Synthetic genetic-interaction map of five NuA4 subunits. Genome-wide SL-SGA screens were performed using query strains for five nonessential NuA4 subunits: the eaf1Δ (YKB622), eaf3Δ (YKB993), eaf5Δ (YKB852), eaf6Δ (YKB623), and eaf7Δ (YKB853) strains. The 199 NuA4-interacting genes are represented by nodes that are color coded based on the GO process. Gray lines indicate genetic interactions (both SS and SL interactions). The 139 interacting genes highlighted in bold text indicate those genes whose interaction with at least one NuA4 query strain was confirmed by tetrad dissection in our lab. The remaining interacting genes in the map represent previously published genetic interactions that were either identified in our SGA screens but were not confirmed by tetrad dissection or not identified in our screens. Genes encoding subunits of a single protein complex are grouped together with black circles.

Eaf1-TAP purifies the NuA4 complex. SDS-PAGE (gradient gel) and silver staining comparing NuA4 that was affinity purified via Eaf1-TAP (YKB1007) and NuA4 from an untagged strain (WT; YPH499), using IgG-coated magnetic beads that bind the PrA component of the TAP tag. IgG heavy and light chains coelute upon heating. Protein bands were identified by MS as indicated. Eaf6 was not efficiently silver stained. The * indicates a non-NuA4, copurifying protein, Fks1. Anti-TAP Western blotting demonstrates the expression of Eaf1-TAP in the WCE. This gel is representative of those from three purification experiments. Numbers at the left indicate molecular masses (in kDa).

NuA4 function impacts vesicle-mediated transport. (A) eaf1Δ, yaf9Δ, yng2Δ, and esa1(L245P) cells display vacuolar-morphology defects. Vacuole morphology was examined in WT (YPH499) cells, in strains deficient in each of the seven nonessential NuA4 subunits (eaf1Δ [YKB44], eaf3Δ [YKB654], eaf5Δ [YKB658], eaf6Δ [YKB662], eaf7Δ [YKB853], yaf9Δ [YKB464], and yng2Δ [YKB494]), and in a temperature-sensitive point mutant of the catalytic subunit [esa1(L245P) (LPY3500)] using the dye FM4-64. The WT and deletion mutants were grown at 30°C, while esa1(L245P) mutants were grown at 37°C for 2 h after FM4-64 treatment. Images shown were taken after the merger of fluorescent light and transmitted light. (B) htz1-ND cells (YKL35) do not show vacuolar-morphology defects, while htz1Δ (YKB625) cells display defects similar to those of the eaf1Δ, yaf9Δ, yng2Δ, and esa1(L245P) mutants. Vacuole morphology was examined in mutant cells with FM4-64 as described for panel A. (C) htz1Δ (YKB500), esa1(L245P) (LPY3500), and eaf1Δ (YKB622) mutants (indicated by the red dots) genetically interact with a subset of vesicle-mediated transport genes (indicated by green dots) identified in the NuA4 SGA screens. Lines connect genes with synthetic genetic interactions.

NuA4 physically interacts with Msn4 but does not regulate Msn4 binding to the HSP12 promoter or heat shock induction of HSP12. (A) NuA4 interacts with Msn4 in vivo. Protein extracts expressing the indicated tagged proteins (Msn4-TAP [YKB1035], Eaf7-MYC [YKB518], Msn4-TAP/Eaf7-MYC [YKB1069]) or an untagged WT control (YPH499) were immunoprecipitated with magnetic beads coated with IgG antibodies that bind the PrA component of the TAP tag. Total protein extracts (WCE) and immunoprecipitates (α-TAP IP) were resolved by 7.5% SDS-PAGE and subjected to Western blot analysis with anti-Myc and anti-TAP antibodies (α-MYC and α-TAP, respectively), as indicated at the right side of the panels. (B) NuA4 and Msn4 interact in vitro. WCE from cells expressing Esa1-TAP (YKB440) were used to purify the NuA4 complex using IgG-coated magnetic beads, and results were compared to those for an untagged strain (WT; YPH499). IgG-coated magnetic beads complexed with NuA4 (lane Esa1-TAP), background yeast proteins (lane WT), or beads alone (lane Beads) were incubated with equivalent amounts of either full-length GST-Msn4 or GST. Resulting immunocomplexes were eluted with heat, resolved by 10% SDS-PAGE, and subjected to Western blotting with anti-TAP and anti-GST antibodies, as indicated. Twenty-five percent of the input GST or GST-Msn4 fusion protein was run alongside the pull-down experiments as an input control (lane Input). (C) Msn4 occupancy at the HSP12 promoter is independent of NuA4. Modified ChIP (see Materials and Methods) was performed using an untagged strain (WT; YPH499) and the Msn4-TAP (YKB1035) and Msn4-TAP eaf1Δ (YKB1091) strains. Immunoprecipitated (ChIP lanes) or WCE (Input lanes) DNA was subjected to multiplex PCR amplification using primers specific to the promoter region of HSP12 and an intergenic region on chromosome V (no ORF). The results of this ChIP was representative of three experiments. (D) Deletion of EAF1 causes derepression of HSP12 but does not inhibit the heat shock induction of HSP12. WT (YPH499), eaf1Δ (YKB44), and eaf1Δ msn2Δ msn4 Δ (YKB1097) cells were grown in YPD at 25°C to mid-log phase, and samples were collected at 25°C and after 30 min of heat shock treatment at 39°C. Northern blots were probed with labeled DNA fragments of the HSP12 gene. The signal was quantitated using AlphaEase FC (Alpha Innotech) and normalized to the ACT1 signal. The values are averages from three independent RNA preparations, and error bars indicate standard deviations.

Eaf1 is required for NuA4 complex integrity. SDS-PAGE (gradient gel) and silver staining of NuA4 affinity purified via Esa1-TAP (YKB440) compared to an untagged strain (WT; YPH499) and Esa1-TAP purified from mutant strain backgrounds of without all seven nonessential subunits (eaf1Δ [YKB855], eaf3Δ [YKB765], eaf5Δ [YKB854], eaf6Δ [YKB766], eaf7Δ [YKB964], yaf9Δ [YKB966], and yng2Δ [YKB967] strains). Eaf6, as it does not stain well with silver, was subjected to a longer exposure (middle panel). Proteins bands in the Esa1-TAP lane were identified by MS as indicated. Anti-TAP Western blotting demonstrated the expression of Esa1-TAP in the WCE of all strains tested. Arrowheads point to proteins eliminated by subunit deletion, while squares specify additional subunits lost. The * indicates a non-NuA4, copurifying protein, Fks1. Numbers at the left are molecular masses (in kDa). The gel is representative of three purification experiments.

Eaf5 and Eaf7 form a subcomplex within NuA4. (A) Two-dimensional, hierarchical clustering of the NuA4 synthetic genetic interactions. Rows display NuA4 query genes, columns indicate the interacting deletion mutant array genes, and a red box indicates genetic interaction. The cluster includes genetic interactions for the esa1(L245P) mutant that were directly tested against all eaf1Δ genetic interactions. (B) Eaf7 interacts with NuA4 through Eaf5. Shown are results with protein extracts prepared from strains expressing the indicated tagged proteins (Esa1-TAP [YKB440], Eaf7-Myc [YKB518], Esa1-TAP Eaf7-Myc [YKB1054], and Esa1-TAP Eaf7-Myc eaf5Δ [YKB1043]) or an untagged WT (YPH499); immunoprecipitations were performed using magnetic beads coated with IgG antibodies that bind the PrA component of the TAP tag. Total protein extracts (WCE) and anti-TAP immunoprecipitates (IP) were then resolved by 7.5% SDS-PAGE and subjected to Western blot analysis with anti-Myc and anti-TAP antibodies, as indicated on the right side of the panels. α, anti. (C) Eaf7 and Eaf5 form a subcomplex in the absence of Eaf1. SDS-PAGE (gradient gel) and silver staining of NuA4 affinity purified with Eaf7-TAP in the presence (YKB442) and absence (eaf1Δ) of Eaf1 (YKB1034). Anti-TAP Western blotting was carried out on WCE as well as on 10% of the IP eluate. An anti-histone H3 (α-H3) Western blot analysis of WCE demonstrates equal protein loadings. The results are representative of three purification experiments.