Growth of wild type (YPH499) strain (OD₆₀₀) as a function of time in hours. Wild type yeast cells were grown in YPD liquid culture with or without C16:0 PAF or C16:0 lyso-PAF as indicated. Growth curves were performed in triplicate and the error bars represent 1 standard deviation.

(A) Silver-stained 4-12% NuPAGE gels of the protein network associated with Srf1-TAP (YKB2270). The arrow indicates the position of Srf1-TAP, the * indicates the position of Spo14 and IgG indicates the position of the immunoglobin. (B) An aliquot of the purification was used for Western blot analysis using anti-TAP-tagged antibodies (α-TAP). (C) Summary of mass spectrometry analysis of the proteins identified in the Srf1-TAP mChIP with high confidence. Spo14 and Srf1-TAP were the two most abundant proteins identified in the Srf1-TAP purification.

(A) Extracts of the particulate fraction were prepared from wild type (YPH500), spo14Δ (YKB2076), srf1Δ (YKB1164) and srf1Δspo14Δ (YKB2080) cells and incubated with BODIPY labeled glycerophosphocholine (BPC) as described in Materials and Methods. Purified Streptomyces chromofuscus (S. chromofuscus) PLD was included as a positive control for the production of phosphatidic acid (PA). In addition, since Spo14, but not S. chromofuscus PLD, can convert n-butanol to phosphatidylbutanol (PBt), PLD activity was also assessed by including n-butanol (1% v/v) in the reaction mixture. Reactions were allowed to proceed for 40 min at 30°C before separating reaction products by TLC. The absence of Srf1 resulted in a complete loss of detectable PLD activity similar to that observed in spo14Δ mutant strains. (B) Strains listed above were transformed with either pRS415, an empty vector (vector), or pME940, a CEN vector expressing HA-SPO14 (SPO14) [24]. Particulate (P) and cytosolic (C) fractions were assessed for PLD as described above. Deletion of SRF1 does not result in altered partitioning of PLD activity into the cytosolic fraction and expression of HA-Spo14 does not rescue PLD activity in srf1Δ strains. (C) Particulate and cytosolic fractions prepared as in (B) were separated by SDS-PAGE and analyzed by immunoblotting using anti-HA antibodies. HA-Spo14 remained associated with the particulate fraction although protein levels were observed to be moderately reduced in srf1Δ strains. Representative images are shown (n = 3).

Undifferentiated N2a cells were treated with vehicle (0.1% DMSO + 0.1% EtOH), or PLD inhibitor (5 µM VUO155056 + 0.1% DMSO) in serum-free media containing 0.025% BSA and cell survival assessed by Live/Dead assay at 24 h. Data are expressed as % survival standardized to control cultures maintained in complete media (dotted line). There was no significant impact of treatment conditions, vehicle treatment, or PLD inhibition on N2a survival (Student's t-test, p>0.05, insert). To assess impact of PLD inhibition on C16:0 PAF sensitivity, cultures were treated with vehicle (0.1% DMSO + 0.01% EtOH), PAF (1 µM C6:0 PAF + 0.1% DMSO), or PAF + PLD inhibitor (1 µM C16:0 PAF + 5 µM VUO155056). Data are expressed as % survival standardized to vehicle-treated cultures. C16:0 PAF sensitivity was significantly enhanced by PLD inhibition (ANOVA, post-hoc Student-Newman-Keuls multiple comparisons test, *p<0.05, **p<0.01).

(A) Chemical genetic interaction network for C16:0 PAF and C16:0 lyso-PAF. Deletion mutants that displayed a hypersensitivity to either 40 µM C16:0 PAF and/or C16:0 lyso-PAF (Bonferroni corrected p-value <0.04) are represented by nodes that are color coded according to their SGD cellular roles and/or through review of literature. Interactions are represented by edges. (B) Plot of the differential sensitivity (in mins)  =  α_g-PAF−α_g-L-PAF for wild type (wt) along with the 11 strains identified that had differential sensitivity to one of the PAF species (Bonferroni corrected p-value <0.04). Strains that are hypersensitive to C16:0 PAF have positive differential sensitivity, while strains that are hypersensitive to C16:0 lyso-PAF have negative differential sensitivity.

(A) Sensitivity of srf1Δ to C16:0 PAF is suppressed by spo14Δ. Wild type (YPH500), spo14Δ (YKB2076), srf1Δ (YKB1164) and srf1Δspo14Δ (YKB2080) cells were plated in five-fold serial dilution onto YPD or YPD supplemented with C16:0 PAF as indicated. The plates were incubated for 3 days at 30°C. (B) Overexpression of SPO14 does not alleviate the sensitivity of srf1Δ cells to C16:0 PAF. Wild type (YPH500), srf1Δ (YKB1164), and spo14Δ (YKB2076) cells transformed with pRS415 (vector), pME962 (SPO14), pME1096 (GFP-SPO14) or pME1130 (GFP-SPO14^K–H)[24] were plated in five-fold serial dilution onto SD-Leu or SD-Leu supplemented with C16:0 PAF as indicated. The plates were incubated for 2 days at 30^oC. The growth of all strains tested was moderately improved on minimal media compared with YPD at equivalent concentrations of C16:0 PAF. This is reflected by the observed differences in growth for both the wild type and srf1Δ strain when comparing panels (A) and (B).

(A) In untreated (Untx) live mid-logarithmic phase cells grown at 30°C GFP-Spo14, expressed from a 2 µ plasmid (pME1096), was found to localize diffusely within the cytoplasm with modest accumulation at the peripheral membrane in both wild type (WT, YPH500) and srf1Δ (YKB1164) cells. Addition of vehicle (Ethanol, EtOH 0.4% v/v) did not result in any changes in GFP-Spo14 localization in comparison to untreated cells. In contrast 15 minutes of C16:0 PAF (PAF, 40 µM) treatment resulted in a loss of peripheral membrane staining and increased aggregation of GFP-Spo14 into distinct foci in wild type (WT) but not srf1Δ cells. (B) Foci were quantified in wild type (YAM282-2) or srf1Δ (YKB2472) cells expressing GFP-Spo14 under the control of the CUP1 promoter as described in Materials and Methods and expressed as a percentage of the total number of cells. A minimum of 300 cells for each condition from two independent experiments (n = 2) were assessed and presented as the mean +/− SD. (C) C16:0 PAF treatment (PAF) resulted in redistribution of a GFP-tagged phosphatidic acid binding protein, GFP-Q2 [31]. Reduced signal was associated with plasma membrane following PAF treatment in comparison to untreated (Untx) and ethanol (EtOH) treated cells. (D) Treatment with ethanol (EtOH, 0.4% v/v), C16:0 PAF or C16:0 lyso-PAF (40 µM) for 30 minutes did not significantly impact the hydrolysis of BODIPY labeled glycerophosphocholine (BPC) to phosphatidic acid (PA) or phosphatidylbutanol (PBt) in wild type cells (YPH500) transformed with empty vector (pRS415, vector) or GFP-SPO14 (pME1096, SPO14). PLD activity remained associated with the particulate (P) fraction under all test conditions. C, cytosolic fraction. Representative image of 3 experiments are shown. (E) Treatment with C16:0 PAF did not affect the expression or partitioning of GFP-Spo14. Particulate (P) and cytosolic (C) fractions prepared as in (D) were separated by SDS-PAGE and analyzed by immunoblotting using anti-GFP antibodies (α GFP). Representative images are shown (n = 2).