Dolichol kinase (DK) catalyzes the CTP-dependent phosphorylation of dolichol in the biosynthesis de novo and possibly the recycling of dolichyl monophosphate in yeast and mammals. A cDNA clone from human brain encoding the mammalian homologue, hDKp, of the yeast enzyme has recently been identified. In this study hDK has been overexpressed in Chinese hamster ovary cells and shown to be a polytopic membrane protein localized in the endoplasmic reticulum with an N terminus extended into the lumen and a cytoplasmically oriented C terminus. A conserved sequence, DXXAXXXGXXXGX8KKTXEG, found in several enzymes utilizing CTP as substrate including DKs, phytol kinases, and several CDP-diacylglycerol synthetases has been identified, and the possibility that it is part of the CTP-binding domain of hDKp has been investigated. Topological studies indicate that the loop between transmembrane domains (TMD) 11 and TMD12 of hDKp, containing the putative CTP binding domain, faces the cytoplasm. Deletion of the loop between TMD11-12, hDK(Δ459-474), or mutation of selected conserved residues within the cytoplasmic loop results in either a partial or total loss of activity and significant reductions in the affinity for CTP. In addition, the SEC59 gene in the yeast DK mutant was sequenced, and a G420D substitution was found. Conversion of the corresponding residue Gly-443 in hDKp to aspartic acid resulted in inactivation of the mammalian enzyme. These results extend the information on the topological arrangement of hDKp and indicate that the cytoplasmic loop between TMDs 11-12, containing the critical conserved residues, lysine 470 and lysine 471 in the 470KKTXEG475 motif, is part of the CTP-binding site in hDK. Dolichol kinase (DK) catalyzes the CTP-dependent phosphorylation of dolichol in the biosynthesis de novo and possibly the recycling of dolichyl monophosphate in yeast and mammals. A cDNA clone from human brain encoding the mammalian homologue, hDKp, of the yeast enzyme has recently been identified. In this study hDK has been overexpressed in Chinese hamster ovary cells and shown to be a polytopic membrane protein localized in the endoplasmic reticulum with an N terminus extended into the lumen and a cytoplasmically oriented C terminus. A conserved sequence, DXXAXXXGXXXGX8KKTXEG, found in several enzymes utilizing CTP as substrate including DKs, phytol kinases, and several CDP-diacylglycerol synthetases has been identified, and the possibility that it is part of the CTP-binding domain of hDKp has been investigated. Topological studies indicate that the loop between transmembrane domains (TMD) 11 and TMD12 of hDKp, containing the putative CTP binding domain, faces the cytoplasm. Deletion of the loop between TMD11-12, hDK(Δ459-474), or mutation of selected conserved residues within the cytoplasmic loop results in either a partial or total loss of activity and significant reductions in the affinity for CTP. In addition, the SEC59 gene in the yeast DK mutant was sequenced, and a G420D substitution was found. Conversion of the corresponding residue Gly-443 in hDKp to aspartic acid resulted in inactivation of the mammalian enzyme. These results extend the information on the topological arrangement of hDKp and indicate that the cytoplasmic loop between TMDs 11-12, containing the critical conserved residues, lysine 470 and lysine 471 in the 470KKTXEG475 motif, is part of the CTP-binding site in hDK. Dolichyl monophosphate (Dol-P) 2The abbreviations used are: Dol-P, dolichyl monophosphate; ER, endoplasmic reticulum; DK, dolichol kinase; hDKp, mammalian homologue of DK; CPY, carboxypeptidase Y; CDS, CDP-diacylglycerol synthase; TMD, transmembrane domain; CHO, Chinese hamster ovary; GFP, green fluorescent protein; PBS, phosphate-buffered protein. 2The abbreviations used are: Dol-P, dolichyl monophosphate; ER, endoplasmic reticulum; DK, dolichol kinase; hDKp, mammalian homologue of DK; CPY, carboxypeptidase Y; CDS, CDP-diacylglycerol synthase; TMD, transmembrane domain; CHO, Chinese hamster ovary; GFP, green fluorescent protein; PBS, phosphate-buffered protein. serves an essential function as a glycosyl carrier lipid in the assembly of N-linked glycoproteins, glycosylphosphatidylinositol anchors, and the C- and O-mannosylation of proteins in the endoplasmic reticulum (ER) of yeast and animal cells (1Schenk B. Fernandez F. Waechter C.J. Glycobiology. 2001; 11: 61-70Crossref PubMed Scopus (142) Google Scholar, 2Helenius A. Aebi M. Annu. Rev. Biochem. 2004; 73: 1019-1049Crossref PubMed Scopus (1609) Google Scholar). The enzymatic transfer of phosphoryl groups from CTP to dolichol catalyzed by microsomal fractions from mammalian cells was first detected more than 20 years ago (3Allen Jr., C.M. Kalin J.R. Sack J. Verizzo D. Biochemistry. 1978; 17: 5020-5026Crossref PubMed Scopus (72) Google Scholar, 4Burton W.A. Scher M.G. Waechter C.J. J. Biol. Chem. 1979; 254: 7129-7136Abstract Full Text PDF PubMed Google Scholar). Developmental changes in dolichol kinase (DK) activity corresponding to an increased capacity for protein N-glycosylation have been reported for sea urchin embryos (5Rossignol D.P. Lennarz W.J. Waechter C.J. J. Biol. Chem. 1981; 256: 10538-10542Abstract Full Text PDF PubMed Google Scholar), estrogen-treated chick oviducts (6Burton W.A. Lucas J.J. Waechter C.J. J. Biol. Chem. 1981; 256: 632-635Abstract Full Text PDF PubMed Google Scholar), Dictyostelium discoideum (7Rossler H.H. Zimpfer A. Risse H.-J. Mol. Cell. Biochem. 1982; 48: 183-189Crossref PubMed Scopus (8) Google Scholar), and pig (8Scher M.G. Sumbilla C.M. Waechter C.J. J. Biol. Chem. 1985; 260: 13742-13746Abstract Full Text PDF PubMed Google Scholar) and rat brain (9Volpe J.J. Sakakihara Y. Rust R.S. Brain Res. 1987; 31: 193-200Crossref Scopus (20) Google Scholar, 10Bhat N.R. Frank D.W. Wolf M.J. Waechter C.J. J. Neurochem. 1991; 56: 339-344Crossref PubMed Scopus (13) Google Scholar). If the enzymatic reduction of the α-isoprene unit in dolichol occurs at the free polyprenol level as proposed by Sagami et al. (11Sagami H. Kurisaki A. Ogura K. J. Biol. Chem. 1993; 268: 10109-10113Abstract Full Text PDF PubMed Google Scholar), DK would catalyze the final step in the de novo pathway for Dol-P biosynthesis (1Schenk B. Fernandez F. Waechter C.J. Glycobiology. 2001; 11: 61-70Crossref PubMed Scopus (142) Google Scholar). In 1992 Heller et al. (12Heller L. Orlean P. Adair Jr., W.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7013-7016Crossref PubMed Scopus (52) Google Scholar) identified the SEC59 gene product as a protein essential for the expression of yeast DK. Recently (13Fernandez F. Shridas P. Jiang S. Aebi M. Waechter C.J. Glycobiology. 2002; 12: 555-562Crossref PubMed Scopus (25) Google Scholar) we have identified a cDNA clone from a human brain library that encodes the mammalian homologue of DK (hDKp). The identification of the mammalian homologue of SEC59 was based on its ability to 1) complement the growth defect, 2) increase DK activity and, consequently, Dol-P levels in vivo, and 3) restore normal N-glycosylation of carboxypeptidase Y (CPY) at the restrictive temperature in the temperature-sensitive mutant sec59-1. The observations that the CTP-mediated phosphorylation of diacylglycerol was not affected by either the temperature-sensitive mutation in the sec59-1 strain or the overexpression of the SEC59 gene or the mammalian homologue, hDK, under conditions that changed the level of DK activity, demonstrated that diacylglycerol was not phosphorylated by Sec59p in yeast. The hDK cDNA has an open reading frame that encodes a protein with 538 amino acids and a molecular weight of 59,268. Analyses by several hydropathy plots suggest that brain hDKp is a very hydrophobic protein with as many as 13 membrane-spanning domains. In this regard, the prokaryotic enzyme counterpart of hDKp, which phosphorylates undeca-prenol in bacteria, is also apparently extremely hydrophobic, as it can be solubilized and partially purified in an active form in n-butanol (14Higashi Y. Strominger J.L. J. Biol. Chem. 1970; 245: 3691-3696Abstract Full Text PDF PubMed Google Scholar, 15Sandermann Jr., H. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 2441-2443Crossref PubMed Scopus (48) Google Scholar). Although the brain enzyme is enriched in heavy microsomes (16Scher M.G. Devries G.H. Waechter C.J. Arch. Biochem. Biophys. 1984; 231: 293-302Crossref PubMed Scopus (28) Google Scholar), it lacks a C-terminal KKXX motif for ER retention (17Nilsson T. Jackson M. Peterson P. Cell. 1989; 58: 707-718Abstract Full Text PDF PubMed Scopus (367) Google Scholar). In this study N- and C-terminally FLAG-tagged constructs were studied to gain new information about the topological arrangement of hDK in the ER the used by and S. Y. H. T. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) to the membrane of the a of glycosylphosphatidylinositol ER enzyme. a motif has been identified in hDK and SEC59 that is conserved in several enzymes utilizing CTP as a including and CDP-diacylglycerol M. A. M. A. S. B. PubMed Scopus Google Scholar, S. K. A. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, H. C.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L. B. M. PubMed Scopus Google Scholar, D. L. S. P. K. H. Mol. Biochem. PubMed Scopus Google Scholar) and phytol from and K. F. B. B. Cell. PubMed Scopus Google Scholar). is a part of the domain of and yeast DK gene this is a to be part of the CTP-binding studies with hDK were to the of the conserved motif in its In addition, the mutation in the temperature-sensitive yeast sec59-1 mutant has been identified. The that the cytoplasmically oriented loop between TMDs containing the conserved motif is part of the CTP-binding site is and strain was used for The strain used in this study was with or with or the of hDK. and of hDK and the in and and hDK were in yeast as (13Fernandez F. Shridas P. Jiang S. Aebi M. Waechter C.J. Glycobiology. 2002; 12: 555-562Crossref PubMed Scopus (25) Google Scholar). FLAG-tagged hDK was by as a and the and with and and The product was into of the mammalian expression The site of the is for expression of the protein with an of the hDK gene was by the the of as in to substitution for the amino acids in the mutation and mutation and mutation and mutation and mutation and mutation and mutation and mutation and mutation and mutation and The containing an and were used as the for the in mammalian and yeast The were by the the of the protein of hDK, the gene from was into and of the The were used for the hDK with a in the loop between and TMD12 and and the of SEC59 and in a the constructs were first in as and into and of cells were at in a in with were in and with the containing gene the were selected with of from and fractions from cells were as for cells (13Fernandez F. Shridas P. Jiang S. Aebi M. Waechter C.J. Glycobiology. 2002; 12: 555-562Crossref PubMed Scopus (25) Google Scholar). the cells were by with PBS, and in containing and at the The cells were by and the were from the by The membrane was with the by and in the at a final protein of fractions from S. were as by Fernandez et al. (13Fernandez F. Shridas P. Jiang S. Aebi M. Waechter C.J. Glycobiology. 2002; 12: 555-562Crossref PubMed Scopus (25) Google Scholar), and protein was by the of et al. F. M. H. Biochem. 1989; PubMed Scopus Google Scholar) of Dolichol in fractions from and yeast cells were as The for DK activity was as (13Fernandez F. Shridas P. Jiang S. Aebi M. Waechter C.J. Glycobiology. 2002; 12: 555-562Crossref PubMed Scopus (25) Google Scholar) that the protein was 20 of dolichol in final and in a total of for 20 at the enzymatic transfer of from to Dol-P was by a Waechter C.J. 1985; PubMed Scopus Google Scholar). The were with to for 20 and The for CTP was at CTP from to for the and and for and for dolichol were by dolichol from to in the the of of of from yeast cells at for and to for were by with as M. J. H. S. Glycobiology. PubMed Scopus Google Scholar). and of FLAG-tagged expression was by 1970; PubMed Scopus Google Scholar) and of the FLAG-tagged proteins by the cells were for 20 with the cells were in in for 20 at more with PBS, the membrane was with at in for on and including the endoplasmic were with in for at containing and was to The cells were with the or in the for 20 at temperature and The cells were an 20 at temperature with the in the and with The were on with were on a was used as the of SEC59 in sec59-1 was from yeast sec59-1 cells by the of was used for the first cDNA a first cDNA with in the in a final of of the cDNA was used to for mutant SEC59 the as (13Fernandez F. Shridas P. Jiang S. Aebi M. Waechter C.J. Glycobiology. 2002; 12: 555-562Crossref PubMed Scopus (25) Google Scholar). of the SEC59 gene and of the were by the cDNA in of Topological of hDK in the ER with N- and C-terminal FLAG-tagged on for hydropathy plots S. T. 12: PubMed Scopus Google Scholar), hDKp is to have 13 TMDs with a oriented N terminus and a cytoplasmically oriented C terminus If this topological arrangement is would be cytoplasmic The possibility that the motif conserved DKs, from and phytol from and (12Heller L. Orlean P. Adair Jr., W.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7013-7016Crossref PubMed Scopus (52) Google Scholar, F. Shridas P. Jiang S. Aebi M. Waechter C.J. Glycobiology. 2002; 12: 555-562Crossref PubMed Scopus (25) Google Scholar, M. A. M. A. S. B. PubMed Scopus Google Scholar, S. K. A. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, H. C.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L. B. M. PubMed Scopus Google Scholar, D. L. S. P. K. H. Mol. Biochem. PubMed Scopus Google Scholar, K. F. B. B. Cell. PubMed Scopus Google Scholar), is part of the CTP-binding site was of this is in the loop between and TMD12 A study that DK activity was enriched in the heavy microsomal from brain (16Scher M.G. Devries G.H. Waechter C.J. Arch. Biochem. Biophys. 1984; 231: 293-302Crossref PubMed Scopus (28) Google Scholar). In the study hDK cDNA was in the and in cells to that the enzyme was in the ER by in the hDKp a to the ER with the results from a study with brain (16Scher M.G. Devries G.H. Waechter C.J. Arch. Biochem. Biophys. 1984; 231: 293-302Crossref PubMed Scopus (28) Google Scholar). of hDK containing or C-terminal were also in cells to the topological arrangement and the of the N and C of the enzyme in the of the N or C not DK enzyme activity not of the constructs used in topological studies were to by the were on and with to the membrane or with to including the cells FLAG-tagged hDK were with and by the protein was not that the extended into the of the with the ER and the was not by in cells by In to this the C-terminal FLAG-tagged of hDK was by a be in that the C terminus was cytoplasmically oriented that the N terminus in the cells were with to the ER and by a the FLAG-tagged of hDK be by by the results in the C-terminal of hDK also be detected in In addition, the protein was that of the cells with the membrane and the ER, These results for the topological arrangement in The in CTP as were conserved or in enzymes utilizing CTP as a the hDKp was in a of at the for J. Mol. Biol. PubMed Scopus Google Scholar). A of the amino acid of hDK, yeast phytol from and and CDS, which catalyze the biosynthesis of CDP-diacylglycerol from and a conserved motif a of the protein In the phytol the of hDK is by a on the results part of the motif residues in was in the cytoplasmic loop between TMDs 11 and of hDK and it has been found in the that were this motif was to be a part of the CTP-binding The between and of the and for DK that the loop between TMDs 11 and containing part of the conserved sequence, was cytoplasmically as in a of hDK was with a between and was into the cells were and with or as in the The FLAG-tagged enzyme was by in that the loop containing part of the putative CTP-binding site faces the as in the in the was not by that the of cells with not the of the ER not with the that the loop part of the CTP-binding of residues in the loop between and TMD12 resulted in a total loss of DK activity in microsomes from and sec59-1 cells normal or of in a new of of on and to in and in sec59-1 residues in the conserved motif essential for activity, studies were The conserved residues, and were as for CTP binding and and were by The hDK as as the were with an in yeast sec59-1 cells temperature-sensitive yeast mutant for DK and cells and as in overexpression of hDK resulted in a increase in DK activity in the sec59-1 The and in kinase activity to the level in sec59-1 cells at the A was for the substitution in the yeast In to results the and not a reduction in DK The temperature-sensitive sec59-1 mutation in yeast results in a reduction in the growth and protein N-glycosylation at the temperature study has shown that overexpression of hDK in the mutant cells the growth and (13Fernandez F. Shridas P. Jiang S. Aebi M. Waechter C.J. Glycobiology. 2002; 12: 555-562Crossref PubMed Scopus (25) Google Scholar). The ability to complement a to the of on hDK this the of the of residues in the conserved were Although the and mutant DK activity, the mutant were to restore growth of sec59-1 cells the and the were not to complement the growth of sec59-1 with the mutant a growth at with the cells with hDK. cells with the or mutant enzymes for These observations indicate that residues and the of in the conserved critical for a kinase of hDK and the in of the of conserved residues in hDK on the overexpression of the proteins was also in cells to the results of the corresponding in yeast. of the hDK in cells resulted in a increase in DK the results in it can be that the hDK(Δ459-474), and levels of DK activity to hDK. the substitution a reduction in the yeast this mutation was more in an reduction to the hDK in of FLAG-tagged hDK and the resulted in the of protein at the molecular weight of as detected by with the level of kinase activity not to be to levels of the mutant in the yeast as the and the not the level of DK The and the of hDK for CTP selected of the amino acid residues, were to changes in kinase activity and its affinity for the CTP and by the for CTP and dolichol in the hDK and the mutant The and of and resulted in significant changes in the for from to to the enzyme. The substitution resulted in a increase in the for CTP Although the and affected the affinity for the of the mutant enzymes were to growth at the temperature of hDK activity in microsomes from cells with and mutant hDK in a new The and that the growth in the yeast mutant not significant changes in the affinity for CTP. of the changes in the for with the that the and residues part of the CTP-binding domain in the loop between TMDs 11 and studies by that the mutant a ER as the hDK, the possibility that the mutation a of DK not of the in the DK from sec59-1 yeast DK gene was also of the sec59-1 mutant to the mutation for the temperature-sensitive The a in the of to aspartic acid within were detected in of the SEC59 was and into sec59-1 the mutant gene to complement growth and at the restrictive temperature A and with the G420D mutation resulted in a loss of DK activity at the temperature the of this mutation of the corresponding residue in hDK also its activity in cells at temperature 3) as as its ability to complement the yeast in growth and not expression of the protein was by in microsomes from yeast sec59-1 cells with SEC59 or the mutant and with hDK or in a new If the polyprenol is the substrate for the enzymatic reduction of the α-isoprene unit of dolichol (11Sagami H. Kurisaki A. Ogura K. J. Biol. Chem. 1993; 268: 10109-10113Abstract Full Text PDF PubMed Google Scholar), DK would catalyze the final step in the de novo pathway for Dol-P biosynthesis (1Schenk B. Fernandez F. Waechter C.J. Glycobiology. 2001; 11: 61-70Crossref PubMed Scopus (142) Google Scholar). The CTP-mediated kinase also be in the recycling of the glycosyl carrier lipid it is as protein N-glycosylation is that the Dol-P, by on the F. Aebi M. Waechter C.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Jiang S. S. Waechter C.J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), is and to the cytoplasmic of the ER as the free (1Schenk B. Fernandez F. Waechter C.J. Glycobiology. 2001; 11: 61-70Crossref PubMed Scopus (142) Google Scholar). DK would also catalyze the of Dol-P for of lipid In this a recently cDNA encoding hDK (13Fernandez F. Shridas P. Jiang S. Aebi M. Waechter C.J. Glycobiology. 2002; 12: 555-562Crossref PubMed Scopus (25) Google Scholar) has been used to more for the ER and topological arrangement of the enzyme in the hDK to the ER of in with studies with brain (16Scher M.G. Devries G.H. Waechter C.J. Arch. Biochem. Biophys. 1984; 231: 293-302Crossref PubMed Scopus (28) Google Scholar). on with N- and C-terminal FLAG-tagged constructs or of hDK is a polytopic protein with a oriented N terminus and a C terminus on the cytoplasmic indicate that DK has 13 In addition, the loop between and proposed to part of the CTP-binding domain has been shown to be on the cytoplasmic of the In an Adair and Jr., W.L. Biophys. PubMed Scopus Google Scholar) demonstrated that DK activity in rat microsomes was to with and These results with the of the ER to CTP CTP have been detected in the ER as of this indicate that at part of the site of the enzyme is on the cytoplasmic of the of this was to the CTP-binding site and to the that the CTP-mediated phosphorylation on the cytoplasmic of the A of the to the identification of a conserved motif the from the a of from and and the recently identified phytol kinase from and (12Heller L. Orlean P. Adair Jr., W.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7013-7016Crossref PubMed Scopus (52) Google Scholar, F. Shridas P. Jiang S. Aebi M. Waechter C.J. Glycobiology. 2002; 12: 555-562Crossref PubMed Scopus (25) Google Scholar, M. A. M. A. S. B. PubMed Scopus Google Scholar, S. K. A. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, H. C.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L. B. M. PubMed Scopus Google Scholar, D. 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Neurochem. PubMed Scopus Google Scholar, K. F. J. PubMed Google Scholar). this motif is in or which proteins in the of A. Jackson S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, A. K. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), in to which catalyze the of the The and have the and in CTP which in and S. Full Text Full Text PDF PubMed Scopus Google Scholar, P. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). mutation of aspartic acid and of the the ability of the enzyme to complement the growth and in the temperature-sensitive sec59-1. The mutant a growth of sec59-1 to the enzyme at the with and part of the CTP-binding the for CTP increased in the mutant and and significant changes in a affinity for CTP. of results with the that the residues, and a in CTP be that of the of the hDK were by on the growth and in the sec59-1 mutant and the level of enzyme activity in microsomes from the yeast mutant and more be by the ability of the to a also be to the to be identified CTP-mediated diacylglycerol kinase from S. has a motif containing the lysine residues A. L. Arch. Biochem. Biophys. PubMed Scopus Google Scholar). In to the residues in the CTP-binding a G420D substitution has been found in the mutant enzyme in sec59-1 The of this residue was by the that the corresponding mutation in hDK, resulted in a loss of DK activity in The activity was to the mutation affected the binding of its within the affinity of the enzyme for the hydrophobic In the studies have extended the information on the topological and the of a conserved motif that is proposed to be part of a putative CTP-binding site in and phytol from as as of the by and the mutation found in the yeast enzyme be the for as to be identified of of H.H. Aebi M. Biol. PubMed Scopus Google Scholar). studies be at more about the of the loop between TMDs 11 and and cytoplasmically oriented ER retention in hDK, and the hydrophobic domains a is also be of to the motif in hDK is also in the CTP-mediated in the of and to the in and Waechter C.J. Biochem. Biophys. Res. PubMed Scopus Google Scholar, L. J. J. Waechter C.J. Proc. Natl. Acad. Sci. U. S. A. 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