The Kap60-Kap95 Karyopherin Complex Directly Regulates Phosphatidylcholine Synthesis

Phosphatidylcholine is the major phospholipid in eukaryotic cells. There are two main pathways for the synthesis of phosphatidylcholine: the CDP-choline pathway present in all eukaryotes and the phosphatidylethanolamine methylation pathway present in mammalian hepatocytes and some single celled eukaryotes, including the yeast Saccharomyces cerevisiae. In S. cerevisiae, the rate-determining step in the synthesis of phosphatidylcholine via the CDP-choline pathway is catalyzed by Pct1. Pct1 converts phosphocholine and CTP to CDP-choline and pyrophosphate. In this study, we determined that Pct1 is in the nucleoplasm and at endoplasmic reticulum and nuclear membranes. Pct1 directly interacts with the α-importin Kap60 via a bipartite basic region in Pct1, and this region of Pct1 was required for its entry into the nucleus. Pct1 also interacted with the β-importin Kap95 in cell extracts, implying a model whereby Pct1 interacts with Kap60 and Kap95 with this tripartite complex transiting the nuclear pore. Exclusion of Pct1 from the nucleus by elimination of its nuclear localization signal or by decreasing Kap60 function did not affect the level of phosphatidylcholine synthesis. Diminution of Kap95 function resulted in almost complete ablation of phosphatidylcholine synthesis under conditions where Pct1 was extranuclear. The β-importin Kap95 is a direct regulator membrane synthesis. Phosphatidylcholine is the major phospholipid in eukaryotic cells. There are two main pathways for the synthesis of phosphatidylcholine: the CDP-choline pathway present in all eukaryotes and the phosphatidylethanolamine methylation pathway present in mammalian hepatocytes and some single celled eukaryotes, including the yeast Saccharomyces cerevisiae. In S. cerevisiae, the rate-determining step in the synthesis of phosphatidylcholine via the CDP-choline pathway is catalyzed by Pct1. Pct1 converts phosphocholine and CTP to CDP-choline and pyrophosphate. In this study, we determined that Pct1 is in the nucleoplasm and at endoplasmic reticulum and nuclear membranes. Pct1 directly interacts with the α-importin Kap60 via a bipartite basic region in Pct1, and this region of Pct1 was required for its entry into the nucleus. Pct1 also interacted with the β-importin Kap95 in cell extracts, implying a model whereby Pct1 interacts with Kap60 and Kap95 with this tripartite complex transiting the nuclear pore. Exclusion of Pct1 from the nucleus by elimination of its nuclear localization signal or by decreasing Kap60 function did not affect the level of phosphatidylcholine synthesis. Diminution of Kap95 function resulted in almost complete ablation of phosphatidylcholine synthesis under conditions where Pct1 was extranuclear. The β-importin Kap95 is a direct regulator membrane synthesis. Phosphatidylcholine (PC) 4The abbreviations used are: PC, phosphatidylcholine; PE, phosphatidylethanolamine; TAP, tandem affinity purification; ER, endoplasmic reticulum; BD, DNA binding domain of Gal4; AD, activation domain of Gal4; GFP, green fluorescent protein; RFP, red fluorescent protein; MS, mass spectrometry; DAPI, 4′,6-diamidino-2-phenylindole; ORF, open reading frame; NLS, nuclear localization signal. is the major lipid in eukaryotic cell membranes, comprising approximately half of the phospholipid content. PC is important for providing organellar and cellular impermeability barriers and has crucial functions in cells beyond this, since it serves as a source of numerous second messengers, including diacylglycerol for the activation of protein kinases, arachidonic acid for metabolism to prostaglandins and leukotrienes, and lysophosphatidylcholine to provide a chemotactic signal for lymphocytes and macrophages (1McMaster C.R. Jackson T.R. Daum G. Topics in Current Genetics: Lipid Metabolism and Membrane Biogenesis. Springer-Verlag, Berlin2004: 5-88Google Scholar, 2Newton A.C. Trends Pharmacol. Sci. 2004; 25: 175-177Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 3Serhan C.N. Yacoubian S. Yang R. Annu. Rev. Pathol. 2008; 3: 279-312Crossref PubMed Scopus (407) Google Scholar, 4Peter C. Waibel M. Radu C.G. Yang L.V. Witte O.N. Schulze-Osthoff K. Wesselborg S. Lauber K. J. Biol. Chem. 2008; 283: 5296-5305Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). 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Biol. 2005; PubMed Scopus Google Scholar). Diminution of the function of Kap60 or Kap95 Pct1 nuclear bipartite basic region in Pct1 was required for Pct1 to with Kap60 and for its into the nucleus. of the nuclear localization signal from Pct1 resulted in its localization to the did not affect the level of PC synthesis. The of PC synthesis the CDP-choline pathway was when Pct1 into the nucleus was by decreasing Kap60 Kap95 function resulted in almost complete ablation of PC synthesis via the CDP-choline pathway under conditions where Pct1 was extranuclear. and from from and and from and and from used fluorescent protein and fluorescent protein and and and yeast and used in this are in and The by from DNA of from the yeast gene by used of the for the open reading and of the and and The was into cells and and by to the of the into the PCT1 for the of the used in this H. D.B. J. J. Biol. 2005; PubMed Scopus Google H. D.B. J. J. Biol. 2005; PubMed Scopus Google C. 283: PubMed Scopus Google in a used in this or low P. PubMed Google low P. PubMed Google P. PubMed Google with PCT1 under of with PCT1 under of with PCT1 under of with PCT1 under of with PCT1 under of 8Howe A.G. Zaremberg V. McMaster C.R. J. Biol. Chem. 2002; 277: 44100-44107Abstract Full Text Full Text PDF PubMed Scopus (30) Google yeast with yeast with with in with a in in with a in in with a in the for acid in with a in the for acid in with a in the for acid in with a in DNA binding domain C. 283: PubMed Scopus Google activation domain C. 283: PubMed Scopus Google in acid in in in in C. 283: PubMed Scopus Google in C. 283: PubMed Scopus Google in a was was with and as and of under of a was with of with was as with the B.J. C. 2003; PubMed Scopus Google Scholar, F. G. B. M. B. 2001; PubMed Scopus Google Scholar). PCT1 or the at the of the open reading to in of at at for at in and A. by a at The of was by of was at for at The was to in for at and by and the with of and with of The with of and of for at The was by with binding to the with of binding and of The was for at into a and with of binding by with of a by and and and by mass of and of with a was from cells as in and to at was and to for at for at cells complete by and The was at for at and the was to and for at in was and with of was was to the and yeast cell protein was to or for of by and for by mass in with with and with with The was under and the in was an and to the source of a mass a at a of of of and the was as to and for at B. The was with a The was with a of and the was to the The two most from the for tandem MS, and the was the mass of the as determined by the and The yeast and all the mass and mass of and and as PCT1 open reading and the PCT1 open reading its nuclear localization signal acid in with the DNA binding domain in The open reading for and in with the DNA activation domain in into the and and to for and to C. 283: PubMed Scopus Google Scholar). in with the DNA binding domain and in with the activation domain used as for as a with a a cells GFP, or as and in cells to at and a for cells in for by of cells in that of and used at used at and by with or for mass with or for to membrane and and by PC synthesis the CDP-choline or methylation pathways, or was to at was to for when and and was to the for with a to for to of Pct1 was by at for in a and the of as (8Howe A.G. Zaremberg V. McMaster C.R. J. Biol. Chem. 2002; 277: 44100-44107Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 9McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: 14776-14783Abstract Full Text PDF PubMed Google Scholar). The of was as The was and in and a of of of and of The was and for a into and was determined by of Kap95 and Kap60 as that directly to Pct1 or its mammalian CCTα to be is a major in the PC synthesis and its with cell for of was to two to both In the Pct1 a at the of the PCT1 open reading for of PCT1 from the and under of its was was and was in was by a second in whereby from yeast that to Pct1 protein The of was determined by and the both yeast and all and The Kap95 was in both the in and in Kap95 is of several that of the nuclear pore. that Kap95 to the nucleus to the α-importin and we Kap60 as a in the binding of Kap60 to the Kap95 is and the tripartite protein complex the nuclear and the nucleus A. F. M. Annu. Rev. Biochem. 2007; PubMed Scopus Google Scholar, M.C. G. 2006; PubMed Scopus Google Scholar, H. D.B. J. J. Biol. 2005; PubMed Scopus Google Scholar, A. M. J. Biol. Chem. 2007; 282: Full Text Full Text PDF PubMed Scopus Google Scholar). of Pct1, and Kap95 and of the of Pct1 for a basic resulting in the of Kap95 and of a that nuclear A. F. M. Annu. Rev. Biochem. 2007; PubMed Scopus Google Scholar, A. M. J. Biol. Chem. 2007; 282: Full Text Full Text PDF PubMed Scopus Google Scholar). Pct1 two basic at its and basic are required for Pct1 with Kap60 or a was and did cells with the and as Pct1 interacts with Kap60 and did not implying that of Pct1 with in of Kap95 to of and in a the of Pct1 the basic and was required for Pct1 nuclear Pct1 with a this was to a that localizes to the (8Howe A.G. Zaremberg V. McMaster C.R. J. Biol. Chem. 2002; 277: 44100-44107Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) or with the basic or both basic of under of a to the that protein was at the to the nucleus in a to Pct1, was The for Pct1 is the acid of its The Kap95 and the Kap60 for Pct1 was from its in with from low and The with Pct1 or into a with an PCT1 gene to for the of the in the of Pct1. that protein of the of in cells not in cells Pct1 The was as determined by PC synthesis via with The protein was and was that be an of Pct1 was found in the as by with the the of with DAPI, was a of to Pct1 is an enzyme to membranes with increased this most the of an organellar a low was into yeast organellar with the (the domain of Young S. The J. Biol. 2007; PubMed Scopus (91) Google Scholar) in the not with at the with both membrane and complex that Pct1 to and nuclear membranes. Pct1 did not with for or the of Kap60 and Kap95 in Pct1 entry into the we yeast of the and The PCT1 gene was in and and the resulting and with the the open reading of PCT1 under of a with a at the of Pct1 The the was in to of to at the for function of the and half of the cells to to Kap60 of was in both the and by from to to the nucleus in cells at the for the and was in cells at the The the was to a as the was a of in the nucleus and the nucleus is in with with this that the of the encoded Kap95 was at H. D.B. J. J. Biol. 2005; PubMed Scopus Google Scholar). cells to the for the was the nucleus. the to in and of Pct1 the was into the yeast and the cells to the was to the nucleus at both and that increased not in of from the nucleus. There was fluorescent signal in cells with Pct1 the or in cells with that the fluorescent signal was to of the protein not Kap60 and Kap95 are required for nuclear of Pct1. Kap95 PC Pct1 the CDP-choline pathway for PC synthesis when Pct1 and its into cells. to and of and was by of cells in was to the and its into CDP-choline pathway and PC was Pct1 its synthesized PC at a level that was that of cells Pct1 PC be synthesized Pct1 is from the nucleus by of its NLS, at a the of Kap60 and Kap95 in PC synthesis. and cells to at and half of was to for to Kap60 or Kap95 function to the of for the localization of Pct1 was to that in as determined by localization of the nucleoplasm and with and nuclear not In the of PC synthesis was to at both and in was a in the of PC synthesis at and the of function of Kap95 results in a in the synthesis of also PC synthesis in cells Kap60 or Kap95 function when Pct1 was from the nucleus. The and cells with the and to at and half of the was to to Kap60 or Kap95 to to of Pct1 to the of Pct1 was from the nucleus to of was a in PC synthesis the Pct1 is from the nucleus at in cells the since this is a for function of this and was a in PC synthesis at this Pct1 was from the nucleus to of Kap95 at PC synthesis was to a level that in cells with an PCT1 with a in in the substrate for Pct1 the Exclusion of Pct1 from the nucleus by of Kap95 PC synthesis the CDP-choline pathway at the Pct1 be from the nucleus by of its or by of Kap60 or of Kap95 resulted in a in PC synthesis when Pct1 was essentially and an almost complete ablation of PC synthesis when Pct1 was from the nucleus. Kap60 directly to Pct1 the region of Pct1 that is required for Pct1 entry into the nucleus. a for this in of PC synthesis, the as was used in and cells its under of a the for the PC synthesis was with at the of PC synthesis was that of a for function of the PC synthesis was to of that of and at to all Kap95 function resulted in synthesis of in the substrate for Pct1. the Kap60 and Pct1 resulted in a of Pct1 in the of the inability to synthesize PC the CDP-choline pathway in at was to a in of at we the second pathway for PC synthesis, the PE methylation pathway. with and the of the into PC was under conditions used to The cells or the synthesized PC via PE methylation at levels to cells at both and of Kap95 function not cells In this study, we determined that Pct1, the rate-determining enzyme in the CDP-choline pathway for the synthesis of the major membrane lipid PC, directly interacts with the α-importin The of Pct1 was required for Pct1 to with Kap60 and for Pct1 to the nucleus. Pct1 was found to with both Kap60 and Kap95 in protein from yeast cell and of Kap60 or Kap95 function Pct1 from the nucleus. Pct1 the nucleus via the route of binding the α-importin Kap60 the basic acid Pct1, by of the β-importin with the tripartite protein complex by the nuclear for into the nucleus. Kap95 function PC this was most when Pct1 was from the nucleus. of the region of Pct1 that interacts with Kap60 the of PC synthesis. as we are this is the of directly a pathway. Pct1 protein enzyme in Kent C. 2001; PubMed Scopus Google and Kap60 or Kap95 are not required for in the of the Kap95 in is required for Pct1 be in this Kap95 Pct1 in the of the or in the of Kap95 Pct1 is by some cellular to nuclear Kap95 be to Pct1 with the Kap60 and Kap95 from Kap60 and Kap95 are of the nucleus to of nuclear the of Kap60 and Kap95 for of Pct1 function when Pct1 is extranuclear. The step in the synthesis of PC via the CDP-choline pathway is catalyzed by A.L. McMaster C.R. Biochem. 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