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. 2017 May 25;6:e23689. doi: 10.7554/eLife.23689

Figure 5. The order of Cdc3 oligomerization is controlled by the Cdc3 N-terminal extension.

(A) Violin plot showing bud neck fluorescence for the indicated GFP-tagged alleles of Cdc10 or Cdc3 expressed from plasmids in WT cells (strain BY4741, plasmids pLA10, pCdc10-1-GFP, pCdc3-GFP, and YCpL-Cdc3(D289N)-GFP) cultured at 22°C to mid-log phase prior to imaging. From left to right, n = 14, 11, 14, and 10. (B) As in (A), but including plasmids (pCdc3-GFP, YCpL-Cdc3(D289N)-GFP, YCpL-Cdc3(Δ1–56)-GFP, YCpL-Cdc3(D289N, Δ1–56)-GFP, YCpL-Cdc3(Δ1100)-GFP, or YCpL-Cdc3(D289N, Δ1–100)-GFP) encoding Cdc3-GFP alleles with D289N and/or the indicated truncations of the NTE. From left to right, n = 35, 30, 25, 16, 28, and 27. (C) Dilution series of cdc10∆ cells (strain JTY5104 w/ Cdc3 covering plasmid pMVB100) carrying the plasmids from (B) grown on rich medium incubated at the indicated temperature. (D–E) CSD-BiFC experiment and interpretation as in Figure 2B, but with different strains (YEF5692, MMY0191, or 12-VN/10-CN) and Cdc3-VC or Cdc3(ΔNTE)-VC expressed from plasmids (YCpHU-Cdc3-VC or YCpHU-Cdc3(Δ1–100)-VC). (D) From left to right, n = 22, 37, 43, and 33. (E) From left to right, n = 37, 66, 42, and 57.

DOI: http://dx.doi.org/10.7554/eLife.23689.014

Figure 5.

Figure 5—figure supplement 1. Proposed G-occlusion model of Cdc3 NTE function in septin oligomerization.

Figure 5—figure supplement 1.

While monomeric, Cdc3’s long NTE occludes the Cdc3 GTP-binding pocket and G interface, as evidenced by the ability of the NTE to mask mutations at the G interface from exclusion upon co-expression with WT Cdc3 (see Figure 5B). Monomeric Cdc3 thus interacts with a septin binding partner (e.g. Cdc12) at its ‘open’ NC interface, but not its occluded G interface. Following NC oligomerization with a Cdc12–Cdc11 or Cdc12–Shs1 dimer, Cdc12 recruits the Cdc3 NTE out of its G-occluding conformation, allowing Cdc3–Cdc10 interaction across the G interface.
DOI: http://dx.doi.org/10.7554/eLife.23689.015
Figure 5—figure supplement 2. Predicted disorder and conservation in the Cdc3 NTE.

Figure 5—figure supplement 2.

(A) The full Cdc3 sequence was analyzed by DISOPRED3 for predicted disorder. Plots show confidence scores for predicted disorder for each residue. The shaded area of the plot at left is expanded in the plot at right. (B) Sequence alignment of the first 97 amino acids of S. cerevisiae Cdc3 (ScCdc3) with Cdc3 homologs from other fungal species that have an obvious NTE. Average DISOPRED3 scores for each NTE (residues Nterminal to the conserved motif) are given in the column labeled ‘Diso’. The percentage value at far right gives the percent identity compared to ScCdc3 residues 88–520. Blue and red indicate similar and identical residues, respectively. Species and accession numbers: Sk, S. kudriavzevii, EJT44570.1; Nc, Naumovozyma castellii, XP_003675929.1; Zb, Zygosaccharomyces bailii, CDF90019.1; Td, Torulaspora delbrueckii, XP_003682181.1; Ag, Ashbya gossypii, NP_985658.2; Cg,Candida glabrata, KTA97432.1; Lm, Lachancea mirantina, SCV04072.1; Km, Kluyveromyces marxianus, BAO38302.1; Lq, Lachancea quebecensis, CUS21187.1; Kl, Kluyveromyces lactis XP_453247.1; Ho, Hanseniaspora osmophila, OEJ91001.1. (C) As in (B), but with fungal species that lack an NTE. Consequently, no DISOPRED3 score is given. Species and accession numbers: Mb, Metschnikowia bicuspidata, XP_018712407.1; Cm, Candida maltosa, EMG46918.1; Ct, C. tropicalis, XP_002550438.1; Ca, C. albicans, P39826.1; Pm, Pichia membranifaciens, XP_019015360.1; Ta, Tilletiaria anomala, XP_013243186.1. (D) The remainder of the ScCdc3 protein sequence is given with the color scheme as in (B) showing similarity and identity with the species in (B). Notable structural features are indicated above the sequence.
DOI: http://dx.doi.org/10.7554/eLife.23689.016
Figure 5—figure supplement 3. In silico structural modeling of G occlusion by the Cdc3 NTE.

Figure 5—figure supplement 3.

(A) An ‘apo Cdc3’ structural model generated by threading the Cdc3 sequence through the crystal structure of nucleotide-free, truncated Cdc11 (PDB 5AR1), highlighting the lack of secondary structure for the Sep4-motif-proximal β7/8 hairpin (compare with ribbon diagrams in other figures). (B) A peptide comprised of residues 57–82 of Cdc3 was docked onto the ‘apo Cdc3’ model allowing full conformational freedom using Autodock Vina. The residues between the docked peptide and the α0 helix (83-100) were then modeled using MODELLER.
DOI: http://dx.doi.org/10.7554/eLife.23689.017