Abstract
Separase is a highly conserved protease required for chromosome segregation. Although observations that separase also regulates membrane trafficking events have been made, it is still not clear how separase achieves this function. Here, we present an extensive ENU mutagenesis suppressor screen aimed at identifying suppressors of sep-1(e2406), a temperature-sensitive maternal effect embryonic lethal separase mutant that primarily attenuates membrane trafficking rather than chromosome segregation. We screened nearly a million haploid genomes and isolated 68 suppressed lines. We identified 14 independent intragenic sep-1(e2406) suppressed lines. These intragenic alleles map to seven SEP-1 residues within the N-terminus, compensating for the original mutation within the poorly conserved N-terminal domain. Interestingly, 47 of the suppressed lines have novel mutations throughout the entire coding region of the pph-5 phosphatase, indicating that this is an important regulator of separase. We also found that a mutation near the MEEVD motif of HSP-90, which binds and activates PPH-5, also rescues sep-1(e2406) mutants. Finally, we identified six potentially novel suppressor lines that fall into five complementation groups. These new alleles provide the opportunity to more exhaustively investigate the regulation and function of separase.
Original language | English |
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Pages (from-to) | 695-705 |
Number of pages | 11 |
Journal | G3: Genes, Genomes, Genetics |
Volume | 8 |
Issue number | 2 |
DOIs | |
State | Published - Feb 1 2018 |
Externally published | Yes |
Funding
We would like to thank Aude Peden for performing the ENU screen and isolating suppressors; Chris Turpin for helping with the screen and discussion of the manuscript; Andy Golden from the National Institute of Diabetes and Digestive and Kidney Disease at the National Institutes of Health (NIH) for his scientific discussion and guidance; and Kevin O’Connell and Bruce McKee for providing critical feedback on the manuscript. We especially thank WormBase and the Caenorhabditis Genetics Center (CGC). WormBase is supported by grant U41 HG002223 from the National Human Genome Research Institute at the NIH, the UK Medical Research Council, and the UK Biotechnology and Biological Sciences Research Council. The CGC (St. Paul, MN), is funded by the NIH Office of Research Infrastructure Programs (P40 OD-010440). This work was funded by startup funds from University of Tennessee, Knoxville and by NIH grant R01 GM-114471. Funding for open access to this research was provided by University of Tennessee’s Open Publishing Support Fund.
Keywords
- C. elegans
- HSP-90
- PPH-5
- Separase
- Suppressors