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PubMed2000

Structures of type 2 peroxisomal targeting signals in two trypanosomatid aldolases.

Trypanosomatids, unicellular organisms responsible for several global diseases, contain unique organelles called glycosomes in which the first seven glycolytic enzymes are sequestered. We report the crystal structures of glycosomal fructose-1,6-bisphosphate aldolase from two major tropical pathogens, Trypanosoma brucei and Leishmania mexicana, the causative agents of African sleeping sickness and one form of leishmaniasis, respectively. Unlike mammalian aldolases, the T. brucei and L. mexicana aldolases contain nonameric N-terminal type 2 peroxisomal targeting signals (PTS2s) to direct their import into the glycosome. In both tetrameric trypanosomatid aldolases, the PTS2s from two different subunits form two closely intertwined structures. These "PTS2 dimers", which have very similar conformations in the two aldolase structures, are the first reported conformations of a glycosomal or peroxisomal PTS2, and provide opportunities for the design of trypanocidal compounds.

Amino Acid Sequence↗

PubMed2001

Functional and protein chemical characterization of the N-terminal domain of the rat corticotropin-releasing factor receptor 1.

Rat corticotropin-releasing factor receptor 1 (rCRFR1) was produced either in transfected HEK 293 cells as a complex glycosylated protein or in the presence of the mannosidase I inhibitor kifunensine as a high mannose glycosylated protein. The altered glycosylation did not influence the biological function of rCRFR1 as demonstrated by competitive binding of rat urocortin (rUcn) or human/rat corticotropin-releasing factor (h/rCRF) and agonist-induced cAMP accumulation. The low production rate of the N-terminal domain of rCRFR1 (rCRFR1-NT) by transfected HEK 293 cells, was increased by a factor of 100 in the presence of kifunensine. The product, rCRFR1-NT-Kif, bound rUcn specifically (K(D) = 27 nM) and astressin (K(I) = 60 nM). This affinity was 10-fold lower than the affinity of full length rCRFR1. However, it was sufficiently high for rCRFR1-NT-Kif to serve as a model for the N-terminal domain of rCRFR1. With protein fragmentation, Edman degradation, and mass spectrometric analysis, evidence was found for the signal peptide cleavage site C-terminally to Thr(23) and three disulfide bridges between precursor residues 30 and 54, 44 and 87, and 68 and 102. Of all putative N-glycosylation sites in positions 32, 38, 45, 78, 90, and 98, all Asn residues except for Asn(32) were glycosylated to a significant extent. No O-glycosylation was observed.

Amino Acid Sequence↗

PubMed1993

Isolation and characterization of an antifreeze protein precursor from transgenic Drosophila: evidence for partial processing.

Transgenic Drosophila melanogaster that contain a winter flounder antifreeze protein (AFP) gene fused to the transcriptional and translational control sequences of the host heat shock protein 70 gene express the transgene under heat shock conditions. They secrete into the hemolymph small quantities of a protein that reacts with antisera to AFP and is of a similar size to the proAFP precursor. To facilitate purification and characterization of this precursor, transformed fly lines homozygous for inserts on the 2nd, 3rd and X chromosomes were crossed together to generate a line with five and six AFP genes present in males and females, respectively. AFP production in the multi-gene line was approximately equal to the sum of that observed in the three starting lines and was just sufficient to perturb the growth of ice crystals. The AFP component was purified from heat-denatured hemolymph of this line by cation- and anion-exchange chromatography, followed by reverse-phase HPLC. Edman degradation sequencing of the purified protein showed that its N-terminus began two amino acids in from the predicted signal peptide cleavage point. An additional amino acid sequence was present that began two amino acids further into the 'pro' sequence. These AFP products are consistent with processing of the proAFP in Drosophila by a type IV dipeptidyl aminopeptidase, as has been suggested for processing in flounder.

Amino Acid Sequence↗

PubMed1997

Trafficking of wheat gluten proteins in transgenic tobacco plants: gamma-gliadin does not contain an endoplasmic reticulum-retention signal.

Wild-type and mutated forms of the wheat (Triticum aestivum L.) storage protein gamma-gliadin were expressed in transgenic tobacco (Nicotiana tabacum L. cv. NVS) under the control of the 35S cauliflower mosaic virus (CaMV) promoter in order to determine what, if any, endogenous targeting signals are present in the wild-type gamma-gliadin protein. The mutant forms of the protein were modified by the addition of a KDEL or HDEL C-terminal endoplasmic reticulum-retention signal, or the addition of a C-terminal propeptide from barley lectin which has been shown to be necessary and sufficient for targeting to the vacuole. Only modified forms of the protein accumulated in leaves of transgenic tobacco, although the transcript levels were similar for all the constructs. Pulse-chase analysis indicated that whereas the wild-type gamma-gliadin was rapidly turned over in tobacco leaves, KDEL and HDEL forms were highly stable. The vacuolar-signal mutant protein accumulated in tobacco leaves, but migrated on sodium dodecyl sulphate-polyacrylamide gel electrophoresis with a lower mobility than wild-type gamma-gliadin, due in part to glycosylation of the C-terminal propeptide. The vacuolar-signal mutant protein was turned over slowly in tobacco, perhaps indicating a poor level of transport competence. When pulse-chase analysis was carried out on protoplasts isolated from tobacco plants expressing wild-type gamma-gliadin, but in the presence of Brefeldin A, gamma-gliadin was seen to accumulate. Taken together, these results indicate that gamma-gliadin is targeted to the vacuole in transgenic tobacco plants and does not contain any structural determinants which confer retention in the endoplasmic reticulum.

Brefeldin A↗

PubMed2005

Characterisation of a highly stable alpha-amylase from the halophilic archaeon Haloarcula hispanica.

Intracellular and extracellular proteins from halophilic archaea face very saline conditions and must be able to maintain stability and functionality at nearly saturated salt concentrations. Haloarchaeal proteins contain specific adaptations to prevent aggregation and loss of activity in such conditions, but these adaptations usually result in a lack of stability in the absence of salt. Here, we present the characterisation of a secreted alpha-amylase (AmyH) from the halophilic archaeon Haloarcula hispanica. AmyH was shown to be very halophilic but, unusually for a halophilic protein, it retained activity in the absence of salt. Intrinsic fluorescence measurements and activity assays showed that AmyH was very stable in high-salt buffer and even maintained stability upon the addition of urea. Urea-induced denaturation was only achieved in the absence of NaCl, demonstrating clearly that the stability of the protein was salt-dependent. Sequencing of the amyH gene showed an amino acid composition typical of halophilic proteins and, moreover, the presence of a signal peptide containing diagnostic features characteristic of export via the Twin-arginine translocase (Tat). Analysis of the export of AmyH showed that it was translocated post-translationally, most likely in a folded and active conformation, confirming that AmyH is a substrate of the Tat pathway.

Amino Acid Sequence↗

PubMedDate not supplied

The Tat pathway in bacteria and chloroplasts (review).

Both in prokaryotic organisms and in chloroplasts, a specialized protein transport pathway exists which is capable of translocating proteins in a fully folded conformation. Transport is mediated in both instances by signal peptides harbouring a twin-arginine consensus motif (twin-arginine translocation (Tat) pathway). The Tat translocase comprises the three functionally different membrane proteins TatA, TatB, and TatC. While TatB and TatC are involved in the specific recognition of the substrate, TatA might be the major pore-forming component. Current evidence suggests that a functional Tat translocase is assembled from separate TatBC and TatA assemblies only on demand, i.e., in the presence of transport substrate and a transmembrane H+-motive force.

Bacterial Proteins↗

PubMed2001

Mitochondrial precursor signal peptide induces a unique permeability transition and release of cytochrome c from liver and brain mitochondria.

This study tested the hypothesis that mitochondrial precursor targeting peptides can elicit the release of cytochrome c from both liver and brain mitochondria by a mechanism distinct from that mediated by the classical, Ca2+-activated permeability transition pore. Human cytochrome oxidase subunit IV signal peptide (hCOXIV1-22) at concentrations from 15 to 100 microM induced swelling, a decrease in membrane potential, and cytochrome c release in both types of mitochondria. Although cyclosporin A and bongkrekic acid were without effect, dibucaine, propanolol, dextran, and the uncoupler FCCP were each able to inhibit signal peptide-induced swelling and cytochrome c release. Adenylate kinase was coreleased with cytochrome c, arguing against a signal peptide-induced cytochrome c-specific pathway of efflux across the outer membrane. Taken together, the data indicate that a human mitochondrial signal peptide can evoke the release of cytochrome c from both liver and brain mitochondria by a unique permeability transition that differs in several characteristics from the classical mitochondrial permeability transition.

Adenosine Triphosphate↗

PubMed2005

Carbohydrate- and conformation-dependent cargo capture for ER-exit.

Some secretory proteins leave the endoplasmic reticulum (ER) by a receptor-mediated cargo capture mechanism, but the signals required for the cargo-receptor interaction are largely unknown. Here, we describe a novel targeting motif that is composed of a high-mannose type oligosaccharide intimately associated with a surface-exposed peptide beta-hairpin loop. The motif accounts for lectin ERGIC-53-assisted ER-export of the lyososomal enzyme procathepsin Z. The second oligosaccharide chain of procathepsin Z exhibits no binding activity for ERGIC-53, illustrating the selective lectin properties of ERGIC-53. Our data suggest that the conformation-based motif is only present in fully folded procathepsin Z and that its recognition by ERGIC-53 reflects a quality control mechanism that acts complementary to the primary folding machinery in the ER. A similar oligosaccharide/beta-hairpin loop structure is present in cathepsin C, another cargo of ERGIC-53, suggesting the general nature of this ER-exit signal. To our knowledge this is the first documentation of an ER-exit signal in soluble cargo in conjunction with its decoding by a transport receptor.

Amino Acid Motifs↗

PubMed2002

A twin arginine signal peptide and the pH gradient trigger reversible assembly of the thylakoid [Delta]pH/Tat translocase.

The thylakoid DeltapH-dependent/Tat pathway is a novel system with the remarkable ability to transport tightly folded precursor proteins using a transmembrane DeltapH as the sole energy source. Three known components of the transport machinery exist in two distinct subcomplexes. A cpTatC-Hcf106 complex serves as precursor receptor and a Tha4 complex is required after precursor recognition. Here we report that Tha4 assembles with cpTatC-Hcf106 during the translocation step. Interactions among components were examined by chemical cross-linking of intact thylakoids followed by immunoprecipitation and immunoblotting. cpTatC and Hcf106 were consistently associated under all conditions tested. In contrast, Tha4 was only associated with cpTatC and Hcf106 in the presence of a functional precursor and the DeltapH. Interestingly, a synthetic signal peptide could replace intact precursor in triggering assembly. The association of all three components was transient and dissipated upon the completion of protein translocation. Such an assembly-disassembly cycle could explain how the DeltapH/Tat system can assemble translocases to accommodate folded proteins of varied size. It also explains in part how the system can exist in the membrane without compromising its ion and proton permeability barrier.

Arginine↗

PubMed2004

Prediction of the subcellular localization of eukaryotic proteins using sequence signals and composition.

A tool called Locfind for the sequence-based prediction of the localization of eukaryotic proteins is introduced. It is based on bidirectional recurrent neural networks trained to read sequentially the amino acid sequence and produce localization information along the sequence. Systematic variation of the network architecture in combination with an efficient learning algorithm lead to a 91% correct localization prediction for novel proteins in fivefold cross-validation. The data and evaluation procedure are the same as the non-plant part of the widely used TargetP tool by Emanuelsson et al. The Locfind system is available on the WWW for predictions (http://www.stepc.gr/~synaptic/locfind.html).

Algorithms↗

PubMed2003

Structure, function and evolution of the signal recognition particle.

The signal recognition particle (SRP) is a ribonucleoprotein particle essential for the targeting of signal peptide-bearing proteins to the prokaryotic plasma membrane or the eukaryotic endoplasmic reticulum membrane for secretion or membrane insertion. SRP binds to the signal peptide emerging from the exit site of the ribosome and forms a ribosome nascent chain (RNC)-SRP complex. The RNC-SRP complex then docks in a GTP-dependent manner with a membrane-anchored SRP receptor and the protein is translocated across or integrated into the membrane through a channel called the translocon. Recently considerable progress has been made in understanding the architecture and function of SRP.

Amino Acid Sequence↗

PubMed2002

Solution structure of intracellular signal-transducing peptide derived from human beta(2)-adrenergic receptor.

The structure of intracellular third loop peptide (betaIII-2: RRSSKFCLKEKKALK) was studied by CD and NMR spectroscopy. According to the CD study, this peptide forms a helix in the TFE solution. The three-dimensional molecular structure in TFE was determined by the 2D (1)H NMR spectroscopy. The structure consists of a positive charge cluster on the C-terminal side of the peptide chain. This part will be an active site of the peptide interacting with the G-protein.

Amino Acid Sequence↗

PubMed2003

Differential interactions between a twin-arginine signal peptide and its translocase in Escherichia coli.

The twin-arginine translocation (Tat) machinery of the Escherichia coli inner membrane is dedicated to the export of proteins harboring a conserved SRRxFLK motif in their signal sequence. TatA, TatB, and TatC are the functionally essential constituents of the Tat machinery, but their precise function is unknown. Using site-specific crosslinking, we have analyzed interactions of the twin-arginine precursor preSufI with the Tat proteins upon targeting to inner membrane vesicles. TatA association is observed only in the presence of a transmembrane H(+) gradient. TatB is found in contact with the entire signal sequence and adjacent parts of mature SufI. Interaction of TatC with preSufI is, however, restricted to a discrete area around the consensus motif. The results reveal a hierarchy in targeting of a Tat substrate such that for the primary interaction, TatC is both necessary and sufficient while a subsequent association with TatB likely mediates transfer from TatC to the actual Tat pore.

Cell Membrane↗

PubMed2006

A novel fusion protein system for the production of native human pepsinogen in the bacterial periplasm.

Human pepsinogen is the secreted inactive precursor of pepsin. Under the acidic conditions present in the stomach it is autocatalytically cleaved into the active protease. Pepsinogen contains three consecutive disulfides, and was used here as a model protein to investigate the production of aspartic proteases in the Escherichia coli periplasm. Various N-terminal translocation signals were applied and several different expression vectors were tested. After fusion to pelB, dsbA or ompT signal peptides no recombinant product could be obtained in the periplasm using the T7 promoter. As a new approach, human pepsinogen was fused to E. coli ecotin (E. coli trypsin inhibitor), which is a periplasmic homodimeric protein of 142 amino acids per monomer containing one disulfide bridge. The fusion protein was expressed in pTrc99a. After induction, the ecotin-pepsinogen fusion protein was translocated into the periplasm and the ecotin signal peptide was cleaved. Upon acid treatment, the fusion protein was converted into pepsin, indicating that pepsinogen was produced in its native form. In shake flasks experiments, the amount of active fusion protein present in the periplasm was 100 microg per litre OD 1, corresponding to 70 microg pepsinogen. After large scale cultivation, the fusion protein was isolated from the periplasmic extract. It was purified to homogeneity with a yield of 20%. The purified protein was native. Acid-induced activation of the fusion protein proceeded very fast. As soon as pepsin was present, the ecotin part of the fusion protein was rapidly digested, followed by a further activation of pepsinogen.

Escherichia coli↗

PubMed2004

Beyond the signal sequence: protein routing in health and disease.

Receptors, hormones, enzymes, ion channels, and structural components of the cell are created by the act of protein synthesis. Synthesis alone is insufficient for proper function, of course; for a cell to operate effectively, its components must be correctly compartmentalized. The mechanism by which proteins maintain the fidelity of localization warrants attention in light of the large number of different molecules that must be routed to distinct subcellular loci, the potential for error, and resultant disease. This review summarizes diseases known to have etiologies based on defective protein folding or failure of the cell's quality control apparatus and presents approaches for therapeutic intervention.

Animals↗

PubMed1990

Cloning and characterization of the human colipase cDNA.

Pancreatic lipase hydrolyzes dietary triglycerides to monoglycerides and fatty acids. In the presence of bile salts, the activity of pancreatic lipase is markedly decreased. The activity can be restored by the addition of colipase, a low molecular weight protein secreted by the pancreas. The action of pancreatic lipase in the gut lumen is dependent upon its interaction with colipase. As a first step in elucidating the molecular events governing the interaction of lipase and colipase with each other and with fatty acids, a cDNA encoding human colipase was isolated from a lambda gt11 cDNA library with a rabbit polyclonal anti-human colipase antibody. The full-length 525 bp cDNA contained an open reading frame encoding 112 amino acids, including a 17 amino acid signal peptide. The predicted protein sequence contains 100% of the published protein sequence for human colipase determined by chemical methods, but predicts the presence of five additional NH2-terminal amino acids and four additional COOH-terminal amino acids. Comparison of the predicted protein sequence with the known sequences of colipase from other species reveals regions of extensive identity. In vitro translation of mRNA transcribed from the cDNA gave a protein of the expected molecular size that was processed by pancreatic microsomal membranes. Sequence analysis of the in vitro translation product after processing demonstrated signal peptide cleavage and the presence of a human procolipase, as exists in the pig and horse colipases. DNA blot analysis was consistent with the presence of a single gene for colipase. RNA blot analysis demonstrated tissue-specific expression of colipase mRNA in the pancreas. Thus, we report, for the first time, a cDNA for colipase.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1983

Transient involvement of signal recognition particle and its receptor in the microsomal membrane prior to protein translocation.

We have shown that neither the signal recognition particle (SRP) nor the SRP receptor is directly involved in the maintenance of a ribosome-membrane junction for the translocation of secretory proteins. We found that the purified SRP receptor releases the signal-sequence-induced and SRP-mediated elongation arrest of synthesis by displacing SRP from the ribosome. This SRP displacement was not accompanied by binding of the SRP receptor to the ribosome. Using intact microsomal membranes as a source of the SRP receptor, we found that both SRP displacement and binding of the elongation-arrested ribosome to the membrane can occur at 0 degrees C, by a mechanism that is independent of chain elongation.

Animals↗

PubMed2004

Monooxygenase X, a member of the copper-dependent monooxygenase family localized to the endoplasmic reticulum.

Based on sequence comparisons, MOX (monooxygenase X), is a member of the copper monooxygenase family that includes dopamine beta-monooxygenase (DBM) and peptidylglycine alpha-hydroxylating monooxygenase (PHM). MOX has all of the residues expected to be critical for copper binding, and its cysteine residues can yield the intramolecular disulfide bond pattern observed in DBM. Although DBM and PHM function within the lumen of the secretory pathway, the published sequence for human MOX lacks a signal sequence, suggesting that it does not enter this compartment. We identified an upstream exon that encodes the signal sequence of human MOX. A retained intron yields minor amounts of transcript encoding MOX without a signal sequence. MOX transcripts are widely expressed, with the highest levels in the salivary gland and ovary and moderate levels in brain, pituitary, and heart. Despite the presence of a signal sequence, exogenous MOX is not secreted, and it localizes throughout the endoplasmic reticulum in both endocrine or nonendocrine cells. Neither appending green fluorescent protein to its C terminus nor deleting the hydrophobic domain near its C terminus facilitates secretion of MOX. MOX is N-glycosylated, is tightly membrane-associated, and forms oligomers that are not disulfide-linked. Based on its sequence and localization, MOX is predicted to hydroxylate a hydrophobic substrate in the endoplasmic reticulum.

Amino Acid Sequence↗