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PubMed2006

Roles for the Drs2p-Cdc50p complex in protein transport and phosphatidylserine asymmetry of the yeast plasma membrane.

Drs2p, a P-type adenosine triphosphatase required for a phosphatidylserine (PS) flippase activity in the yeast trans Golgi network (TGN), was first implicated in protein trafficking by a screen for mutations synthetically lethal with arf1 (swa). Here, we show that SWA4 is allelic to CDC50, encoding a membrane protein previously shown to chaperone Drs2p from the endoplasmic reticulum to the Golgi complex. We find that cdc50Delta exhibits the same clathrin-deficient phenotypes as drs2Delta, including delayed transport of carboxypeptidase Y to the vacuole, mislocalization of resident TGN enzymes and the accumulation of aberrant membrane structures. These trafficking defects precede appearance of cell polarity defects in cdc50Delta, suggesting that the latter are a secondary consequence of disrupting Golgi function. Involvement of Drs2p-Cdc50p in PS translocation suggests a role in restricting PS to the cytosolic leaflet of the Golgi and plasma membrane. Annexin V binding and papuamide B hypersensitivity indicate that drs2Delta or cdc50Delta causes a loss of plasma membrane PS asymmetry. However, clathrin and other endocytosis null mutants also exhibit a comparable loss of PS asymmetry, and studies with drs2-ts and clathrin (chc1-ts) conditional mutants suggest that loss of plasma membrane asymmetry is a secondary consequence of disrupting protein trafficking.

Amino Acid Sequence↗

PubMed2006

Cell type-specific transgene expression of the prion protein in Xenopus intermediate pituitary cells.

The cellular form of prion protein (PrPC) is anchored to the plasma membrane of the cell and expressed in most tissues, but predominantly in the brain, including in the pituitary gland. Thus far, the biosynthesis of PrPC has been studied only in cultured (transfected) tumour cell lines and not in primary cells. Here, we investigated the intracellular fate of PrPCin vivo by using the neuroendocrine intermediate pituitary melanotrope cells of the South-African claw-toed frog Xenopus laevis as a model system. These cells are involved in background adaptation of the animal and produce high levels of its major secretory cargo proopiomelanocortin (POMC) when the animal is black-adapted. The technique of stable Xenopus transgenesis in combination with the POMC gene promoter was used as a tool to express Xenopus PrPC amino-terminally tagged with the green fluorescent protein (GFP-PrPC) specifically in the melanotrope cells. The GFP-PrPC fusion protein was expressed from stage-25 tadpoles onwards to juvenile frogs, the expression was induced on a black background and the fusion protein was subcellularly located mainly in the Golgi apparatus and at the plasma membrane. Pulse-chase metabolic cell labelling studies revealed that GFP-PrPC was initially synthesized as a 45-kDa protein that was subsequently stepwise glycosylated to 48-, 51-, and eventually 55-kDa forms. Furthermore, we revealed that the mature 55-kDa GFP-PrPC protein was sulfated, anchored to the plasma membrane and cleaved to a 33-kDa product. Despite the high levels of transgene expression, the subcellular structures as well as POMC synthesis and processing, and the secretion of POMC-derived products remained unaffected in the transgenic melanotrope cells. Hence, we studied PrPC in a neuroendocrine cell and in a well-defined physiological context.

Animals↗

PubMed1989

Intracellular targeting and structural conservation of a prohormone-processing endoprotease.

The prohormone-processing endoprotease (KEX2 gene product) of the yeast Saccharomyces cerevisiae is a membrane-bound, 135,000-dalton glycoprotein, which contains both asparagine-linked and serine- and threonine-linked oligosaccharide and resides in a secretory compartment. Analysis of mutant kex2 genes truncated at their 3' end indicates that carboxyl terminal domains of the enzyme are required for its proper localization within the cell. A human gene product, "furin," shares 50% identity with the catalytic domain of Kex2 protease and is, therefore, a candidate for a human prohormone-processing enzyme.

Amino Acid Sequence↗

PubMed1997

Modulation of Ras and a-factor function by carboxyl-terminal proteolysis.

Prenylated proteins contain a covalently linked cholesterol intermediate near their carboxyl-termini. Maturation of most prenylated proteins involves proteolytic removal of the last three amino acids. Two genes in Saccharomyces cerevisiae, RCE1 and AFC1, were identified that appear to be responsible for this processing. The Afc1 protein is a zinc protease that participates in the processing of yeast a-factor mating pheromone. The Rce1 protein contributes to the processing of both Ras protein and a-factor. Deletion of both AFC1 and RCE1 resulted in the loss of proteolytic processing of prenylated proteins. Disruption of RCE1 led to defects in Ras localization and signaling and suppressed the activated phenotype associated with the allele RAS2val19.

Cell Membrane↗

PubMed1991

The secretion leader of Mucor pusillus rennin which possesses an artificial Lys-Arg sequence directs the secretion of mature human growth hormone by Saccharomyces cerevisiae.

The prepro-peptide of fungal aspartic proteinase, Mucor pusillus rennin, is useful as a secretion leader for efficient secretion of human growth hormone (HGH) from Saccharomyces cerevisiae. For secretion by yeast cells of HGH with the same NH2 terminus as native HGH, an artificial Lys-Arg linker, which is one of the potential KEX2 recognition sequences, was introduced at the junction between the M. pusillus rennin secretion leader and mature HGH. The HGH directed by this construction was the same size as native HGH, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and amino acid sequencing of its NH2 terminus revealed that the secretion leader peptide was removed correctly at the COOH-terminal side of the Lys-Arg linker. On the other hand, when the same plasmid was expressed in a kex2 mutant strain, unprocessed HGH of a higher molecular weight was secreted, indicating that no proteolytic cleavage at the Lys-Arg site occurred. These results clearly showed that the leader peptide with the Lys-Arg linker was recognized and specifically cleaved by the yeast KEX2 protease. The mature HGH purified from yeast culture medium was indistinguishable from native HGH in biological activity, determined by the adipocyte conversion assay, and in secondary structure, determined by circular dichroism spectroscopy.

Amino Acid Sequence↗

PubMed1999

Thaumatin production in Aspergillus awamori by use of expression cassettes with strong fungal promoters and high gene dosage.

Four expression cassettes containing strong fungal promoters, a signal sequence for protein translocation, a KEX protease cleavage site, and a synthetic gene (tha) encoding the sweet protein thaumatin II were used to overexpress this protein in Aspergillus awamori lpr66, a PepA protease-deficient strain. The best expression results were obtained with the gdhA promoter of A. awamori or with the gpdA promoter of Aspergillus nidulans. There was good correlation of tha gene dosage, transcript levels, and thaumatin secretion. The thaumatin gene was expressed as a transcript of the expected size in each construction (1.9 or 1.4 kb), and the transcript levels and thaumatin production rate decayed at the end of the growth phase, except in the double transformant TB2b1-44-GD5, in which secretion of thaumatin continued until 96 h. The recombinant thaumatin secreted by a high-production transformant was purified to homogeneity, giving one major component and two minor components. In all cases, cleavage of the fused protein occurred at the KEX recognition sequence. This work provides new expression systems in A. awamori that result in very high levels of thaumatin production.

Actins↗

PubMed2000

Characterization of the kexin-like maturase of Aspergillus niger.

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.

Aspergillus niger↗

PubMed2006

Saccharomyces cerevisiae a-factor mutants reveal residues critical for processing, activity, and export.

The Saccharomyces cerevisiae mating pheromone a-factor provides a paradigm for understanding the biogenesis of prenylated fungal pheromones. The biogenesis of a-factor involves multiple steps: (i) C-terminal CAAX modification (where C is cysteine, A is aliphatic, and X is any residue) which includes prenylation, proteolysis, and carboxymethylation (by Ram1p/Ram2p, Ste24p or Rce1p, and Ste14p, respectively); (ii) N-terminal processing, involving two sequential proteolytic cleavages (by Ste24p and Axl1p); and (iii) nonclassical export (by Ste6p). Once exported, mature a-factor interacts with the Ste3p receptor on MATalpha cells to stimulate mating. The a-factor biogenesis machinery is well defined, as is the CAAX motif that directs C-terminal modification; however, very little is known about the sequence determinants within a-factor required for N-terminal processing, activity, and export. Here we generated a large collection of a-factor mutants and identified residues critical for the N-terminal processing steps mediated by Ste24p and Axl1p. We also identified mutants that fail to support mating but do not affect biogenesis or export, suggesting a defective interaction with the Ste3p receptor. Mutants significantly impaired in export were also found, providing evidence that the Ste6p transporter recognizes sequence determinants as well as CAAX modifications. We also performed a phenotypic analysis of the entire set of isogenic a-factor biogenesis machinery mutants, which revealed information about the dependency of biogenesis steps upon one another, and demonstrated that export by Ste6p requires the completion of all processing events. Overall, this comprehensive analysis will provide a useful framework for the study of other fungal pheromones, as well as prenylated metazoan proteins involved in development and aging.

ATP-Binding Cassette Transporters↗

PubMed2003

A single-copy gene encodes Kex1, a serine endoprotease of Pneumocystis jiroveci.

We have cloned and characterized the kex1 gene of Pneumocystis jiroveci. Unlike the case for Pneumocystis carinii, in which the homologous PRT-1 genes are multicopy, kex1 is a single-copy gene encoding a protein homologous to fungal serine endoproteases, which localize to the Golgi apparatus. Thus, substantial biological differences can be seen among Pneumocystis species.

Amino Acid Sequence↗

PubMed1999

Identification of two mannoproteins released from cell walls of a Saccharomyces cerevisiae mnn1 mnn9 double mutant by reducing agents.

In this report, we present the identification of the main polypeptides that are extracted from purified cell walls of a Saccharomyces cerevisiae mnn1 mnn9 strain by reducing agents. Treatment of the purified cell walls of this strain with beta-mercaptoethanol releases several mannoproteins, of which three, with apparent sizes of 120, 45, and 40 kDa, are the most abundant. Analysis of the amino-terminal sequences revealed that the 120-kDa mannoprotein is Bar1p, the protease involved in the so-called barrier activity in yeast cells, and that the 45- and 40-kDa mannoproteins are the Kex2-unprocessed and Kex2-processed forms of the gene product of open reading frame (ORF) YJL158c, an ORF that belongs to the PIR (protein with internal repeats) family of genes, composed thus far of PIR1, PIR2/HSP150, and PIR3. Accordingly we have named this gene PIR4, and Pir4 denotes the 40-kDa Kex2-processed form of the mannoprotein. We have characterized Pir4 and have shown the feasibility of using it as a fusion partner for the targeting of recombinant proteins to the cell wall.

Amino Acid Sequence↗

PubMed2001

HreP, an in vivo-expressed protease of Yersinia enterocolitica, is a new member of the family of subtilisin/kexin-like proteases.

The role of proteases in pathogenesis is well established for several microorganisms but has not been described for Yersinia enterocolitica. Previously, we identified a gene, hreP, which showed significant similarity to proteases in a screen for chromosomal genes of Y. enterocolitica that were exclusively expressed during an infection of mice. We cloned this gene by chromosome capture and subsequently determined its nucleotide sequence. Like inv, the gene encoding the invasin protein of Y. enterocolitica, hreP is located in a cluster of flagellum biosynthesis and chemotaxis genes. The genomic organization of this chromosomal region is different in Escherichia coli, Salmonella, and Yersinia pestis than in Y. enterocolitica. Analysis of the distribution of hreP between different Yersinia isolates and the relatively low G+C content of the gene suggests acquisition by horizontal gene transfer. Sequence analysis also revealed that HreP belongs to a family of eukaryotic subtilisin/kexin-like proteases. Together with the calcium-dependent protease PrcA of Anabaena variabilis, HreP forms a new subfamily of bacterial subtilisin/kexin-like proteases which might have originated from a common eukaryotic ancestor. Like other proteases of this family, HreP is expressed with an N-terminal prosequence. Expression of an HreP-His(6) tag fusion protein in E. coli revealed that HreP undergoes autocatalytic processing at a consensus cleavage site of subtilisin/kexin-like proteases, thereby releasing the proprotein.

Amino Acid Sequence↗

PubMed1992

Mammalian subtilisin-related proteinases in cleavage activation of the paramyxovirus fusion glycoprotein: superiority of furin/PACE to PC2 or PC1/PC3.

The fusion glycoprotein precursor of Newcastle disease virus is ubiquitously cleaved in the constitutive secretory pathway if it possesses an oligobasic cleavage motif (RRQR/KR), whereas the precursor is refractory to cleavage if the motif is monobasic (GR/KQGR). We examined the cleavage activity of the mammalian subtilisin-related proteinases furin/PACE, PC2, and PC1/PC3, which are thought to be responsible for proprotein processing in either the constitutive (furin/PACE) or the regulated (PC2 and PC1/PC3) secretory pathway, for the viral precursors with different cleavage motifs. Only furin/PACE was fully capable of cleaving the precursors with the oligobasic motif. PC2 and PC1/PC3 were incapable or only partially capable of cleaving at this motif. None of the proteinases cleaved the monobasic motif. These results suggest involvement of furin/PACE in viral protein processing in the constitutive secretory pathway.

Amino Acid Sequence↗

PubMed1994

Proteolytic cleavage of wild type and mutants of the F protein of human parainfluenza virus type 3 by two subtilisin-like endoproteases, furin and Kex2.

The fusion (F) protein of human parainfluenza virus type 3 contains the tribasic cleavage site R-T-K-R, which was altered by site-directed mutagenesis. Wild-type F protein and various mutants were expressed by recombinant vaccinia viruses. The endogenous endoprotease present in CV-1 cells cleaves F variants containing the furin recognition motif R-X-K/R-R but not variants containing the dibasic site K-R or a single R at the cleavage site. A similar cleavage pattern was obtained when the subtilisin-like endoproteases Kex2 and furin were coexpressed with the wild type and mutants of the F protein. Peptidylchloromethylketone inhibitors mimicking basic cleavage sites prevent cleavage of the precursor Fo by the endogenous protease only when the furin-specific motif is present in the peptidyl portion. The data support the concept that furin is a cellular protease responsible for the activation of the F protein of human parainfluenza virus type 3.

Amino Acid Sequence↗

PubMed1994

Proprotein-processing endoproteases PC6 and furin both activate hemagglutinin of virulent avian influenza viruses.

Among the proprotein-processing subtilisin-related endoproteases, furin has been a leading candidate for the enzyme that activates the hemagglutinin (HA) of virulent avian influenza viruses. In the present study, we examined the cleavage activity of two other recently isolated ubiquitous subtilisin-related proteases, PACE4 and PC6, using wild-type HA of A/turkey/Ireland/1378/83 (H5N8) and a series of its mutant HAs. Vaccinia virus-expressed wild-type HA was not cleaved in human colon adenocarcinoma LoVo cells, which lack active furin. This processing defect was corrected by the expression of furin and PC6 but not of PACE4 and a control wild-type vaccinia virus. PC6 showed a sequence specificity similar to that with the endogenous proteases in cultured cells. When LoVo cells were infected with a virulent avian virus, A/turkey/Ontario/7732/66 (H5N9), only noninfectious virions were produced because of the lack of HA cleavage. However, when the cells were coinfected with vaccinia virus that expressed either furin or PC6, the avian virus underwent multiple cycles of replication, indicating that both furin and PC6 specifically cleave the virulent virus HA at the authentic site. These data suggest that PC6, as well as furin, can activate virulent avian influenza viruses in vivo, implying the presence of multiple HA cleavage enzymes in animals.

Amino Acid Sequence↗

PubMed2003

Crimean-Congo hemorrhagic fever virus glycoprotein proteolytic processing by subtilase SKI-1.

Crimean-Congo hemorrhagic fever (CCHF) virus is a tick-borne member of the genus Nairovirus, family Bunyaviridae. The mature virus glycoproteins, Gn and Gc (previously referred to as G2 and G1), are generated by proteolytic cleavage from precursor proteins. The amino termini of Gn and Gc are immediately preceded by tetrapeptides RRLL and RKPL, respectively, leading to the hypothesis that SKI-1 or related proteases may be involved (A. J. Sanchez, M. J. Vincent, and S. T. Nichol, J. Virol. 76:7263-7275, 2002). In vitro peptide cleavage data show that an RRLL peptide representing the Gn processing site is efficiently cleaved by SKI-1 protease, whereas an RKPL peptide representing the Gc processing site is cleaved at negligible levels. The efficient cleavage of RRLL peptide is consistent with the known recognition sequences of SKI-1, including the sequence determinants involved in the cleavage of the Lassa virus (family Arenaviridae) glycoprotein precursor. These in vitro findings were confirmed by expression of wild-type or mutant CCHF virus glycoproteins in CHO cells engineered to express functional or nonfunctional SKI-1. Gn processing was found to be dependent on functional SKI-1, whereas Gc processing was not. Gn processing occurred in the endoplasmic reticulum-cis Golgi compartments and was dependent on an R at the -4 position within the RRLL recognition motif, consistent with the known cleavage properties of SKI-1. Comparison of SKI-1 cleavage efficiency between peptides representing Lassa virus GP2 and CCHF virus Gn cleavage sites suggests that amino acids flanking the RRLL may modulate the efficiency. The apparent lack of SKI-1 cleavage at the CCHF virus Gc RKPL site indicates that related proteases, other than SKI-1, are likely to be involved in the processing at this site and identical or similar sites utilized in several New World arenaviruses.

Amino Acid Sequence↗

PubMed2003

Endoproteolytic processing of the lymphocytic choriomeningitis virus glycoprotein by the subtilase SKI-1/S1P.

The envelope glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) is posttranslationally cleaved into two subunits. We show here that this endoproteolytic processing is not required for transport to the cell surface but is essential for LCMV GP to mediate infectivity of pseudotyped retroviral vectors. By systematic mutational analysis of the LCMV GP cleavage site, we determined that the consensus motif R-(R/K/H)-L-(A/L/S/T/F)(265) is essential for the endoproteolytic processing. In agreement with the identified consensus motif, we show that the cellular subtilase SKI-1/S1P cleaves LCMV GP.

Animals↗

PubMed2006

Role of disulfide bridges formed in the luminal domain of ATF6 in sensing endoplasmic reticulum stress.

ATF6 is a membrane-bound transcription factor activated by proteolysis in response to endoplasmic reticulum (ER) stress to induce the transcription of ER chaperone genes. We show here that, owing to the presence of intra- and intermolecular disulfide bridges formed between the two conserved cysteine residues in the luminal domain, ATF6 occurs in unstressed ER in monomer, dimer, and oligomer forms. Disulfide-bonded ATF6 is reduced upon treatment of cells with not only the reducing reagent dithiothreitol but also the glycosylation inhibitor tunicamycin, and the extent of reduction correlates with that of activation. Although reduction is not sufficient for activation, fractionation studies show that only reduced monomer ATF6 reaches the Golgi apparatus, where it is cleaved by the sequential action of the two proteases S1P and S2P. Reduced monomer ATF6 is found to be a better substrate than disulfide-bonded forms for S1P. ER stress-induced reduction is specific to ATF6 as the oligomeric status of a second ER membrane-bound transcription factor, LZIP/Luman, is not changed upon tunicamycin treatment and LZIP/Luman is well cleaved by S1P in the absence of ER stress. This mechanism ensures the strictness of regulation, in that the cell can only process ATF6 which has experienced the changes in the ER.

Activating Transcription Factor 6↗

PubMed1994

RNA polymerase II cofactor PC2 facilitates activation of transcription by GAL4-AH in vitro.

We have isolated from a crude Hela cell cofactor fraction (USA) a novel positive cofactor that cooperates with the general transcription machinery to effect efficient stimulation of transcription by GAL4-AH, a derivative of the Saccharomyces cerevisiae regulatory factor GAL4. PC2 was shown to be a 500-kDa protein complex and to be functionally and biochemically distinct from native TFIID and previously identified cofactors. In the presence of native TFIID and other general factors, PC2 was necessary and sufficient for activation by GAL4-AH. Cofactor function was specific for transcriptional activation domains of GAL4-AH. The repressor histone H1 further potentiated but was not required for activation of transcription by GAL4-AH. On the basis of the observation that PC2 exerts entirely positive effects on transcription, we propose a model in which PC2 increases the activity of the preinitiation complex in the presence of an activator, thereby establishing a specific pathway during activation of RNA polymerase II.

Blotting, Western↗