(A) eaf1Δ mutants exhibit elongated buds that can be suppressed by deletion of the Swe1 morphogenesis checkpoint. Wild type (YKB779), eaf1Δ (YKB42), swe1Δ (YKB1807), and eaf1Δ swe1Δ (YKB1266) cells were grown to mid-log phase in YPD at 25°C, fixed, and bud morphology was scored for at least 100 cells of each type. The average of three replicates is presented. (B) Deletion of SWE1 rescues the temperature sensitivity of eaf1Δ cells. The strains used in (A) were spotted in ten-fold serial dilutions and incubated at the temperatures indicated on YPD plates. (C) Overexpression of SWE1 is toxic to eaf1Δ cells. Wild type (YKB779), eaf1Δ (YKB42), pGAL-SWE1 (YKB1648), and eaf1Δ pGAL-SWE1 (YKB1806) cells were spotted on complete medium (yeast extract, peptone) in 10-fold serial dilutions containing glucose or galactose as the sugar source at 25°C for 2 days.

Cells expressing Cdc11-GFP, in which the sole source of histone H4 was expressed from a plasmid encoding a wild type (WT H4, YKB1260) or a mutant gene (H4-NΔ, YKB1261), were grown to mid-log phase at 25°C in YPD medium supplemented with adenine. Cells were fixed with paraformaldehyde prior to imaging by fluorescence microscopy. H4-NΔ encodes a protein in which amino acids 4 to 19 are deleted. Representative images of WT H4 and H4-ΔN cells are shown in (A) and (B), respectively. The yellow arrow points to an ectopic septin ring and the red arrows point to cells with septin in both the bud tip and neck. (C) Quantification of the defects observed for Cdc11-GFP localization in the cells represented in (A) and (B). The experiment was performed in triplicate scoring more than 100 cells in each replicate and the average is presented.

(A) Shs1-HA₃ exhibits decreased acetylation in vivo. Immunopurified septins were analyzed as in Figure 5. Lanes 1 and 2: Cells expressing CDC11-GFP and lacking genomic SHS1 (Δ) transformed with an empty vector (emp, YKB2254), or plasmid encoding SHS1-HA₃ (HA₃, YKB2255). Lanes 3 and 4: Cells expressing genomic CDC11-GFP and SHS1 as an untagged protein (+, YKB1312) or SHS1–HA₃ fusion protein (HA₃, YKB2518). Antibodies are described in Figure 5. (B) C-terminal truncation abolishes Shs1 acetylation. Lane 1: Cells expressing genomic CDC11-GFP and SHS1 (YKB 1312). Lane 2: Untagged control (YKB780). Lanes 3–6: Cells expressing CDC11-GFP and lacking SHS1 (Δ) transformed with an empty vector (emp, lane 3, YKB2519), plasmid encoding wild type SHS1 (WT, lane 4, YKB2520), C-terminal shs1 truncation (66Δ, lane 5, YKB2521), or shs1 lysine-to-arginine mutant (All-R: K16,K19,K57,K82,K204,K443,K478,K488,K536, lane 6, YKB2523). Intervening lanes are cropped. (C and D) Schematic outlining Shs1 domains and acetylation sites. Domains are noted on top in black. Acetylated lysine residues identified by mass spectrometry are in red along the bottom. Additional lysines used in mutational analyses are in black. 66Δ: site of C-terminal Shs1. (E) C-terminal Shs1 lysine mutations reduce in vivo acetylation and cell fitness. Lane 1: untagged control (YKB780). Lanes 2-7: Cells expressing CDC11-GFP and lacking SHS1 (Δ) transformed with an empty vector (emp, lane 2, YKB2519), plasmid encoding wild type SHS1 (WT, lane 3, YKB2520), C-terminal shs1 truncation (66Δ, lane 4, YKB2521), C-terminal shs1 lysine-to-arginine point mutants (8R: K488,K492,K500,K505,K509,K515,K535,K536, lane 6, YKB2747; 9R (8R + K478 lane 7, YKB2748). Representative of three experimental replicates. Bottom panels: strains spotted in ten-fold serial dilutions on YPD plates. (F) Bud morphology and septin localization in shs1 mutants. Cells were grown at 30°C and fixed. Genomic SHS1 was expressed as wild type (SHS1; YKB2712), C-terminal truncation (shs1-66Δ; YKB2713), fusion protein (SHS1-HA₃; YKB2518), or deleted from the genome (shs1Δ; YKB2173). Red arrow: mislocalized septins within an elongated bud.

(A) The NuA4 SDL genetic interaction network. Black nodes represent NuA4 genes, which are organized based on sub-complexes within the NuA4 complex (EAF1: complex integrity; EAF3, EAF5, EAF7, YAF9: recruitment module; EAF6, YNG2: Piccolo NuA4, PicNuA4). Interacting genes are represented by nodes colour-coded according to functional annotation as listed in the legend, and organized into groups based on the number of interactions with NuA4 sub-complexes. For instance, nodes located in the centre of the figure interact with one or more NuA4 mutants from each of the three sub-complexes. Small nodes denote genes that interact with only one NuA4 mutant. Edges indicate SDL or SDS genetic interactions (see also Table S3). (B) Comparison of the NuA4 SDL and SL genetic interactions identified by genome-wide screens for the five non-essential NuA4 subunits EAF1, EAF3, EAF5, EAF6, EAF7 (see Table S4 for the compiled list of SL interactions). (C) Overexpression of the septin genes CDC11 and SHS1 (yellow nodes) is toxic to most non-essential NuA4 deletion mutants (black nodes). Black and grey edges represent SDL or SDS interactions, respectively. (D) Overexpression of the septin genes CDC11 and SHS1 cause death in the absence of EAF1 or YNG2. Isogenic wild type (YKB779), eaf1Δ (YKB44) and yng2Δ (YKB494) cells containing galactose-inducible, plasmid-borne copy of galactose inducible CDC10, SHS1, CDC11, CDC12, or an empty vector control (pRS416) were spotted in serial ten-fold dilutions on minimal media lacking uracil and containing either glucose or galactose at 25°C for 3 or 5 days, respectively.

Time lapse imaging of cells expressing Cdc11-GFP in the presence (WT, YKB1312) or absence of EAF1 (eaf1Δ, YKB1310). Cells were grown to mid-log phase in YPD supplemented with adenine at 25°C, and transferred onto synthetic complete agarose pads for imaging at room temperature. Images were collected at five or fifteen minute intervals for wild type and eaf1Δ cells, respectively. Representative images of cells over time are shown in panel (A) for wild type cells and panels (B–C) for eaf1Δ cells. Red arrows point to septins mislocalized into the bud. The yellow arrow points out initially mislocalized septins that re-localize to the bud neck, followed cell division. (D) Images of three different eaf1Δ cells exhibiting septins misorganized to one side of the neck. Quantification of live cell imaging is presented in Table 1.

(A) Cdc10 and Shs1 are acetylated in vivo. Septin protein complexes were immunopurified through the GFP tag and separated by SDS-PAGE (7.5%) in cells expressing Cdc11-GFP (YKB1312), Shs1-GFP (YKB1878), or an untagged strain (YKB780; no tag). Left: silver stain to indicate the relative migration of septins. Right: Western blots using anti-acetyl lysine (α-Ac K) antibodies from Cell Signaling (Cell Sig.) or Upstate. Intervening lanes are cropped. (B) Acetylation of Shs1 and Cdc10 is dependent on Esa1 KAT activity in vivo. Septin proteins were immunopurified from cells grown at 25°C or 37°C for four hours through Cdc11-GFP in a wild type ESA1 (YKB1312) or mutant esa1-L254P background (YKB1804). Immunopurified (IP) products and whole cell extract (WCE) samples were separated by SDS-PAGE, silver stained or subjected to Western blot analysis. Shs1 and Cdc10 acetylation was assessed using antibodies in (A). Expression and immunopurification of Cdc11-GFP and Act1 was assessed with α-GFP and α-actin antibodies. Esa1 catalytic activity was assessed through histone H4 acetylation levels (α-Ac K H4). Glyceraldehyde-6-phosphate dehydrogenase (α-G6PDH) is a loading control. Red star: actin. This figure is representative of five experimental replicates. Intervening lanes are cropped. (C) NuA4 acetylates septins in vitro. In vitro KAT assays were carried out on septins immunopurified through Cdc10-TAP (YKB1443), Cdc11-TAP (YKB1455), or Shs1-TAP (YKB1466), relative to an untagged control (YKB779; no tag) using NuA4 purified from yeast and [³H]-acetyl coenzyme A. Reactions were separated by SDS-PAGE (7.5%), visualized by Coomassie staining, treated for fluorography, and visualized on film. The migration of NuA4 subunits is shown on the left. Acetylation of chicken core histones monitors NuA4's activity in vitro. Plus sign: shift in mobility on the gel due to the presence of the calmodulin binding peptide. Red circles: septin proteins that are acetylated in vitro. This figure is representative of two experimental replicates.