Single site suppressors of a fission yeast temperature-sensitive mutant in Cdc48 identified by whole genome sequencing

4Citations
Citations of this article
21Readers
Mendeley users who have this article in their library.

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.

Cite

CITATION STYLE

APA

Marinova, I. N., Engelbrecht, J., Ewald, A., Langholm, L. L., Holmberg, C., Kragelund, B. B., … Hartmann-Petersen, R. (2015). Single site suppressors of a fission yeast temperature-sensitive mutant in Cdc48 identified by whole genome sequencing. PLoS ONE, 10(2). https://doi.org/10.1371/journal.pone.0117779

Register to see more suggestions

Mendeley helps you to discover research relevant for your work.

Already have an account?

Save time finding and organizing research with Mendeley

Sign up for free