Transit peptide cleavage sites of integral thylakoid membrane proteins

A set of 58 nuclearly encoded thylakoid-integral membrane proteins from four plant species was identified, and their amino termini were assigned unequivocally based upon mass spectrometry of intact proteins and peptide fragments. The dataset was used to challenge the Web tools ChloroP, TargetP, SignalP, PSORT, Predotar, and MitoProt II for predicting organelle targeting and transit peptide proteolysis sites. ChloroP and TargetP reliably predicted chloroplast targeting but only reliably predicted transit peptide cleavage sites for soluble proteins targeted to the stroma. SignalP (eukaryote settings) accurately predicted the transit peptide cleavage site for soluble proteins targeted to the lumen. SignalP (Gram-negative bacteria settings) reliably predicted peptide cleavage of integral thylakoid proteins inserted into the membrane via the "spontaneous" pathway. The processing sites of more common thylakoid-integral proteins inserted by the signal recognition peptide-dependent pathway were not well predicted by any of the programs. The results suggest the presence of a second thylakoid processing protease that recognizes the transit peptide of integral proteins inserted via the spontaneous mechanism and that this mechanism may be related to the secretory mechanism of Gram-negative bacteria.     

Conserved residues of the leader peptide are essential for cleavage by leader peptidase

Gene 8 of bacteriophage M13 codes for procoat, the precursor of its major coat protein. Gene 8 has been cloned into a plasmid and mutagenized. We have isolated mutants of this gene in which procoat is synthesized but is not processed to coat protein. We now describe mutants in the leader region of procoat, at positions -6, -3, and -1 with respect to the leader peptidase cleavage site. These positions are quite conserved among the leader peptides of various pre-proteins. Each of these mutant procoats is synthesized at a normal rate and inserts correctly into the plasma membrane, as judged by its accessibility to protease in intact spheroplasts. Procoat accumulates, largely in its transmembrane form, and is not cleaved to coat. In detergent extracts, the mutant procoats are very poor substrates for added leader peptidase. We conclude that these 3 residues are not conserved for insertion across the membrane but are part of an essential recognition site for the leader peptidase.     

The sites of synthesis of the principal thylakoid membrane polypeptides in Chlamydomonas reinhardtii

The sites of synthesis of the major thylakoid membrane polypeptides have been studied in the green alga Chlamydomonas reinhardtii by pulse labeling of cells with [14C]acetate in the presence of inhibitors specific for chloroplast and cytoplasmic protein synthesis. The labeled membrane polypeptides were separated by an improved method of sodium dodecyl sulfate (SDS) gradient gel electrophoresis, and autoradiographs were made of the dried gels. The results demonstrate that of the 33 polypeptides resolved in the gels, at least nine are made on chloroplast ribosomes. Two of these (polypeptides 2 and 6) are associated with the reaction centers of photosystems I and II. Another polypeptide (polypeptide 5) appears from genetic data to be coded by chloroplast DNA. Experiments with a mutant whose chloroplast ribosomes are resistant to spectinomycyn (spr-u-1-6-2) show that polypeptides whose synthesis takes place on chloroplast ribosomes are made in the presence of spectinomycin in the mutant although their synthesis is blocked by this antibiotic in wild type cells.     

Light-dependent phosphorylation of thylakoid membrane polypeptides.

The phosphorylation of thylakoid membrane proteins was studied using isolated chloroplasts from $\text{Euglena gracilis}$. We have found, using [³²P] labelling, that this phenomenon was light-driven, reversible in the dark, and completely inhibited by Carbonyl cyanide m-chlorophenyl-hydrazone (CCCP). Polyacrylamide gel electrophoresis containing SDS has revealed five main bands which have been found to be proteins. Amino acid analysis of the bands has shown that [³²P] is incorporated into phosphothreonine.     

Targeting of proteins to the thylakoid lumen by the bipartite transit peptide of the 33 kd oxygen-evolving protein.

Various chimeric precursors and deletions of the 33 kd oxygen-evolving protein (OEE1) were constructed to study the mechanism by which chloroplast proteins are imported and targeted to the thylakoid lumen. The native OEE1 precursor was imported into isolated chloroplasts, processed and localized in the thylakoid lumen. Replacement of the OEE1 transit peptide with the transit peptide of the small subunit of ribulose-1,5-bisphosphate carboxylase, a stromal protein, resulted in redirection of mature OEE1 into the stromal compartment of the chloroplast. Utilizing chimeric transit peptides and block deletions we demonstrated that the 85 residue OEE1 transit peptide contains separate signal domains for importing and targeting the thylakoid lumen. The importing domain, which mediates translocation across the two membranes of the chloroplast envelope, is present in the N-terminal 58 amino acids. The thylakoid lumen targeting domain, which mediates translocation across the thylakoid membrane, is located within the C-terminal 27 residues of the OEE1 transit peptide. Chimeric precursors were constructed and used in in vitro import experiments to demonstrate that the OEE1 transit peptide is capable of importing and targeting foreign proteins to the thylakoid lumen.     

Prediction of neuropeptide cleavage sites in insects

MOTIVATION: The production of neuropeptides from their precursor proteins is the result of a complex series of enzymatic processing steps. Often, the annotation of new neuropeptide genes from sequence information outstrips biochemical assays and so bioinformatics tools can provide rapid information on the most likely peptides produced by a gene. Predicting the final bioactive neuropeptides from precursor proteins requires accurate algorithms to determine which locations in the protein are cleaved. RESULTS: Predictive models were trained on Apis mellifera and Drosophila melanogaster precursors using binary logistic regression, multi-layer perceptron and k-nearest neighbor models. The final predictive models included specific amino acids at locations relative to the cleavage sites. Correct classification rates ranged from 78 to 100% indicating that the models adequately predicted cleaved and non-cleaved positions across a wide range of neuropeptide families and insect species. The model trained on D.melanogaster data had better generalization properties than the model trained on A. mellifera for the data sets considered. The reliable and consistent performance of the models in the test data sets suggests that the bioinformatics strategies proposed here can accurately predict neuropeptides in insects with sequence information based on neuropeptides with biochemical and sequence information in well-studied species.     

Proteome-derived, database-searchable peptide libraries for identifying protease cleavage sites

We introduce human proteome-derived, database-searchable peptide libraries for characterizing sequence-specific protein interactions. To identify endoprotease cleavage sites, we used peptides in such libraries with protected primary amines to simultaneously determine sequence preferences on the N-terminal (nonprime P) and C-terminal (prime P') sides of the scissile bond. Prime-side cleavage products were tagged with biotin, isolated and identified by tandem mass spectrometry, and the corresponding nonprime-side sequences were derived from human proteome databases using bioinformatics. Identification of hundreds to over 1,000 individual cleaved peptides allows the consensus protease cleavage site and subsite cooperativity to be readily determined from P6 to P6'. For the highly specific GluC protease, >95% of the 558 cleavage sites identified displayed the canonical selectivity. For the broad-specificity matrix metalloproteinase 2, >1,200 peptidic cleavage sites were identified. Profiling of HIV protease 1, caspase 3, caspase 7, cathepsins K and G, elastase and thrombin showed that this approach is broadly applicable to all mechanistic classes of endoproteases.     

Requirements of the engineered leader peptide of nisin for inducing modification, export, and cleavage

Nisin A is a pentacyclic peptide antibiotic produced by Lactococcus lactis. The leader peptide of prenisin keeps nisin inactive and has a role in inducing NisB- and NisC-catalyzed modifications of the propeptide and NisT-mediated export. The highly specific NisP cleaves off the leader peptide from fully modified and exported prenisin. We present here a detailed mutagenesis analysis of the nisin leader peptide. For alternative cleavage, we successfully introduced a putative NisP autocleavage site and sites for thrombin, enterokinase, Glu-C, and factor Xa in the C-terminal part of the leader peptide. Replacing residue F-18 with Trp or Thr strongly reduced production. On the other hand, D-19A, F-18H, F-18M, L-16D, L-16K, and L-16A enhanced production. Substitutions within and outside the FNLD box enhanced or reduced the transport efficiency. None of the above substitutions nor even an internal 6His tag from positions -13 to -8 had any effect on the capacity of the leader peptide to induce NisB and NisC modifications. Therefore, these data demonstrate a large mutational freedom. However, simultaneous replacement of the FNLD amino acids by four alanines strongly reduced export and even led to a complete loss of the capacity to induce modifications. Reducing the leader peptide to MSTKDFNLDLR led to 3- or 4-fold dehydration. Taken together, the FNLD box is crucial for inducing posttranslational modifications.     

LumenP--a neural network predictor for protein localization in the thylakoid lumen

We report the development of LumenP, a new neural network-based predictor for the identification of proteins targeted to the thylakoid lumen of plant chloroplasts and prediction of their cleavage sites. When used together with the previously developed TargetP predictor, LumenP reaches a significantly better performance than what has been recorded for previous attempts at predicting thylakoid lumen location, mostly due to a lower false positive rate. The combination of TargetP and LumenP predicts around 1.5%-3% of all proteins encoded in the genomes of Arabidopsis thaliana and Oryza sativa to be located in the lumen of the thylakoid.     

Adaptive encoding neural networks for the recognition of human signal peptide cleavage sites

MOTIVATION: Data representation and encoding are essential for classification of protein sequences with artificial neural networks (ANN). Biophysical properties are appropriate for low dimensional encoding of protein sequence data. However, in general there is no a priori knowledge of the relevant properties for extraction of representative features. RESULTS: An adaptive encoding artificial neural network (ACN) for recognition of sequence patterns is described. In this approach parameters for sequence encoding are optimized within the same process as the weight vectors by an evolutionary algorithm. The method is applied to the prediction of signal peptide cleavage sites in human secretory proteins and compared with an established predictor for signal peptides. CONCLUSION: Knowledge of physico-chemical properties is not necessary for training an ACN. The advantage is a low dimensional data representation leading to computational efficiency, easy evaluation of the detected features, and high prediction accuracy. Availability: A cleavage site prediction server is located at the Humboldt University http://itb.biologie.hu-berlin.de/ approximately jo/sig-cleave/ACNpredictor.cgi Contact: jo@itb.hu-berlin.de; berndj@zedat.fu-berlin.de     

SPEPlip: the detection of signal peptide and lipoprotein cleavage sites

SUMMARY: SPEPlip is a neural network-based method, trained and tested on a set of experimentally derived signal peptides from eukaryotes and prokaryotes. SPEPlip identifies the presence of sorting signals and predicts their cleavage sites. The accuracy in cross-validation is similar to that of other available programs: the rate of false positives is 4 and 6%, for prokaryotes and eukaryotes respectively and that of false negatives is 3% in both cases. When a set of 409 prokaryotic lipoproteins is predicted, SPEPlip predicts 97% of the chains in the signal peptide class. However, by integrating SPEPlip with a regular expression search utility based on the PROSITE pattern, we can successfully discriminate signal peptide-containing chains from lipoproteins. We propose the method for detecting and discriminating signal peptides containing chains and lipoproteins. AVAILABILITY: It can be accessed through the web page at http://gpcr.biocomp.unibo.it/predictors/     

Prediction of protein signal sequences and their cleavage sites

Protein signal sequences play a central role in the targeting and translocation of nearly all secreted proteins and many integral membrane proteins in both prokaryotes and eukaryotes. The knowledge of signal sequences has become a crucial tool for pharmaceutical scientists who genetically modify bacteria, plants, and animals to produce effective drugs. However, to effectively use such a tool, the first important thing is to find a fast and effective method to identify the "zipcode" entity; this is also evoked by both the huge amount of unprocessed data available and the industrial need to find more effective vehicles for the production of proteins in recombinant systems. In view of this, a sequence-encoded algorithm was developed to identify the signal sequences and predict their cleavage sites. The rate of correct prediction for 1,939 secretory proteins and 1,440 nonsecretory proteins by self-consistency test is 90.14% and that by jackknife test is 90.13%. The encouraging results indicate that the signal sequences share some common features although they lack similarity in sequence, length, and even composition and that they are predictable to a considerably accurate extent.     

Co-translational,intraribosomal cleavage of polypeptides by the foot-and-mouth disease virus 2A peptide

During co-translational protein import into the endoplasmic reticulum ribosomes are docked onto the translocon. This prevents inappropriate exposure of nascent chains to the cytosol and, conversely, cytosolic factors from gaining access to the nascent chain. We exploited this property of co-translational translocation to examine the mechanism of polypeptide cleavage by the 2A peptide of the foot-and-mouth disease virus. We find that the scission reaction is unaffected by placing 2A into a co-translationally targeted protein. Moreover, the portion of the polypeptide C-terminal to the cleavage site remains in the cytosol unless it contains its own signal sequence. The pattern of cleavage is consistent with the proposal that the 2A-mediated cleavage reaction occurs within the ribosome itself. In addition, our data indicate that the ribosome-translocon complex detects the break in the nascent chain and prevents any downstream protein lacking a signal sequence from gaining access to the endoplasmic reticulum.     

The location of phosphorylation sites and Ca2+-dependent proteolytic cleavage sites on the major neurofilament polypeptides from Myxicola infundibulum.

1. When axoplasm is incubated with [32P]Pi the main phosphorylated components are the neurofilament polypeptides. 2. Activation with Ca2+ of the proteinase present in axoplasm causes degradation of these neurofilaments and the peptides produced by this reaction have been analysed by fingerprinting. 3. Fingerprinting shows that initially the Ca2+-activated proteinase cleaves the neurofilament polypeptides at three major sites producing polypeptides with mol.wts. 70,000, 50,000 and 47,000. 4. These polypeptides sediment with filaments, originate from the tail-region of the molecule and contain a little radioactive label. 5. As these polypeptides are produced, other polypeptides that come from the head-region of the molecule are liberated as soluble products that contain the bulk of the radioactivity. 6. Fingerprinting therefore shows that at least two regions on the molecule are phosphorylated and that the major one is located towards the head-end of the polypeptides.     

叶绿体类囊体腔蛋白功能研究进展

【目的】类囊体是叶绿体光合作用中光反应进行的重要场所。类囊体腔是由类囊体膜包围形成的一个狭小空间。在类囊体腔中存在多种不同的蛋白家族,包括高叶绿素荧光(high chlorophyll fluorescence, HCF)蛋白、亲免蛋白、放氧复合物(oxygen-evolving complex, OEC)蛋白、PsbP类蛋白等,它们对植物的光合作用、核酸代谢以及氧化还原反应等都起着重要作用。【评论】文章分类综述了参与光合作用调控的类囊体腔蛋白在光系统组装、植物生长发育调节和高光逆境响应等生理活动中发挥的重要作用。【展望】文章可为未来研究类囊体腔蛋白的生理功能提供理论参考。     

Vectorial discharge of nascent polypeptides attached to chloroplast thylakoid membranes.

Chloroplast thylakoids with attached ribosomes were isolated from Chlamydomonas reinhardti. They were allowed to incorporate labeled amino acids into polypeptides. Labeled membranes were recovered from the reaction mixture, and a portion was treated with puromycin. The amount of labeled polypeptides released to the medium, and to the membranes by puromycin was determined by comparing radioactivity in soluble protein before, and after untreated, and puromycin-treated membranes were solubilized with the detergent Nonidet P-40. About 20% of the radioactive protein associated with the membranes was in nascent chains which were terminated by puromycin. Essentially all of terminated nascent chains remained with the membranes, and thus, were vectorially released. The results support the hypothesis that polypeptides which are synthesized by thylakoid-bound ribosomes are being incorporated into the membranes as they are synthesized.     

Prediction of protein cleavage sites by the barley cysteine endoproteases EP-A and EP-B based on the kinetics of synthetic peptide hydrolysis

Hordeins, the natural substrates of barley (Hordeum vulgare) cysteine endoproteases (EPs), were isolated as protein bodies and degraded by purified EP-B from green barley malt. Cleavage specificity was determined by synthesizing internally quenched, fluorogenic tetrapeptide substrates of the general formula 2-aminobenzoyl-P(2)-P(1)-P(1)'-P(2)' 1-tyrosine(NO(2))-aspartate. The barley EPs preferred neutral amino acids with large aliphatic and nonpolar (leucine, valine, isoleucine, and methionine) or aromatic (phenylalanine, tyrosine, and tryptophan) side chains at P(2), and showed less specificity at P(1), although asparagine, aspartate, valine, and isoleucine were particularly unfavorable. Peptides with proline at P(1) or P(1)' were extremely poor substrates. Cleavage sites with EP-A and EP-B preferred substrate sequences are found in hordeins, their natural substrates. The substrate specificity of EP-B with synthetic peptides was used successfully to predict the cleavage sites in the C-terminal extension of barley beta-amylase. When all of the primary cleavage sites in C hordein, which occur mainly in the N- and C-terminal domains, were removed by site-directed mutagenesis, the resulting protein was degraded 112 times more slowly than wild-type C hordein. We suggest that removal of the C hordein terminal domains is necessary for unfolding of the beta-reverse turn helix of the central repeat domain, which then becomes more susceptible to proteolytic attack by EP-B.     

Selection of functional signal peptide cleavage sites from a library of random sequences.

The export of proteins to the periplasmic compartment of bacterial cells is mediated by an amino-terminal signal peptide. After transport, the signal peptide is cleaved by a processing enzyme, signal peptidase I. A comparison of the cleavage sites of many exported proteins has identified a conserved feature of small, uncharged amino acids at positions -1 and -3 relative to the cleavage site. To determine experimentally the sequences required for efficient signal peptide cleavage, we simultaneously randomized the amino acid residues from positions -4 to +2 of the TEM-1 beta-lactamase enzyme to form a library of random sequences. Mutants that provide wild-type levels of ampicillin resistance were then selected from the random-sequence library. The sequences of 15 mutants indicated a bias towards small amino acids. The N-terminal amino acid sequence of the mature enzyme was determined for nine of the mutants to assign the new -1 and -3 residues. Alanine was present in the -1 position for all nine of these mutants, strongly supporting the importance of alanine at the -1 position. The amino acids at the -3 position were much less conserved but were consistent with the -3 rules derived from sequence comparisons. Compared with the wild type, two of the nine mutants have an altered cleavage position, suggesting that sequence is more important than position for processing of the signal peptide.     

Incorporation of polypeptides into thylakoid membranes of Chlamydomonas reinhardtii. Cyclic variations

A purified fraction of unstacked thylakoid membranes (TMF1u) has been obtained from homogenates of Chlamydomonas reinhardtii (wild type 137+) by using repeated centrifugates in sucrose density gradients and low salt treatment. The contaminants of the fraction are reduced to a few mitochondria (approximately 3% of the total mitochondrial population), a few osmiophilic granules, and fragments of chloroplast envelopes. By SDS-polyacrylamide gel electrophoresis the polypeptide components of TMF1u were resolved into at least 30 bands. To determine the relative rates of assembly of newly synthesized polypeptides into thylakoid membranes, synchronized algal cells were doubly labeled in vivo with L-[14C] and L-[3H]arginine--used for long- and short-term labeling, respectively. TMF1u's were isolated from the labeled cells at selected time points during the cycle and the distribution of radioactivity was assayed in the gel electrophoretograms of their solubilized polypeptides. Incorporation of newly synthesized polypeptides into the bands of the gels was found to occur continuously but differentially throughout the cycle. Maximal rates of incorporation for the majority of the polypeptides were detected shortly after cell division (6D-7D; equivalent to early G1 phase). The rates of radioactive labeling decreased gradually to a low level at the end of the dark period and then rose slightly at the beginning of the next light period. The findings suggest that, in addition to the light/dark control postulated in the past, assembly of newly synthesized proteins into thylakoid membranes is activated by signals at work in the early G1 phase.