Single Site Suppressors of a Fission Yeast Temperature-Sensitive Mutant in <i>cdc48</i> Identified by Whole Genome Sequencing

<div><p>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 <i>cdc48⁺</i> 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 <i>cdc48-353</i> fission yeast strain. In total, 29 independent pseudo revertants that had lost the temperature-sensitive growth defect of the <i>cdc48-353</i> 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 <i>cdc48-353</i> 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 <i>cdc48</i> 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 <i>ufd1</i> 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.</p></div

Growth phenotypes of the <i>cdc48–353</i> and <i>cdc48–353</i>^sup strains.

<p>(A) Cell growth of wild type, <i>cdc48–353</i> and <i>cdc48–353-flag</i> strains on YES media was compared at the indicated temperatures. Note that the <i>cdc48–353</i> strain does not form colonies at 36°C. (B) Growth of wild type (wt), the <i>cdc48–353</i> parental strain (par) and the 28 different <i>cdc48–353</i>^sup strains on YES media was compared at the indicated temperatures. For each strain 10⁶ cells were applied to the media. (C) Cell growth of wild type, <i>cdc48–353</i> and the <i>cdc48–353</i>^<i>sup</i><i>-29</i> strain on YES media was compared at the indicated temperatures.</p

The <i>cdc48–353</i>^sup<i>-29</i> suppressor mutation maps to <i>ufd1</i>.

<p>(A) Schematic representation of the Ufd1 domain organization. The Ufd1-domain is shown in green, the SHP box and SIM in red and blue, respectively. The effect of the frame shift mutation and early stop codon in suppressor 29 (sup29) is shown in the lower panel. (B) Multiple sequence alignment of the Ufd1 protein from human (Hs), mouse (Mm), plant (At), fission yeast (Sp) and budding yeast (Sc). The UFD1 domain is boxed in green, the SHP box in red and the SIM in blue. The position of the frame shift mutation is marked with an arrow. Identical and homologues residues have been marked with (*) and (:), respectively.</p

Whole genome sequencing of selected <b><i>cdc48–353</i></b>^<b>sup</b> mutants.

<p>Whole genome sequencing of selected <b><i>cdc48–353</i></b>^<b>sup</b> mutants.</p