The Phosphatidylglycerol/Cardiolipin Biosynthetic Pathway Is Required for the Activation of Inositol Phosphosphingolipid Phospholipase C, Isc1p, during Growth of Saccharomyces cerevisiae

Abstract

Inositolsphingolipid phospholipase C (Isc1p) is the Saccharomyces cerevisiae member of the extended family of neutral sphingomyelinases that regulates the generation of bioactive ceramides. Recently, we reported that Isc1p is post-translationally activated in the post-diauxic phase of growth and that it localizes to mitochondria (Vaena de Avalos, S., Okamoto, Y., and Hannun, Y. A. (2004) J. Biol. Chem. 279, 11537–11545). In this study the in vivo mechanisms of activation and function of Isc1p were investigated. Deletion of ISC1 resulted in markedly lower growth in non-fermentable carbon sources. Interestingly, the growth defect of isc1Δ strains resembled that of pgs1Δ strains, lacking the committed step in the synthesis of phosphatidylglycerol (PG) and cardiolipin (CL), which were shown to activate Isc1p in vitro. Therefore, the role of Pgs1p in activation of Isc1p in vivo was investigated. The results showed that in the pgs1Δ strain, the growth-dependent activation of Isc1p was impaired as was the ISC1-dependent increase in the levels of phytoceramide during the post-diauxic phase, demonstrating that the activation of Isc1p in vivo is dependent on PGS1 and on the mitochondrial phospholipids PG/CL. Mechanistically, loss of Isc1p resulted in lower levels of mitochondrial cytochrome c oxidase subunits cox3p and cox4p, previously established targets of both PG and CL (Ostrander, D. B., Zhang, M., Mileykovskaya, E., Rho, M., and Dowhan, W. (2001) J. Biol. Chem. 276, 25262–25272), thus suggesting that Isc1p mediates at least some functions downstream of PG/CL. This study provides the first evidence for the mechanism of in vivo activation and function of Isc1p. A model with endogenous PG/CL as the in vivo activator of Isc1p is proposed. Inositolsphingolipid phospholipase C (Isc1p) is the Saccharomyces cerevisiae member of the extended family of neutral sphingomyelinases that regulates the generation of bioactive ceramides. Recently, we reported that Isc1p is post-translationally activated in the post-diauxic phase of growth and that it localizes to mitochondria (Vaena de Avalos, S., Okamoto, Y., and Hannun, Y. A. (2004) J. Biol. Chem. 279, 11537–11545). In this study the in vivo mechanisms of activation and function of Isc1p were investigated. Deletion of ISC1 resulted in markedly lower growth in non-fermentable carbon sources. Interestingly, the growth defect of isc1Δ strains resembled that of pgs1Δ strains, lacking the committed step in the synthesis of phosphatidylglycerol (PG) and cardiolipin (CL), which were shown to activate Isc1p in vitro. Therefore, the role of Pgs1p in activation of Isc1p in vivo was investigated. The results showed that in the pgs1Δ strain, the growth-dependent activation of Isc1p was impaired as was the ISC1-dependent increase in the levels of phytoceramide during the post-diauxic phase, demonstrating that the activation of Isc1p in vivo is dependent on PGS1 and on the mitochondrial phospholipids PG/CL. Mechanistically, loss of Isc1p resulted in lower levels of mitochondrial cytochrome c oxidase subunits cox3p and cox4p, previously established targets of both PG and CL (Ostrander, D. B., Zhang, M., Mileykovskaya, E., Rho, M., and Dowhan, W. (2001) J. Biol. Chem. 276, 25262–25272), thus suggesting that Isc1p mediates at least some functions downstream of PG/CL. This study provides the first evidence for the mechanism of in vivo activation and function of Isc1p. A model with endogenous PG/CL as the in vivo activator of Isc1p is proposed. Ceramide is a bioactive lipid that in eukaryotic cells functions as a mediator of a variety of extracellular signals through the regulation of several downstream effectors which in turn control basic cellular functions such as cell growth, cell cycle arrest, apoptosis, and senescence (1Vaena de Avalos S. Jones J.A. Hannun Y.A. Bioactive Lipids: The Oily Press. PJ Barnes & Associates, Bridgwater, England2004: 135-167Google Scholar, 2Hannun Y.A. Obeid L.M. J. Biol. Chem. 2002; 277: 25847-25850Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar, 3Merrill Jr., A.H. J. Biol. Chem. 2002; 277: 25843-25846Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar, 4Spiegel S. Milstien S. J. Biol. Chem. 2002; 277: 25851-25854Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 5Perry D.K. Kolesnick R.N. Cancer Treat. Res. 2003; 115: 345-354Crossref PubMed Google Scholar, 6Andrieu-Abadie N. Gouaze V. Salvayre R. Levade T. Free Radic. Biol. Med. 2001; 31: 717-728Crossref PubMed Scopus (230) Google Scholar). Sphingomyelinases, which hydrolyze the phosphodiester linkage of sphingomyelin (SM) 1The abbreviations used are: SM, sphingomyelin; ISC1, inositol phosphosphingolipid phospholipase C; PGS1, phosphatidylglycerophosphate synthase; CLS1, cardiolipin synthase; CL, cardiolipin; PG, phosphatidylglycerol; cox, cytochrome c oxidase; NFCS, non-fermentable carbon sources; MUSC, Medical University of South Carolina; NBD, 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)); wt, wild type; Cls1p, cardiolipin synthase; GFP, green fluorescent protein; SD, synthetic minimal medium; MES, 4-morpholineethanesulfonic acid. to produce ceramide and phosphorylcholine, function as key regulators of the intracellular levels of ceramide in mammalian cells. Sphingolipids are also important regulatory molecules in yeast where they are known to be required for viability (7Dickson R.C. Wells G.B. Schmidt A. Lester R.L. Mol. Cell. 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Although Saccharomyces cerevisiae do not contain SM, they do contain inositol phosphoceramides, and recently we identified a homologue of neutral sphingomyelinase in S. cerevisiae, Isc1p, which acts on phosphoceramides to generate ceramide. This enzyme displays ∼30% identity to neutral sphingomyelinase 2 and shares with it several common features (12Vaena de Avalos S. Okamoto Y. Hannun Y.A. J. Biol. Chem. 2004; 279: 11537-11545Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) including hydrolytic activity on SM, the requirement of Mg2+ for optimal activity, optimal neutral pH, and the presence of a newly discovered domain that is conserved in the entire family of sphingomyelinases, the P-loop-like domain (13Okamoto Y. Vaena de Avalos S. Hannun Y.A. Biochemistry. 2003; 42: 7855-7862Crossref PubMed Scopus (23) Google Scholar), which appears to be important for substrate binding and/or catalysis. In addition, both enzymes demonstrate an absolute requirement for anionic phospholipids for in vitro activation. Thus, Isc1p is activated selectively in vitro by cardiolipin (CL), phosphtidylglycerol (PG), or phosphatidylserine. In a recent study we demonstrated that the enzyme binds these anionic phospholipids, and we identified the second transmembrane domain and the C terminus of Isc1p as required for this binding, and it was proposed that this interaction plays a critical role in enzyme function through a novel tethering mechanism of enzyme activation by lipid cofactors (14Okamoto Y. Vaena De Avalos S. Hannun Y.A. J. Biol. Chem. 2002; 277: 46470-46477Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Given these specific requirements for anionic phospholipids in vitro, it became important to determine whether the enzyme requires phospholipids for activation in vivo. Our attention was particularly directed to the possible roles of PG and CL as the deletion of PGS1, the gene that encodes the enzyme responsible for the synthesis of PG phosphate, and subsequent synthesis of PG and CL was shown to cause a late defect during growth in glucose media similar to that of ISC1. Moreover, these lipids are almost exclusively present in mitochondria, and we have shown recently that Isc1p localizes preferentially to mitochondria in the post-diauxic phase of growth. Synthesis of CL in yeast requires three sequential reactions. The first step is the synthesis of phosphatidylglycerophosphate from CDP diacylglycerol and glycerol This by phosphatidylglycerophosphate is the committed and PG is to PG is to CL by cardiolipin with CDP diacylglycerol as CL to be important for the function of mitochondria PubMed Scopus Google Scholar, D. 1990; PubMed Scopus Google Scholar, de D. Biochemistry. 34: PubMed Scopus Google Scholar), to and the of V. de de B. PubMed Google Scholar), a of cytochrome c oxidase the the of PG of of PGS1 was shown to cause mitochondrial W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. 42: PubMed Scopus Google Scholar) and to to a growth defect on non-fermentable carbon suggesting a key role for PG in mitochondrial function W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C. PubMed Scopus Google Scholar, W. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Recently, a role for Pgs1p in the of was proposed on the that the pgs1Δ a in of W. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). Recently, Isc1p was to activated and to to the mitochondria during the in an increase in the levels of phytoceramide during growth of yeast cells (12Vaena de Avalos S. Okamoto Y. Hannun Y.A. J. Biol. Chem. 2004; 279: 11537-11545Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). the for enzyme activation In the present study the in vivo mechanism of Isc1p activation was investigated. and were from was from and were from was from was in the at the Medical University of South as A. Hannun Y.A. 2000; PubMed Scopus Google Scholar). and were from lipids and and were from the at were from and were from minimal and were from and yeast wild and the deletion H. Okamoto Y. C. C. A. N. Hannun Y.A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) were used in this and strains were from In addition, the wild and strains for of the cardiolipin used in this study were previously W. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar), and W. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The of ISC1 and ISC1 in the control of the was previously (14Okamoto Y. Vaena De Avalos S. Hannun Y.A. J. Biol. Chem. 2002; 277: 46470-46477Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). were in yeast or yeast carbon or as were cells from the strains as C. Obeid L.M. Biochem. J. PubMed Scopus (107) Google Scholar). were in SD, glucose and were in media and at in a at were in media for in to the phase, and cells were with strains that not in media glucose and was were by of the with of media to a of and for the at Sphingolipids in or as were to and the of was which was not for the growth of wild or cells. were by the at of Isc1p isc1Δ cells present sphingomyelinase activity and the activity of enzyme from cells was as previously (14Okamoto Y. Vaena De Avalos S. Hannun Y.A. J. Biol. Chem. 2002; 277: 46470-46477Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), cell were used to determine Isc1p activity as with (14Okamoto Y. Vaena De Avalos S. Hannun Y.A. J. Biol. Chem. 2002; 277: 46470-46477Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). cell were in of of of SM, and of at for the of of and of were to the of J. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and the in a of the phase was with for and of in of in mitochondrial or of were in in a and to were and was as previously (12Vaena de Avalos S. Okamoto Y. Hannun Y.A. J. Biol. Chem. 2004; 279: 11537-11545Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). was of of were and with and lipids were the of and J. Biochem. PubMed Google Scholar). The phase was and for or phytoceramide the diacylglycerol was and for D.K. A. Hannun Y.A. 2000; PubMed Google Scholar). of cells were as and in of synthetic media with and glucose as a carbon were to by of to growth and cells were for phospholipids were by and by a were to and PG, CL, and were of were at for to the of mitochondria was on PubMed Scopus Google Scholar, 2000; PubMed Scopus Google Scholar) which reported that with the mitochondrial be in by the of the PubMed Scopus Google Scholar). were in A phosphate, and in the of the cellular was and mitochondrial were as by and PubMed Scopus Google Scholar). were on a with and the cells were with a for The for was and was were a with a and were was recently shown that isc1Δ a growth defect as a in media to wild strains, during the (12Vaena de Avalos S. Okamoto Y. Hannun Y.A. J. Biol. Chem. 2004; 279: 11537-11545Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Isc1p was shown to to the mitochondria and to be activated with a increase in the levels of intracellular phytoceramide (12Vaena de Avalos S. Okamoto Y. Hannun Y.A. J. Biol. Chem. 2004; 279: 11537-11545Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). of these it became important to the role of Isc1p during Thus, the of this to in media non-fermentable as the carbon was with the wild strain, isc1Δ showed a defect in growth glycerol was used as the carbon and this defect in growth was with the growth defect in glucose In addition, growth of isc1Δ was markedly cells were in such as or as to the glucose or Although the in or were these were with the sources. Therefore, these results the role for Isc1p in growth, and they to that the growth of isc1Δ at the glucose is and is to a defect of this to in of carbon on growth of or cells were for the of in media or as the carbon and the was The results are the of The results shown are of The results with results demonstrating activation of Isc1p in mitochondria during the (12Vaena de Avalos S. Okamoto Y. Hannun Y.A. J. Biol. Chem. 2004; 279: 11537-11545Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) to the mechanisms the regulation of Isc1p in growth in the roles of CL and PG, the anionic phospholipids in the growth of isc1Δ was with that of a for PG the enzyme responsible for synthesis of PG and The of isc1Δ was similar to that of pgs1Δ both strains showed growth similar to wild strains in glucose to and from to a in the for isc1Δ and pgs1Δ was and and In growth of isc1Δ and pgs1Δ was for the entire to The the with the pgs1Δ and the isc1Δ strains a the in vivo role of and the role of Isc1p during growth such that Isc1p and Pgs1p and/or or downstream in the in of the or in cell growth. these were the activation of Isc1p during growth of the strains was as previously (12Vaena de Avalos S. Okamoto Y. Hannun Y.A. J. Biol. Chem. 2004; 279: 11537-11545Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Isc1p specific activity in and and from the wild Isc1p showed the specific activity at the the phase with activation at and in the growth-dependent activation of Isc1p was markedly in the pgs1Δ with a increase in the activity of Isc1p at the post-diauxic phase results that of PGS1 and of PG/CL the activation of Isc1p during growth. the showed Isc1p activation similar to the strain, that the of and of CL not the activation of Isc1p. it shown that Isc1p from to mitochondria the post-diauxic (12Vaena de Avalos S. Okamoto Y. Hannun Y.A. J. Biol. Chem. 2004; 279: 11537-11545Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), it became of to determine whether Pgs1p a role on the of Isc1p. from wild or strains were with and by at and strains showed similar of the during the phase of growth and the mitochondrial of a the post-diauxic suggesting that the of is not in these Although this for mitochondria is a was to the of Isc1p during the in cells that are for the PG/CL Thus, and mitochondrial were as previously PubMed Scopus Google Scholar) from wild or pgs1Δ cells for and to for Isc1p, and as and mitochondrial three were in the and Isc1p were in the mitochondrial from the pgs1Δ cells which was similar to the wild that the mitochondrial of Isc1p is not in the pgs1Δ results a role for Pgs1p in the regulation of Isc1p to the mitochondria during growth. evidence for a function of Pgs1p of Isc1p in the levels of the lipid of Isc1p, were have previously shown that phytoceramide levels increase during growth, and this increase was dependent on ISC1 (12Vaena de Avalos S. Okamoto Y. Hannun Y.A. J. Biol. Chem. 2004; 279: 11537-11545Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Therefore, the levels of phytoceramide were in from or isc1Δ cells for or and the levels of phytoceramide were by the diacylglycerol The levels of phytoceramide in the wild in post-diauxic phase cells previously (12Vaena de Avalos S. Okamoto Y. Hannun Y.A. J. Biol. Chem. 2004; 279: 11537-11545Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), the levels of phytoceramide not to increase in the isc1Δ with a wild also the levels of phytoceramide in the were with the wild not the levels of phytoceramide were markedly lower in pgs1Δ cells at the with wild or to the phase cells results evidence that in vivo the levels of phytoceramide are in yeast lacking PGS1, the that the lipid of PG, and CL a role of Isc1p and that PG and CL are required for the of phytoceramide during growth we the that ISC1 function of Pgs1p is responsible for the synthesis of both PG and CL, we the levels of these lipids in wild cells or cells lacking Isc1p as previously W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The showed similar levels of PG and of CL in wild and isc1Δ first and second and this was by the of in the PG and CL were and the levels were to The of was and was that these strains have similar levels of CL and Therefore, it is that the of Isc1p not the levels of the lipid of and Isc1p not of a role for PG and CL in the of the subunits was recently reported W. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar), it became of to determine whether Isc1p as a mediator of the of PG/CL on the of these mitochondrial Therefore, the levels of and subunits were by mitochondrial from wild or isc1Δ cells as for a of cox, a specific of the was in wild the levels of cox3p were in isc1Δ cells with wild the for the mitochondrial of Isc1p, that this is not a of was previously shown that isc1Δ growth defect in glucose be by of ISC1. Therefore, we used the to whether the of Isc1p in this with the lower levels of cox3p in the isc1Δ strain, the levels of this were almost to the levels of wild ISC1 was results were the were and for the in this a specific of was in the wild and a of the was in the to isc1Δ which was by of ISC1 not Thus, deletion of ISC1 the of PGS1 in the regulation of the levels of the results a role for Isc1p of we we the of PGS1 deletion by phytoceramide to yeast thus the of the PGS1 deletion to cause the activation of Isc1p. be that of ISC1 in pgs1Δ not the defect of this to in not which was an the of Pgs1p and to be required for activation of Isc1p. endogenous phytoceramide are by yeast cells and are by enzymes of (1Vaena de Avalos S. Jones J.A. Hannun Y.A. Bioactive Lipids: The Oily Press. PJ Barnes & Associates, Bridgwater, England2004: 135-167Google Scholar). or strains were for in the presence of or in glucose The of to the media of isc1Δ cells a increase in growth at This was specific for phytoceramide of growth was cells were with ceramide for the the wild cells were with was pgs1Δ cells also to the of an increase in growth at the The of phytoceramide on the growth of isc1Δ in media demonstrated and not the increase in growth to phytoceramide was at of in media increase in growth to was to in a was and and the and were at in glucose media not and at in media The pgs1Δ also showed a growth-dependent increase of in non-fermentable carbon media that a of the growth defect of this in this was lower in the isc1Δ at is that the late of to cells that were a of growth 2 as to a was at results that the is required at or the not Therefore, these results that the growth defect to of isc1Δ or pgs1Δ be by the lipid of Isc1p. The results from this study on the in vivo mechanisms of activation of Isc1p, role the role of in regulation of growth, and to the of synthesis of the anionic phospholipids PG and The results also evidence for the function of PG/CL as a bioactive of the ISC1 gene in S. cerevisiae resulted in a on carbon for growth. Thus, Isc1p a critical role in yeast to mitochondrial that is required in the post-diauxic cells are on Interestingly, the growth in both and non-fermentable carbon that of the pgs1Δ not of the strain, the pgs1Δ showed a critical on carbon the with on the growth of pgs1Δ and C. PubMed Scopus Google Scholar, W. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, W. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). This of growth as as the results demonstrating a requirement for in vitro activation of Isc1p by the of the that endogenous PG be required for activation of Isc1p in vivo. of evidence are to such a role for Pgs1p and PG loss of function by of PGS1 resulted in the loss of the growth activation of Isc1p that is in wild cells and the post-diauxic of growth. The of Pgs1p were specific for the activation of Isc1p as the of Isc1p to the mitochondria to be the of phytoceramide during the post-diauxic phase of growth was by the of PGS1, demonstrating that the regulation of Isc1p by the function of the gene responsible for the synthesis of PG/CL in vivo. the of phytoceramide was to the growth of both isc1Δ and pgs1Δ Isc1p not function of Pgs1p PG/CL levels at the in an The results from this study also an important role for yeast the of Isc1p, in the of Isc1p on growth. Thus, the growth defect of the isc1Δ in both glucose the post-diauxic and media of the and of it was not possible to that growth was by phytoceramide the of phytoceramide were specific in that not cell growth, and the of phytoceramide were not for the wild strains, which an increase in endogenous ceramide during the a on the growth of the growth of this results with the requirement of Isc1p for yeast growth in non-fermentable carbon the that the is a key of growth on non-fermentable carbon sources. A growth of pgs1Δ and isc1Δ was also such that the isc1Δ to and in glycerol by the pgs1Δ This that Isc1p not functions of Pgs1p and lipid downstream in to Isc1p. Mechanistically, the results demonstrate at least role for Isc1p in mitochondrial function through the regulation of levels of of the mitochondrial of the levels of mitochondrial or subunits of in mitochondrial from cells in which Isc1p is showed a of the levels of these with the wild results that Isc1p regulates the of these mitochondrial the of subunits in yeast showed regulation at the of W. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, V. 2001; PubMed Google Scholar), it demonstrated that the of subunits is in pgs1Δ strains W. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar), and that Isc1p is by Pgs1p in it is to that the of cox3p and is by Isc1p. of the ISC1 gene the defect in levels of the results Isc1p Pgs1p and the regulation of levels and optimal growth. Thus, a novel PG Isc1p phytoceramide is proposed that to in the roles of Pgs1p and Isc1p in mitochondrial and Although the results demonstrating a requirement for PGS1 in activation of Isc1p in vivo a role for endogenous PG as the activator of Isc1p, a role for CL be at this lipids are of Isc1p in vitro, with a for Moreover, it is that PG levels were reported to be is the PG for some functions of Thus, the of the PGS1 deletion to activation of Isc1p be to loss of PG or CL, the of the deletion which the levels of subunits are not C. PubMed Scopus Google to Isc1p activation be to the of a role for CL in vivo activation of Isc1p or to the for the loss of CL by the be that PG not for of CL for the of of the mitochondrial of and or W. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, K. V. C. H. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) is not in the levels of on the results in this a possible role for phytoceramide as a bioactive lipid is the of phytoceramide a cellular of and this of phytoceramide lipid the levels of phytoceramide are the generation of phytoceramide at this phase of growth is by in both the regulation of the generation of phytoceramide and of activation of Isc1p are by and Isc1p and phytoceramide downstream targets of subunits of cytochrome c as these results with a role for PG as a bioactive it for a bioactive the levels of PG increase during the phase, PG Isc1p in vitro, PG is required for in and loss of PG synthesis results in a growth defect on non-fermentable sources. Thus, in PG levels on a as previously is required for important In addition, have roles for PG and CL in and In the results from this study novel mechanisms of of PG in cells bioactive mechanisms of regulation of Isc1p in and the role of Isc1p in growth of yeast in non-fermentable carbon and during the post-diauxic phase of growth. the Cancer and J. A. Jones for the and for the used in this the and the at the also C. and H. for on yeast and J. A. A. K. and Obeid for also H. and K. for this