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First published online February 12, 2004
doi: 10.1242/10.1242/jcs.00943

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

Philippe Collin^*, Pascale B. Beauregard, Aram Elagöz and Luis A. Rokeach

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

View larger version (19K): [in a new window]   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 + 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.

View larger version (17K): [in a new window]   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 + 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.

View larger version (39K): [in a new window]   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 + phcd_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 + 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 + pcnx1+ (lanes 2 and 7), SP3222: cnx1 + phcd_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 (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 + phcd_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 + pcnx1+ (lane 2), SP3222: cnx1 + phcd_cnx1 (lane 3), SP7188: Cin (lane 4) and SP7202: Cin + pcnx1+ (lane 5), were hybridised as described by Jannatipour and Rokeach (Jannatipour and Rokeach, 1995) 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 + pcnx1+ (lane 2), SP3222 SP cnx1 + phcd_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., 1999). Perpendicular, thick black arrows in A, C, D, F and G indicate the lanes corresponding to analyses of the Leu–/Ura–(Cin) strain.

View larger version (81K): [in a new window]   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 + 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.

View larger version (33K): [in a new window]   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=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 + 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.

View larger version (16K): [in a new window]   Fig. 5. The Cin state can be transmitted by transformation and requires a proteinaceous factor. Extracts from Cin (SP7188) and cnx1 + 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 + 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.

View larger version (11K): [in a new window]   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 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 + 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.