Supplemental Materials

This article contains the following supporting material:

• Supplemental Figure S1 - (A) Cells containing the indicated BIR1 deletions (pUD243, 286, 326-335, 423-425, and pRS315; see plasmid table, supplemental data) were grown at 30°C on 5-fluoroorotic acid containing plates (see Material and Methods). (B) The chart represents a summary of cell growth experiments (as grown in A) of strains carrying the indicated BIR1 alleles (pUD325-335, 423-425): + = wildtype growth, +/- = reduced growth, -/+ = very slow growth (see text). (C) KSC 1476 cells containing bir1^{A931E, I935E} (pUD425), BIR1 (pUD243) or empty vector (pRS315) were plated on SD leu- dextrose plates with 5-FOA to clear the wildtype BIR1 covering plasmid to check for the appearance of suppressors under these conditions. Two-hybrid experiments were conducted on SD Leu- Trp- His- dextrose plates at 30°C, except for plates in (E) which also contained 6mM 3'-amino-1,2,4-triazole (3AT; see Materials and Methods). As summarized in Figure 1, the growth of yeast two-hybrid strains containing (D) BD (GAL4-DNA Binding Domain)-NDC10 (pUD256), (E) BD-SLI15 (pUD364), and (F) BD-IPL1 (pUD404) and the indicated AD (GAL4 Activation Domain)-BIR1 constructs is shown (pUD257, 347, 413, 414).
• Supplemental Figure S3 - Glycerol gradient sedimentation of extracts from strains expressing (A) Bir1p-13Myc and Sli15p-3HA or (B) Bir1p-13Myc and Ipl1p-3HA were analyzed as in Figure 2 and compared with the results from the triple-tagged strain (13Myc-Bir1p, Sli15p-13Myc and Ipl1p-3HA; overlaid grey lines) described in the text and Figure 2 (13Myc-Bir1p ( ), Sli15p-13Myc ( ) and Ipl1p-3HA ( )). (C) Extracts were prepared from cells containing GAL-3HA-CTF13 and grown in galactose containing medium and then shifted for 3 hours to dextrose containing medium. Sli15p was immunoprecipitated and Bir1p co-immunoprecipitation was analyzed (see materials and methods for details). The bands indicated loading control are non-specific bands from αHA immunoblot. (D) Protein levels in (C) are quantified using a LiCor Odyssey fluorescence reader (see Material and Methods). Samples were normalized to the R/G sample for the load control, to the Sli15p-R/G sample and Bir1p-R/G sample for the Sli15p and Bir1p inputs respectively, and to the Sli15p-R/G sample for all of the IP samples.
• Supplemental Figure S3 - (A) Wildtype cells expressing Cdc11p-GFP containing either pGAL-SLI15^1-229 (pUD499) or pGAL (pUD108) were arrested in α-factor and then released into the cell cycle in the presence of galactose and imaged. Representative fluorescent (GFP) or DIC and fluorescent overlaid (GFP/DIC) images are presented. (B) The percentage of cells from (A) with septin abnormalities was quantified in large budded cells as they emerged from -factor release. (C) As done in (A), the percentage of large budded cells with mono-attached CEN IV-GFP was determined as they emerged from α-factor arrest (~3-5 hours) in the cells containing pGAL-SLI15^1-229 (pUD499) and compared to ipl1-321 cells at 37°C. (D) Comparison of mutant Bir1p and Sli15p^1-229 cell viability. Viability was determined for cells expressing BIR1 (pUD286), bir1^W901A (pUD424), and bir1^{A931E, I935E} (pUD425) grown on SD Leu- dextrose plates grown at 30°C. Cells expressing either pGAL-empty (pUD108) or pGAL-SLI15^1-229 (pUD499) were grown to log phase in liquid SD Leu- dextrose media and plated to either SD Leu- dextrose or SD Leu- raffinose and galactose and grown at 30 °C (see Materials and Methods for details). The viability of cells containing the pGAL-empty vector and grown in dextrose was set at 100%. All scale bars are 4 μm.
• Movie 01
• Movie 02
• Movie 03
• Table Plasmid List
• Table Strain List