ARTICLE ADDENDUM Plant Signaling & Behavior 10:10, e1065367; October 2015; © 2015 Taylor and Francis Group, LLC
Regulation of endomembrane biogenesis in arabidopsis by phospatidic acid hydrolase Christian P Craddocky, Nicolette Adamsz, Fiona M Bryant, Smita Kurup, and Peter J Eastmond* Department of Plant Biology and Crop Science; Rothamsted Research, Harpenden; Hertfordshire, UK y
Present address: Horticultural Plant Biology and Metabolomics Research Center; Fujian Agriculture and Forestry University; Fuzhou, China
z
Present address: Center for Proteomic and Genomic Research; Cape Town, South Africa
C
Keywords: Arabidopsis, cell cycle, membrane biogenesis, phosphatidic acid phosphohydrolase, phosphatidylcholine biosynthesis *Correspondence to: Peter J Eastmond; Email: peter. [email protected] Submitted: 06/01/2015 Accepted: 06/18/2015
oordination of membrane lipid biosynthesis is important for cell function during plant growth and development. Here we summarize our recent work on PHOSPHATIDIC ACID PHOSPHOHYDROLASE (PAH) which suggests that this enzyme is a key regulator of phosphaticylcholine (PC) biosynthesis in Arabidopsis thaliana. Disruption of PAH activity elevates phosphatidic acid (PA) levels and stimulates PC biosynthesis and biogenesis of the endoplasmic reticulum (ER). Furthermore, the activity of PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE (CCT), which is the key enzyme controlling the rate of PC biosynthesis, is directly stimulated by PA and expression of a constitutively active version of CCT replicates the effects of PAH disruption. Hence PAH activity can control the abundance of PA, which in turn can modulate CCT activity to govern the rate of PC biosynthesis. Crucially it is not yet clear how PAH activity is regulated in Arabidopsis but there is evidence that PAH1 and PAH2 are both phosphorylated and further work will be required to investigate whether this is functionally significant.
http://dx.doi.org/10.1080/15592324.2015.1065367 Addendum to: Craddock CP, Adams N, Bryant FM, Kurup S, Eastmond PJ. PHOSPHATIDIC ACID PHOSPHOHYDROLASE regulates phosphatidylcholine biosynthesis in Arabidopsis by phosphatidic acid-mediated activation of CTP: PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE activity. Plant Cell 2015; 27:1251–1264
www.tandfonline.com
Phospholipids are the major structural components of most plant membranes, where their amphipathic properties contribute to the formation of lipid bilayers.1 The rate of phospholipid biosynthesis in plant cells must be tightly regulated so as to respond to fluctuations in demand. Plant Signaling & Behavior
Cell expansion and proliferation (division) are the 2 most important mechanisms underlying plant growth and both rely on membrane biogenesis. For example cell expansion requires production of plasma membrane while, during the mitotic phase of the cell cycle, division of the nucleus also demands rapid production of nuclear-endoplasmic reticulum (ER) membrane.2,3 Our current understanding of how phospholipid biosynthesis and membrane biogenesis are regulated in plant cells is relatively rudimentary and there has been little focus on how these processes are coordinated with growth or are integrated with the environmental and developmental signaling pathways that govern it. In contrast to plants, regulation of phospholipid biosynthesis and nuclear-ER membrane biogenesis has been studied relatively extensively in the yeast Saccharomyces cerevisiae. In this system it has been shown to be regulated by several kinases including the cyclin-dependent kinases (CDKs), cell division cycle 282 (Cdc28p) and phosphate metabolism 853 (Pho85p), which together allow for regulation of a host of cellular processes in response to environmental stimuli. Cdc28p and Pho85p phosphorylate and inactivate a key regulator of phospholipid biosynthesis called phosphatidic acid phosphohydrolase (Pah1p).2,3 Pah1p is an amphitropic enzyme that can interconvert between a phosphorylated soluble inactive form and a dephosphorylated membrane-bound active form2. Recruitment of phosphorylated Pah1p to the nuclear-ER membrane e1065367-1
Figure 1. Role of PAH in Arabidopsis ER membrane biogenesis. (A) Morphology of the ER in root cortical cells of wild type and pah1 pah2 double mutant imaged by laser-scanning confocal microscopy of a lumen-targeted GFP marker.8 Scale bar D 5 mm. (B) A model depicting how PC biosynthesis is linked to PAH activity.8 Repression of PAH activity leads to accumulation of its substrate PA. The increase in PA abundance relative to PC stimulates CCT activity which provides more CDP-Cho substrate for PC biosynthesis.8 CDP-Cho and not DG is most limiting for PC biosynthesis under normal circumstances and therefore the rate increases.8 P-Cho provision for CCT also increases because depletion leads to de-repression of PEAMT.8 PEAMT, PHOSPHOETHANOLAMINE N-METHYLTRANSFERASE; PAH, PHOSPHATIDIC ACID PHOSPHOHYDROLASE; G3P, glycerol 3 phosphate; LPA, lysophosphatidic acid; PA, phosphatidic acid; DG, diacylglycerol; PC, phosphatidylcholine; acyl-CoA, fatty acyl-coenzymeA; CDP-Cho, cytidine diphosphate-choline; P-Cho, phosphocholine; P-EA, phosphoethanolamine.
depends on the nuclear envelope morphology 1 – sporulation 7 (Nem1p– Spo7p) protein phosphatase complex, which dephosphorylates the enzyme and allows it to associate with the membrane via a short N-terminal amphipathic helix.2,4 Pah1p membrane disassociation and inactivation, resulting from phosphorylation by Cdc28p and Pho85p leads to elevated levels of the enzymes’ substrate phosphatidic acid (PA), expansion of the nuclear-ER membrane and enhanced expression of key phospholipid biosynthetic genes.2,3,4 These genes contain upstream activating sequence inositolresponsive (UASINO) elements in their promoters.5 Their expression is induced by interaction of the inositol requiring 2 – inositol requiring 4 (Ino2p-Ino4p) transcription factor complex with UASINO elements, but blocked by interaction of the repressor protein overproducer of inositol 1 (Opi1p) with Ino2p.5 PA levels control the Opi1p-mediated repression of gene expression because PA tethers Opi1p at the nuclear–ER membrane, together with a vesicle-associated protein homolog called suppressor of choline sensitivity 2.6
e1065367-2
In the model plant Arabidopsis thaliana, we have shown that disruption of PAH activity also stimulates net phosphatidylcholine (PC) biosynthesis and massive proliferation of the ER (Fig. 1A), and that this response positively correlates with an accumulation of the enzymes’ substrate PA.7,8 However, we have also found that changes in PAH activity in Arabidopsis have a very limited effect on global gene expression.8 The only differentially expressed gene that is currently known to play a role in PC biosynthesis is PHOSPHOETHANOLAMINE N-METHYLTRANSFERASE1 (PEAMT1).7,8 In Arabidopsis, choline is made by the sequential methylation of ethanolamine (EA), predominantly in its phosphorylated form (P-EA), to produce phosphocholine (P-Cho), which is then converted to PC by the nucleotide pathway using cytidine triphosphate (CTP) phosphocholine cytidylyltransferase (CCT) and aminoalcohol phosphotransferase.9 (Fig. 1B). The sequential methylation of P-EA catalyzed by PEAMT is the first committed step in PC biosynthesis.9 We have shown that PEAMT1 is induced at the level of transcription in leaves but not roots of PAHdeficient Arabidopsis plants and therefore
Plant Signaling & Behavior
cannot account for the enhanced PC biosynthesis observed in both tissues.7,8 PEAMT1 is negatively regulated by PCho both at the level of translation10 and enzyme activity.9 This provides a mechanism to balance supply of P-Cho with demand for PC biosynthesis that does not rely on transcription (Fig. 1B). Nevertheless PEAMT1 does still respond transcriptionally to PAH disruption in leaves and further work will be required to uncover the mechanism. At present it is not known whether this response depends on the catalytic activity of PAH or on the protein directly.8 In plants radiolabeling experiments have suggested that CCT activity is most likely to be rate-limiting for PC biosynthesis.7 We found that disruption of CCT activity suppresses the increase in PC biosynthesis caused by disruption of PAH activity.8 We also found that CCT activity is increased when PAH is disrupted but transcript and protein levels change very little.8 CCT protein also remains associated with microsomal membranes.8 In mammals CCTa is an amphitropic enzyme that can interconvert between a soluble inactive form and a membranebound active form.11 Furthermore, when
Volume 10 Issue 10
CCTa is membrane-bound, its activity is stimulated by the presence of free fatty acids (FFA) and ionic lipids such as PA.11 We found that the activity of recombinant Arabidopsis CCT1 is also stimulated by PC vesicles containing FFA or PA and that this increases the Vmax of the enzyme with little effect on the Km for either substrate (P-Cho and CTP).8 Given that disruption of PAH activity leads to accumulation of PA we hypothesized that direct activation of CCT by membranes enriched with PA could be the prime mechanism that stimulates PC biosynthesis in PAH-deficient Arabidopsis plants.8 (Fig. 1B). In mammalian CCTa, removal of the amphipathic C-terminal lipid binding domain (‘M’) stops membrane interaction and also causes the enzyme to become lipid insensitive and constitutively active.12,13 Arabidopsis CCT1 contains a shorter predicted amphipathic a-helical region that has been suggested to play a similar role. We found that removal of this domain from CCT1 also resulted in an enzyme that is constitutively active and unaffected by vesicles containing FFA or PA.8 Expression of this truncated CCT1 protein in Arabidopsis resulted in an increase in PC biosynthesis and ER membrane proliferation while expression of the intact CCT1 protein did not.8 These data supported the hypothesis that CCT activity modulates the production of PC in response to the relative abundance of PA (and possibly other anionic lipids) in the ER. A change in PAH activity therefore provides a potential mechanism to regulate membrane biogenesis in Arabidopsis.8 (Fig. 1). In yeast there is already evidence to show that Pah1p activity is regulated by CDKs and that its phosphorylation status modulates phospholipid biosynthesis and nuclear-ER membrane biogenesis through control of PA levels.2,3,4 A key question that remains to be answered in Arabidopsis is whether PAH activity is also regulated to govern membrane biogenesis and if so how is this achieved? Yeast Cdc28p phosphorylates 3 sites on Pah1p while Pho85p targets 7 and exerts a much stronger effect on PAH activity.2,3 Arabidopsis contains a number of kinases that share sequence similarity with Pho85 but CDKA;1 is the closest homolog to both
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Cdc28p and Pho85p. Furthermore, CDKA;1 is the only Arabidopsis kinase to contain the PSTAIRE cyclin-binding motif.14 that is also found in both Cdc28p and Pho85p. CDKA;1 has been shown to complement the cdc28D mutant.14 CDKA;1 activity is greatest during the entry into mitosis, suggesting a major function at this stage in the cell cycle in Arabidopsis.14 Unlike yeast cdc28D, null mutants in CDKA;1 are viable but display cell cycle defects, particularly in S phase entry, and exhibit severely retarded growth.14 Phospho-proteomics studies have found that PAH1 and PAH2 are both phosphorylated at multiple sites.15 Interestingly this includes possible CDKA;1 target sites with the minimal consensus S/T-P (S162 in PAH1 and S524 in PAH2).15 In yeast Pah1p phosphorylation status controls membrane association and abundance, as well as catalytic activity.2,3 It is not known whether this is the case in Arabidopsis. We have previously shown that N-terminal green fluorescent protein (GFP) fusion proteins of PAH1 and PAH2 are predominantly cytosolic.7 However, work in yeast has subsequently established that the N-terminus of Pah1p contains a short amphipathic helix that’s responsible for membrane binding.2,3 Hence the cytosolic localization of N-terminal GFP-PAH fusions may be misleading. Indeed sequence analysis suggests that the amphipathic helix found in yeast Pah1p might also be conserved in Arabidopsis. Arabidopsis also contains proteins with weak homology to the Nem1p catalytic subunit of the Nem1p–Spo7p protein phosphatase complex, which recruits Pah1p to the nuclear-ER membrane in yeast.2,3 In conclusion changes in PAH activity can strongly affect the rate of phospholipid synthesis and membrane proliferation in both yeast.2,3,4 and Arabidopsis.7,8 In both systems PAH exerts this effect through controlling the level of PA.5,8 In yeast PA regulates the expression of multiple genes involved in phospholipid metabolism,5,6 while in Arabidopsis the impact on global transcription is comparatively very limited.8 Instead, PA appears to regulate PC biosynthesis at the biochemical level in Arabidopsis by directly stimulating the activity of CCT, which is the rate-
Plant Signaling & Behavior
limiting enzyme for the pathway.8 In yeast Pah1p activity is highly regulated by its phosphorylation status and the protein is a substrate for the CDKs Cdc28p and Pho85p. This provides a mechanism to couple phospholipid synthesis and membrane biogenesis directly to cell cycle regulation. How PAH is regulated in Arabidopsis is currently not known, but might also involve phosphorylation by CDKs. Addressing this question will be important for our fundamental understanding of membrane biogenesis in plants.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Funding
Support for this work was provided by the UK Biotechnology and Biological Sciences Research Council through grant BB/G009724/1 and BBS/E/C/00005207. References 1. Ohlrogge J, Browse J. Lipid biosynthesis. Plant Cell 1995; 7:957-70; PMID:7640528; http://dx.doi.org/ 10.1105/tpc.7.7.957 2. Choi HS, Su WM, Morgan JM, Han GS, Xu Z, Karanasios E, Siniossoglou S, Carman GM. Phosphorylation of phosphatidate phosphatase regulates its membrane association and physiological functions in Saccharomyces cerevisiae: identification of SER(602), THR(723), AND SER(744) as the sites phosphorylated by CDC28 (CDK1)-encoded cyclin-dependent kinase. J Biol Chem 2011; 286:1486-98; PMID:21081492; http://dx.doi.org/10.1074/jbc.M110.155598 3. Choi HS, Su WM, Han GS, Plote D, Xu Z, Carman GM. Pho85p-Pho80p phosphorylation of yeast Pah1p phosphatidate phosphatase regulates its activity, location, abundance, and function in lipid metabolism. J Biol Chem 2012; 287:11290-301; PMID:22334681; http://dx.doi.org/10.1074/jbc.M112.346023 4. Karanasios E, Han GS, Xu Z, Carman GM, Siniossoglou S. A phosphorylation-regulated amphipathic helix controls the membrane translocation and function of the yeast phosphatidate phosphatase. Proc Natl Acad Sci USA 2010; 107:17539-44; PMID:20876142; http://dx.doi.org/10.1073/pnas.1007974107 5. Carman GM, Henry SA. Phosphatidic acid plays a central role in the transcriptional regulation of glycerophospholipid synthesis in Saccharomyces cerevisiae. J Biol Chem 2007; 282:37293-7; PMID:17981800; http://dx.doi.org/10.1074/jbc.R700038200 6. Loewen CJ, Gaspar ML, Jesch SA, Delon C, Ktistakis NT, Henry SA, Levine TP. Phospholipid metabolism regulated by a transcription factor sensing phosphatidic acid. Science 2004; 304:1644-7; PMID:15192221; http://dx.doi.org/10.1126/science.1096083 7. Eastmond PJ, Quettier A-L, Kroon JT, Craddock C, Adams N, Slabas AR. Phosphatidic acid phosphohydrolase 1 and 2 regulate phospholipid synthesis at the endoplasmic reticulum in Arabidopsis. Plant Cell 2010;
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22:2796-811; PMID:20699392; http://dx.doi.org/ 10.1105/tpc.109.071423 8. Craddock CP, Adams N, Bryant FM, Kurup S, Eastmond PJ. Phosphatidic acid phosphohydrolase regulates phosphatidylcholine biosynthesis in arabidopsis by phosphatidic acid-mediated activation of ctp: phosphocholine cytidylyltransferase activity. Plant Cell 2015; 27:1251-64; PMID:25862304; http://dx.doi.org/ 10.1105/tpc.15.00037 9. Keogh MR, Courtney PD, Kinney AJ, Dewey RE. Functional characterization of phospholipid N-methyltransferases from Arabidopsis and soybean. J Biol Chem. 2009; 284:15439-47; PMID:19366698; http:// dx.doi.org/10.1074/jbc.M109.005991 10. Tabuchi T, Okada T, Azuma T, Nanmori T. Yasuda T. Posttranscriptional regulation by the upstream open
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reading frame of the phosphoethanolamine N-methyltransferase gene. Biosci Biotechnol Biochem 2006; 70:2330-4; PMID:16960350; http://dx.doi.org/ 10.1271/bbb.60309 11. Cornell RB, Northwood IC. Regulation of CTP:phosphocholine cytidylyltransferase by amphitropism and relocalization. Trends Biochem Sci 2000; 25:441-7; PMID:10973058; http://dx.doi.org/10.1016/S09680004(00)01625-X 12. Wang Y, Kent C. Identification of an inhibitory domain of CTP:phosphocholine cytidylyltransferase. J Biol Chem 1995; 270:18948-52; PMID:7642553; http://dx.doi.org/10.1074/jbc.270.32.18948 13. Friesen JA, Campbell HA, Kent C. Enzymatic and cellular characterization of a catalytic fragment of CTP: phosphocholine cytidylyltransferase a. J Biol Chem
Plant Signaling & Behavior
1999; 274:13384-9; PMID:10224101; http://dx.doi. org/10.1074/jbc.274.19.13384 14. Nowack MK, Harashima H, Dissmeyer N, Zhao X, Bouyer D, Weimer AK, De Winter F, Yang F, Schnittger A. Genetic framework of cyclin-dependent kinase function in Arabidopsis. Dev Cell 2012; 22:1030-40; PMID:22595674; http://dx.doi.org/ 10.1016/j.devcel.2012.02.015 15. Durek P, Schmidt R, Heazlewood JL, Jones A, Maclean D, Nagel A, Kersten B, Schulze WX. PhosPhAt: the Arabidopsis thaliana phosphorylation site database. An update. Nucleic Acids Res 2010; 38:D828-34; PMID:19880383; http://dx.doi.org/10.1093/nar/ gkp810
Volume 10 Issue 10
Regulation of endomembrane biogenesis in arabidopsis by phospatidic acid hydrolase Christian P Craddocky, Nicolette Adamsz, Fiona M Bryant, Smita Kurup, and Peter J Eastmond* Department of Plant Biology and Crop Science; Rothamsted Research, Harpenden; Hertfordshire, UK y
Present address: Horticultural Plant Biology and Metabolomics Research Center; Fujian Agriculture and Forestry University; Fuzhou, China
z
Present address: Center for Proteomic and Genomic Research; Cape Town, South Africa
C
Keywords: Arabidopsis, cell cycle, membrane biogenesis, phosphatidic acid phosphohydrolase, phosphatidylcholine biosynthesis *Correspondence to: Peter J Eastmond; Email: peter. [email protected] Submitted: 06/01/2015 Accepted: 06/18/2015
oordination of membrane lipid biosynthesis is important for cell function during plant growth and development. Here we summarize our recent work on PHOSPHATIDIC ACID PHOSPHOHYDROLASE (PAH) which suggests that this enzyme is a key regulator of phosphaticylcholine (PC) biosynthesis in Arabidopsis thaliana. Disruption of PAH activity elevates phosphatidic acid (PA) levels and stimulates PC biosynthesis and biogenesis of the endoplasmic reticulum (ER). Furthermore, the activity of PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE (CCT), which is the key enzyme controlling the rate of PC biosynthesis, is directly stimulated by PA and expression of a constitutively active version of CCT replicates the effects of PAH disruption. Hence PAH activity can control the abundance of PA, which in turn can modulate CCT activity to govern the rate of PC biosynthesis. Crucially it is not yet clear how PAH activity is regulated in Arabidopsis but there is evidence that PAH1 and PAH2 are both phosphorylated and further work will be required to investigate whether this is functionally significant.
http://dx.doi.org/10.1080/15592324.2015.1065367 Addendum to: Craddock CP, Adams N, Bryant FM, Kurup S, Eastmond PJ. PHOSPHATIDIC ACID PHOSPHOHYDROLASE regulates phosphatidylcholine biosynthesis in Arabidopsis by phosphatidic acid-mediated activation of CTP: PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE activity. Plant Cell 2015; 27:1251–1264
www.tandfonline.com
Phospholipids are the major structural components of most plant membranes, where their amphipathic properties contribute to the formation of lipid bilayers.1 The rate of phospholipid biosynthesis in plant cells must be tightly regulated so as to respond to fluctuations in demand. Plant Signaling & Behavior
Cell expansion and proliferation (division) are the 2 most important mechanisms underlying plant growth and both rely on membrane biogenesis. For example cell expansion requires production of plasma membrane while, during the mitotic phase of the cell cycle, division of the nucleus also demands rapid production of nuclear-endoplasmic reticulum (ER) membrane.2,3 Our current understanding of how phospholipid biosynthesis and membrane biogenesis are regulated in plant cells is relatively rudimentary and there has been little focus on how these processes are coordinated with growth or are integrated with the environmental and developmental signaling pathways that govern it. In contrast to plants, regulation of phospholipid biosynthesis and nuclear-ER membrane biogenesis has been studied relatively extensively in the yeast Saccharomyces cerevisiae. In this system it has been shown to be regulated by several kinases including the cyclin-dependent kinases (CDKs), cell division cycle 282 (Cdc28p) and phosphate metabolism 853 (Pho85p), which together allow for regulation of a host of cellular processes in response to environmental stimuli. Cdc28p and Pho85p phosphorylate and inactivate a key regulator of phospholipid biosynthesis called phosphatidic acid phosphohydrolase (Pah1p).2,3 Pah1p is an amphitropic enzyme that can interconvert between a phosphorylated soluble inactive form and a dephosphorylated membrane-bound active form2. Recruitment of phosphorylated Pah1p to the nuclear-ER membrane e1065367-1
Figure 1. Role of PAH in Arabidopsis ER membrane biogenesis. (A) Morphology of the ER in root cortical cells of wild type and pah1 pah2 double mutant imaged by laser-scanning confocal microscopy of a lumen-targeted GFP marker.8 Scale bar D 5 mm. (B) A model depicting how PC biosynthesis is linked to PAH activity.8 Repression of PAH activity leads to accumulation of its substrate PA. The increase in PA abundance relative to PC stimulates CCT activity which provides more CDP-Cho substrate for PC biosynthesis.8 CDP-Cho and not DG is most limiting for PC biosynthesis under normal circumstances and therefore the rate increases.8 P-Cho provision for CCT also increases because depletion leads to de-repression of PEAMT.8 PEAMT, PHOSPHOETHANOLAMINE N-METHYLTRANSFERASE; PAH, PHOSPHATIDIC ACID PHOSPHOHYDROLASE; G3P, glycerol 3 phosphate; LPA, lysophosphatidic acid; PA, phosphatidic acid; DG, diacylglycerol; PC, phosphatidylcholine; acyl-CoA, fatty acyl-coenzymeA; CDP-Cho, cytidine diphosphate-choline; P-Cho, phosphocholine; P-EA, phosphoethanolamine.
depends on the nuclear envelope morphology 1 – sporulation 7 (Nem1p– Spo7p) protein phosphatase complex, which dephosphorylates the enzyme and allows it to associate with the membrane via a short N-terminal amphipathic helix.2,4 Pah1p membrane disassociation and inactivation, resulting from phosphorylation by Cdc28p and Pho85p leads to elevated levels of the enzymes’ substrate phosphatidic acid (PA), expansion of the nuclear-ER membrane and enhanced expression of key phospholipid biosynthetic genes.2,3,4 These genes contain upstream activating sequence inositolresponsive (UASINO) elements in their promoters.5 Their expression is induced by interaction of the inositol requiring 2 – inositol requiring 4 (Ino2p-Ino4p) transcription factor complex with UASINO elements, but blocked by interaction of the repressor protein overproducer of inositol 1 (Opi1p) with Ino2p.5 PA levels control the Opi1p-mediated repression of gene expression because PA tethers Opi1p at the nuclear–ER membrane, together with a vesicle-associated protein homolog called suppressor of choline sensitivity 2.6
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In the model plant Arabidopsis thaliana, we have shown that disruption of PAH activity also stimulates net phosphatidylcholine (PC) biosynthesis and massive proliferation of the ER (Fig. 1A), and that this response positively correlates with an accumulation of the enzymes’ substrate PA.7,8 However, we have also found that changes in PAH activity in Arabidopsis have a very limited effect on global gene expression.8 The only differentially expressed gene that is currently known to play a role in PC biosynthesis is PHOSPHOETHANOLAMINE N-METHYLTRANSFERASE1 (PEAMT1).7,8 In Arabidopsis, choline is made by the sequential methylation of ethanolamine (EA), predominantly in its phosphorylated form (P-EA), to produce phosphocholine (P-Cho), which is then converted to PC by the nucleotide pathway using cytidine triphosphate (CTP) phosphocholine cytidylyltransferase (CCT) and aminoalcohol phosphotransferase.9 (Fig. 1B). The sequential methylation of P-EA catalyzed by PEAMT is the first committed step in PC biosynthesis.9 We have shown that PEAMT1 is induced at the level of transcription in leaves but not roots of PAHdeficient Arabidopsis plants and therefore
Plant Signaling & Behavior
cannot account for the enhanced PC biosynthesis observed in both tissues.7,8 PEAMT1 is negatively regulated by PCho both at the level of translation10 and enzyme activity.9 This provides a mechanism to balance supply of P-Cho with demand for PC biosynthesis that does not rely on transcription (Fig. 1B). Nevertheless PEAMT1 does still respond transcriptionally to PAH disruption in leaves and further work will be required to uncover the mechanism. At present it is not known whether this response depends on the catalytic activity of PAH or on the protein directly.8 In plants radiolabeling experiments have suggested that CCT activity is most likely to be rate-limiting for PC biosynthesis.7 We found that disruption of CCT activity suppresses the increase in PC biosynthesis caused by disruption of PAH activity.8 We also found that CCT activity is increased when PAH is disrupted but transcript and protein levels change very little.8 CCT protein also remains associated with microsomal membranes.8 In mammals CCTa is an amphitropic enzyme that can interconvert between a soluble inactive form and a membranebound active form.11 Furthermore, when
Volume 10 Issue 10
CCTa is membrane-bound, its activity is stimulated by the presence of free fatty acids (FFA) and ionic lipids such as PA.11 We found that the activity of recombinant Arabidopsis CCT1 is also stimulated by PC vesicles containing FFA or PA and that this increases the Vmax of the enzyme with little effect on the Km for either substrate (P-Cho and CTP).8 Given that disruption of PAH activity leads to accumulation of PA we hypothesized that direct activation of CCT by membranes enriched with PA could be the prime mechanism that stimulates PC biosynthesis in PAH-deficient Arabidopsis plants.8 (Fig. 1B). In mammalian CCTa, removal of the amphipathic C-terminal lipid binding domain (‘M’) stops membrane interaction and also causes the enzyme to become lipid insensitive and constitutively active.12,13 Arabidopsis CCT1 contains a shorter predicted amphipathic a-helical region that has been suggested to play a similar role. We found that removal of this domain from CCT1 also resulted in an enzyme that is constitutively active and unaffected by vesicles containing FFA or PA.8 Expression of this truncated CCT1 protein in Arabidopsis resulted in an increase in PC biosynthesis and ER membrane proliferation while expression of the intact CCT1 protein did not.8 These data supported the hypothesis that CCT activity modulates the production of PC in response to the relative abundance of PA (and possibly other anionic lipids) in the ER. A change in PAH activity therefore provides a potential mechanism to regulate membrane biogenesis in Arabidopsis.8 (Fig. 1). In yeast there is already evidence to show that Pah1p activity is regulated by CDKs and that its phosphorylation status modulates phospholipid biosynthesis and nuclear-ER membrane biogenesis through control of PA levels.2,3,4 A key question that remains to be answered in Arabidopsis is whether PAH activity is also regulated to govern membrane biogenesis and if so how is this achieved? Yeast Cdc28p phosphorylates 3 sites on Pah1p while Pho85p targets 7 and exerts a much stronger effect on PAH activity.2,3 Arabidopsis contains a number of kinases that share sequence similarity with Pho85 but CDKA;1 is the closest homolog to both
www.tandfonline.com
Cdc28p and Pho85p. Furthermore, CDKA;1 is the only Arabidopsis kinase to contain the PSTAIRE cyclin-binding motif.14 that is also found in both Cdc28p and Pho85p. CDKA;1 has been shown to complement the cdc28D mutant.14 CDKA;1 activity is greatest during the entry into mitosis, suggesting a major function at this stage in the cell cycle in Arabidopsis.14 Unlike yeast cdc28D, null mutants in CDKA;1 are viable but display cell cycle defects, particularly in S phase entry, and exhibit severely retarded growth.14 Phospho-proteomics studies have found that PAH1 and PAH2 are both phosphorylated at multiple sites.15 Interestingly this includes possible CDKA;1 target sites with the minimal consensus S/T-P (S162 in PAH1 and S524 in PAH2).15 In yeast Pah1p phosphorylation status controls membrane association and abundance, as well as catalytic activity.2,3 It is not known whether this is the case in Arabidopsis. We have previously shown that N-terminal green fluorescent protein (GFP) fusion proteins of PAH1 and PAH2 are predominantly cytosolic.7 However, work in yeast has subsequently established that the N-terminus of Pah1p contains a short amphipathic helix that’s responsible for membrane binding.2,3 Hence the cytosolic localization of N-terminal GFP-PAH fusions may be misleading. Indeed sequence analysis suggests that the amphipathic helix found in yeast Pah1p might also be conserved in Arabidopsis. Arabidopsis also contains proteins with weak homology to the Nem1p catalytic subunit of the Nem1p–Spo7p protein phosphatase complex, which recruits Pah1p to the nuclear-ER membrane in yeast.2,3 In conclusion changes in PAH activity can strongly affect the rate of phospholipid synthesis and membrane proliferation in both yeast.2,3,4 and Arabidopsis.7,8 In both systems PAH exerts this effect through controlling the level of PA.5,8 In yeast PA regulates the expression of multiple genes involved in phospholipid metabolism,5,6 while in Arabidopsis the impact on global transcription is comparatively very limited.8 Instead, PA appears to regulate PC biosynthesis at the biochemical level in Arabidopsis by directly stimulating the activity of CCT, which is the rate-
Plant Signaling & Behavior
limiting enzyme for the pathway.8 In yeast Pah1p activity is highly regulated by its phosphorylation status and the protein is a substrate for the CDKs Cdc28p and Pho85p. This provides a mechanism to couple phospholipid synthesis and membrane biogenesis directly to cell cycle regulation. How PAH is regulated in Arabidopsis is currently not known, but might also involve phosphorylation by CDKs. Addressing this question will be important for our fundamental understanding of membrane biogenesis in plants.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Funding
Support for this work was provided by the UK Biotechnology and Biological Sciences Research Council through grant BB/G009724/1 and BBS/E/C/00005207. References 1. Ohlrogge J, Browse J. Lipid biosynthesis. Plant Cell 1995; 7:957-70; PMID:7640528; http://dx.doi.org/ 10.1105/tpc.7.7.957 2. Choi HS, Su WM, Morgan JM, Han GS, Xu Z, Karanasios E, Siniossoglou S, Carman GM. Phosphorylation of phosphatidate phosphatase regulates its membrane association and physiological functions in Saccharomyces cerevisiae: identification of SER(602), THR(723), AND SER(744) as the sites phosphorylated by CDC28 (CDK1)-encoded cyclin-dependent kinase. J Biol Chem 2011; 286:1486-98; PMID:21081492; http://dx.doi.org/10.1074/jbc.M110.155598 3. Choi HS, Su WM, Han GS, Plote D, Xu Z, Carman GM. Pho85p-Pho80p phosphorylation of yeast Pah1p phosphatidate phosphatase regulates its activity, location, abundance, and function in lipid metabolism. J Biol Chem 2012; 287:11290-301; PMID:22334681; http://dx.doi.org/10.1074/jbc.M112.346023 4. Karanasios E, Han GS, Xu Z, Carman GM, Siniossoglou S. A phosphorylation-regulated amphipathic helix controls the membrane translocation and function of the yeast phosphatidate phosphatase. Proc Natl Acad Sci USA 2010; 107:17539-44; PMID:20876142; http://dx.doi.org/10.1073/pnas.1007974107 5. Carman GM, Henry SA. Phosphatidic acid plays a central role in the transcriptional regulation of glycerophospholipid synthesis in Saccharomyces cerevisiae. J Biol Chem 2007; 282:37293-7; PMID:17981800; http://dx.doi.org/10.1074/jbc.R700038200 6. Loewen CJ, Gaspar ML, Jesch SA, Delon C, Ktistakis NT, Henry SA, Levine TP. Phospholipid metabolism regulated by a transcription factor sensing phosphatidic acid. Science 2004; 304:1644-7; PMID:15192221; http://dx.doi.org/10.1126/science.1096083 7. Eastmond PJ, Quettier A-L, Kroon JT, Craddock C, Adams N, Slabas AR. Phosphatidic acid phosphohydrolase 1 and 2 regulate phospholipid synthesis at the endoplasmic reticulum in Arabidopsis. Plant Cell 2010;
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Volume 10 Issue 10
