Fig 7. Cdc45-PIP box is essential for S. pombe cell survival.

a. Upper panel: Sequence alignment of Schizosaccharomyces pombe Cdc45 against human Cdc45 generated by Phyre2. PIP boxes highlighted in red. Lower left panel: Human Cdc45 ribbon representation derived from crystal structure (PDB ID: 5DGO; [9]). Navy blue region indicates α12 helix, red region depicts PIP box, sequence of PIP box indicated below structure. Lower right panel: Ribbon representation (pink) of 3D model of Schizosaccharomyces pombe Cdc45 modelled with Phyre2 using human Cdc45 (PDB ID: 5DGO) as template. Black arrowheads and associated numbers indicate amino acid stretches that have been excluded by Phyre2 during modeling. Navy blue region indicates α12 helix. Red region indicates PIP box. Sequence of PIP box indicated below structure. b. Western blot analysis of whole cell lysates isolated from sna1^goa1 transformants expressing LdCdc45 (wild type or PIP mutant) proteins. Two clones of each type were analyzed, using anti-FLAG antibodies (1:1000 dil). c. Complementation assays. sna1^goa1 transformants expressing LdCdc45 (wild type or PIP mutant) proteins were streaked on EMM2 plates (with necessary supplements) and incubated at permissive and non-permissive temperatures to assess functional complementation. Transformant expressing SpCdc45 served as positive control for complementation. Both clones of each transformant were streaked out on the same plate, one on each half. The experiment was done twice with comparable results; one set is presented here. d. Western blot analysis of whole cell lysates isolated from sna1^goa1 transformants expressing SpCdc45 (non-tagged wild type or His-tagged PIP mutant) proteins. Two clones of SpCdc45-PIP were analyzed, using anti-His antibodies (1:10000 dil). e. Complementation assays. sna1^goa1 transformants expressing SpCdc45 (wild type or PIP mutant) proteins were streaked on EMM2 plates and incubated at permissive and non-permissive temperatures. Both clones were streaked out on the same plate, one on each half. The experiment was done twice with comparable results; one set is presented here.