, and 97 kDa. These proteins designated, respectively, the alpha, beta, and gamma subunits are present in a molar ratio of 1:1:1 and are also unable to bind HA when reduced. The 175-kDa protein and all three subunits of the 300-kDa species contain N-linked oligosaccharides, as indicated by increased migration in SDS-PAGE after treatment with N-glycosidase F. Both of the deglycosylated, nonreduced HA receptor proteins still bind HA.     

Biosynthesis of the adenosine deaminase-binding protein in human fibroblasts and hepatoma cells

The adenosine deaminase-binding protein has previously been localized to the cell surface of human fibroblasts (Andy, R. J., and Kornfeld, R. (1982) J. Biol. Chem. 257, 7922-7925). In this study we examine the biosynthesis of binding protein in human fibroblasts, human hepatoma HepG2 cells, and a human kidney tumor cell line. Binding protein immunoprecipitated from radioiodinated detergent-extracted fibroblast membranes has a molecular weight of 120,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An additional band of Mr 100,000 is also present which we believe is a result of proteolysis of the 120,000 band. Purified soluble kidney binding protein has an Mr of 112,000. Binding protein from fibroblasts pulse-labeled with [35S]methionine for 15 min migrates as a 110-kDa band on sodium dodecyl sulfate-polyacrylamide gels. Within 30-60 min of chase, the intensity of the 110-kDa band is diminished, and a 120-kDa band has appeared. Binding protein reaches the cell surface of fibroblasts within 30-60 min of chase. The same results are obtained with the other cell lines studied. Thus, binding protein is initially synthesized as a precursor of 110 kDa which chases into a 120-kDa mature form. The shift of 10 kDa is probably due to processing of its oligosaccharide chains since soluble kidney-binding protein contains 7-9 complex N-linked chains. Upon endoglycosidase H treatment, the 110,000 precursor shifts to a Mr of 89,000 while the 120,000 mature band shifts to 115,000, consistent with the presence of 7-9 high mannose chains on the precursor and 1-2 high mannose chains on the mature form. These results and the presence of complex N-linked chains on binding protein were confirmed by lectin affinity chromatography of glycopeptides derived from [2-3H]mannose-labeled binding protein. Analysis of [6-3H]glucosamine-labeled binding protein indicates the presence of 1 sialic acid residue per chain.     

C. botulinum neurotoxin types A and E: isolated light chain breaks down into two fragments. Comparison of their amino acid sequences with tetanus neurotoxin

The flaccid paralysis in the neuromuscular disease botulism appears to depend on the coordinated roles of the approximately 50 kDa light and approximately 100 kDa heavy chain subunits of the approximately 150 kDa neurotoxic protein produced by Clostridium botulinum (J. Biol. Chem. (1987) 262, 2660 and Eur. J. Biochem. (1988) 177, 683). We observed that the light chain after separation from its conjugate heavy chain, in the presence of dithiothreitol and 2 M urea, begins to split into approximately 28 and approximately 18 kDa fragments. The other subunit-the approximately 100 kDa heavy chain following its isolation-and the parent approximately 150 kDa dichain neurotoxin do not break down under comparable conditions. This cleavage was examined in the neurotoxin serotypes A and E. The cleavage does not appear to be due to a protease. Partial amino acid sequences established that: i) the approximately 28-kDa and approximately 18-kDa fragments comprise the N- and C-terminal regions of the light chain, respectively; ii) the light chain of the neurotoxin serotypes A and E break down at precise peptide bonds; iii) the peptide bonds cleaved in serotypes A and E are five residues apart; and iv) the portions of the approximately 18 kDa fragments of serotype A and E neurotoxin sequenced so far are highly homologous to the corresponding region of tetanus neurotoxin produced by Clostridium tetani. The partial N-terminal sequence of the approximately 28 kDa fragment matches with the N-terminal sequence of the intact L chain. The 47 residues of the approximately 18-kDa fragment of type A sequenced from its N-terminal are: -Y.E.M.S.G.L.E.V.S.F.E.E.L.R.T.F.G.G.H.D.A.K.F.I.D.S.L.Q.E.N.E.F.R.L.Y.Y .Y. N.K.F.K. D.I.A.S.T.L.-. These align with those of tetanus neurotoxin beginning at its residue #259 (Tyr); the 18 underlined residues of the above 47 residues (i.e. 38%) are identical in positions between the two proteins. The 41 residues sequenced from the approximately 18 kDa fragment of type E botulinum neurotoxin are: -K.G.I.N.I.E.E.F.L. T.F.G.N.N.D.L.N.I.I.T.V.A.Q.Y.N.D.I.Y.T.N.L.L.N.D.Y.R. K.I.A.X.K. L.-.(ABSTRACT TRUNCATED AT 250 WORDS)     

A 10S particle released from deoxyribonuclease-sensitive regions of HeLa cell nuclei contains the 86-kilodalton-70-kilodalton protein complex

Digestion of HeLa cell nuclei with micrococcal nuclease or deoxyribonuclease I (DNase I) released the 86-kilodalton-70-kilodalton (kDa) protein complex in particles sedimenting at approximately 10 S in sucrose density gradients. Immunoaffinity-purified 32P-labeled complexes contained 86- and 70-kDa polypeptides with phosphorylated serine residues and DNA fragments, of which the largest was 110 base pairs long. Digestion of nick-translated nuclei with micrococcal nuclease released 32P-labeled 10S particles that were immunoaffinity-purified; they contained labeled 110-base-pair DNA fragments. The micrococcal nuclease digests were analyzed by two-dimensional electrophoresis, which separated nucleosomes in the first dimension and the associated proteins in the second. Western blots of the separated proteins showed that the 86-kDa-70-kDa complex was associated with the mono-, di-, and trinucleosomes. A more extensive electrophoretic separation revealed that the 10S particle from nick-translated nuclei migrated with a subfraction of the mononucleosomes that lacked H1 histones. These results suggest that the 10S particle which contains the 86-kDa-70-kDa complex is associated with an unfolded nucleosome that is present in DNase I sensitive regions.     

Cleavage of Treponema denticola PrcA polypeptide to yield protease complex-associated proteins Prca1 and Prca2 is dependent on PrtP

Analysis of potential virulence factors of oral spirochetes focuses on surface and secreted proteins. The Treponema denticola chymotrypsin-like protease (CTLP) is implicated in degradation of host cell molecules and contributes to tissue invasion. The CTLP complex, composed of the 72-kDa PrtP protein and two auxiliary proteins with molecular masses of approximately 40 and 30 kDa, is also involved in localization and oligomerization of the T. denticola major surface protein (Msp). The larger auxiliary protein was reported to be encoded by an open reading frame (ORF2) directly upstream of prtP. The deduced 39-kDa translation product of ORF2 contains a sequence matching the N-terminal sequence determined from one of the CTLP complex proteins. No proteins with significant homology are known, nor was information available on the third protein of the complex. DNA sequence analysis showed that ORF2 extended an additional 852 bp upstream of the reported sequence. The complete gene, designated prcA, encodes a predicted N-terminally-acylated polypeptide of approximately 70 kDa. Isogenic mutants with mutations in prtP, prcA, and prcA-prtP all lacked CTLP protease activity. The prcA mutant lacked all three CTLP proteins. The prcA-prtP mutant produced only a C-terminally-truncated 62-kDa PrcA protein. The prtP mutant produced a full-length 70-kDa PrcA. Immunoblot analysis of recombinant PrcA constructs confirmed that PrcA is cleaved to yield the two smaller proteins of the CTLP complex, designated PrcA1 and PrcA2. These data indicate that PrtP is required for cleavage of PrcA and suggest that this cleavage may be required for formation or stability of outer membrane complexes.     

Characterization of the periplasmic flagellum proteins of Leptospira interrogans.

The structure and composition of periplasmic flagella (PF) from Leptospira interrogans serovar pomona type kennewicki were characterized. Electron microscopic observations showed that leptospiral PF were complex structures composed of an 11.3-nm-diameter core surrounded by two sheath layers with 21.5- and 42-nm diameters. Two-dimensional gel electrophoresis of isolated PF showed the presence of seven different proteins ranging in mass from 31.5 to 36 kDa. Rabbit polyclonal and mouse monoclonal antibodies against PF proteins were prepared and were used to localize specific proteins to portions of the PF structure by immunoelectron microscopy. A 34-kDa protein was associated with the 11.3-nm-diameter core filament, while a 36-kDa protein was associated with a PF sheath (21.5-nm-diameter filament). The amino termini of the 34- and 35.5-kDa proteins were homologous to PF core proteins of other spirochetes. The experimental data suggested that L. interrogans PF contains 2 proteins (34 and 35.5 kDa) in the PF core.