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1.
In vivo topological analysis of Ste2, a yeast plasma membrane protein, by using beta-lactamase gene fusions. 总被引:7,自引:5,他引:2
Gene fusions were constructed between Ste2, the receptor for the Saccharomyces cerevisiae alpha-factor, and beta la, the secreted form of beta-lactamase encoded by the bla gene of pBR322. The Ste2 and beta la components were linked by a processing fragment (P) from the yeast killer preprotoxin containing a C-terminal lysine-arginine site for cleavage by the Golgi-associated Kex2 protease. Ste2 is predicted to have a rhodopsinlike topology, with an external N terminus and seven transmembrane segments. Fusions to three of the four Ste2 domains predicted to be external resulted in beta la secretion from yeast cells. A fusion at a site just preceding the first transmembrane segment was an exception; the product was cell associated, indicating that the first 44 residues of Ste2 are insufficient to direct secretion of beta la; translocation of this domain presumably requires the downstream transmembrane segment. Expression of fusions located in two domains predicted to be cytoplasmic failed to result in beta la secretion. Following insertion of the preprotoxin signal peptide (S) between the Ste2 and P components of these cytoplasmic fusions, secretion of beta la activity occurred, which is consistent with inversion of the orientation of the beta la reporter. Conversely, insertion of S between Ste2 and P in an external fusion sharply reduced beta la secretion. Complementary information about both cytoplasmic and external domains of Ste2 was therefore provided, and most aspects of the predicted topology were confirmed. The steady-state levels of beta la detected were low, presumably because of efficient degradation of the fusions in the secretory pathway; levels, however, were easily detectable. This method should be valuable in the analysis of in vivo topologies of both homologous and foreign plasma membrane proteins expressed in yeast cells. 相似文献
2.
H Zhu H Bussey D Y Thomas J Gagnon A W Bell 《The Journal of biological chemistry》1987,262(22):10728-10732
The carboxyl-terminal sequences of the two polypeptide chains of the Saccharomyces cerevisiae K1 killer toxin were determined by protein sequencing and amino acid analysis of peptide fragments generated from the mature, secreted toxin. The COOH-terminal amino acid of the beta chain is histidine 316, the final residue encoded by the precursor gene. The COOH terminus of the alpha chain is at alanine 147 of the preprotoxin. Amino acid composition data for the purified toxin are consistent with that predicted from the gene sequence of the preprotoxin where the alpha and beta subunits consist of amino acid residues 45-147 and 234-316, respectively. The molecular weight of the mature alpha beta dimer is about 20,658. The COOH-terminal sequence determination completes the location of the toxin subunits in the precursor, and its configuration may be represented as prepropeptide-Pro-Arg-alpha-Arg-Arg-gamma-Lys-Arg-beta, where gamma represents the interstitial glycosylated peptide. The COOH terminal side of the paired basic residues (Arg-148 Arg-149 and Lys-232 Arg-233 of preprotoxin) are endoproteolytic processing sites for the product of the KEX2 gene (Julius, D., Brake, A., Blair, L., Kunisawa, R., and Thorner, J. (1984) Cell 37, 1075-1089), and thus maturation of the alpha subunit of killer toxin apparently requires a carboxypeptidase B-like activity. A possible candidate for this activity is the product of the KEX1 gene (Dmochowska, A., Dignard, D., Henning, D., Thomas, D.Y., and Bussey, H. (1987) Cell, in press). 相似文献
3.
We cloned and sequenced a cDNA from a library of mouse pituitary AtT-20 cells which are known to cleave an endogenous and various foreign prohormones at dibasic sites. This cDNA encodes a novel 753-residue protein, named PC3, which is structurally related to the yeast Kex2 protease involved in precursor cleavage at dibasic sites and to recently identified mammalian Kex2-like proteins, furin and PC2. Among examined cell lines and tissues, PC3 mRNA was only detected in AtT-20 cells. The substrate specificity of PC3 expressed in mammalian cells was similar to that observed in AtT-20 cells. We conclude that PC3 is a resident prohormone processing endoprotease in AtT-20 cells. 相似文献
4.
Processing and secretion of a virally encoded antifungal toxin in transgenic tobacco plants: evidence for a Kex2p pathway in plants. 总被引:3,自引:0,他引:3
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Ustilago maydis is a fungal pathogen of maize. Some strains of U. maydis encode secreted polypeptide toxins capable of killing other susceptible strains of U. maydis. We show here that one of these toxins, the KP6 killer toxin, is synthesized by transgenic tobacco plants containing the viral toxin cDNA under the control of a cauliflower mosaic virus promoter. The two components of the KP6 toxin, designated alpha and beta, with activity and specificity identical to those found in toxin secreted by U. maydis cells, were isolated from the intercellular fluid of the transgenic tobacco plants. The beta polypeptide from tobacco was identical in size and N-terminal sequence to the U. maydis KP6 beta polypeptide. Processing of the KP6 preprotoxin in U. maydis requires a subtilisin-like processing protease, Kex2p, which is present in both animal and fungal cells and is required for processing of (among other things) small secreted polypeptide hormones and secreted toxins. Our findings present evidence for Kex2p-like processing activity in plants. The systemic production of this viral killer toxin in crop plants may provide a new method of engineering biological control of fungal pathogens in crop plants. 相似文献
5.
Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity. 总被引:110,自引:17,他引:110
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A Ullrich A Gray A W Tam T Yang-Feng M Tsubokawa C Collins W Henzel T Le Bon S Kathuria E Chen et al. 《The EMBO journal》1986,5(10):2503-2512
To identify structural characteristics of the closely related cell surface receptors for insulin and IGF-I that define their distinct physiological roles, we determined the complete primary structure of the human IGF-I receptor from cloned cDNA. The deduced sequence predicts a 1367 amino acid receptor precursor, including a 30-residue signal peptide, which is removed during translocation of the nascent polypeptide chain. The 1337 residue, unmodified proreceptor polypeptide has a predicted Mr of 151,869, which compares with the 180,000 Mr IGF-I receptor precursor. In analogy with the 152,784 Mr insulin receptor precursor, cleavage of the Arg-Lys-Arg-Arg sequence at position 707 of the IGF-I receptor precursor will generate alpha (80,423 Mr) and beta (70,866 Mr) subunits, which compare with approximately 135,000 Mr (alpha) and 90,000 Mr (beta) fully glycosylated subunits. 相似文献
6.
Y Bourbonnais A Danoff D Y Thomas D Shields 《The Journal of biological chemistry》1991,266(20):13203-13209
The peptide somatostatin (SRIF) exists as two different molecular species. In addition to the most common form, which is a 14-residue peptide, there is also a 14-amino acid amino-terminally extended form of the tetradecapeptide, SRIF-28. Both peptides are synthesized as larger precursors containing paired basic and monobasic amino acids at their processing sites, which, upon cleavage, generate either SRIF-14 or -28, respectively. In mammals a single prepro-SRIF molecule undergoes tissue-specific processing to generate the mature hormone whereas in some species of fish separate genes encode two distinct but homologous precursors prepro-SRIF-I and -II that give rise to SRIF-14 and -28, respectively. To investigate the molecular basis for differential processing of the prohormones we introduce their cDNAs into yeast cells (Saccharomyces cerevisiae). The signal peptides of both precursors were poorly recognized by the yeast endoplasmic reticulum translocation apparatus, consequently only low levels of SRIF peptides were synthesized. To circumvent this problem a chimeric precursor consisting of the alpha-factor signal peptide plus 30 residues of the proregion was fused to pro-SRIF-II. This fusion protein was efficiently transported through the yeast secretory pathway and processed to SRIF-28 exclusively, which is identical to the processing of the native precursor in pancreatic islet D-cells. Most significantly, cleavage of the precursor to SRIF-28 was independent of the Kex 2 endoprotease since processing occurred efficiently in a kex 2 mutant strain. We conclude that in addition to the Kex 2 protease, yeast possess a distinct prohormone converting enzyme with specificity toward monobasic processing sites. 相似文献
7.
8.
Activation of interleukin-1 beta by a co-induced protease 总被引:13,自引:0,他引:13
The proteolytic generation of mature interleukin-1 beta (IL-1 beta) from its inactive precursor does not proceed by a conventional pathway for hormonal processing. Pro-IL-1 beta is found dispersed in the cytoplasm, and there are no basic amino acid residues or other commonly recognized processing sites adjoining the mature N-terminus. Processing appears to occur during release of the hormone. In the present study, we have identified a specific protease that generates mature IL-1 beta from the precursor. This enzyme is co-induced with the hormone, and it differs in its cleavage specificity and inhibitor sensitivity from all known proteases. 相似文献
9.
Specific modulation of Kex2/furin family proteases by potassium 总被引:6,自引:0,他引:6
Kex2 protease is the prototype for a family of proteases responsible for endoproteolytic cleavage at multi-basic motifs in the eukaryotic secretory pathway. Here we demonstrate that potassium ion can act as a modulator of Kex2 activity with an apparent affinity of approximately 20 mm. Other monovalent cations (Li(+), Na(+), etc.) display similar effects, but affinities are all over 20-fold lower. Potassium ion binding stimulates turnover at physiologically relevant Lys-Arg cleavage sites but reduces turnover with at least one incorrect sequence. Furthermore, the mammalian Kex2 homolog furin displays similar effects. In contrast, the neuroendocrine homolog PC2 is inhibited by potassium ion with all substrates examined. The pre-steady-state behavior of Kex2 is also altered upon binding of potassium ion, with opposite effects on acylation and deacylation rates. These biochemical data indicate that potassium ion concentration may function as a regulator of processing protease specificity and activity in the eukaryotic secretory pathway, with such enzymes potentially encountering compartments high in potassium ion caused by the action of antiporters such as yeast NHX1 (VPS44) or the mammalian NHE7. 相似文献
10.
Jalving R van de Vondervoort PJ Visser J Schaap PJ 《Applied and environmental microbiology》2000,66(1):363-368
Secreted yields of foreign proteins may be enhanced in filamentous fungi through the use of translational fusions in which the target protein is fused to an endogenous secreted carrier protein. The fused proteins are usually separated in vivo by cleavage of an engineered Kex2 endoprotease recognition site at the fusion junction. We have cloned the kexin-encoding gene of Aspergillus niger (kexB). We constructed strains that either overexpressed KexB or lacked a functional kexB gene. Kexin-specific activity doubled in membrane-protein fractions of the strain overexpressing KexB. In contrast, no kexin-specific activity was detected in the similar protein fractions of the kexB disruptant. Expression in this loss-of-function strain of a glucoamylase human interleukin-6 fusion protein with an engineered Kex2 dibasic cleavage site at the fusion junction resulted in secretion of unprocessed fusion protein. The results show that KexB is the endoproteolytic proprotein processing enzyme responsible for the processing of (engineered) dibasic cleavage sites in target proteins that are transported through the secretion pathway of A. niger. 相似文献
11.
Identification of a human insulinoma cDNA encoding a novel mammalian protein structurally related to the yeast dibasic processing protease Kex2 总被引:44,自引:0,他引:44
We have identified a human insulinoma cDNA (PC2) that encodes a protein homologous to the precursor processing Kex2 endoprotease of yeast by using a polymerase chain reaction to detect and amplify conserved sequences within the catalytic site. The 638-residue amino acid sequence of PC2 begins with a cleavable signal peptide, indicating that it enters the secretory pathway, and contains a 282-residue domain that is homologous to the catalytic modules of both Kex2 and the related bacterial subtilisins. Within this region 49 and 27% of the amino acids are identical to those in the aligned Kex2 and subtilisin BPN' sequences, respectively, and the catalytically essential Asp, His, and Ser residues are all conserved. Northern blot analysis revealed the presence of 2.8- and 5.0-kilobase hybridizing bands in mRNA from the insulinoma. The PC2 protein also shows great similarity to the incomplete NH2-terminal sequence of the human furin gene product, a putative membrane-inserted receptor-like molecule. We propose that PC2 is a member of a family of mammalian Kex2/subtilisin-like proteases that includes members involved in a number of specific proteolytic events within cells, including the processing of prohormones. 相似文献
12.
13.
BACKGROUND: Members of the subtilisin family of serine proteases usually have a conserved asparagine residue that stabilizes the oxyanion transition state of peptide-bond hydrolysis. Yeast Kex2 protease is a member of the subtilisin family that differs from the degradative subtilisin proteases in its high substrate specificity, it processes pro-alpha-factor, the precursor of the alpha-factor mating pheromone of yeast, and also removes the pro-peptide from its own precursor by an intramolecular cleavage reaction. Curiously, the mammalian protease PC2, a Kex2 homolog that is likely to be required for pro-insulin processing, has an aspartate in place of asparagine at the 'oxyanion hole'. RESULTS: We have tested the effect of making substitutions of the conserved oxyanion-hole asparagine (Asn 314) of the Kex2 protease. To do this, we have developed a rapid method of site-directed mutagenesis, involving homologous recombination of a polymerase chain reaction product in yeast. Using this method, we have substituted alanine or aspartate for Asn 314 in a form of Kex2 engineered for secretion. Transformants expressing the two mutant enzymes could be identified by failure either to produce mature alpha-factor or to mate. The Ala 314 enzyme was unstable but the Asp 314 enzyme accumulated to a high level, so that it could be purified and its activity towards various substrates tested in vitro. We found that, with three peptides that are good substrates of wild-type Kex2, the k(cal) of the Asp 314 enzyme was reduced approximately 4500-fold and its K(M) approximately 4-fold, relative to the wild-type enzyme. For the peptide substrate corresponding to the cleavage site of pro-alpha-factor, however, k(cat) of the Asp 314 enzyme was reduced only 125-fold, while the K(m) was increased 3-fold. Despite its reduced catalytic activity, however, processing of the mutant enzyme in vivo - by the intramolecular cleavage that removes its amino-terminal pro-domain - occurs at an unchanged rate. CONCLUSIONS: The effects of the Asn 314-Asp substitution reveal contributions to the reaction specificity of the Kex2 protease of substrate residues amino-terminal to the pair of basic residues at the cleavage site. Aspartate at the oxyanion hole appears to confer k(caf) discrimination between substrates by raising the energy barrier for productive substrate binding: this may have implications for pro-insulin processing by the PC2 protease, which has an aspartate at the equivalent position. The rate of intramolecular cleavage of pro-Kex2 may be limited by a step other than catalysis, presumably protein folding. 相似文献
14.
Molecular and enzymatic properties of furin, a Kex2-like endoprotease involved in precursor cleavage at Arg-X-Lys/Arg-Arg sites. 总被引:13,自引:0,他引:13
We have recently shown that furin, a mammalian homologue of the yeast precursor-processing endoprotease Kex2, is involved in precursor cleavage at sites marked by the Arg-X-Lys/Arg-Arg motif within the constitutive secretory pathway. In this study, we analyzed molecular and enzymatic properties of furin expressed in Chinese hamster ovary cells using gene transfer techniques. COOH-terminal truncation analyses indicate that the polypeptide region significantly conserved among the Kex2 family members is required for the endoprotease activity of furin, while the COOH-terminal unconserved region containing the Cys-rich domain and the transmembrane domain is dispensable. A mutant of furin truncated up to the transmembrane domain from the COOH-terminus was secreted into the culture medium as an active form. The sequence requirements for precursor cleavage of this truncated furin determined in vitro were similar to those of wild-type furin determined by expression studies in cultured cells. It had a strong resemblance to the Kex2 protease in the inhibitor profile and pH dependency. These observations support the notion that furin is the endogenous endoprotease involved in precursor cleavage at Arg-X-Lys/Arg-Arg sites. 相似文献
15.
Ruud Jalving Peter J. I. van de Vondervoort Jaap Visser Peter J. Schaap 《Applied microbiology》2000,66(1):363-368
Secreted yields of foreign proteins may be enhanced in filamentous fungi through the use of translational fusions in which the target protein is fused to an endogenous secreted carrier protein. The fused proteins are usually separated in vivo by cleavage of an engineered Kex2 endoprotease recognition site at the fusion junction. We have cloned the kexin-encoding gene of Aspergillus niger (kexB). We constructed strains that either overexpressed KexB or lacked a functional kexB gene. Kexin-specific activity doubled in membrane-protein fractions of the strain overexpressing KexB. In contrast, no kexin-specific activity was detected in the similar protein fractions of the kexB disruptant. Expression in this loss-of-function strain of a glucoamylase human interleukin-6 fusion protein with an engineered Kex2 dibasic cleavage site at the fusion junction resulted in secretion of unprocessed fusion protein. The results show that KexB is the endoproteolytic proprotein processing enzyme responsible for the processing of (engineered) dibasic cleavage sites in target proteins that are transported through the secretion pathway of A. niger. 相似文献
16.
Complementary deoxyribonucleic acid cloning of a messenger ribonucleic acid encoding transforming growth factor beta 4 from chicken embryo chondrocytes 总被引:19,自引:0,他引:19
S B Jakowlew P J Dillard M B Sporn A B Roberts 《Molecular endocrinology (Baltimore, Md.)》1988,2(12):1186-1195
Using a human transforming growth factor beta 1 (TGF beta) cDNA probe, we have detected an RNA species migrating at about 1.7 kilobases in cultured primary chicken embryo chondrocytes that is distinct from chicken TGF beta 1. The cloning and sequencing of cDNAs corresponding to this chondrocyte RNA demonstrate that it represents a new member of the TGF beta family, which we have named TGF beta 4. Unlike previously described TGF beta which are 390 to 414 amino acids long, the predicted precursor protein of TGF beta 4 is only 304 amino acids and does not appear to contain a signal peptide. Also unique to this new TGF beta is an insertion of two amino acids near the N-terminus of the processed peptide which would result in a 114 amino acid mature protein after cleavage from the precursor at a tetrabasic arg-arg-arg-arg site. The nine cysteine residues characteristic of all TGF beta are conserved. TGF beta 4 shows 82%, 64%, and 71% identity with the amino acid sequences of processed TGF beta 1, 2, and 3, respectively. 相似文献
17.
In vivo evidence for posttranslational translocation and signal cleavage of the killer preprotoxin of Saccharomyces cerevisiae. 总被引:6,自引:3,他引:3
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A full-length cDNA of the M1 double-stranded RNA killer preprotoxin coding region successfully directed the synthesis of secreted K1 toxin when expressed in Saccharomyces cerevisiae from a plasmid vector. Three protein species immunoreactive with antitoxin antiserum were detected intracellularly in transformants harboring this killer cDNA plasmid. These toxin precursor species were characterized by using secretory-defective hosts, by comparative electrophoretic mobilities, and by tunicamycin susceptibility. Such studies indicate that these three protein species represent intermediates generated by signal cleavage of the preprotoxin and its subsequent glycosylation and provide evidence that these events occur posttranslationally. 相似文献
18.
Endocytotic uptake and retrograde transport of a virally encoded killer toxin in yeast 总被引:1,自引:0,他引:1
We demonstrate that a virally encoded yeast 'killer' toxin is entering its eukaryotic target cell by endocytosis, subsequently travelling the yeast secretory pathway in reverse to exhibit its lethal effect. The K28 killer toxin is a secreted alpha/beta heterodimer that kills sensitive yeasts in a receptor-mediated fashion by blocking DNA synthesis in the nucleus. In vivo processing of the toxin precursor results in a protein whose beta-C-terminus carries the endoplasmic reticulum (ER) retention signal HDEL, which, as we show here, is essential for retrograde toxin transport. Yeast end3/4 mutants as well as cells lacking the HDEL receptor (Deltaerd2) or mutants defective in Golgi-to-ER protein recycling (erd1) are toxin resistant because the toxin can no longer enter and/or retrograde pass the cell. Site-directed mutagenesis further indicated that the toxin's beta-HDEL motif ensures retrograde transport, although in a toxin-secreting yeast the beta-C-terminus is initially masked by an R residue (beta-HDELR) until Kex1p cleavage uncovers the toxin's targeting signal in a late Golgi compartment. Prevention of Kex1p processing results in high-level secretion of a biologically inactive protein incapable of re-entering the secretory pathway. Finally, we present evidence that ER-to-cytosol toxin export is mediated by the Sec61p translocon and requires functional copies of the lumenal ER chaperones Kar2p and Cne1p. 相似文献
19.
Zygocin, a monomeric protein toxin secreted by a virus-infected killer strain of the osmotolerant spoilage yeast Zygosaccharomyces bailii, kills a broad spectrum of human and phytopathogenic yeasts and filamentous fungi by disrupting cytoplasmic membrane function. The toxin is encoded by a double-stranded (ds)RNA killer virus (ZbV-M, for Z. bailii virus M) that stably persists within the yeast cell cytosol. In this study, the protein toxin was purified, its N-terminal amino acid sequence was determined, and a full-length cDNA copy of the 2.1 kb viral dsRNA genome was cloned and successfully expressed in a heterologous fungal system. Sequence analysis as well as zygocin expression in Schizosaccharomyces pombe indicated that the toxin is in vivo expressed as a 238-amino-acid preprotoxin precursor (pptox) consisting of a hydrophobic N-terminal secretion signal, followed by a potentially N-glycosylated pro-region and terminating in a classical Kex2p endopeptidase cleavage site that generates the N-terminus of the mature and biologically active protein toxin in a late Golgi compartment. Matrix-assisted laser desorption mass spectrometry further indicated that the secreted toxin is a monomeric 10.4 kDa protein lacking detectable post-translational modifications. Furthermore, we present additional evidence that in contrast with other viral antifungal toxins, zygocin immunity is not mediated by the toxin precursor itself and, therefore, heterologous pptox expression in a zygocin-sensitive host results in a suicidal phenotype. Final sequence comparisons emphasize the conserved pattern of functional elements present in dsRNA killer viruses that naturally infect phylogenetically distant hosts (Saccharomyces cerevisiae and Z. bailii) and reinforce models for the sequence elements that are in vivo required for viral RNA packaging and replication. 相似文献
20.
Semisynthesis of human ghrelin: condensation of a Boc-protected recombinant peptide with a synthetic O-acylated fragment 总被引:1,自引:0,他引:1
Makino T Matsumoto M Suzuki Y Kitajima Y Yamamoto K Kuramoto M Minamitake Y Kangawa K Yabuta M 《Biopolymers》2005,79(5):238-247
The creation of peptide using a combination of recombinant expression and chemical synthesis can be a powerful tool for the production of a wide variety of polypeptides modified by phosphorylation, glycosylation, etc. We have developed a new method for the preparation of a recombinant peptide with a free N(alpha)-amino group and protected N(epsilon)-amino groups, and have used this method in the semisynthesis of human ghrelin. Ghrelin, a natural ligand for growth hormone secretagogue receptor, is a 28-residue peptide with an essential n-octanoyl modification on Ser3. A 7-residue N-terminal fragment of ghrelin containing the octanoyl modification was prepared by Fmoc chemistry. In the preparation of it, all reactions were performed on the 2-chlorotrityl resin. Additionally, TBDMS and tBu turned out to be the most effective protection groups for the Ser3 and the Ser2, Ser6, respectively. For preparation of a 21-residue C-terminal fragment, we established a two-step protease processing method for the partially protected segment. A recombinant precursor peptide was Boc protected and subsequently cleaved using two distinct proteases, OmpT and Kex2. The peptides were then coupled to each other and, after deprotection, resulted in fully active human ghrelin. 相似文献