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Srf1 is a novel regulator of phospholipase D activity and is essential to buffer the toxic effects of C16:0 platelet activating factor.

Kennedy MA, Kabbani N, Lambert JP, Swayne LA, Ahmed F, Figeys D, Bennett SA, Bryan J, Baetz K - PLoS Genet. (2011)

Bottom Line: As C16:0 PAF is a naturally occurring lipid involved in cellular signaling, it is likely that mechanisms exist to protect cells against its toxic effects.Deletion of YDL133w, a previously uncharacterized gene which we have renamed SRF1 (Spo14 Regulatory Factor 1), resulted in the greatest differential sensitivity to C16:0 PAF over C16:0 lyso-PAF.Though C16:0 PAF treatment does not impact hydrolysis of phosphatidylcholine in yeast, C16:0 PAF does promote delocalization of GFP-Spo14 and phosphatidic acid from the cell periphery.

View Article: PubMed Central - PubMed

Affiliation: Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Canada.

ABSTRACT
During Alzheimer's Disease, sustained exposure to amyloid-β₄₂ oligomers perturbs metabolism of ether-linked glycerophospholipids defined by a saturated 16 carbon chain at the sn-1 position. The intraneuronal accumulation of 1-O-hexadecyl-2-acetyl-sn-glycerophosphocholine (C16:0 PAF), but not its immediate precursor 1-O-hexadecyl-sn-glycerophosphocholine (C16:0 lyso-PAF), participates in signaling tau hyperphosphorylation and compromises neuronal viability. As C16:0 PAF is a naturally occurring lipid involved in cellular signaling, it is likely that mechanisms exist to protect cells against its toxic effects. Here, we utilized a chemical genomic approach to identify key processes specific for regulating the sensitivity of Saccharomyces cerevisiae to alkyacylglycerophosphocholines elevated in Alzheimer's Disease. We identified ten deletion mutants that were hypersensitive to C16:0 PAF and five deletion mutants that were hypersensitive to C16:0 lyso-PAF. Deletion of YDL133w, a previously uncharacterized gene which we have renamed SRF1 (Spo14 Regulatory Factor 1), resulted in the greatest differential sensitivity to C16:0 PAF over C16:0 lyso-PAF. We demonstrate that Srf1 physically interacts with Spo14, yeast phospholipase D (PLD), and is essential for PLD catalytic activity in mitotic cells. Though C16:0 PAF treatment does not impact hydrolysis of phosphatidylcholine in yeast, C16:0 PAF does promote delocalization of GFP-Spo14 and phosphatidic acid from the cell periphery. Furthermore, we demonstrate that, similar to yeast cells, PLD activity is required to protect mammalian neural cells from C16:0 PAF. Together, these findings implicate PLD as a potential neuroprotective target capable of ameliorating disruptions in lipid metabolism in response to accumulating oligomeric amyloid-β₄₂.

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Srf1 regulates phospholipase D catalytic activity in mitotic cells.(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).
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pgen-1001299-g005: Srf1 regulates phospholipase D catalytic activity in mitotic cells.(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).

Mentions: Despite the limited impact on sporulation, there is a possibility that Srf1 may regulate PLD activity in mitotic cells. Therefore, we sought to examine whether Srf1 could modify Spo14 catalytic activity or localization in mitotic cells. The former possibility was directly assessed by measuring PLD activity in particulate fractions prepared from wild type and mutant strains using a previously described methodology employing a fluorescently labeled phosphatidylcholine derivative as a PLD substrate [28], [29]. Production of PA and phosphatidyl butanol (PBt), a product of transphosphatidylation, was evident in wild type particulate preparations but was completely absent in srf1Δ, spo14Δ and srf1Δspo14Δ mutant strains (Figure 5A). This result indicates that Srf1 may contribute to particulate-associated PLD catalytic activity in mitotic cells. As we have demonstrated that Spo14 physically interacts with Srf1, a predicted transmembrane protein, we sought to determine whether the deletion of SRF1 promotes the loss of Spo14 from the particulate fraction. To test this, particulate and cytosolic fractions were prepared from strains transformed with either an empty vector control or a plasmid expressing HA-tagged SPO14 [24]. The absence of PLD activity in srf1Δ strains is not a consequence of altered partitioning of Spo14 catalytic activity between particulate and cytosolic fractions in these cells as catalytic activity was absent from both fractions (Figure 5B). Furthermore, western blot analysis demonstrates that HA-Spo14 remains associated with the particulate fraction independent of Srf1 (Figure 5C). Interestingly, HA-Spo14 protein levels are consistently reduced in srf1Δ mutants (∼30% less HA-Spo14 as determined by densitometry). However, the absence of detectable PLD activity cannot be fully explained by the exclusion of Spo14 from the particulate fraction or a reduction in Spo14 protein levels (Figure 5C) thereby further implicating a biological role for Srf1 in regulating Spo14 catalytic activity during mitosis.


Srf1 is a novel regulator of phospholipase D activity and is essential to buffer the toxic effects of C16:0 platelet activating factor.

Kennedy MA, Kabbani N, Lambert JP, Swayne LA, Ahmed F, Figeys D, Bennett SA, Bryan J, Baetz K - PLoS Genet. (2011)

Srf1 regulates phospholipase D catalytic activity in mitotic cells.(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).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037409&req=5

pgen-1001299-g005: Srf1 regulates phospholipase D catalytic activity in mitotic cells.(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).
Mentions: Despite the limited impact on sporulation, there is a possibility that Srf1 may regulate PLD activity in mitotic cells. Therefore, we sought to examine whether Srf1 could modify Spo14 catalytic activity or localization in mitotic cells. The former possibility was directly assessed by measuring PLD activity in particulate fractions prepared from wild type and mutant strains using a previously described methodology employing a fluorescently labeled phosphatidylcholine derivative as a PLD substrate [28], [29]. Production of PA and phosphatidyl butanol (PBt), a product of transphosphatidylation, was evident in wild type particulate preparations but was completely absent in srf1Δ, spo14Δ and srf1Δspo14Δ mutant strains (Figure 5A). This result indicates that Srf1 may contribute to particulate-associated PLD catalytic activity in mitotic cells. As we have demonstrated that Spo14 physically interacts with Srf1, a predicted transmembrane protein, we sought to determine whether the deletion of SRF1 promotes the loss of Spo14 from the particulate fraction. To test this, particulate and cytosolic fractions were prepared from strains transformed with either an empty vector control or a plasmid expressing HA-tagged SPO14 [24]. The absence of PLD activity in srf1Δ strains is not a consequence of altered partitioning of Spo14 catalytic activity between particulate and cytosolic fractions in these cells as catalytic activity was absent from both fractions (Figure 5B). Furthermore, western blot analysis demonstrates that HA-Spo14 remains associated with the particulate fraction independent of Srf1 (Figure 5C). Interestingly, HA-Spo14 protein levels are consistently reduced in srf1Δ mutants (∼30% less HA-Spo14 as determined by densitometry). However, the absence of detectable PLD activity cannot be fully explained by the exclusion of Spo14 from the particulate fraction or a reduction in Spo14 protein levels (Figure 5C) thereby further implicating a biological role for Srf1 in regulating Spo14 catalytic activity during mitosis.

Bottom Line: As C16:0 PAF is a naturally occurring lipid involved in cellular signaling, it is likely that mechanisms exist to protect cells against its toxic effects.Deletion of YDL133w, a previously uncharacterized gene which we have renamed SRF1 (Spo14 Regulatory Factor 1), resulted in the greatest differential sensitivity to C16:0 PAF over C16:0 lyso-PAF.Though C16:0 PAF treatment does not impact hydrolysis of phosphatidylcholine in yeast, C16:0 PAF does promote delocalization of GFP-Spo14 and phosphatidic acid from the cell periphery.

View Article: PubMed Central - PubMed

Affiliation: Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Canada.

ABSTRACT
During Alzheimer's Disease, sustained exposure to amyloid-β₄₂ oligomers perturbs metabolism of ether-linked glycerophospholipids defined by a saturated 16 carbon chain at the sn-1 position. The intraneuronal accumulation of 1-O-hexadecyl-2-acetyl-sn-glycerophosphocholine (C16:0 PAF), but not its immediate precursor 1-O-hexadecyl-sn-glycerophosphocholine (C16:0 lyso-PAF), participates in signaling tau hyperphosphorylation and compromises neuronal viability. As C16:0 PAF is a naturally occurring lipid involved in cellular signaling, it is likely that mechanisms exist to protect cells against its toxic effects. Here, we utilized a chemical genomic approach to identify key processes specific for regulating the sensitivity of Saccharomyces cerevisiae to alkyacylglycerophosphocholines elevated in Alzheimer's Disease. We identified ten deletion mutants that were hypersensitive to C16:0 PAF and five deletion mutants that were hypersensitive to C16:0 lyso-PAF. Deletion of YDL133w, a previously uncharacterized gene which we have renamed SRF1 (Spo14 Regulatory Factor 1), resulted in the greatest differential sensitivity to C16:0 PAF over C16:0 lyso-PAF. We demonstrate that Srf1 physically interacts with Spo14, yeast phospholipase D (PLD), and is essential for PLD catalytic activity in mitotic cells. Though C16:0 PAF treatment does not impact hydrolysis of phosphatidylcholine in yeast, C16:0 PAF does promote delocalization of GFP-Spo14 and phosphatidic acid from the cell periphery. Furthermore, we demonstrate that, similar to yeast cells, PLD activity is required to protect mammalian neural cells from C16:0 PAF. Together, these findings implicate PLD as a potential neuroprotective target capable of ameliorating disruptions in lipid metabolism in response to accumulating oligomeric amyloid-β₄₂.

Show MeSH
Related in: MedlinePlus