(A) Effect of cerulenin on the growth of SCS3 and YFT2 gene-deletion strains. Strains were inoculated in duplicate into YPD containing cerulenin (3.2 µM) and grown with shaking at 30°C. Optical density was recorded using a Bioscreen Analyzer C at 15 min intervals. Cerulenin inhibition of fatty acid synthesis limited yeast growth and viability resulting in low cell density at saturation and slow apparent doubling times during log phase growth: Wt, 233±9 min (black); scs3Δ, 309±75 min (green); yft2Δ, 197±6 min (red); scs3Δ yft2Δ, 1088±2 min (blue). Doubling times of all strains in YPD+DMSO averaged 92±4 min and all strains reached a comparably high cell density at saturation (data not shown). (B–D) Plate phenotypes of scs3Δ, yft2Δ and double mutant (ΔΔ) strains. Equal cell numbers from 10-fold serial dilutions were spotted onto media containing drugs or inorganic compounds and scored for growth after 2 to 3 days at 30°C. Rich medium (YPD) or synthetic complete medium (SC) contained drug delivery solvent or (B) fenpropimorph (0.1 µM), (C) paromomycin (4 mg/ml), (D) CdCl₂ (25 µM). (E) Serial dilutions of scs3Δ, yft2Δ and double mutant (ΔΔ) strains were spotted on defined synthetic medium in the presence or absence of inositol (75 µM). Complementation of the Ino- phenotype in the scs3Δ yft2Δ strain is shown by an integrated copy of the human FIT2 gene under the control of the yeast TDH3 promoter. Images are from non-contiguous regions of the same plates.

(A) Genetic interaction scores (ε values) for all strains in all screens show a normal distribution centered on zero (no bias in the multiplicative model). Epsilon values determined for each array gene-deletion in combination with scs3Δ or yft2Δ or scs3Δ yft2Δ were combined, binned (bin size = 0.025) and fit to a Gaussian function. The fitted curve was centered at 0.004±6.7E-4 with σ = 0.08 and an adjusted r² = 0.997. Similar quality fits were obtained when data from individual query screens were analyzed separately. (B) Overlapping genetic interactions between SCS3 and YFT2. The Venn diagram shows the intersection of genes exhibiting aggravating or alleviating interactions in screens with the three query strains. The stringent criteria for inclusion in this comparison required a size difference between query and control screens of >40 pixels and a p value<0.01 (see Material and Methods). Interactions with 221 genes were found in two or more screens. (C) Pairwise comparison of genetic interaction scores within the set of 62 genetic interactions identified in screens of the individual SCS3 and YFT2 deletion strains (see panel b). Similar comparisons for the other two pairwise combinations are shown in Figure S4.

The frequencies of 17 broad GO biological process terms [42] represented among the genes on the deletion array were compared with the 636 genes that interacted with SCS3 and/or YFT2. Functional enrichment was calculated by hypergeometric distribution.

(A) The heat map shows array genes that interact genetically with deletions of SCS3, YFT2 or both genes. The strength of the interactions is indicated by the colorbar. Array genes are grouped according to the transcription complexes or activities/pathways that define their function as indicated above the heat map. Supporting interactions that are not represented in the high stringency set were identified using relaxed criteria and include UME6 and LGE1 (p = 0.02), DEP1, which had a 39 pixel size difference just below the 40 pixel cutoff and UBP8 (ε = −0.09 in the triple mutant). Genetic interactions with EAF1 were identified with OPI7 which encodes a dubious ORF that overlaps the N-terminus and promoter of EAF1. (B) Genetic relationships between transcription components that interact with SCS3 and YFT2 (panel A) were determined by searching the DryGin database [78]. This resulted in the identification of three linked triplet genetic motifs. Each edge connecting the complexes in this network represents a minimum of three interactions, either all alleviating (red) or all aggravating (green). The same color scheme is used for nodes which indicate the type of interaction of the different complexes with the SCS3 YFT2 double deletion strain (as in panel A). (C) Analysis of the unfolded protein response (UPR) pathway in SCS3 and YFT2 gene-deletion strains. Northern analysis of HAC1 mRNA (reporting both unspliced Hac1^u and spliced Hac1ⁱ forms), stable U3 snRNA and RPL28 mRNA was performed on RNA from early log phase cultures (SC-medium) of wild-type and double deletion strains before and after DTT treatment. The Hac1 band intensities were used to calculate the distribution between spliced and unspliced forms. Normalized HAC1 mRNA band intensities were used to determine the level of transcription relative to the wild-type untreated strain. The RPL28 mRNA levels are included to report the global cellular stress response to DTT treatment. Results from duplicate independent samples of wild-type and double deletion strains are reported under the representative autoradiogram.

(A) Wild-type and gene-deletion strains were crossed to an array of temperature-sensitive strains (gift from Charlie Boone) and haploid single, double or triple mutant strains generated by SGA methods were printed at the permissive temperature of 22°C. Images from the final selection plates show quadruplicate colonies for each array strain including a wild-type SEC13 control strain (left panels). Strains containing the sec13-1 mutation are boxed in red and are flanked on either side by other temperature-sensitive strains that are not synthetic with SCS3 or YFT2. (B) Wild-type and gene-deletion strains were spotted onto SC media with no inositol, 100 µM inositol, and 100 µM inositol plus 2.5 mM DTT. Plates were photographed at 2 days.

(A) HEK293 (human embryonic kidney cells) were transfected with empty pcDNA3.1 vector (Mock), or expression vectors for H. sapiens FIT2 (hFIT2), S. cerevisiae SCS3, or S. cerevisiae YFT2. Cells were stained for lipid droplets (LDs, green) using BODIPY 493/503 and nuclei using Hoechst 33342 (blue). Images are representative of two different experiments. Scale bar, 10 µm. (B) Early log phase cultures of a wild-type yeast strain and a strain lacking both SCS3 and YFT2 were fixed with formaldehyde and stained with BODIPY 493/503 (0.5 µg/ml) in PBS. Fluorescence was recorded using optical sectioning (100× magnification) and the images were combined into a single projection. Analysis of ∼300 cells of each strain yielded 7.1±2.5 and 7.9±3.6 LDs/cell for the wild-type and double mutant strains, respectively. Similar numbers were obtained for the single mutant strains.

Clustergrams show interactions representing unique, shared and antagonizing functions of SCS3 and YFT2 in the set of 636 genes defined by stringent selection criteria (see text). (A) Aggravating interactions identified for SCS3 but not YFT2 (ε>−0.05). (B) Aggravating interactions identified for YFT2 but not SCS3 (ε>−0.05). (C) Aggravating interactions identified for the SCS3 YFT2 double mutant but not for either gene individually (ε>−0.05). (D) Alleviating interactions identified for the SCS3 YFT2 double mutant where the individual genes showed an aggravating or no interaction (ε<0).

Cross-talk between the different pathways is illustrated by the central role of PI synthesis and DAG production/utilization. The arrangement of the steps in the Kennedy and CDP-DAG pathways for the synthesis of PC is modified from Carman and Han [47]. The role of the ERMES complex in transporting PS from the ER to the mitochondria for conversion to PE and its return to the ER [50] is represented by a red box. Genes encoding all four components of the complex (MDM10, MDM12, MDM34 and MMM1) exhibited alleviating interactions when both SCS3 and YFT2 were deleted (Table S1; the alleviating interaction between MDM10 and the SCS3 YFT2 double deletion was identified with relaxed criteria, 38 pixel size difference, p = 0.006, ε = 0.18). The synthesis of PE and PC from lysoPE and lyso PC is omitted for clarity. The steps in the synthesis of inositol phosphates are modified from York [52]. The pathway for the synthesis of complex sphingolipids is modified from Dickson [53]. Steps involving genes that interact genetically with SCS3 and/or YFT2 show the gene name above three boxes which indicate the relative strength and sign of the interactions. Aggravating (green) and alleviating (red) interactions are ordered from left to right for the scs3Δ, yft2Δ and scs3Δ yft2Δ strains using the values in Table S1. A colorbar shows the intensity of the interactions. Phosphatidic acid, PA; diacylglycerol, DAG; triglyceride, TG; lipid droplet, LD, Gro, glycerol; DHAP, dihydroxyacetone phosphate; Glu, glucose; Ins, inositol; PME, phosphatidyl monomethylethanolamine; PDE, phosphatidyl dimethylethanolamine; Etn, ethanolamine; Cho, choline., dihydrosphingosine, DHS; phytosphingosine, PHS; inositolphosphoceramide, IPC; mannosyl-inositolphosphoceramide, MIPC; mannosyl-diinositolphosphoceramide, M(IP)2.

(A) The heat map shows ribosomal protein genes that interact genetically with deletions of SCS3, YFT2 or both genes (data were extracted from Table S1). The strength of the interactions is indicated by the colorbar. (B) Triplet genetic motif representing mutually alleviating interactions (red nodes and edges) between multiple RP genes, SCS3/YFT2 and CHO2. The thickness of the edges reflects the number of interactions. They include 25 alleviating or suppressing interactions between RP genes and SCS3/YFT2 (from panel A), five of which were also alleviating with CHO2 (i.e. RPS29B, RPS18B, RPS1B, RPL43A and RPS19B [42]) and the interaction of SCS3/YFT2 with CHO2 (see Figure 6).

(A) Northern analysis of wild-type and gene-deletion strains grown in low inositol media. Cells were grown overnight to early log phase at 30°C in the presence of 10 µM inositol before RNA preparation. RNA from the same strains grown in 100 µM inositol is included for comparison. Detection and quantitation of INO1 and HAC1 mRNAs and U3 snRNA was as described in Figure 7c. The amount of INO1 mRNA normalized for U3 snRNA is expressed relative to the wild-type strain as indicated under each lane. The extent of Hac1 splicing is expressed as % of total HAC1 mRNA, normalized for U3 snRNA and is indicated under each lane. Note that deletion of OPI1 results in constitutive INO1 transcription and inositol prototrophy for the SCS3 and YFT2 deletion strains (see also Figure 9b), thereby alleviating the UPR response to low inositol (i.e. no Hac1 splicing in opi1Δ strains). (B) Data from three independent low inositol experiments, as shown above, are plotted ± standard deviation. The left panel shows the relative level of Ino1 mRNA and the right panel the extent of HAC1 mRNA splicing for each strain.