(A) Model of the subunit arrangement in the yeast septin hetero-octamer (McMurray & Thorner, 2008); Cdc3 (blue), Cdc10 (brown); Cdc11 (pink); Cdc12 (green). (B) Sequence conservation of the Trp contact residue in the G interface. (C) Cultures of the indicated genotypes were grown in YPGal, spotted in five-fold serial dilutions onto agar plates containing SCGal-Leu medium in the absence (left) or presence (right) of FOA to select against URA3-containing cells, and photographed after incubation at RT for 5–6 dy. Far right, schematic depiction of septin composition in cells that have lost the URA3 plasmid; red asterisk, W267A mutation at G interface of Cdc12. (D) Strains MJY201 and JTY3631 were crossed, generating (after loss of a URA3-marked plasmid) the indicated cdc11Δ0::HIS3MX/CDC11⁺ shs1Δ0::kanMX/SHS1⁺ diploid, which was subjected to sporulation and tetrad dissection. Shown are representative meiotic progeny of the indicated genotypes after incubation on YPD medium at 26° for 4 dy. Far right, representative image of the type of microcolony formed by the cdc11Δ SHS1⁺ spores, which all failed to form a viable clone. (E) As in (C), except that the cdc11Δ cells also lacked Shs1. See also Figure S1.

(A) Representative class averages of negatively-stained particles of purified recombinant Cdc11–Cdc12–Cdc3–Cdc10(Δα0) complexes in 300 mM (top) and 60 mM (bottom) KCl buffer. (B) Growth of representative meiotic progeny of cdc10Δ::kanMX/CDC10⁺ cells (strain 4003482^™) carrying either a URA3-marked plasmid expressing CDC10-GFP (left) or the same vector expressing cdc10(Δα0)-GFP (right) on YPD medium at 26° for 4 dy. Far right, images of representative clones of cdc10Δ spores either carrying (top) or lacking (bottom) the Cdc10(Δα0) plasmid. (C) Cultures of strain JTY3993 carrying URA3-marked plasmids expressing either CDC10-GFP or cdc10(Δα0)-GFP from either the CDC10 promoter (P_CDC10) or the Met-repressible MET17 promoter (P_MET17), as indicated, were grown in SCGlc-Ura and ten-fold serial dilutions were plated on the same medium either containing (top) or lacking (bottom) Met, and the plates were incubated at 26° C for 3 dy. (D) Strain JTY3993, which expresses CDC10-mCherry from the chomosomal CDC10 locus, and also carrying the URA3-marked P_MET17-cdc10(Δα0)-GFP plasmid (pJT3456), was grown in SCGlc-Ura in the presence of Met, washed, spotted on an agarose pad containing the same medium lacking Met, incubated under a coverslip at RT, and examined periodically using DIC optics (top) and by epifluorescence microscopy using the appropriate filters to visualize GFP (middle) and mCherry (bottom). (E) JTY3993 cells carrying either the P_MET17-CDC10-GFP plasmid (pJT2696) or the P_MET17-cdc10(Δα0)-GFP plasmid (pJT3456) were grown in SCGlc-Ura in the presence of Met to mid-exponential phase, then exposed in the same medium to the ribonucleotide reductase (and DNA replication) inhibitor hydroxyurea (HU; 200 mM final concentration) and incubated for 2 h (left columns), then transferred to Met-free HU-containing medium and incubated for 7 h (center columns), and finally transferred to Met-free medium lacking HU for 1 h (right columns). Arrows, normal split rings; arrowheads, abnormal septin structures. See also Figure S3.

Cultures of the indicated genotype were grown in YPGal, spotted in five-fold serial dilutions onto agar plates containing SCGal-Leu medium in the absence (left) or presence (right) of FOA, and photographed after incubation at RT for 5–6 dy. Far right, schematic depiction of septin composition in cells that have lost the URA3 plasmid; red asterisk, Δα0 mutation at the NC interface of Cdc11; grey spheres, septin subunits of complexes where Cdc11 normally occupies the terminal position; blue spheres, septin subunits of complexes where Shs1 normally occupies the terminal position. See also Figure S4.

(A) As in 1C, for cells of the indicated genotype; red asterisk, W364A mutation at G interface of Cdc3. (B) Morphology of representative cells of isogenic CDC3⁺ (left) or cdc3(W364A) strains (right) (derived from JTY5071) grown at 30°C, examined by differential interference contrast (DIC) microscopy (left panels) and co-expressing Cdc10-GFP (from plasmid pLA10), whose localization was determined by epifluorescence microscopy (right panels). See also Figure S2.

(A) Sequence alignment (left) of human SEPT2 with WT Cdc3 and Cdc3(G261V). Highlighting (Phe, pink; Gly, orange; His, blue) refers to same residues shown (right) in the close-up of the G homodimer interface of GppNHp-bound mouse SEPT2 (PDB accession no. 3FTQ) (Sirajuddin et al., 2009). (B) As in 1C, except grown on SCGlc-Leu and incubated for 3 (left) or 5 dy (right); green asterisks, cdc3(G261V) mutation. (C) Purified recombinant Cdc11–Cdc12–Cdc3(G261V) complexes were examined by EM in 300 mM salt buffer in the absence (solid bars) and presence (open bars) of 10 mM GTP; frequencies of particle classes observed (representative examples on the right) plotted as histograms (left). All 824 particles (100%) in the absence of GTP were trimers; 3358 particles in the presence of GTP were 97 dimers (3%), 2831 trimers (84%), 376 tetramers (11%), 46 pentamers (1%), and 8 hexamers (0.2%). Asterisk, classes whose appearance depended on presence of GTP at a statistically significant level (P < 0.0001, according to a two-tailed Fisher’s exact test).

Cells of the genotype shown, expressing from either CEN plasmids (in brackets) or from the chomosomal locus (for CDC10-mCherry) the indicated fluorescently-tagged proteins, were grown to mid-exponential phase and examined by DIC (top or left) and by epifluorescence microscopy with appropriate filters to visualize either Cdc12-CFP, in presence and absence of Cdc10-mCherry, or Cdc12-GFP, and either Sec3-GFP or Chs2-GFP (bottom panels). Strains were JTY3992 (A), 4003482^™ (B), FOA-resistant derivative of JTY4944 (C), and JTY5140 (D and E). Plasmids were pSEC3-GFP3-1, YCpH-CDC12-CFP, YCp-CHS2-iGFP, and pLP17 (CDC12-GFP). Arrowheads, split septin rings; Arrows, bud neck localization of non-septin proteins. See also Figure S5.