Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M604087200 on August 21, 2006

J. Biol. Chem., Vol. 281, Issue 42, 31696-31704, October 20, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/42/31696    most recent
M604087200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shridas, P.
Right arrow Articles by Waechter, C. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shridas, P.
Right arrow Articles by Waechter, C. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Human Dolichol Kinase, a Polytopic Endoplasmic Reticulum Membrane Protein with a Cytoplasmically Oriented CTP-binding Site*

Preetha Shridas and Charles J. Waechter1

From the Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536

Received for publication, April 28, 2006 , and in revised form, August 8, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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({Delta}459-474), 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Dolichyl monophosphate (Dol-P)2 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 (1, 2). 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 (3, 4). Developmental changes in dolichol kinase (DK) activity corresponding to an increased capacity for protein N-glycosylation have been reported for sea urchin embryos (5), estrogen-treated chick oviducts (6), Dictyostelium discoideum (7), and pig (8) and rat brain (9, 10). If the enzymatic reduction of the {alpha}-isoprene unit in dolichol occurs at the free polyprenol level as proposed by Sagami et al. (11), DK would catalyze the final step in the de novo pathway for Dol-P biosynthesis (1).

In 1992 Heller et al. (12) identified the SEC59 gene product as a protein essential for the expression of yeast DK. Recently (13) 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 (14, 15). Although the brain enzyme is enriched in heavy microsomes (16), it lacks a C-terminal KKXX motif for ER retention (17).

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 following the same basic experimental approach used by Menon and co-workers (18) to characterize the membrane topology of the Gaa1 protein, a subunit of glycosylphosphatidylinositol transamidase, another ER enzyme. Moreover, a DXXAXXXGXXXGX8KKTXEG motif has been identified in hDK and SEC59 that is conserved in several other enzymes utilizing CTP as a substrate, including human, rat, yeast, Drosophila, and Plasmodium falciparum CDP-diacylglycerol synthases (CDSs) (19-23) and phytol kinases from Arabidopsis and Synechocystis (24). This sequence is a part of the PFAM domain of cytidylyltransferases and yeast DK gene (CTP_transf_1, PFAM accession number PF01148). Thus, this sequence is a plausible candidate to be part of the CTP-binding site. Site-directed mutagenesis studies with hDK were conducted to explore the role of the conserved DXXAXXXGXXXGX8KKTXEG motif in its catalytic function. In addition, the point mutation in the temperature-sensitive yeast sec59-1 mutant has been identified. The evidence that the cytoplasmically oriented loop between TMDs 11-12 containing the conserved motif is part of the CTP-binding site is discussed.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Culture Conditions—Escherichia coli strain DH5{alpha} was used for all cloning procedures. The Saccharomyces cerevisiae strain used in this study was sec59-1, YG 0736 MAT{alpha} ade2 his3{Delta}200 ura3-52, transformed with plasmid YEp352, pSEC59, or with phDK or the mutants of hDK.

Construction and Expression of hDK and the Mutants in Yeast and CHO Cells—SEC59 and hDK were constructed in yeast shuttle vector YEp352 as described earlier (13). N-terminal FLAG-tagged hDK was constructed by PCR using hDK-YEp352 as a template and the following forward and reverse primers with SalI and XbaI restriction sites, respectively: 5'-TAG, CGT CGA CAT GAC CCG A-3' (F) and 5'-TCC TCT AGA CTA GGC CAT CAG CAA TAT-3' (R). The PCR product was cloned into similar restriction sites of the mammalian expression vector pME18Sf. The restriction site of the plasmid is designed for expression of the protein with an N-terminal FLAG epitope tag. Site-directed mutagenesis of the hDK gene was performed by the QuikChange method (Stratagene, La Jolla, CA) using the set of primers as follows (codons indicated in bold letters correspond to alanine substitution for the respective amino acids in the wild type): mutation G475A, 5'-AAA AAG ACT TTT GAG GCG ACC ATG ACA TCT ATA-3' (F) and 5'-TAT AGA TGT CAT GGT CGC CTC AAA AGT CTT TTT-3' (R); mutation K470A/K471A, 5'-CGCTGGCCTGGAACCGCAGCGACTTTTGAGGGGACC-3' (F) and 5'-GGTCCCCTCAAAAGTCGCTGCGGTTCCAGGCCAGCG-3' (R); mutation K470A, 5'-CGCTGGCCTGGAACCGCAAAGACTTTTGAGGGG-3' (F) and 5'-CCCCTCAAAAGTCTTTGCGGTTCCAGGCCAGCG-3' (R); mutation K471A, 5'-TGGCCTGGAACCAAAGCGACTTTTGAGGGGACC-3' (F) and 5'-GGTCCCCTCAAAAGTCGCTTTGGTTCCAGGCCA-3' (R); mutation E474A, 5'-ACCAAAAAGACTTTTGCGGGGACCATGACATCT-3' (F) and 5'-AGATGTCATGGTCCCCGCAAAAGTCTTTTTGGT-3' (R); mutation D451A, 5'-GCT GTG GGT GTG GGT GCT ACT GTG GCC TCC ATC-3' (F) and 5'-GAT GGA GGC CAC AGT AGC ACC CAC ACC CAC AGC-3' (R); mutation T472A, 5'-CCT GGA ACC AAA AAG GCT TTT GAG GGG ACC ATG-3' (F) and 5'-CAT GGT CCC CTC AAA AGC CTT TTT GGT TCC AGG-3' (R); mutation G443D, 5'-CTC GTC CCC TAT GCC GAT GTC CTG GCT GTG GGT-3' (F) and 5'-ACC CAC AGC CAG GAC ATC GGC ATA GGG GAC GAG-3' (R); mutation hDK({rho}459-474), 5'-GCC TCC ATC TTC GGT ACC ATG ACA TCT ATA-3' (F) and 5'-TAT AGA TGT CAT GGT ACC GAA GAT GGA GGC-3' (R); mutation SEC59(G420D), 5'-AAT AAC TCT CCA ATG GAT CTA ATA GGA TTG GGA-3' (F) and 5'-TCC CAA TCC TAT TAG ATC CAT TGG AGA GTT ATT-3' (R).

The hDK-pME18Sf construct containing an N-terminal FLAG tag and hDK-YEp352 were used as the templates for constructing the mutants in mammalian vector pME18Sf and yeast shuttle vector YEp352, respectively. The mutations were verified by sequencing the entire coding region (Davis Sequencing). For the construction of the N-terminal GFP fusion protein of hDK, the gene from pME18Sf was subcloned into SalI and XbaI sites of the plasmid pEGFP-C1.

The following primers were used for the hDK construct with a FLAG tag in the loop between TMD11 and TMD12 (Pro467 and Gly468, Fig. 1A): 5'-GAG ATC CGC TGG CCT GAC TAC AAG GAC GAC GAT GAC AAG GGA ACC AAA AAG ACT-3' (F) and 5'-AGT CTT TTT GGT TCC CTT GTC ATC GTC GTC CTT GTA GTC AGG CCA GCG GAT CTC-3' (R). For the construction of SEC59 and SEC59(G420D) in a single copy plasmid pRS316, the constructs were first made in Yep352 as described above and then subcloned into pRS316.

Cell Culture and Transfection of CHO Cells—CHO cells were grown at 37 °C in a 5% CO2 atmosphere in F-12 nutrient mixture (Ham's medium) (Invitrogen) supplemented with 10% (v/v) fetal bovine serum. Cells were plated in 100-mm dishes and transfected with the plasmid containing appropriate gene using Lipofectamine 2000 (Invitrogen) following the manufacture's protocol. Stable transformants were selected with 10 µg/ml blasticidin S HCl (Invitrogen).

Preparation of Crude Microsomal Fractions from CHO and Yeast Cells—Microsomal fractions from CHO cells were prepared as described earlier for Sf9 cells (13). Briefly, the cells were harvested by centrifugation, washed once with chilled PBS, and suspended in buffer containing 50 mM Tris-HCl (pH 7.4), 0.25 M sucrose, 0.5 mM EDTA, 5 mM 2-mercaptoethanol, and Complete Mini protease inhibitor mixture (Roche Applied Science) tablet at the recommended concentration (suspension buffer). The cells were disrupted by sonication, and the membranes were sedimented from the lysate by centrifugation (100,000 x g, 25 min, 4 °C). The crude membrane fraction was washed once with the suspension buffer, sedimented by centrifugation (100,000 x g, 15 min, 4 °C), and resuspended in the suspension buffer at a final protein concentration of 10-20 mg/ml. Microsomal fractions from S. cerevisiae were prepared as described by Fernandez et al. (13), and protein was determined by the method of Rodriquez-Vico et al. (25) using BCA (Pierce).

Assay of Dolichol Kinase Activity in Vitro—Membrane fractions from CHO and yeast cells were prepared as described above. The assay for DK activity was carried out essentially as described previously (13) except that the protein concentration was 50 µg/0.05 ml. Standard reaction mixtures contained 50 mM Tris-HCl (pH 7.4), 0.125 M sucrose, 0.5 mM EDTA, 20 mM UTP, 5 µg of dolichol (dispersed in Triton X-100, final concentration = 0.05%), 30 mM CaCl2, 5mM mercaptoethanol, and 40 µM [{gamma}-32P]CTP (500 cpm/pmol) in a total volume of 50 µl. After incubation for 20 min at 37 °C, the enzymatic transfer of 32P from [{gamma}-32P]CTP to Dol-P was assayed essentially by a procedure described elsewhere (26). The reaction rates were linear with respect to time for 20 min and reflect initial rates. The Km value for CTP was determined at varying CTP concentrations from 0.2 to 4.0 µM for the wild-type, K470A, and E474A and 5-50 µM for K470A/K471A and K471A. Km values for dolichol were determined by varying dolichol concentrations from 5 to 40 µM in the assay mixture. Values are the average of three independent experiments.


Figure 1
View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 1.
Predicted topological arrangement of hDKp in the ER based on the SOSUI system (29). The N and C termini face the luminal and the cytoplasmic side of the membrane, respectively. The amino acids conserved among DKs and CDSs are indicated by solid circles (loop between TMDs 11-12, panel A). Residues in the loop between TMD11 and TMD12 are indicated in the box (panel B). Conserved residues are shaded.

 
Detection of Glycoforms of CPY—Total cell lysates from yeast cells grown at 30 °C (permissive temperature) for 7 h and shifted to 37 °C (nonpermissive temperature) for 10 h were analyzed by Western blotting with anti-CPY antisera after 7% SDS-PAGE as described previously (27).

SDS-PAGE and Detection of FLAG-tagged Proteins—Protein expression was analyzed by 10% SDS-PAGE (28) and detection of the FLAG-tagged fusion proteins by Western blot analysis using anti-FLAG M2 monoclonal antibody.

Immunofluorescence Microscopy—For immunostaining, the pertinent cells were plated onto glass coverslips fixed for 20 min with cold methanol. For topology studies, the cells were fixed in 4% paraformaldehyde in PBS for 20 min at room temperature. After 3 more washes with PBS, the plasma membrane was selectively permeabilized with digitonin at 3 µg/ml in PBS for 5 min on ice. Alternatively, plasma membranes and intracellular membranes, including the endoplasmic reticulum, were permeabilized with 0.3% Triton X-100 in PBS for 10 min at room temperature. PBS containing 5% bovine calf serum and 5% horse serum was then added to prevent nonspecific antibody interactions. The cells were incubated with primary antibodies raised against the FLAG epitope or calnexin in the same buffer for 20 min at room temperature and washed 3 times. The cells were re-incubated an additional 20 min at room temperature with the appropriate antibody in the same buffer and washed again three times with PBS. The coverslips were mounted on glass slides with Vectashield mounting solution. Cells were viewed on a Nikon Eclipse E600 microscope. Texas Red-conjugated bovine anti-mouse antibody (Vector Laboratories, Burlingame, CA) was used as the secondary antibody.

Sequencing of Mutant SEC59 Gene in sec59-1 Cells—Total RNA was isolated from yeast sec59-1 cells by RNA isolation kit (Qiagen, Valencia, CA) following the manufacturer's protocol. Five µg of RNA was used for the first strand cDNA synthesis using a first strand cDNA synthesis kit (Amersham Biosciences) with random hexamers provided in the kit in a final volume of 15 µl. Ten µl of the cDNA mixture was used to amplify mRNA for mutant SEC59 using the gene-specific primers as described previously (13). Sequence of the defective SEC59 gene and 225 bases of the 5'-untranslated promoter regions were confirmed by sequencing (Davis Sequencing, Davis, CA) the cDNA isolated in two separate sets of experiments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Topological Arrangement of hDK in the ER Assessed with N- and C-terminal FLAG-tagged Constructs—Based on theoretical algorithms for hydropathy plots (29), hDKp is predicted to have 13 TMDs with a luminally oriented N terminus and a cytoplasmically oriented C terminus (Fig. 1A). If this topological arrangement is correct, there would be five major cytoplasmic loops. The possibility that the motif DXXAXXXGXXXG-X8KKTXEG, conserved among DKs, CDSs from various sources, and phytol kinases from Arabidopsis and Synechocystis (12, 13, 19-24), is part of the CTP-binding site was investigated (see below). Part of this sequence is located in the cytosolic loop between TMD11 and TMD12 (Fig. 1B).

A previous subcellular fractionation study indicated that DK activity was enriched in the heavy microsomal fraction from calf brain (16). In the current study N-terminal GFP-tagged hDK cDNA was constructed in the vector pEGFP-C1 and stably transfected in CHO cells to verify that the enzyme was indeed located in the ER by immunofluorescence. As seen in Fig. 2, the GFP-tagged hDKp exhibited a reticular pattern identical to the ER marker, calnexin, consistent with the earlier results from a subcellular fractionation study with calf brain (16).

Constructs of hDK containing N-terminal or C-terminal FLAG tags were also expressed in CHO cells to examine the topological arrangement and the orientation of the N and C termini of the enzyme in the ER. Epitope-tagging of the N or C termini did not affect DK enzyme activity (data not shown). All of the constructs used in these topological studies were stable transformants to avoid any mislocalization produced by the transfection agent, Lipofectamine. Cells were grown on coverslips, fixed, and treated with digitonin (3 µg/ml) to permeabilize the plasma membrane selectively or with 0.3% Triton X-100 to permeabilize intracellular membranes including the ER. When cells expressing N-terminal FLAG-tagged hDK were permeabilized with digitonin and examined by immunofluorescence (Fig. 3A), the epitope-tagged protein was not detected, indicating that the N-terminus extended into the luminal compartment of the ER. Consistent with the ER remaining sealed and intact, the luminal protein, calnexin, was similarly not detectable by immunofluorescence in cells permeabilized by digitonin (Fig. 3B). In contrast to this result, when the C-terminal FLAG-tagged construct of hDK was examined by immunofluorescence, a reticular pattern could be observed in digitonin-permeabilized cells, indicating that the C terminus was cytoplasmically oriented (Fig. 3B).


Figure 2
View larger version (9K):
[in this window]
[in a new window]
  		FIGURE 2.
ER localization of hDKp containing an N-terminal GFP-tag (center panel) in CHO cells by immunofluorescence. Calnexin (left panel) was used as an ER marker protein, and the patterns for hDK and calnexin are seen to overlap in the merged images (right panel).

 
To obtain additional evidence that the N terminus was, indeed, exposed in the luminal compartment, cells were permeabilized with 0.3% Triton X-100 to disrupt the ER and examined by immunofluorescence. Supporting a luminal orientation, the N-terminal FLAG-tagged construct of hDK could now be observed by immunofluorescence (Fig. 3A). As predicted by the results in (Fig. 3B), the C-terminal FLAG tag of hDK could also be detected in these cells. In addition, the luminal protein marker, calnexin, was now detectable, corroborating that treatment of the cells with 0.3% Triton X-100 unsealed the plasma membrane and the ER, exposing luminal components. These results provide evidence for the topological arrangement illustrated in Fig. 1.

The DXXAXXXGXXXGX8K/NKTXEG Sequence Is Conserved in Several Enzymes Utilizing CTP as Substrate—To determine if there were any conserved regions or sequence similarity in other enzymes utilizing CTP as a substrate, the hDKp sequence was tested in a BLAST search of sequence databases at the National Center for Biotechnology Information (30). A comparison of the deduced amino acid sequences of hDK, yeast SEC59, phytol kinases from Arabidopsis and Synechocystis and CDS, which catalyze the biosynthesis of CDP-diacylglycerol from phosphatidate and CTP, revealed a highly conserved sequence motif DXXAXXXGXXXGX8K/NKTXEG over a 25-residue span of the protein sequences (Fig. 4). In the phytol kinases the 471KTXEG475 sequence of hDK is replaced by a similar KSXAG sequence. Based on the results presented above, part of the DXXAXXXGXXXGX8K/NKTXEG motif (shaded residues in Fig. 1B) was contained in the cytoplasmic loop between TMDs 11 and 12 of hDK (Fig. 1B), and it has been found in all the CDSs that were analyzed. Thus, this motif was considered to be a potential part of the CTP-binding domain.

The Loop between TMD11 and TMD12, Containing Part of the DXXAXXXGXXXGX8K/NKTXEG Motif, Is Cytoplasmically Oriented and Required for DK Activity—To obtain proof that the loop between TMDs 11 and 12, containing part of the conserved sequence, was cytoplasmically oriented, as predicted in Fig. 1, a construct of hDK was made with a FLAG tag inserted between Pro467 and Gly468 (Fig. 5). This construct was then transfected into CHO cells; the cells were fixed and permeabilized with Triton X-100 or digitonin as in the experiments described above. The FLAG-tagged enzyme was observed by immunofluorescence in digitonin-permeabilized cells, indicating that the loop containing part of the putative CTP-binding site faces the cytoplasm as predicted in the model illustrated in Fig. 1. Under the same conditions, calnexin was not detectable by immunofluorescence, confirming that the treatment of cells with digitonin did not disrupt the integrity of the ER (data not included). Consistent with the proposal that the loop contains part of the CTP-binding site, truncation of 15 residues in the loop between TMD11 and TMD12 (hDK{rho}459-474) resulted in a total loss of DK activity (Table 1).


View this table:
[in this window]
[in a new window]
  		TABLE 1
DK activities in microsomes from CHO and sec59-1 cells expressing normal or variant forms of hDK

sec59-1 cells expressing the indicated form of hDK were grown in yeast extract/peptone/dextrose media at 30 °C (permissive) for 7 h and then shifted to 37 °C (nonpermissive) for 10 h. CHO cells stably expressing the indicated form of hDK were grown at 37 °C. Microsomes were prepared, and DK activity was assayed for all cells as described under "Experimental Procedures." All results are average values obtained from three separate experiments. nd, not determined.

 
Effects of Site-directed Mutagenesis of Conserved Residues on Activity and Ability to Complement Defects in Growth and Protein N-Glycosylation in sec59-1 Cells—To identify specific residues in the conserved motif essential for catalytic activity, site-directed mutagenesis studies were conducted. The conserved residues, Asp-451, Lys-470, Lys-471, Thr-472, Glu-474, and Gly-475 were chosen as candidates for CTP binding and catalysis and were mutated by alanine substitutions. The wild type hDK as well as the different mutants were then expressed with an N-terminal FLAG tag in yeast sec59-1 cells (a temperature-sensitive yeast mutant for DK activity) and CHO cells using YEp352 and pME18Sf vectors, respectively.

First, as seen in Table 1, overexpression of hDK resulted in a 5-fold increase in DK activity in the sec59-1 cells. The mutants D451A, K470A/K471A, K470A, and K471 produced lower increases in kinase activity relative to the low background level in sec59-1 cells at the nonpermissive temperature. A minor reduction, ~7%, was seen for the T472A substitution in the yeast background. In contrast to these results the mutations, G475A and E474A did not cause a reduction in DK activity.

The temperature-sensitive sec59-1 mutation in yeast results in a substantial reduction in the growth rate and protein N-glycosylation at the nonpermissive temperature (37 °C). Our earlier study has shown that overexpression of hDK in the mutant cells complements the growth and hypoglycosylation defects (13). The ability to complement these defects provides a simple approach to evaluating the effects of mutations on hDK activity. Using this strategy, the effects of the various mutations of residues in the conserved sequence were evaluated further.

Although the K470A and T472A mutant forms had reduced DK activity, the mutant forms were still able to restore growth of sec59-1 cells (Fig. 6A). However, the doubly mutated enzyme, K470A/K471A, and the single mutation, D451A, were not able to complement the growth defect of sec59-1 cells. Cells transformed with the K471A mutant exhibited a significantly reduced growth rate at 37 °C compared with the cells transformed with wild type hDK. Similarly, cells transformed with the K471A, K470A/K471A, D451A, or T472A mutant enzymes exhibited hypoglycoform patterns for CPY (Fig. 6B). These observations indicate that residues Asp-451, Thr-472, and the combination of Lys-470/471 in the conserved sequence are all critical for a fully functional kinase activity.


Figure 3
View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 3.
hDKp has a luminally oriented N terminus and a C terminus facing the cytoplasm. hDKp containing a FLAG-epitope tag (position denoted by black circle) at N terminus (A) or C terminus (B) were expressed in CHO cells. The cells were then fixed in 4% paraformaldehyde, selectively permeabilized with digitonin (3 µg/ml) or 0.3% Triton X-100, and stained with anti-FLAG antibodies followed by Texas Red-conjugated secondary antibodies.

 
Overexpression of hDK and the Mutant Enzymes in CHO Cells—The effect of the mutations of conserved residues in hDK on the overexpression of the variant proteins was also evaluated in CHO cells to compare the results of the corresponding mutations in yeast. Overexpression of the wild type hDK in CHO cells resulted in a 16-fold increase in DK activity. From the results in Table 1 it can be seen that the mutations, hDK({Delta}459-474), D451A, K470A/K471A, K470A, K471A, and T472A produced reduced levels of DK activity relative to wild type hDK. Whereas the T472A substitution produced a relatively small reduction (7%) in the yeast background, this mutation was considerably more disruptive, resulting in an 83% reduction relative to the wild type hDK when expressed in CHO cells.

Overexpression of N-terminal FLAG-tagged hDK and the pertinent mutants resulted in the appearance of comparable protein bands at the expected molecular weight of around 59 as detected by Western blotting with anti-FLAG antibodies. Thus, the reduced level of kinase activity does not appear to be due to lower levels of the mutant proteins. As seen in the yeast background as well, the E474A and the G475A substitutions did not diminish the level of DK activity.

The Double Mutant K470A/K471A, K471A, and T472A Substitutions Alter the Affinity of hDK for CTP but Not Dolichol—After selected mutagenesis of the pertinent amino acid residues, kinetic analyses were performed to assess any changes in kinase activity and its affinity for the two substrates, CTP and dolichol, by comparing the apparent Km values for CTP and dolichol in the wild type hDK and the mutant enzymes. The single and double substitutions of K471A and K740A/K471A resulted in significant changes in the apparent Km values for CTP, ranging from 20- to 27-fold increases relative to the wild type enzyme. The T472A substitution resulted in a 7.5-fold increase in the Km for CTP (Table 2). Although the single substitutions K470A and T472A significantly affected the affinity for CTP, the reduced catalytic efficiencies of the mutant enzymes were still sufficient to support modest growth at the nonpermissive temperature (Fig. 6).


View this table:
[in this window]
[in a new window]
  		TABLE 2
Kinetic analyses of hDK activity in microsomes from CHO cells transfected with wild type and mutant hDK cDNAs

Initial rates of DK activity were assayed as described under "Experimental Procedures." Cat. eff., catalytic efficiency.

 
The mutants G475A and E474A that still complemented the growth defect in the yeast mutant (Fig. 6A) did not exhibit any significant changes in the affinity for CTP. None of the mutations tested produced any changes in the apparent Km for dolichol, consistent with the conclusion that the Lys-470 and Lys-471 residues are part of the CTP-binding domain in the cytosolic loop between TMDs 11 and 12. Localization studies by immunofluorescence indicated that the double mutant K470A/K471A exhibited a similar ER pattern as the wild type hDK, excluding the possibility that the double mutation caused a mislocalization of DK (data not included).

Identification of the Mutation in the DK Gene from sec59-1 Cells—The yeast DK gene was also sequenced after RT-PCR of the sec59-1 mutant to identify the mutation responsible for the temperature-sensitive phenotype. The sequence revealed a single base change, resulting in the conversion of Gly-420 to aspartic acid within TMD11. No mutations were detected in 225 bases of the 5'-untranslated promoter region.

When wild type SEC59 was similarly mutated (G420D) and transformed into sec59-1 cells, the mutant gene failed to complement growth and hypoglycosylation at the restrictive temperature (Fig. 7, A and B). Consistent with these results, the G420D mutation resulted in a loss of DK activity when expressed at the nonpermissive temperature 37 °C (Table 3), confirming the importance of this residue. Moreover, mutation of the corresponding residue in hDK (G443D) also abolished its activity when expressed in CHO cells at nonpermissive temperature (37 °C) (Table 3) as well as its ability to complement the yeast defects in growth and glycosylation (data not shown) even though expression of the variant protein was confirmed by Western blot analysis.


View this table:
[in this window]
[in a new window]
  		TABLE 3
DK activities in microsomes from yeast sec59-1 cells transformed with wild type SEC59 or the mutant SEC59(G420D) cDNAs and CHO cell lines transfected with wild type hDK or hDK(G443D)

sec59-1 cells expressing the indicated form of hDK were grown in yeast extract/peptone/dextrose media at 30°C (permissive) for 7 h and then shifted to 37°C (non-permissive) for 10 h. CHO cells stably expressing the indicated form of hDK were grown at 37°C. Microsomes were prepared, and DK activity was assayed for all cells as described under "Experimental Procedures."

 


Figure 4
View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 4.
Amino acid sequence alignment of the conserved sequence motif carrying the putative CTP-binding domain in various enzymes utilizing CTP as a substrate. 1, DK from Schizosaccharomyces pombe (NP_587983); 2, human brain DK (NM_014908); 3, DK SEC59 from S. cerevisiae (P20048); 4, phytol kinase from Arabidopsis thaliana; 5, phosphatidate cytidylyltransferases from Helicobacter pylori (AAD05785); 6, Wigglesworthia brevipalpis (NP_715347); 7, Campylobacter jejuni (NP_282493); 8, E. coli (NP_415927); 9, Staphylococcus aureus (BAB 57423); 10, A. thaliana (O04928); 11, Solanum tuberosum (O04940); 12, Bacillus subtilis (O031752); 13, Aquifex aeolicus (O67292); 14, CDS from Trichodesmium erythraeum (O0325923); 15, S. cerevisiae (O09585); 16, Homo sapiens (O95674); 17, Mus musculus (NP_775546).

 


Figure 5
View larger version (40K):
[in this window]
[in a new window]
  		FIGURE 5.
Cytosolic orientation of the proposed CTP binding motif of hDK on the ER membrane. hDK with a FLAG tag (indicated by an arrow) inserted between Pro467 and Gly468 of the proposed CTP binding domain between TMD11 and TMD12 was expressed in CHO cells. The cells were then fixed in 4% paraformaldehyde, selectively permeabilized with digitonin (3 µg/ml) or 0.3% Triton X-100, and stained with anti-FLAG antibodies followed by Texas Red-conjugated secondary antibodies.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
If the fully unsaturated polyprenol intermediate is the substrate for the enzymatic reduction of the {alpha}-isoprene unit of dolichol (11), DK would catalyze the final step in the de novo pathway for Dol-P biosynthesis (1). The CTP-mediated kinase may also be involved in the recycling of the glycosyl carrier lipid after it is discharged as Dol-P-P during protein N-glycosylation reactions. It is plausible that the Dol-P, produced by Dol-P-P phosphatase on the luminal surface (31, 32), is dephosphorylated and diffuses back to the cytoplasmic leaflet of the ER as the free polyisoprenol (1). DK would then also catalyze the reformation of Dol-P for further rounds of lipid intermediate biosynthesis. In this study, a recently cloned cDNA encoding hDK (13) has been used to obtain more direct proof for the ER localization and topological arrangement of the enzyme in the ER.


Figure 6
View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 6.
Complementation of growth (panel A) and CPY hypoglycosylation (panel B) defects of sec59-1, the temperature-sensitive yeast mutant, by hDK and the various point mutants. Transformation of the mutant with hDK and the point mutants G475A, K470A, T472A, and E474A were able to complement growth of sec59-1 at 37 °C, whereas the hDK single point mutant D451A or double mutant K470A/K471A could not support growth at the nonpermissive temperature (37 °C). Transformation with the hDK mutant, K471A, resulted in much slower growth than wild type hDK at 37 °C. Overexpression of hDK corrects the hypoglycosylation defect of CPY in yeast sec59-1 cells, whereas the hDK mutants, D451A, K471A, K470A/K471A, and T472A were unable to restore normal N-glycosylation. The yeast cells were grown at 30 °C for 7 h, shifted to 37 °C for 10 h, and analyzed by SDS-PAGE and immunoblotting using anti-CPY serum. The positions of mature CPY (mCPY) and hypoglycosylated glycoforms lacking one to four N-linked oligosaccharide chains (-1 to -4) are indicated.

 
Immunofluorescence analyses localize hDK to the ER of CHO cells, in accord with earlier subcellular fractionation studies with calf brain (16). Based on experiments performed with N- and C-terminal FLAG-tagged constructs after complete or semipermeabilization of membranes, hDK is a polytopic protein with a lumenally oriented N terminus and a C terminus exposed on the cytoplasmic face. Predictive algorithms indicate that DK has 13 TMDs. In addition, the loop between TMD11 and TMD12, proposed to contain part of the CTP-binding domain (see below), has been shown to be exposed on the cytoplasmic surface of the ER. In an earlier related report, Adair and Cafmeyer (33) demonstrated that DK activity in sealed rat liver microsomes was sensitive to treatment with trypsin and mercury-dextran, two impermeant reagents. These results along with the impermeability of the ER to CTP (no CTP transporters have been detected in the ER as of this writing) strongly indicate that at least part of the reactive site of the enzyme is exposed on the cytoplasmic surface of the ER.

Another primary objective of this investigation was to begin mapping the CTP-binding site and to strengthen the evidence that the CTP-mediated phosphorylation reaction occurred on the cytoplasmic face of the ER. A search of the data base led to the identification of a conserved sequence motif shared among the DKs from all the available sources, a large number of CDSs from human, rat, yeast, and plant origin, and the recently identified phytol kinase genes from Arabidopsis and Synechocystis, respectively (12, 13, 19-24). CDSs are key enzymes in membrane phospholipid biosynthesis and phototransduction in Drosophila and are essential for cell growth in S. cerevisiae (21, 22, 34, 35). This sequence is a part of the PFAM domain of cytidylyltransferases and yeast DK gene (CTP_transf_1, PFAM accession number PF01148).

Part of the motif DXXAXXXGXXXGX8KKTXEG is located between TMD11 and TMD12 of the topological model for hDK. This motif is reported to be one of the two most highly conserved regions among human CDS1 and CDS2 and Drosophila eye CDS (19, 36). DKs, phytol kinases, and CDSs utilize CTP as one of their substrates, although the catalytic activities are different mechanistically, making it likely that these conserved amino acids are involved in CTP binding. Interestingly, the phytol kinases, which can also utilize UTP as the phosphoryl donor (24), have a KSWAG sequence instead of the similar 471KTXEG sequence in hDK. A future goal will be to determine if the substitution of the phytol kinase sequence would allow hDK to use UTP as a substrate.

As for DKs, CDSs from yeast are localized to the ER predominantly and do not contain any conspicuous ER retention sequences (36, 37). Interestingly, this motif is absent in cytidylyltransferases like CTP:phosphocholine cytidylyltransferase, CTP:glycerol-3-phosphate cytidyltransferase, or ethanolamine-phosphate cytidylyltransferases, which are soluble proteins involved in the formation of water-soluble products (34, 38, 39), in contrast to CDSs, which catalyze the formation of the liponucleotide intermediate, CDP-diacylglycerol. The related cytidylyltransferases (CTP:phosphocholine cytidylyltransferase, CTP:glycerol-3-phosphate cytidyltransferase, and ethanolamine-phosphate cytidylyltransferases) have the signature sequences HXGH and RTEGISTT involved in CTP binding, which are absent in DKs and CDSs (40, 41).


Figure 7
View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 7.
Complementation of growth (panel A) and CPY hypoglycosylation (panel B) defects of sec59-1, the temperature-sensitive yeast mutant, by wild type SEC59 and SEC59(G420D). Upon transformation, wild-type SEC59 was able to complement growth of sec59-1 at the nonpermissive temperature (37 °C), whereas the empty plasmid (pRS316) and the mutant G420D could not support growth at the nonpermissive temperature (37 °C). Yeast cells were grown at 30 °C for 7 h, shifted to 37 °C for 10 h, and analyzed by SDS-PAGE and immunoblotting using anti-CPY serum (panel B). Shown are sec59-1 cells transformed with the wild-type SEC59 gene (lane 1), sec59-1 cells transformed with empty plasmid pRS316 (lane 2), and sec59-1 transformed with the variant gene, SEC59(G420D) (lane 3). The positions of mature CPY (mCPY) and hypoglycosylated glycoforms lacking one to four N-linked oligosaccharide chains (-1 to -4) are indicated.

 
Single point mutation of aspartic acid 451 and double mutations of both the lysines (Lys-470/471) abrogated the ability of the enzyme to complement the growth and hypoglycosylation defects in the temperature-sensitive mutant, sec59-1. The K471A mutant produced a much slower growth rate of sec59-1 relative to the wild type enzyme at the nonpermissive temperature. Consistent with Lys-470/471 and Thr-472 being part of the CTP-binding site, the apparent Km for CTP increased 8-27-fold in the K470A/K471A double mutant and K471A and T472A mutants without significant changes in Vmax, indicating a substantially reduced affinity for CTP. All of these results are consistent with the conclusion that the two positively charged residues, Lys-470 and -471 play a role in CTP binding. It should be noted that all of the effects of the hDK mutations were evaluated by assessing their effects on the growth and hypoglycosylation phenotypes in the sec59-1 mutant and the level of enzyme activity in microsomes from the yeast mutant and CHO cells. However, more definitive proof may be obtained by testing the ability of the various mutants to rescue a sec59 null mutant. It will also be interesting to see if the yet to be identified CTP-mediated diacylglycerol kinase from S. cerevisiae has a similar motif containing the neighboring lysine residues (42).

In addition to the residues implicated in the CTP-binding site, a G420D substitution has been found in the mutant enzyme in sec59-1 cells. The importance of this glycine residue was supported by the finding that the corresponding mutation in hDK, G443D, resulted in a drastic loss of DK activity when expressed in CHO cells. The activity was too low to determine if the mutation affected the binding of CTP, but its position within TMD11 could affect the affinity of the enzyme for the hydrophobic substrate, dolichol.

In summary, the structure-function studies presented here have extended the information on the localization, topological arrangement, and the identity of a conserved motif that is proposed to be part of a putative CTP-binding site in DKs and phytol kinases from Arabidopsis as well as CDSs. All of the deleterious mutations produced by site-directed mutagenesis and the mutation found in the yeast enzyme could potentially be the genetic basis for as yet to be identified cases of congenital disorders of glycosylation (43). Future studies will be aimed at learning more about (a) the interaction of the loop between TMDs 11 and 12 and other cytoplasmically oriented domains, (b) potential ER retention signals in hDK, and (c) the hydrophobic domains where dolichol, a relatively large aliphatic substrate, is bound. It will also be of interest to see if the DXXAXXXGXXXGX8KKTXEG motif in hDK is also present in the CTP-mediated kinases involved in the conversion of t,t-farnesol and t,t,t,-geranylgeraniol to the allylic pyrophosphate intermediates in mammals and plants (44, 45) and the phosphorylation of galactolipids in chloroplast envelopes (46).


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant GM36035 (to C. J. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, BBSRB, 741 S. Limestone St., Lexington, KY 40536. E-mail: waechte{at}uky.edu.

2 The 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. Back


   ACKNOWLEDGMENTS
 
We thank Myung-Hee Kim, Jeff Rush, and Sergey Matveev for helpful suggestions and carefully editing the manuscript. We also thank Susan Wang for technical assistance during the early part of this work.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Schenk, B., Fernandez, F., and Waechter, C. J. (2001) Glycobiology 11, 61-70
  2. Helenius, A., and Aebi, M. (2004) Annu. Rev. Biochem. 73, 1019-1049[CrossRef][Medline] [Order article via Infotrieve]
  3. Allen, C. M., Jr., Kalin, J. R., Sack, J., and Verizzo, D. (1978) Biochemistry 17, 5020-5026[CrossRef][Medline] [Order article via Infotrieve]
  4. Burton, W. A., Scher, M. G., and Waechter, C. J. (1979) J. Biol. Chem. 254, 7129-7136[Free Full Text]
  5. Rossignol, D. P., Lennarz, W. J., and Waechter, C. J. (1981) J. Biol. Chem. 256, 10538-10542[Free Full Text]
  6. Burton, W. A., Lucas, J. J., and Waechter, C. J. (1981) J. Biol. Chem. 256, 632-635[Abstract/Free Full Text]
  7. Rossler, H. H., Zimpfer, A., and Risse, H.-J. (1982) Mol. Cell. Biochem. 48, 183-189[CrossRef][Medline] [Order article via Infotrieve]
  8. Scher, M. G., Sumbilla, C. M., and Waechter, C. J. (1985) J. Biol. Chem. 260, 13742-13746[Abstract/Free Full Text]
  9. Volpe, J. J., Sakakihara, Y., and Rust, R. S. (1987) Brain Res. 31, 193-200[CrossRef]
  10. Bhat, N. R., Frank, D. W., Wolf, M. J., and Waechter, C. J. (1991) J. Neurochem. 56, 339-344[CrossRef][Medline] [Order article via Infotrieve]
  11. Sagami, H., Kurisaki, A., and Ogura, K. (1993) J. Biol. Chem. 268, 10109-10113[Abstract/Free Full Text]
  12. Heller, L., Orlean, P., and Adair, W. L., Jr. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7013-7016[Abstract/Free Full Text]
  13. Fernandez, F., Shridas, P., Jiang S., Aebi M., and Waechter C. J. (2002) Glycobiology 12, 555-562[Abstract/Free Full Text]
  14. Higashi, Y., and Strominger, J. L. (1970) J. Biol. Chem. 245, 3691-3696[Abstract/Free Full Text]
  15. Sandermann, H., Jr., and Strominger, J. L. (1971) Proc. Natl. Acad. Sci. U. S. A. 68, 2441-2443[Abstract/Free Full Text]
  16. Scher, M. G., Devries, G. H., and Waechter, C. J. (1984) Arch. Biochem. Biophys. 231, 293-302[CrossRef][Medline] [Order article via Infotrieve]
  17. Nilsson, T., Jackson, M., and Peterson, P. (1989) Cell 58, 707-718[CrossRef][Medline] [Order article via Infotrieve]
  18. Vainauskas, S., Maeda, Y., Kurniawan, H., Kinoshita, T., and Menon, A. K. (2002) J. Biol. Chem. 277, 30535-30542[Abstract/Free Full Text]
  19. Volta, M., Bulfone, A., Gattuso, C., Rossi, E., Mariani, M., Consalez, G.G., Zuffardi, O., Ballabio, A., Banfi, S., and Franco, B. (1999) Genomics 55, 68-77[CrossRef][Medline] [Order article via Infotrieve]
  20. Saito, S., Goto, K., Tonosaki, A., and Kondo, H. (1997) J. Biol. Chem. 272, 9503-9509[Abstract/Free Full Text]
  21. Shen, H., Heacock, P. N., Clancey, C. J., and Dowhan, W. (1996) J. Biol. Chem. 271, 789-795[Abstract/Free Full Text]
  22. Wu, L., Niemeyer, B., Colley, N., Socolich, M., and Zuker, C.S., (1995) Nature 373, 216-222[CrossRef][Medline] [Order article via Infotrieve]
  23. Martin, D., Gannoun-Zaki, L., Bonnefoy, S., Eldin, P., Wengelnik, K., and Vial, H. (2000) Mol. Biochem. Parasitol. 110, 93-105[CrossRef][Medline] [Order article via Infotrieve]
  24. Valentin, H. E., Lincoln, K., Moshriri, F., Jensen, P. K., Qi, Q., Venkatesh, T. V., Karunanandaa, B., Baszis, S. R., Norris, S. R., Savidge, B., Gruys, K. J., and Last, R. L. (2006) Plant Cell 18, 212-224[Abstract/Free Full Text]
  25. Rodriquez-Vico, F., Martinez-Cayeula, M., Garcia-Peregrin, E., and Ramirez, H. (1989) Anal. Biochem. 183, 275-278[CrossRef][Medline] [Order article via Infotrieve]
  26. Sumbilla, C., and Waechter, C. J. (1985) Methods Enzymol. 111, 471-482[Medline] [Order article via Infotrieve]
  27. Aebi, M., Gassenhuber, J., Domdey, H., and Te Heesen, S. (1996) Glycobiology 6, 439-444[Abstract/Free Full Text]
  28. Laemmli, U. K. (1970) Nature 227, 680-685[CrossRef][Medline] [Order article via Infotrieve]
  29. Mitaku, S., and Hirokawa, T. (1999) Protein Eng. 12, 953-957[Abstract/Free Full Text]
  30. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410[CrossRef][Medline] [Order article via Infotrieve]
  31. Fernandez, F., Rush, J. S., Toke, D. A., Han, G.-S., Quinn, J. E., Carman, G. M., Choi, J.-Y., Voelker, D. R., Aebi, M., and Waechter, C. J. (2001) J. Biol. Chem. 276, 41455-41464[Abstract/Free Full Text]
  32. Rush, J. S., Cho, S., Jiang, S., Hoffman, S., and Waechter, C. J. (2002) J. Biol. Chem. 277, 45226-45234[Abstract/Free Full Text]
  33. Adair, W. L., Jr., and Cafmeyer, N. (1983) Biochim. Biophys. Acta 751, 21-26[Medline] [Order article via Infotrieve]
  34. Lykidis, A., Jackson, P. D., Rock, C. O., and Jackowski, S. (1997) J. Biol. Chem. 272, 33402-33409[Abstract/Free Full Text]
  35. Halford, S., Dulai, K. S., Daw, S. C., Fitzgibbon, J., and Hunt, D. M. (1998) Genomics 54, 140-144[CrossRef][Medline] [Order article via Infotrieve]
  36. Heacock, A. M., Uhler, M. D., and Agranoff, B. W. (1996) J. Neurochem. 67, 2200-2203[Medline] [Order article via Infotrieve]
  37. Kuchler, K., Daum, G., and Paltauf, F., (1986) J. Bacteriol. 165, 901-910[Abstract/Free Full Text]
  38. Kalmar, G. B., Kay, R. J., Lachance, A., Aebersold, R., and Cornell, R. B. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6029-6033[Abstract/Free Full Text]
  39. Nakashima, A., Hosaka, K., and Nikawa, J. (1997) J. Biol. Chem. 272, 9567-9572[Abstract/Free Full Text]
  40. Weber, C. H., Park, Y. S., Sanker, S., Kent, C., and Ludwig, M. L. (1999) Structure 7, 1113-1124[Medline] [Order article via Infotrieve]
  41. Park, Y. S., Gee, P., Sanker, S., Schurter, E. J., Zuiderweg, E. R. P., and Kent, C. (1997) J. Biol. Chem. 272, 15161-15166[Abstract/Free Full Text]
  42. Szkopinska, A., Nowak, L., Swiezewska, E., and Palamarczyk, G. (1988) Arch. Biochem. Biophys. 266, 124-131[CrossRef][Medline] [Order article via Infotrieve]
  43. Freeze, H. H., and Aebi, M. (2005) Curr. Opin. Struct. Biol. 15, 490-498[CrossRef][Medline] [Order article via Infotrieve]
  44. Crick, D. C., Andres, D. A., and Waechter, C. J. (1997) Biochem. Biophys. Res. Commun. 237, 483-487[CrossRef][Medline] [Order article via Infotrieve]
  45. Thai, L., Rush, J. S., Maul, J., Rodgers, D. L., Chappell, J., and Waechter, C. J. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 13080-13085[Abstract/Free Full Text]
  46. Muller, M.-O., Meylan-Bettex, M., Eckstein, F., Martinoia, E., Siegenthaler, P.-A., and Bovet, L. (2000) J. Biol. Chem. 275, 19475-19481[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
G.-S. Han, L. O'Hara, S. Siniossoglou, and G. M. Carman
Characterization of the Yeast DGK1-encoded CTP-dependent Diacylglycerol Kinase
J. Biol. Chem., July 18, 2008; 283(29): 20443 - 20453.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
G.-S. Han, L. O'Hara, G. M. Carman, and S. Siniossoglou
An Unconventional Diacylglycerol Kinase That Regulates Phospholipid Synthesis and Nuclear Membrane Growth
J. Biol. Chem., July 18, 2008; 283(29): 20433 - 20442.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/42/31696    most recent
M604087200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shridas, P.
Right arrow Articles by Waechter, C. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shridas, P.
Right arrow Articles by Waechter, C. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement