NR ACXJ
AU Coustou,V.; Deleu,C.; Saupe,S.; Begueret,J.
TI The protein product of the het-s heterokaryon incompatibility gene of the fungus Podospora anserina behaves as a prion analog
QU Proceedings of the National Academy of Sciences of the United States of America 1997 Sep 2; 94(18): 9773-8
PT journal article
AB The het-s locus of Podospora anserina is a heterokaryon incompatibility locus. The coexpression of the antagonistic het-s and het-S alleles triggers a lethal reaction that prevents the formation of viable heterokaryons. Strains that contain the het-s allele can display two different phenotypes, [Het-s] or [Het-s*], according to their reactivity in incompatibility. The detection in these phenotypically distinct strains of a protein expressed from the het-s gene indicates that the difference in reactivity depends on a posttranslational difference between two forms of the polypeptide encoded by the het-s gene. This posttranslational modification does not affect the electrophoretic mobility of the protein in SDS/PAGE. Several results suggest a similarity of behavior between the protein encoded by the het-s gene and prions. The [Het-s] character can propagate in [Het-s*] strains as an infectious agent, producing a [Het-s*] -> [Het-s] transition, independently of protein synthesis. Expression of the [Het-s] character requires a functional het-s gene. The protein present in [Het-s] strains is more resistant to proteinase K than that present in [Het-s*] mycelium. Furthermore, overexpression of the het-s gene increases the frequency of the transition from [Het-s*] to [Het-s]. We propose that this transition is the consequence of a self-propagating conformational modification of the protein mediated by the formation of complexes between the two different forms of the polypeptide.
VT
Communicated by David D. Perkins, Stanford University, Stanford, CA, June 16, 1997 (received for review January 24, 1997)
------------------------------------------------------------------------
INTRODUCTION
The het-s locus of the filamentous fungus Podospora anserina is one of the nine known loci controlling heterokaryon incompatibility in that species (for review, see ref. 1). Coexpression of the antagonistic het-s and het-S alleles in the same cytoplasm triggers an adverse reaction that prevents the formation of viable heterokaryotic cells between strains that contain the incompatible alleles (2). This locus encodes a 289-aa protein that is not essential for cell viability or completion of the life cycle of the fungus (3, 4). The proteins encoded by the incompatible alleles differ by 14 amino acid substitutions, but one difference is sufficient for expression of the antagonistic [Het-s] and [Het-S] specificities (5).
It has been reported by Rizet (ref. 2; for review, see ref. 6) that haploid strains of the het-s genotype can exhibit two different phenotypes: they either are incompatible with het-S strains (this phenotype will herein be designated [Het-s]), or they are neutral in incompatibility and display the [Het-s*] phenotype. This latter phenotype is stably maintained during vegetative growth but a transition from [Het-s*] to [Het-s] can occur spontaneously at a very low frequency, estimated under 10[-]7 per nucleus
(7). This phenotypic conversion is, however, invariably induced after anastomosis and cytoplasmic mixing with a [Het-s] strain. This transition cannot be induced by strains that contain het-S or the null alleles het-sx and het-s° The [Het-s] character is dominant and can propagate in [Het-s*] strains in the absence of nuclear transmission. When it has been induced, the [Het-s*] -> [Het-s] transition spreads very rapidly as an infectious process from the region of anastomosis throughout the mycelium. Both the [Het-s] and [Het-s*] characters are transmitted as nonmendelian elements through meiosis. The phenotype of the offsprings of the het-s genotype depends on the phenotype of the female parent, suggesting that these phenotypes are controlled by cytoplasmic elements. It has been proposed from these different results that the expression of the het-s gene might be positively controlled by its protein product (7).
We report here that a polypeptide encoded by het-s is present in similar amounts in [Het-s*] and [Het-s] strains. So the absence of reactivity of [Het-s*] strains in incompatibility does not result from the lack of expression of the het-s-encoded protein but to a posttranslational modification of the protein. We also show that several properties of the protein encoded by the het-s gene suggest that it may have a behavior similar to that of a prion protein: (i) overexpression of the protein increases the rate of the spontaneous [Het-s*] -> [Het-s] transition; (ii) the protein present in [Het-s] and [Het-s*] strains displays different sensitivity to a protease; (iii) a physical interaction between monomers can be detected using the two-hybrid yeast system. Similar properties have already been described for yeast prions [URE3] and [PSI] (8-12). We propose that the protein encoded by the het-s gene can exist under two different conformations, pHET-s and pHET-s*, and that the stable form present in the [Het-s] strain is able to propagate by transmitting its conformation to the form present in [Het-s*] strains.
------------------------------------------------------------------------
MATERIALS AND METHODS
P. anserina Strains.
P. anserina is a filamentous ascomycete. The mycelium has a coenocytic structure. It is formed by multinucleate cells isolated from each other by incomplete cell walls. Life cycle and methods for genetic analysis have been described (13). Compatibility between strains can be determined by confrontation on cornmeal agar medium. Incompatibility results in the formation of a barrage, a dense and unpigmented line in the region where the strains meet (2). Three different alleles of the het-s locus have been found in wild-type isolates: het-s and het-S are reactive, and incompatible alleles, het-sx is neutral in incompatibility; het-sx is compatible with both het-s and het-S. The DNA sequence of these wild-type alleles has been reported (3, 5). The het-s°allele was derived from het-s, the promoter and the 5['] end of the coding sequence having been deleted by gene replacement (4). Strains that display the [Het-s*] phenotype were obtained in the offspring of crosses between het-s and het-S strains. These strains are compatible with both parents. All strains used in this study are isogenic except for the allele present at the het-s and mating type loci.
Transformation of P. anserina.
Protoplasts were prepared and transformed as described (14). The pMOcosX, containing the bacterial hph gene coding for hygromycin resistance, was used in cotransformations (15). Transformants were screened for hygromycin B resistance at 100 µg/ml.
DNA Analysis.
General methods for nucleic acid analysis and vector construction were as described (16). PCR amplification of DNA (17) was achieved in a 50-ml reaction mixture: 10 mM Tris HCl, pH 8.4/5 mM KCl/1.5 mM MgCl2/0.2 mM of each dNTP/100 ng of each primer/2 ng of plasmids pSKPS, which contains the het-s allele, or pSKGS, which contains the het-S allele. After 10 min at 95°, 1 unit of Taq polymerase (AmpliTaq, Cetus) was added, and DNA was amplified for 35 cycles in a Perkin-Elmer/Cetus Thermocycler. The cycling parameters were: denaturation at 95° for 30 sec, annealing at 58° for 2 min, and extension at 72° for 2 min.
Site-specific mutagenesis was carried out using the Transformer Site-Directed Mutagenesis Kit (CLONTECH) according to the manufacturer's protocols. Two primers were used in the mutagenesis. For het-s modification the oligonucleotide (5[']-ACGGTTCTGCCATGGCAGTTTG-3[']) produced a TCA -> GCA modification, which creates a NcoI restriction site overlapping the initiator ATG codon. The second primer produced a mutation in the amp gene, which restores the resistance to ampicillin. This modification was used for the selection of mutant plasmids.
Protein Extraction and Analysis.
For preparation of cell-free extracts, the mycelia were grown at 26° in 100 ml of liquid medium in 1,000-ml Roux bottles. Mycelia from 2-day-old cultures were harvested, rinsed twice with cold distilled water, and frozen at [-]80° for 1 hr. The mycelium then was lyophilized and ground. The powder was resuspended in the extraction buffer (50 mM NaH2PO4, pH 8/10 mM Tris-HCl, pH 8/100 mM NaCl/0.25% Triton/4.3 mM phenylmethylsulfonyl fluoride/1 mM L-1-tosyl amide-2-phenylethyl chloromethyl ketone/1 mM N [alpha]-p-tosyl-L-lysine chloromethyl ketone/5 µg/ml leupeptin/5 µg/ml pepstatin A). The mixture was sonicated for 2 min at 4° by applying 120-watt short pulses of 20 sec each. The homogenate then was incubated at 4° for 1 hr with gentle agitation. The homogenate was centrifuged for 15 min at 10,000 x g. The supernatant constituted the crude extract. Conditions for SDS/PAGE and immunoblotting were as described (5). Sample preparation for proteinase K digestion was as described above except that the extraction buffer was devoid of protease inhibitors. Ten milliliters of crude extract was incubated with 50 µg/ml of proteinase K at 37° for 25 min. The digestion was stopped by addition of final concentrations of 50 mM EDTA and 4.3 mM phenylmethylsulfonyl fluoride after 0- to 25-min incubation. Proteins were precipitated by adding ammonium sulfate to 662 mg/ml to the samples. Proteins were collected by centrifugation for 15 min at 10,000 x g, and the pellet was resuspended and dialyzed overnight in 0.1 M phosphate buffer at pH 8.
Yeast Strains and Methods.
Vectors for construction of GAL4 fusions were pDBT and pTAL (P. Navarro, personal communication) constructed by exchanging the ApaI-BamHI fragment between pPC62 and pPC86 (18). A PCR amplification on pSKPS or pSKGS plasmids with DH1 oligonucleotide (5[']-ACTGCCCCGGGGAACCGTTC-3[']), which creates the SmaI restriction site overlapping the initiator ATG codon, and DH2 oligonucleotide (5[']-ACATTCTAGCGGCCGCCCGTTAAT-3[']), which creates the NotI restriction site downstream to the termination codon, was performed as described above. The 950-bp PCR fragments were ligated in pDBT and pTAL after digestion of fragments and vectors by SmaI and NotI. The fusion vectors carrying the GAL4 DNA-binding domain in-frame with the ORFs of het-s and het-S were designated pDBT-s and pDBT-S, respectively. The fusion vectors carrying the GAL4 transcription activator domain in-frame with the het-s and the het-S ORFs were named pTAL-s and pTAL-S, respectively. These fusion vectors were verified by restriction mapping and sequencing across recombinant joints and PCR products. Yeast strain Y190 (MATa gal4 gal80 his3 trp1-901 ade2-101 ura3-52 leu2-3,-112+URA3::GAL -> lacZ, LYS2::GAL -> HIS3 cyhr) was used in the two-hybrid assay. Yeast strains were grown in yeast extract/peptone/dextrose liquid medium for the transformation step and on supplemented synthetic dextrose medium lacking tryptophan and leucine and supplemented or not with histidine. These media were noted SD-leu-trp and SD-leu-trp+his, respectively. A combination of two fusion vectors was used for each transformation. Transformants were first tested for histidine prototrophy by plating cells on SD-leu-trp containing 0-100 mM 3-amino-1,2,4-triazole. The presence of 3-amino-1,2,4-triazole limits the growth of the recipient strain on minimal medium due to leakage of the HIS3 promoter, so that the interaction between proteins could be estimated by the level of resistance to 3-amino-1,2,4-triazole. The transformants also were assayed for ß-galactosidase activity as described (19). Transformation with pPC76 and pPC79 (18), which contain, respectively, the GAL4-DB in frame with Fos oncoprotein and the GAL4-TA in frame with Jun oncoprotein, were used as positive controls. Combinations of each fusion vector with pDBT or pTAL were used as negative controls.
------------------------------------------------------------------------
RESULTS
Analysis of the Protein Encoded by the het-s Gene in [Het-s] and [Het-s*] Strains.
[Het-s*] strains contain the het-s gene but display a neutral incompatibility phenotype (2). This absence of reactivity against antagonistic het-S strains could result from the lack of expression of the het-s gene. Alternatively, the protein encoded by the het-s gene might be expressed as an inactive form in [Het-s*] strains. The expression of the het-s gene has been examined by Northern and Western blotting in [Het-s] and [Het-s]* strains. The results show that the gene is expressed at the same level in both strains. No significant differences were observed either in the amount or apparent size of the RNA (not shown) or of the protein. A protein with the same electrophoretic mobility was detected in extracts from [Het-s] and [Het-s*] strains (Fig. 1A). Therefore, the difference of reactivity of the two strains in incompatibility against a het-S strain is not due to a differential expression of the het-s gene but to a posttranslational difference. Furthermore, as the two polypeptides display the same mobility in SDS/PAGE, they differ very little or not at all in molecular mass. It can be estimated that this difference in molecular mass, if it exists, cannot be greater than 500 to 1,000 Da. The difference in reactivity cannot be attributed to a maturation process such as an intensive glycosylation or the cleavage of a long peptide. The two polypeptides will be tentatively designated pHET-s and pHET-s*. The presence of the protein in [Het-s*] strains suggests that the phenotypic transition [Het-s*] -> [Het-s] is related to a modification of pHET-s* that can be converted to the pHET-s form.
-----------------------------------------------------------------
Fig. 1. Immunoblot analysis of proteins isolated from [Het-s] and [Het-s*] strains (A) or from strains that contain the het-s ORF under the control of its promoter or under the control of the promoter of the glyceraldehyde 3-phosphate dehydrogenase (G3PD) gene of A. nidulans (B). Markers used for gel calibration were muscle rabbit phosphorylase B (97.4 kDa), BSA (66 kDa), egg albumin (45 kDa), and carbonic anhydrase from bovine erythrocytes (29 kDa). Arrowheads indicate position of protein encoded by the het-s gene.
------------------------------------------------------------------------
Analysis of the Propagation of the [Het-s] Character.
Some aspects of the transmission of the [Het-s] character to [Het-s*] strains have been analyzed using the experimental procedure described by Beisson-Schecroun (7) and shown in Fig. 2. Recipient and donor strains were grown on cellophane pads laid on corn meal agar. Formation of anastomosis between the mycelia was followed under a binocular microscope. Immediately after the fusion of filaments, the cellophane pads were transferred to fresh medium supplemented or not with cycloheximide at a concentration of 25 µg/ml, which is known to inhibit more than 90% of growth of the mycelium (20). Propagation of the [Het-s] character in the recipient strains was measured 15 hr later by sampling small pieces of mycelium on the recipient strain at 3 cm behind the line of contact between the strains. The phenotype of the mycelia regenerated from these samples was determined in a barrage test against a het-S strain. No significant difference was observed in the rate of propagation of the [Het-s*] -> [Het-s] transition whether the medium contained cycloheximide or not (Table 1). As previously described (7), we have verified that the transition is not due to the migration of nuclei from the donor to the recipient. This possibility was eliminated using donor and recipient strains that differ in their mating type. In no case had samples of mycelium taken from the recipient become self-fertile, thus showing that no nuclei had migrated from the donor into the recipient mycelium. We never observed the propagation of the [Het-s] character if the recipient strain contained the null allele het-s° Furthermore, we observed that het-s°strains that have been previously fused to a [Het-s] donor strain cannot induce the [Het-s*] -> [Het-s] transition when they are used as donor strains in confrontation with a [Het-s*] recipient (Table 1). These results suggest that the molecular determinant responsible for the [Het-s] character and for the [Het-s*] -> [Het-s] transition cannot be maintained in the het-s°genetic background and that the presence of the het-s gene is necessary for the propagation of the [Het-s] character. As the propagation of the [Het-s] character in the [Het-s*] strain is independent of protein synthesis, it can be interpreted as the consequence of the conversion of the pre-existing inactive pHET-s* form of the protein into the reactive pHET-s form.
-----------------------------------------------------------------
Fig. 2. Schematic drawing of the experimental device used to analyze the [Het-s*] -> [Het-s] transition. The striped square shows the position where a piece of mycelium was picked up 15 hr after the fusion of donor and recipient mycelia to determine the phenotype.
------------------------------------------------------------------------
Table 1. Analysis of the [Het-s*] -> [Het-s] transition
------------------------------------------------------------------------
Number of strains that have gained the [Het-s] phenotype
----------------------------------------------------------
Donor Donor Donor
strain = [Het-s] strain = [Het-s] strain = het-s
ZR 26 Zitate
IN
Die zwei Stämme des Schlauchpilzes Podospora anserina Het-s* und Het-s unterscheiden sich nur dadurch, dass Het-s kein lebensfähiges Heterokaryon mit dem Podospora anserina Stamm Het-S bilden kann, während Het-s* mit Het-S verschmelzen kann. Interessanterweise kann der Stamm Het-s* gelegentlich spontan in den Stamm Het-s konvertieren und eine 10-fache Überexpression erhöht die Häufigkeit dieser spontanen Umwandlung 20-fach. Außerdem kann HET-s den Stamm HET-s* nach Ausbildung von Plasmabrücken ohne Austausch von Zellkernen so infizieren, dass dieser in die Variante HET-s übergeht und sich diese Form im ganzen Mycel ausbreitet. Den Stamm HET-s°ohne das Protein pHET-s kann der Stamm HET-s dagegen nicht umwandeln.
Die beiden Proteine pHET-s und pHET-s* werden aber gleich stark exprimiert und sind gelelektrophoretisch nicht zu unterscheiden. Allerdings ist pHET-s resistenter gegenüber Protease K als pHET-s*. Während pHET-s* bereits nach 2 Minuten vollständig abgebaut war, überstand ein Teil des pHET-s selbst eine 10-minütige Protease K - Behandlung. Es wurde aber bei pHET-s und pHET-s* etwa gleich große Neigungen zur Aggregation beobachtet.
MH Ascomycota/*genetics; Fungal Proteins/*genetics/metabolism; *Genes, Fungal; Prions/*genetics; Protein Processing, Post-Translational/genetics; Support, Non-U.S. Gov't
AD Virginie Coustou (To whom reprint requests should be addressed.), Carol Deleu (Present address: Laboratoire de Biologie Végétale, Université de Rennes I, 35042 Rennes cedex, France), Sven Saupe (Present address: Centre National de la Recherche Scientifique Equipe en Restructuration 155, Institut de Biologie, 4 bd Henri IV, 34060 Montpellier cedex, France), and Joel Begueret - Laboratoire de Génétique Moléculaire des Champignons Filamenteux, Institut de Biochimie et Génétique Cellulaires, Centre National de la Recherche Scientifique Unité Propre de Recherche 9026, 1, rue Camille Saint Saens, 33077 Bordeaux cedex, France
SP englisch
PO USA
OR Prion-Krankheiten C