JCS040949 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Induction of calnexin-independent cells: Plasmid segregation assay. Cells auxotrophic for leucine and uracil (ura4D18, leu1-32), with a genomic deletion for cnx1⁺ (Δcnx1) and harboring a plasmid encoding calnexin (pcnx1⁺) are used to test for the induction of the Cin state. (1) Cin-inducing genes such as Δhcd_cnx1 or cif1⁺ on expression plasmids are introduced by transformation into this strain. After introduction of the Cin inducers, cells are cultured in non-selective minimal medium for 5-7 days. Under these conditions, Cin cells lose pcnx1⁺ as calnexin is not longer essential for viability in the Cin epigenetic state. To identify the Cin cells, the cultures are plated on non-selective medium, and colonies are restreaked on selective and non-selective medium to check for the presence or absence of the pcnx1⁺ plasmid. For each experiment 400 colonies were streaked, and experiments were performed at least 3 times. (2) In the overexpression screen, the candidate Cin-inducing genes are screened for by transformation of S. pombe cDNA overexpression libraries. (3) In the random insertion screen, to identify candidate Cin-inducing genes, a ura4⁺ linear DNA fragment was transformed and the cassette inserted randomly into the S. pombe genome.

• Supplemental Figure S2 -

  Fig. S2. The shapes of the nucleus and the nucleolus are not affected in the CinΔ_hcd__cnx1 or Cin _cif1 states. (A) Examination of the nuclear shape of WT and both types of Cin cells. Exponentially growing cells containing a plasmid encoding the nuclear protein Hht2p fused with YFP (Riken, Japan) were fixed as described in Material and Methods. Slides were mounted with a DAPI-containing media (1 µg/mL DAPI, 1 mg/mL p-phenylenediamine, 90% glycerol). Cells were examined by fluorescence microscopy. Venus, DAPI and Nomarski show the same fields of cells for each strain. (B) Examination of the nucleolar shape of WT and both types of Cin cells. Exponentially growing cells expressing a plasmid encoding the nucleolar protein Gar2p fused with YFP (Riken, Japan) were fixed, mounted and examined as in panel A.

• Supplemental Figure S3 -

  Fig. S3. Cif1p forms fibers in vitro. Cif1p-his₆ was purified from E. coli under denaturing conditions, and incubated at 30°C in 50 mM Tris-HCl (pH 7.0) 150 mM NaCl for 14 days. The protein solution was centrifuged at 16,000 g for 15 minutes and the supernatant was transferred on MICA for observation by solid-state atomic force microscopy. The diameter (height) of oligomers and fibrils is shown; the full range of the brown scale corresponds to a height of 15 nm. Soluble proteins are not visible, amorphous aggregates form particles of various sizes; organized aggregates form regularly-shaped particles or fibers. Arrows indicate fibrillar structures.

• Supplemental Figure S4 -

  Fig. S4. Leptomycin B does not affect the distribution of Cif1p-KRKR_27-30/_AAAA-Venus. Exponentially growing Δcif1 cells expressing a Cif1p-Venus and a Fib1p-mRFP fusion were treated with 1.6% methanol (vehicle, control) or 100 ng/mL leptomycin B for 6 hours. Following which, cells were fixed, mounted and examined as describe previously. Venus, mRFP, DAPI, merge and Nomarski show the same fields of cells for each strain. Leptomycin B provokes both a deceleration of cell growth and aberrant nuclear shape.

• Supplemental Figure S5 -

  Fig. S5. Cif1p and Cnx1p do not co-immunoprecipitate. Immunoprecipitations were performed using extracts of exponentially growing cells with anti-Cif1p antibodies. Calnexin was detected by immunoblotting using the appropriate antibodies as described in the Materials and Methods.