A non-chromosomal factor allows viability of Schizosaccharomyces pombe lacking the essential chaperone calnexin

Philippe Collin*, Pascale B. Beauregard, Aram Elagöz{ddagger} and Luis A. Rokeach§

Department of Biochemistry, Université de Montréal, Montréal, Québec H3C 3J7, Canada

§ Author for correspondence (e-mail: luis.rokeach{at}umontreal.ca)

Accepted 16 October 2003


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Calnexin is a molecular chaperone playing key roles in protein folding and the quality control of this process in the endoplasmic reticulum. We, and others, have previously demonstrated that cnx1+, the gene encoding the calnexin homologue in Schizosaccharomyces pombe, is essential for viability. We show that a particular cnx1 mutant induces a novel mechanism allowing the survival of S. pombe cells in the absence of calnexin/Cnx1p. Calnexin independence is dominant in diploid cells and is inherited in a non-Mendelian manner. Remarkably, this survival pathway, bypassing the necessity for calnexin, can be transmitted by transformation of cell extracts into a wild-type naive strain, thus implicating a non-chromosomal factor. Nuclease and UV treatments of cells extracts did not obliterate transmission of calnexin independence by transformation. However, protease digestion of extracts did reduce the appearance of calnexin-independent cells, indicating that a protein element is required for calnexin-less viability. We discuss a model in which this calnexin-less survival mechanism would be activated and perpetuated by a protein component acting as a genetic element.

Key words: Non-Mendelian Genetics, Yeast genetics, Protein folding, Fission Yeast


    Introduction
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Proper folding of proteins and the quality control of this process are basic and essential cellular processes. While a number of proteins can acquire their final structures in vitro, a battery of molecular chaperones and foldases assist the in vivo folding of proteins, bringing both the efficiency and the yield of this cellular process to levels compatible with life (reviewed by Fink and Goto, 1998Go; Leitzgen and Haas, 1998Go; Ellis and Hartl, 1999Go; Bukau et al., 2000Go; Fewell et al., 2001Go; Frydman, 2001Go). Inherited alterations in protein folding and/or assembly in the endoplasmic reticulum (ER) constitute the molecular basis for several genetic diseases such as cystic fibrosis, juvenile emphysema, familial hypercholesterolemia, and certain coagulation disorders (Kuznetsov and Nigam, 1998Go). Incorrect folding can also be infectious, as is the case of prion-mediated diseases such as sheep scrapie, bovine spongiform encephalopathy (BSE; mad cow disease), and Creutzfeldt-Jakob Disease (CJD) in humans (reviewed by Prusiner, 1998Go). Indeed, in these diseases, prion propagation is the result of the structural conversion of a native cellular protein that is mainly {alpha}-helical into its prionic conformer, which is rich in ß structure (reviewed by Prusiner, 1998Go). This structural conversion is mediated by the infecting or already existing prionic conformer; therefore prions impose their structure on their native conformers. In fungi, the mechanism of propagation of prions by structural replication constitutes the basis of protein-only inheritance genetics, without involving changes in the nucleic-acid encoded information (reviewed by Serio and Lindquist, 1999Go; Wickner et al., 1999aGo; Wickner et al., 1999bGo; Serio and Lindquist, 2000Go; Uptain and Lindquist, 2002Go; Ter-Avanesyan and Kushnirov, 1999Go; Chernoff et al., 2002Go).

Calnexin is a molecular chaperone of the endoplasmic reticulum (ER) playing key roles in the folding and quality control of numerous secreted and membrane-bound proteins (reviewed by Williams, 1995Go; Parodi, 2000Go; Helenius and Aebi, 2001Go; Fewell et al., 2001Go; Yen et al., 2003Go). To explore the functions exerted by calnexin in vivo, we have established a genetic system in the fission yeast S. pombe. We, and others, have demonstrated that the gene encoding calnexin (cnx1+) in S. pombe is essential for cell viability (Jannatipour and Rokeach, 1995Go; Parlati et al., 1995Go). Calnexin molecules contain a highly conserved central domain (hcd) that has been described as being involved in Ca2+ binding, and as being required for interaction of calnexin with folding substrates and potentially with other chaperones (Vassilakos et al., 1998Go; Schrag et al., 2001Go). In a previous study we determined that, in spite of its conservation and encoded functions, the central domain of calnexin/Cnx1p is dispensable for viability, and that the minimal sequences required for viability could be reduced to the last 123 amino acids at the C terminus (Elagöz et al., 1999Go).

We show that a particular cnx1 mutant, deleted of calnexin's highly conserved domain ({Delta}hcd_cnx1), triggers an unprecedented phenomenon by which cells adapt to live without the essential chaperone calnexin (Cnx1p). We demonstrate that this phenomenon can be transmitted to naive cnx1+ cells by transformation, and we present evidence indicating that this survival mechanism involves a non-chromosomal genetic component, exhibiting some protein-like features.


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Strains and media
The S. pombe strains are described in Table 1 and Fig. 1. Mutant {Delta}hcd_cnx1 was previously designated deleted_cnx1 (Elagöz et al., 1999Go), its name was changed in order to prevent confusions with cnx1{Delta}. DNA transformations into S. pombe cells were performed by the PEG-lithium acetate procedure as previously described (Elbe, 1992Go). Unless otherwise indicated, S. pombe strains were grown at 30°C in minimal medium (EMM, here designated MM) supplemented with required nutrients (Moreno et al., 1991Go).


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Table 1. Strains used in this study

 


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Fig. 1. Mutants tested in plasmid segregation assays. (A) Schematic representation of cnx1 constructs tested. The hatched box represents the highly conserved central domain; dotted line represents deleted sequences; dark grey box next to SP represents N-terminal domain; light grey box following hatched box (or dotted line) represents C-terminal domain within the ER lumen; ADEL represents the ADEL ER-retention signal of S. pombe; black boxes, potential sites of phosphorylation by protein kinase C (PKC); SP, signal peptide of Cnx1p; TM, transmembrane domain. (B) Plasmid segregation assays were carried out by culturing the cnx1{Delta} + pcnx1 + pcnx1 cells in liquid non-selective medium and scoring auxothrophies/protothropies on solid media as described in Materials and Methods. For each line, the first construct mentioned is on the pREP41 (LEU2 marker) vector and the second construct is on pREP42 vector (ura4+ marker). When only one plasmid is denoted, the cnx1 construct is based on the pREP41 vector. a, Average of at least two independent experiments; b, average of at least three experiments.

 

Plasmids and nucleic acid manipulation
pREP41 is an S. pombe expression multicopy vector bearing the S. cerevisiae LEU2 marker and the ars1 origin of replication (Maundrell, 1993Go). pREP42 differs from pREP41 in that it contains the S. pombe ura4+ marker instead of LEU2. Expression of cloned sequences in these plasmids is under control of the thiamine repressible nmt41 promoter (Maundrell, 1993Go). PCR products were cloned using the pCR XL TOPOTM vector according to manufacturer's conditions (Invitrogen). Nucleic acid manipulation and analysis were done as previously described (Sambrook et al., 1989Go).

Polymerase chain reaction
PCR amplifications were carried out using Taq polymerase according to the manufacturer's conditions (Pharmacia Biotech), and 100 ng of S. pombe total DNA of the strains used in this study. Primers used for the amplification of the his3+ gene integrated in the genome at the cnx1 locus were; (1) 5'-CAACTTACCAGATAGGTCTTTC-3' and (2) 5'-GGCTTTTAACAGAGTCGCTAC-3'; for the amplification of the nmt1 promoter and the cloning cassette of the expression vectors pREP41 and pREP42; (3) 5'-CGGCAATGTGCAGCGAAAC-3' and (4) 5'-TATCTCATCTAAACCAC-3' and finally for the amplification of cnx1 or the {Delta}hcd_cnx1 mutant construct; (5) 5'-CCACCCAACACGTGCATATGAAGTACGGAAAG-3' and (6) 5'-CGGGATCCGGCTTTTAACAGAGTCGCTAC-3'. Amplifications with the pair of primers 1/2 and 5/6 were carried out as follows: 94°C for 30 seconds, 50°C for 30 seconds, 72°C for 2 minutes 30 seconds. Amplifications with the primers 3/4 and 3/2 were carried out as follows: 94°C for 30 seconds, 48°C for 30 seconds, 72°C for 1 minute 50 seconds.

Southern and northern blot analyses
Genomic DNA extractions and DNA-DNA hybridisation were carried out as previously described (Moreno et al., 1991Go; Sambrook et al., 1989Go). Two probes were used for Southern blotting. The first was specific to the 400 bp 3' region of cnx1 between the ClaI and BamHI sites, and it was obtained by digestion of pSPCA3261 (Elagöz et al., 1999Go). The second one was the linearised pREP41 vector. RNA preparation and northern blotting was carried out as described previously (Jannatipour and Rokeach, 1995Go), using a 32P-labelled DNA probe encompassing the entire cnx1+-coding region.

Plasmid segregation experiments
S. pombe strains bearing one or two plasmids were grown for 6 days at 30°C in 5 ml liquid MM supplemented with adenine (Ade), uracil (Ura) and leucine (Leu) to chase the containing pREP42-based (ura4+ marker) and/or pREP41-based (LEU2 marker) plasmid(s). Cells were plated onto MM+Ade+Ura+Leu, MM+Ade+Ura, MM+Ade and MM+Ade+Leu. Phenotypes of the cells for uracil and/or leucine auxothrophy were then analysed after 2-3 days of growth at 30°C, and statistical values for plasmid loss were calculated.

Cell-extract preparation and transformation
Protein extracts for immunoblotting were prepared as previously described (Jannatipour et al., 1998Go). Lysates for transformation were made using spheroplasts prepared as described previously (Elagöz et al., 1999Go). Briefly, lysates were prepared by resuspending the cells in 2 ml lysis buffer (0.1 M sorbitol, 20 mM Hepes, 50 mM potassium acetate, pH 7.4) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride: PMSF, 10 mM iodoacetamide, 300 µg/ml pepstatin A, 300 µg/ml leupeptin, 300 mg/ml phenantroline) and homogenising in a Potter-Elvejhem homogeniser 25 times. Clumps of unbroken cells were discarded by centrifugation at 2000 g for 5 minutes at 4°C. The cleared lysate was then centrifuged at 15,000 g, and the supernatant was designated as the soluble fraction. The pellet was resuspended in lysis buffer and was designated as the insoluble fraction. In order to destroy nucleic acid present in the extracts, both the pellets and supernatants were digested with DNaseI (10 µg/ml) and RNaseA (10 µg/ml), at 37°C for 3 hours. Following this, the samples were UV-irradiated at 360 mJ/cm2, which was three times the radiation dosage necessary to inactivate 500 ng of plasmid DNA or 108 pfu of bacteriophages {lambda} or M13. To ensure that these treatments did efficiently destroy the nucleic acid present in the extracts, controls included 500 ng of plasmid DNA in the samples, which were subjected to the same treatment, and transformed into E. coli strain DH5{alpha}, which yielded no transformants. Likewise, 108 pfu of {lambda} or M13 were included in control samples that were subjected to the same treatments before infection of E. coli strain XL-1 Blue (Stratagene), producing no plaques. Moreover, to ascertain that no S. pombe cells survived the preparation of lysates, treated and untreated cell extracts were plated on appropriate media.

S. pombe transformation with protein extracts was carried out using two methods: a variation of the PEG/LiAc protocol (Elbe, 1992Go) and a liposomal method (DOTAP, N-[1-2(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate); according to the manufacturer's instructions (Roche Molecular Biochemicals, #1811177). Soluble and insoluble protein fractions were individually transformed into SP3220 ({Delta}cnx1::his3 + pREP42cnx1+) cells. Briefly, for the LiAc-PEG technique, 1 ml of mid-log SP3220 cells were pelleted, 5 or 15 µg of proteins were added and mixed by gentle vortexing. A 500 µl volume of sterile PLATE medium (40.5% PEG 3350, 100 mM lithium acetate, 10 mM Tris-HCl pH 7.5, 1 mM EDTA) was added and cells were incubated at room temperature for 24-48 hours. Cells were then washed in MM+Ade+Ura+Leu three times and diluted in the same medium containing 2% glucose for plasmid segregation assay. For transformation with the DOTAP liposomal reagent, SP3220 cells (1x106 cells) were spheroplasted, as described above, resuspended in fresh MM+Ade+Ura+Leu, transformed with 100 µl of mixture containing DOTAP reagent and 5 or 15 µg of proteins, followed by incubation for 16 hours. The DOTAP-proteins mixture was obtained by incubating the proteins with 30 µg DOTAP reagent in a 100 µl final volume of HBS buffer (20 mM Hepes, 150 mM NaCl, pH 7.4) at room temperature for 20 minutes. Finally cells were washed and resuspended in fresh MM+Ade+Ura+Leu and plasmid segregation assay was then carried out. For proteinase K digestions, the protein extracts were prepared as described above but using buffer A (25 mM tris-HCl pH 7.5, 50 mM KCl, 10 mM MgCl2, 1 mM EDTA, 5% glycerol) as described by Paushkin et al. (Paushkin et al., 1997Go). Digestions with proteinase K were carried out for 2 hours at 37°C at 4 µg/ml final enzyme concentration. Both the soluble and the pellet fractions produced Cin cells, with either transformation protocol. Since both the soluble and pellet fractions of Cin cell extracts transmitted the Cin state, transformations were carried out with unfractionated lysates.

Calcofluor staining of the cell wall and confocal microscopy
Calcofluor-white staining was carried out as described previously (Elagöz et al., 1999Go). Indirect confocal immunofluorescence was carried out using standard formaldehyde fixation essentially as described on the following website: http://www.bio.uva.nl/pombe/handbook/section4/section4-2.html. Primary antibody was used at a dilution of 1:50 for anti-BiP, or 1:100 for anti-Cnx1p. Secondary antibody FITC-conjugated anti-rabbit IgG at a 1:50 dilution was incubated overnight.


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S. pombe cells can become independent of the essential chaperone calnexin/Cnx1p
To explore the functions of calnexin, cnx1 deletion mutants (see Fig. 1A) were assessed using a plasmid segregation assay. Briefly, the experiment consisted of evaluating the functionality of mutants by determining the frequency at which an S. pombe strain, disrupted for genomic cnx1+ ({Delta}cnx1::his3=cnx1{Delta}), retained the episomal copies of mutant cnx1 (pREP41 vector, LEU2 marker) as compared to cnx1+ on an ura4+ vector (pREP42). After 6 days of liquid culture without selective pressure, cells were scored on selective plates for the presence of either the wild-type (WT) or mutant cnx1 plasmids (for the sake of simplicity, these constructs are denoted here as pcnx1).

As shown in Fig. 1, while strains cnx1{Delta} + pcnx1+, cnx1{Delta} + pmini_cnx1 and cnx1{Delta} + plumenal_cnx1 never gave rise to plasmid-free cells, the mutant cnx1{Delta} + p{Delta}hcd_cnx1 by itself or in the presence of either cnx1+ (wild type) or other cnx1 mutant constructs consistently gave rise to a population of Leu/Ura cells. We deduced that the simplest explanations for the appearance of viable Leu/Ura cells were: (i) integration of the p{Delta}hcd_cnx1 sequences into the genome; or (ii) mutations in the vector's LEU2 selection marker and maintenance of the episomal constructs because of the essentiality of cnx1+. In this regard, however, the frequency of appearance of Leu/Ura cells (87%) was several orders of magnitude higher than the expected rates for illegitimate genomic integration or for spontaneous mutations, which in both cases is estimated to be of about 10–6 (Chua et al., 2000Go; Zeyl and DeVisser, 2001Go). While it could be argued that the high frequency of Leu/Ura clones could be due to some selective growth advantage, this seems, however, unlikely since these cells grow more slowly than cnx1{Delta} + pcnx1 strains under the same culture conditions (see Fig. 3).



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Fig. 3. Cin and Cin + pcnx1+ cells display temperature-sensitive growth. Cells were cultured to saturation and then diluted into 10 ml of fresh MM+Ade+Ura medium to OD595 of 0.02. (A) Growth curves of SP556 (genomic cnx1+), SP3220: cnx1{Delta} + pcnx1+, SP7188: Cin and SP7202: Cin + pcnx1+ cells at 30°C for 45 hours. (B) Growth curves of the same cells grown at 37°C for 72 hours. (C) Histograms of calculated growth rates for each strain, at 30°C and 37°C.

 

Because calnexin/Cnx1p is essential for S. pombe viability, it seemed highly unlikely that these Leu/Ura cells had lost the cnx1 genes along with the markers/plasmids. Nevertheless, we considered it of importance to ascertain that cnx1 sequences were present in these marker-less cells. To this effect, we performed PCR, Southern, northern, and western analyses as summarised in Fig. 2. In the PCR experiments we used different combinations of primers in order to differentiate the amplification of genomic from plasmid DNA. The primer pair 3/4 was specific for the nmt1 promoter and terminator sequences that are found upstream and downstream, respectively, of the multicloning sites of both the pREP41 and pREP42 vectors (Fig. 2B). Therefore, PCR reactions using this pair of primers allowed the amplification of any sequence cloned within the multicloning site of these vectors, in our case the cnx1+ or any of the cnx1 mutants, as well as the nmt1 genomic sequences. As shown in Fig. 2A (lane 1) for one representative of the Leu/Ura clones analysed, PCR using the pair of primers 3/4 did not produce any amplification band, except for the one corresponding the intrinsic nmt1 genomic sequences (approximately 1.2 kb). In contrast, the strains cnx1{Delta} + p{Delta}hcd_cnx1 (SP3222; Fig. 2A, lane 3), SP556 harbouring the pREP41 empty vector (Fig. 2A, lane 2), or the purified pREP41cnx1+ plasmid DNA produced the expected PCR amplifications (Fig. 2A, lane 4). Thus, according to these PCR results, the Leu/Ura clones did not contain cnx1 or other DNA sequences under the control of the nmt1 promoter, either plasmid-borne or integrated in the S. pombe genome. Furthermore, the absence of cnx1 under the control of the nmt1 promoter was assessed by PCR amplification using the primers 3/2 that hybridise respectively to the nmt1 promoter and the 3' untranslated region of cnx1, which are present in all the pREP41cnx1 constructs used in this study (see Fig. 2 A,B).



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Fig. 2. Leu/Ura clones are viable calnexin-independent (Cin) cells. PCR, Southern blot, northern blot and western blot analyses demonstrate the existence of Cin cells. (A) Total DNA from a representative Leu/Ura clone (lanes 1 and 5), SP556 + pREP41 (lanes 2 and 6), SP3222: cnx1{Delta} + p{Delta}hcd_cnx1 (lanes 3 and 7), purified pREP41cnx1+ plasmid DNA (lanes 4 and 8) was subjected to PCR analysis with two sets of primers. The primer set 3/4 amplifies the nmt1 coding sequences in the genome and sequences inserted in the multicloning sites of the pREP41 or pREP42. The primer pair 3/2 specifically detects episomal constructs with cnx1+ or cnx1 mutants. Positions of certain DNA size markers (in kb) (lane 9) are indicated on the right. (B) Schematic representation of the cnx1{Delta} + pcnx1 haploid strains with the annealing sites for the primers used. (C) PCR analyses of cnx1 and his3 sequences. Total DNA from a representative Leu/Ura clone (lanes 4 and 9), SP556 + pREP41 (lanes 1 and 6), SP3220: cnx1{Delta} + pcnx1+ (lanes 2 and 7), SP3222: cnx1{Delta} + p{Delta}hcd_cnx1 (lanes 3 and 8), or purified empty pREP41 vector DNA (lanes 5 and 10), were subjected to PCR analysis with two sets of primers. The primer set 1/2 amplifies the cnx1 coding sequences in the genome of WT cells, or the his3+ marker in the cnx1{Delta} (cnx1::his3+) strains. The primer pair 5/6 specifically detects genomic or episomal cnx1 sequences. Positions of DNA size markers are indicated on the left. (D) Southern blot analysis showing the absence of genomic integration of cnx1 coding sequences. DNA from cnx1{Delta} + p{Delta}hcd_cnx1 (SP3222; lanes 1), a representative Leu/Ura clone (Cin; lanes 2), or the control for genomic cnx1+ strain SP556 + pREP41 (lanes 3) was digested with either BglII or ClaI. After transfer, the membranes were hybridised with either the mini_cnx1 or the pREP41 probes. Positions of DNA size markers are indicated on the right. (E) Schematic representation of the cnx1+ locus relevant to the Southern blot analyses. The cnx1+ open reading frame is indicated by a box with hatching to the right. The 5' and 3' untranslated regions are denoted by boxes with hatching to the left. Only relevant restriction sites and distances in kilobases (kb) are shown. (F) Northern blot analysis probing cnx1. Total RNA from strains SP556 (genomic cnx1+; lane 1), SP3220: cnx1{Delta} + pcnx1+ (lane 2), SP3222: cnx1{Delta} + p{Delta}hcd_cnx1 (lane 3), SP7188: Cin (lane 4) and SP7202: Cin + pcnx1+ (lane 5), were hybridised as described by Jannatipour and Rokeach (Jannatipour and Rokeach, 1995Go) with a 32P-labelled DNA probe encompassing the entire cnx1+ coding sequence. (G) Western blot analyses using rabbit anti-Cnx1p or anti-BiP polyclonal antibodies, as indicated. The anti-Cnx1p antibodies detect epitopes throughout the entire calnexin/Cnx1p molecule. Log phase cultures of SP556 (genomic cnx1+; lane 1), SP3220: cnx1{Delta} + pcnx1+ (lane 2), SP3222 SP cnx1{Delta} + p{Delta}hcd_cnx1 (lane 3), 7188: Cin (lane 4), and SP7202: Cin + pcnx1+ (lane 5) cells were used to prepare total protein extracts as described in Materials and Methods, and 20 µg of material was loaded for fractionated by SDS-PAGE, and incubated with antibodies as described by Elagöz et al. (Elagöz et al., 1999Go). Perpendicular, thick black arrows in A, C, D, F and G indicate the lanes corresponding to analyses of the Leu/Ura(Cin) strain.

 

In addition, the Leu/Ura clones were scrutinised for the presence of cnx1 coding sequences by PCR and Southern blot analyses. To this aim, PCR analyses were carried out with the pair of primers 5/6 that was designed to amplify the Cnx1p coding region (see Fig. 2B). As shown in Fig. 2C, this pair amplified the corresponding bands in DNA extracted from the control strains (lanes 6-8 and 10), but no amplification band could be detected with the Leu/Ura strain (lane 9). As a positive control for this experiment, PCR was carried out with the pair of primers 1/2 that amplifies the his3+ marker disrupting the cnx1 gene in the strains cnx1{Delta} + pcnx1+ (SP3220), cnx1{Delta} + p{Delta}hcd_cnx1 (SP3222), and the Leu/Ura clone (Fig. 2C, lanes 2, 3 and 4, respectively), or cnx1 coding sequences in the strain SP556 (Fig. 2C, lane 1). Finally, Southern blot analysis revealed that, as in contrast to the control strains (Fig. 2D, lanes 1 and 3), no band corresponding to cnx1 sequences was detectable from the Leu/Ura clones (Fig. 2D, lanes 2).

Next it was of interest to investigate whether the Leu/Ura clones were viable because of the possible random integration of cnx1 sequences into the genome and their expression under the control of a spurious promoter. No cnx1 transcript could be detected by northern blot analysis of the Leu/Ura clones (see Fig. 2F, lane 4). Likewise, while BiP was readily observed with anti-BiP antibodies, no Cnx1p was detectable in protein extracts from Leu/Ura clones by immunoblot analysis using anti-Cnx1p polyclonal antibodies (see Fig. 2G, lane 4), whereas the mutant protein {Delta}hcd_Cnx1p in extracts of the corresponding strain was distinctly detected (Fig. 2G, lane 3). Here it is important to stress that the anti-Cnx1p serum used recognises epitopes throughout the entire molecule (Elagöz et al., 1999Go), thus it would have been possible to detect fragments of calnexin/Cnx1p even smaller than the size of {Delta}hcd_Cnx1p.

Therefore, in conclusion, the S. pombe Leu/Ura clones described above do not contain cnx1 sequences in the genome and have lost the pcnx1 episome(s), nevertheless they are viable. Thus these clones have become calnexin-independent through a mechanism triggered by mutant {Delta}hcd_cnx1. One of these clones is hereafter designated as the Cin strain, for calnexin independent (SP7188; see Table 1).

The Cin state is maintained in the presence of the cnx1+ allele on a plasmid
As described in Fig. 1 (rows 3, 7 and 11), the appearance of S. pombe Cin cells was only observed with the mutant cnx1{Delta} + p{Delta}hcd_cnx1 (SP3222). Interestingly, this phenomenon took place even in the presence of cnx1+, as it was possible to lose the WT construct, but only when construct p{Delta}hcd_cnx1 was present in the cell (Fig. 1, row 3). In order to verify whether the phenomenon of calnexin independence (Cin) once initiated could be reversed by the re-introduction of the cnx1+ allele, the Cin strain was transformed with pREP41cnx1+ to generate strain SP7202, and the plasmid segregation assay was repeated. Results from these experiments clearly showed that the cnx1+ plasmid was lost, giving rise again to a population of Cin cells at a frequency of about 70% (Fig. 1, row 13). Thus the Cin state is not reversible under these conditions.

Cin cells display reduced tolerance to temperature and altered morphology
Since calnexin/Cnx1p was shown to be essential for S. pombe viability, it was of interest to examine whether the lack of the calnexin/Cnx1p chaperone affected the ability of Cin cells to cope with environmental stress conditions. As depicted in Fig. 3, when compared to the wild-type strain SP3220 (cnx1{Delta} + pcnx1+), the growth rates of Cin cells was slightly reduced at 30°C (Fig. 3A,C) and considerably slower at 37°C (Fig. 3B,C), thus reflecting a reduction in the cell's capacity to withstand thermal stress. Interestingly, the Cin strain containing the pcnx1+ plasmid (SP7202) presented an intermediate phenotype (Fig. 3B,C), suggesting that the reduced thermal resistance of Cin cells is not exclusively caused by the lack of calnexin/Cnx1p.

Morphological observations using Nomarski-interference microscopy revealed that, like certain previously characterised cnx1 mutants (Elagöz et al., 1999Go), the Cin cells were rounder at 30°C than cnx1+ (Fig. 4A), and this altered morphology was more evident when these cells were grown at 37°C (Fig. 4B). Owing to the lack of calnexin/Cnx1p it could be expected that Cin cells would exhibit altered endoplasmic reticulum (ER) morphology. However, confocal microscopy analysis with anti-BiP antibodies to stain the ER revealed no distinguishable changes in Cin cells as compared to the calnexin-dependent cnx1+ cells (Fig. 4C).



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Fig. 4. Cin cells display thermosensitive altered morphology. (A-C) S. pombe cells exponentially grown at 30°C were incubated for 20 hours in MM+Ade+Ura (2% glucose) at 30°C (A), or at 37°C (B). Cells were then stained with the fluorescent dye Calcofluor White (upper part of A and B) and viewed with Nomarski interference (lower part of panels A and B). Arrows indicate intracellular accumulation of the fluorescent dye. (C) Confocal indirect immunofluorescence analysis on cnx1+ (SP3220: cnx1{Delta} + pcnx1+), Cin (SP7188) and Cin + pcnx1+ (SP7202) cells was carried out with anti-Cnx1p rabbit antibodies (as described in Materials and Methods). For the Cin strain, anti-BiP antibodies were used for the endoplasmic reticulum immunostaining.

 

The Cin state is dominant and transmitted to the meiotic progeny
The results described above showed that the Cin state in haploid cells is maintained even upon re-introduction of the cnx1+ allele on a plasmid. Next we wondered whether the calnexin-independence state could be reversed in a diploid where any possible mutation in the original Cin strain should be complemented, if recessive, by the genome of the partner `naive' strain. Accordingly, the original Cin strain was mated with an isogenic strain to create a diploid Cin/cnx1{Delta} + pcnx1+ (strain SP7571). As controls, we constructed a diploid cnx1+/cnx1{Delta} + pcnx1+ (strain SP9450) that should lose the cnx1+ episome at high frequency because of the presence of genomic cnx1+, and a diploid cnx1{Delta} + pcnx1+/cnx1{Delta} + pcnx1+ (strain SP8394), which should not produce calnexin-less mitotic progeny because calnexin is essential, under normal conditions. As shown in Table 2, positive and negative controls produced the expected results, while plasmid segregation experiments with the Cin/cnx1{Delta} + pcnx1+ strain (SP7571) revealed that calnexin independence is maintained in diploid cells. Therefore, the Cin state is dominant.


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Table 2. The Cin state is dominant

 

To assess whether the Cin trait is transmitted to the meiotic progeny, random spore analysis (RSA) was performed. The diploid strain SP7571, described above, was constructed by selecting for adenine prototrophy using the complementing chromosomal alleles ade6-210 and ade6-216. Since in haploid cells ade6-210 and ade6-216 produce dark pink and light pink colonies, respectively, we used these colour phenotypes as indication of segregation for a chromosomal marker. As expected, these alleles were found at a frequency of approximately 50% each among the germinated spores (not shown). Then, to assess the inheritance ratio of Cin with respect to a chromosomal marker we scored approximately 250 of each, dark pink and light pink colonies, for the presence of the pcnx1+ episome (Leu+). As shown in Table 3, 88.7% of the germinated spores tested were Cin, revealing that calnexin independence segregated to the meiotic progeny at ratios different from the expected 2:2 for a chromosomal marker (such as the ade6 alleles). Furthermore, we assessed the inheritance of the Cin trait among the germinated spores that kept the pcnx1+ episome by culturing 18 of these haploid strains in non-selective liquid medium. In this case, 50% of these strains were in fact Cin (see Table 3). Thus these experiments established that the Cin state is dominant in diploid cells and transmitted to the meiotic progeny in an aberrant manner, compared to a chromosomal genetic trait. The non-Mendelian inheritance of the Cin state is further analysed below (see also Fig. 6).


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Table 3. The Cin state is transmitted to the meiotic progeny

 


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Fig. 6. The Cin state is plasmid independent, maintained in the cnx1+ genomic background and transmitted in a non-Mendelian fashion. (A) Phenotypic analysis of tetrads resulting from sporulation of the cnx1+/Cin diploid SP7501 (SP247/SP7188; see Table 1), in which deletion of the cnx1 genomic copy in the SP7188 haploid is marked with his3+. Cin spores bear the his3+ maker (i.e. cnx1:: his3={Delta}cnx1), thus they can grow on medium lacking histidine (MMAUL), while cnx1+ spores grow only on medium containing histidine (MMaULH). The his3+ marker was inherited in 2:2 ratio. Spores bearing the ade6-210 allele produce dark pink colonies on low adenine medium (MMaULH), while the ade6-210 allele produces light pink colonies. The ade6 markers were inherited in 2:2 ratio. To assess whether the factor encoding calnexin independence was present in the cnx1+ progeny, extracts form the his3 germinated spores were transformed into the cnx1{Delta} + pcnx1+ strain (SP3220; Table 1), and their capacity to generate Cin cells was examined by plasmid segregation. All cnx1+/his3 extracts produced Cin cells, as symbolised by [cif] within a circle. The putative [cif] factor was then inherited in a 4:0 ratio. Tetrads are labelled 1-4, and spores A-D. (B) Western blotting with anti-Cnx1p antibodies confirmed that the his3+ germinated spores were viable in the absence of calnexin. As positive control, the same extracts were immunoblotted with anti-BiP antibodies. Tetrads are labelled 1-4, and spores A-D.

 

The Cin state can be transmitted by transformation and requires a proteinaceous factor
The appearance of Cin cells raised the question of what mechanism could render the essential chaperone calnexin dispensable for viability in S. pombe. From the various features described thus far for the Cin strain it is possible to highlight the following key points. (i) Owing to the high frequency at which the Cin phenomenon occurs it is unlikely to be the result of mutations, rearrangements in the S. pombe genome, or the selection of a particular population. (ii) The phenomenon leading to the loss of episomal copies of cnx1+ is maintained after reintroduction of the cnx1+ allele. (iii) Calnexin independence is dominant in a diploid strain. (iv) The Cin state is transmitted to the meiotic progeny in an aberrant ratio. Taken together, these features suggest that the Cin state behaves as a dominant non-chromosomal genetic trait. Hence, we hypothesised that the Cin state could be encoded by a non-chromosomal genetic determinant such as a cryptic plasmid, or a virus, or a prion-like element (see Wickner, 1996Go; Wickner et al., 1999aGo; Sparrer et al., 2000Go; Maddelein et al., 2002Go). As such, we assumed that such determinant of calnexin independence could be transformable.

To test these hypotheses, we performed an experiment that involved preparing cell extracts depleted or not of nucleic acids from the Cin strain (SP7188) not containing the pcnx1+ plasmid, transforming them into the WT strain cnx1{Delta} + pcnx1+ (SP3220), and subsequently assessing whether it was possible to obtain Cin cells in this manner. To deplete nucleic acids, the cell extracts were extensively treated with DNaseI, RNaseA and UV-irradiated to degrade DNA and RNA in the lysates. In order to verify that these conditions effectively destroy nucleic acids, exogenous pREP42cnx1+ plasmid DNA was added to the lysates and the samples subjected to the treatments, followed by transformation into S. pombe strain SP3220. While untreated control samples yielded approximately 1,500 c.f.u./µg, the DNaseI, RNaseA and UV-irradiated samples produced no colonies. To further ascertain that the conditions used did proficiently degrade plasmid or viral nucleic acid, control experiments were performed with exogenously added plasmid DNA, as well as with the {lambda} or m13 bacteriophages (see Materials and Methods). In these controls, the extracts treated with DNaseI, RNaseA and UV-irradiated containing the added control plasmid did not yield ampicillin-resistant colonies by transformation into E. coli. The latter organism was used in these tests because it is several orders of magnitude more efficient in transformation than S. pombe (~108 versus ~103 c.f.u./µg, respectively). Likewise, the added bacteriophages did not produce plaques by infection of a sensitive E. coli strain. Furthermore, the absence of S. pombe genomic DNA in the treated extracts was confirmed by PCR analysis using primers designed to amplify the genes encoding BiP, Pdi1 and Ppi1 (data not shown). To transform the extracts, we used the Li+/PEG procedure and a spheroplast transformation technique mediated by liposomes (see Materials and Methods). Considering the possibility that a protein could encode the Cin state, purified GFP and fluorescence microscopy was used to confirm that protein transformation into S. pombe cells is indeed feasible using either technique. Fluorescence microscopy analysis revealed that GFP was efficiently transformed with both methods (data not shown). Transformation of untreated extracts from the Cin strain into strain cnx1{Delta} + pcnx1+ (SP3220) resulted in the appearance of Cin cells at about 1.3% of the colonies tested (about 2,000) with either transformation method (not shown). Likewise, the DNaseI, RNaseA and UV-irradiated extracts from the Cin strain consistently provoked the loss of episomal cnx1+ after transformation and resulted in the highly efficient appearance of cells lacking the cnx1+ plasmid, with either transformation method (Fig. 5). In contrast, extracts prepared from the WT strain (cnx1{Delta} + pcnx1+; SP3220) did not induce, in any case, the loss of episomal cnx1+ in the transformed strain. Furthermore, the plating on of treated and untreated lysates ascertained that the clones lacking the pcnx1+ were actual transformants and not survivor cells that were not lysed during preparation of the extracts.



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Fig. 5. The Cin state can be transmitted by transformation and requires a proteinaceous factor. Extracts from Cin (SP7188) and cnx1{Delta} + pcnx1+ (SP3220) cells were prepared and treated with RNaseA, DNaseI and UV, as described in Materials and Methods. Cell extracts were treated with proteinase K, or untreated, as indicated. Five and 15 µg of these extracts were transformed into strain SP3220 (cnx1{Delta} + pcnx1+) using the PEG/LiAc method. After plasmid segregation assay the appearance of Cin cells was scored. The table on the right gives details about the numbers of colonies tested. The frequencies of Cin cells are the mean of two to three independent experiments. In initial experiments, the cell extracts were fractionated by centrifugation into pellet and soluble fractions. Since both the soluble and pellet fractions of Cin (SP7188) cell extracts transmitted the Cin state, transformations were carried out with unfractionated lysates.

 

Two independent clones devoid of the cnx1+ episome, obtained with each transformation method, were further characterised to confirm their Cin state. Retransformation of the pcnx1+ plasmid followed by the plasmid segregation assay in non-selective, liquid medium, showed comparable frequencies of episomal-cnx1+ loss (~70%) to the Cin + pcnx1+ (SP7202) strain described earlier. Furthermore, PCR and western blot analyses ascertained, at the molecular level, that the clones obtained by transformation of nuclease- and UV-treated cell-extracts did not contain calnexin (data not shown).

That calnexin independence was transmissible by transformation indicated that an extrachromosomal genetic element is involved in the inheritance of the Cin state. In addition, because the DNase, RNase and UV treatments used did not reduce the transmission of the Cin state as compared to untreated lysates, it would be unlikely that the Cin state could be encoded by nucleic acid, be it naked or encapsidated as certain yeast viruses. Rather, these observations suggested that a protein could be involved in the transmission of the Cin state by transformation. To further investigate this point, the cell extracts were treated with proteinase K. The transmission of the Cin state was significantly reduced by proteinase K digestion, but not abolished (see Fig. 5), thus indicating the involvement of a protein, partially resistant to protease digestion, in the transmission of the Cin state. Interestingly, calnexin independence was efficiently transmitted by both soluble and insoluble fractions of extracts from Cin cells, suggesting that the extrachromosomal factor allowing viability in the absence of calnexin/Cnx1p may exist in at least two molecular states, one being of higher-order complexity than the other.

The Cin state is maintained in a cnx1+ genomic background and transmitted in a non-Mendelian manner
To assess whether the trait allowing calnexin-independence was stably maintained in a background with an intact cnx1+ genomic copy, the cnx1+/Cin diploid SP7501 (SP247/SP7188; see Table 1) was sporulated and the meiotic progeny analysed. It should be noted that this diploid does not contain a cnx1+ plasmid, and that deletion of the cnx1 genomic copy in the SP7188 haploid is marked with his3+. Analysis of four tetrads revealed that the his3+ marker (i.e. cnx1:: his3=cnx1{Delta}) was inherited in 2:2 ratio, as expected for a chromosomal marker (see Fig. 6A). Western blotting with anti-Cnx1p antibodies confirmed that the his3+ germinated spores were viable in the absence of calnexin (Fig. 6B). To determine whether the trait encoding calnexin independence was present in the cnx1+ progeny, extracts from the his germinated spores were transformed into the cnx1{Delta} + pcnx1+ strain (SP3220; Table 1), and their capacity to generate Cin cells was examined by the plasmid-segregation assay and absence of calnexin/Cnx1p was verified by western blotting. All cnx1+/his extracts produced Cin cells, showing that calnexin independence is encoded by a non-Mendelian, dominant genetic element that is stably maintained in the cnx1+-genomic background. Hereafter, this putative non-chromosomal genetic determinant is designated as [cif], for calnexin-independence factor. Moreover, these observations show that [cif] is maintained in the cnx1+ genomic background, and does not require the initial presence of the pcnx1+ plasmid.

The appearance of Cin cells mediated by the {Delta}hcd_cnx1 mutant requires the cytosolic tail of {Delta}hcd_Cnx1p
As described above and in Fig. 1, Cin cells only occur in the presence of the p{Delta}hcd_cnx1 construct, which is deleted of the segment encoding the calnexin's highly conserved central region (hcd). In contrast, the mutant mini_cnx1 construct, which in addition lacks the region encoding the N-terminal domain of calnexin/Cnx1p, does not elicit the loss of episomal cnx1+. Therefore, it was of interest to test whether it was possible to complement the inability of mini_Cnx1p to induce the appearance of Cin cells by providing in trans N-terminal_Cnx1p. As it is possible to see on Fig. 1 (row 15), the simultaneous presence of mutant mini_Cnx1p and N-terminal_Cnx1p in the same cell did not result in pcnx1+ loss. Therefore, induction of the mechanism leading to calnexin independence requires that the N-terminal and C-terminal parts of calnexin/Cnx1p be associated covalently. To test whether the cytosolic tail of {Delta}hcd_Cnx1p was required for the occurrence of calnexin-less cells by {Delta}hcd_cnx1, a mutant designated s{Delta}hcd_cnx1 was constructed, in which the regions coding for the Cnx1p's transmembrane domain and cytosolic tail were deleted from {Delta}hcd_cnx1. The mutant s{Delta}hcd_cnx1 failed to produce Cin cells in the plasmid segregation assay (Fig. 1, row 14). Therefore, these observations suggest that the transmembrane domain and the cytosolic tail of calnexin/Cnx1p could be involved in the occurrence of Cin cells mediated by {Delta}hcd_cnx1.


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We report the unprecedented phenomenon by which a gene essential for S. pombe viability becomes dispensable. We show that S. pombe cells are able to live in the absence of the essential molecular chaperone calnexin/Cnx1p. Remarkably, this survival mechanism is triggered by the {Delta}hcd_cnx1 mutant, which lacks the region encoding the calnexin's highly conserved domain (hcd), whether when alone in the cell or in the presence of wild type or other cnx1 mutants. Indeed, calnexin independence is dominant both in haploid and diploid cells, and is inherited in a non-Mendelian manner. Furthermore, calnexin independence can be transmitted by transformation using cell extracts from Cin cells into naive wild-type cells, which are calnexin dependent. This indicates that a non-chromosomal genetic element is implicated in the transmission of Cin state, and thus also explains its non-Mendelian inheritance. We designated this putative non-chromosomal element [cif], for calnexin-independence factor. That calnexin independence was efficiently transmitted by both soluble and insoluble fractions of cell extracts suggests that this factor may exist in at least two molecular states, one being of higher-order complexity than the other.

Since treatments with nuclease and UV that efficiently destroy nucleic acids did not obliterate the transmissibility of the Cin state by transformation, it appears improbable that a plasmid or a virus could be the genetic factor encoding calnexin independence. However, the reduction of transmissibility of calnexin-independence by protease treatment of the Cin cell extracts suggests the involvement of a protein in the inheritance of the Cin state. Therefore, we propose that a protein is at least an important component of [cif], the putative non-chromosomal element carrying the genetic information to transmit the Cin state. As mentioned above, in fungi prions are proteins that act as genetic elements, capable of propagating themselves, perpetuating the phenotypes they cause, and able to infect by transformation (Serio and Lindquist, 1999Go; Wickner et al., 1999aGo; Wickner et al., 1999bGo; Serio and Lindquist, 2000Go; Sparrer et al., 2000Go; Uptain and Lindquist, 2002Go; Ter-Avanesyan and Kushnirov, 1999Go; Maddelein et al., 2002Go; Chernoff et al., 2002Go).

Remarkably, the factor encoding the Cin state does exhibit some of the features of yeast prions, such as dominance in diploid cells, non-Mendelian inheritance, partial sensitivity/resistance to proteases, and infectivity by transformation. Whether the factor encoding calnexin independence is actually a prion or not awaits its identification and characterisation; we propose two models based on the proteinaceous, non-Mendelian, and metastable features of [cif].

Two models for the appearance and survival of Cin cells
Both models are based on the notion that the conversion of S. pombe cells, which are normally calnexin dependent, into calnexin-independent cells (Cin) implies the existence of an inducible pathway replacing the essential function of calnexin/Cnx1p. The second premise is that once suppression of calnexin's essentiality is induced in Cin cells, this state is dominant and maintained through generations, therefore implying an inheritance mechanism.

The first and simpler model comprises a single gene designated cif1, for calnexin-independence factor, encoding the protein Cif1p of unknown function, which can covert under special conditions (see below) into the [cif] conformer. The structural conversion into the [cif] conformer would entail the gain by this molecule of a function replacing calnexin's vital role, thereby allowing calnexin/Cnx1p to become dispensable for viability and give rise to Cin cells. Importantly, the [cif] conformer would be able to propagate by `structural replication' to other Cif1p molecules present in the cell, and thus being inherited through generations, acting then as a proteinaceous extrachromosomal factor able to transmit the Cin state.

Alternatively, one could envisage a second model comprising two key elements (see Fig. 7): (1) a suppressor gene for calnexin/Cnx1p essentiality (scx1, for suppressor of cnx1), and (2) a regulatory, proteinaceous, non-chromosomal genetic factor [cif], encoded by the cif1 gene as described above. In calnexin-dependent cells, the protein encoded by cif1 would normally exist in a conformational state, designated Cif1p, that would negatively act on the function of scx1. However, under particular conditions to be discussed below, this protein could convert into the alternative conformer [cif], which would be unable to negatively act on scx1. According to this model, in calnexin-dependent cells WT calnexin/Cnx1p exerts its essential function by acting on its essential target `?', while scx1 action on `?' is blocked by Cif1p. However, the presence of {Delta}hcd_Cnx1p in the cell would induce directly or indirectly the structural change of Cif1p into the [cif] conformer. As above, the [cif] conformer would then propagate by `structural replication' to other Cif1p molecules present in the cell, entailing the functional depletion of the Cif1p activity. As a result this would constitutively activate scx1, thereby allowing calnexin/Cnx1p to become dispensable for viability and give rise to Cin cells.



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Fig. 7. A two-component model for the survival mechanism of Cin cells. The model presented comprises two key elements: (1) a suppressor gene for calnexin/Cnx1p essentiality (scx1+, for suppressor of cnx1+); and (2) a regulator of scx1 activity that is designated Cif1p, for calnexin-independence factor). In the calnexin-dependent state, the protein encoded by cif1 is present in the cell in its native conformer Cif1p, negatively regulating scx1 activity on cnx1+ essential function on its putative target (symbolised as `?'). However, under particular conditions, such as the presence of {Delta}hcd_Cnx1p, the Cif1p protein could convert into an alternative conformer [cif] unable to inhibit scx1 activity. Under these latter conditions, scx1 could complement the essential function of calnexin/Cnx1p on its target `?'. This would allow the loss of episomal cnx1+ in a cnx1{Delta} + pcnx1+ strain when grown under non-selective conditions since cnx1+ activity on target `?' would be no longer vital, and thereby giving rise to calnexin-independent cells (Cin). The [cif] conformer would provoke further conversion of Cif1p molecules into the [cif] form by an `autocatalytic' process, such as structural replication, which can be inherited during mitosis and meiosis, and that can be transmitted by transformation. These features of [cif] would constitute the basis for the dominance and the inheritance of the Cin state. Details of this model are given in the Discussion section.

 

In any case, the [cif]-scx1 pathway may be normally a functional back-up to Cnx1p/calnexin that could be reversibly turned-on under certain conditions, such as during protein-folding stress. Under these folding-stress conditions scx1 activity would be likely to occur in a reversible and modulated fashion in the cnx1+ background. However. in the case of mutant {Delta}hcd_Cnx1p, the activation of scx1 would be prolonged. This might be because the 3D structure of the {Delta}hcd_Cnx1p mutant could mimic a long-lasting calnexin/Cnx1p-substrate interaction, as perhaps during folding stress, facilitating the conversion of Cif1p into [cif]. In this regard, it is interesting that the mini_cnx1 and N-terminal_cnx1 mutants alone or in concert do not trigger calnexin independence. So both portions of calnexin/Cnx1p have to be covalently attached for calnexin independence to occur. While the mechanism of induction remains to be elucidated, it is remarkable that the {Delta}hcd_Cnx1p mutant elicits the emergence of Cin cells at a very high frequency (~7x10–1). For instance, while the spontaneous appearance of the yeast prions [URE3] and [PSI+] is of about 10–6-10–5, overexpression of these yeasts prion proteins or the presence of an unrelated prion in the cell can increase the emergence frequency up to ~6,000 fold (Chernoff et al., 1995Go; Wickner, 1994Go; Masison and Wickner, 1995Go; Derkatch et al., 1997Go; Crist et al., 2003Go; Osherovich and Weissman, 2001Go; Derkatch et al., 2001Go), which is about the level of Cin appearance. Titration of a common cellular inhibitor, like a chaperone, has been proposed as a possible mechanism for the induction of prion appearance mediated by an unrelated prion (Osherovich and Weissman, 2001Go; Derkatch et al., 2001Go). While {Delta}hcd_Cnx1p is unlikely to be a prion, it is tempting to speculate that this non-functional chaperone (data not shown) could titrate a cellular factor and thereby mediate the emergence of the Cin state. We will use a combination of genetic and biochemical approaches in order to delineate the components of the [cif]-scx1 pathway, and map their circuitry.


    Acknowledgments
 
We wish to express our gratitude to Anne-Marie Sdicu and Dr Howard Bussey (Department of Biology, McGill University, Montreal) for help with the dissection of tetrads. We thank Drs John Armstrong, Pascal Chartrand, Jonathan Dinman, Gerardo Ferbeyre, Arthur Horwich, Reed B. Wickner, Sandra Wolin, and the members of the Rokeach lab for the critical reading of the manuscript. This work was supported by a grant from The Medical Research Council of Canada (L.A.R.), and by bridging funds from the Department of Biochemistry, the Faculty of Medicine and the Université de Montréal.


    Footnotes
 
* Present address: ISREC; Ch. des Boveresses 155, CH-1066 Epalinges sur Lausanne, Switzerland Back

{ddagger} Present address: AstraZeneca R&D Montreal, 7171 Frederick Banting, Saint-Laurent, Quebec, H4S 1Z9 Canada Back


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