title: Single site suppressors of a fission yeast temperature-sensitive mutant in cdc48 identified by whole genome sequencing.
abstract: The protein called p97 in mammals and Cdc48 in budding and fission yeast is a homo-hexameric, ring-shaped, ubiquitin-dependent ATPase complex involved in a range of cellular functions, including protein degradation, vesicle fusion, DNA repair, and cell division. The cdc48+ gene is essential for viability in fission yeast, and point mutations in the human orthologue have been linked to disease. To analyze the function of p97/Cdc48 further, we performed a screen for cold-sensitive suppressors of the temperature-sensitive cdc48-353 fission yeast strain. In total, 29 independent pseudo revertants that had lost the temperature-sensitive growth defect of the cdc48-353 strain were isolated. Of these, 28 had instead acquired a cold-sensitive phenotype. Since the suppressors were all spontaneous mutants, and not the result of mutagenesis induced by chemicals or UV irradiation, we reasoned that the genome sequences of the 29 independent cdc48-353 suppressors were most likely identical with the exception of the acquired suppressor mutations. This prompted us to test if a whole genome sequencing approach would allow us to map the mutations. Indeed genome sequencing unambiguously revealed that the cold-sensitive suppressors were all second site intragenic cdc48 mutants. Projecting these onto the Cdc48 structure revealed that while the original temperature-sensitive G338D mutation is positioned near the central pore in the hexameric ring, the suppressor mutations locate to subunit-subunit and inter-domain boundaries. This suggests that Cdc48-353 is structurally compromized at the restrictive temperature, but re-established in the suppressor mutants. The last suppressor was an extragenic frame shift mutation in the ufd1 gene, which encodes a known Cdc48 co-factor. In conclusion, we show, using a novel whole genome sequencing approach, that Cdc48-353 is structurally compromized at the restrictive temperature, but stabilized in the suppressors.
authors: Department of Biology, University of Copenhagen, Copenhagen, Denmark.
authors: Irina N
authors: IN
authors: Marinova
authors: Department of Biology, University of Copenhagen, Copenhagen, Denmark.
authors: Jacob
authors: J
authors: Engelbrecht
authors: Department of Biology, University of Copenhagen, Copenhagen, Denmark.
authors: Adrian
authors: A
authors: Ewald
authors: Department of Biology, University of Copenhagen, Copenhagen, Denmark.
authors: Lasse L
authors: LL
authors: Langholm
authors: Department of Biology, University of Copenhagen, Copenhagen, Denmark.
authors: Christian
authors: C
authors: Holmberg
authors: Department of Biology, University of Copenhagen, Copenhagen, Denmark.
authors: Birthe B
authors: BB
authors: Kragelund
authors: MRC Human Genetics Unit, Western General Hospital, Edinburgh, Scotland, United Kingdom.
authors: Colin
authors: C
authors: Gordon
authors: Department of Biology, University of Copenhagen, Copenhagen, Denmark.
authors: Olaf
authors: O
authors: Nielsen
authors: Department of Biology, University of Copenhagen, Copenhagen, Denmark.
authors: Rasmus
authors: R
authors: Hartmann-Petersen
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics*; D003080:Cold Temperature; D016681:Genome, Fungal / Q000235:genetics*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D017434:Protein Structure, Tertiary; D012568:Schizosaccharomyces / Q000145:classification / Q000235:genetics* / Q000254:growth & development; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D000074405:Valosin Containing Protein
chemical_list: D018797:Cell Cycle Proteins; D029702:Schizosaccharomyces pombe Proteins; D000251:Adenosine Triphosphatases; D000074405:Valosin Containing Protein