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Volume 270, Number 1, Issue of January 6, 1995 pp. 354-360
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of the Nuclear Localization Signal of Rat Liver CTP:Phosphocholine Cytidylyltransferase (*)

(Received for publication, July 27, 1994; and in revised form, October 19, 1994)

Yuli Wang,  James I. S. MacDonald,  Claudia Kent (§)

From the Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0606

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

CTP:phosphocholine cytidylyltransferase (CT) is a major regulatory enzyme in phosphatidylcholine synthesis in mammalian cells. CT is found in both soluble and particulate forms, both of which are nuclear. We report here the identification of a 21-residue sequence at the amino terminus of CT, ^8KVNSRKRRKEVPGPNGATEED, which was sufficient to direct beta-galactosidase into the cell nucleus. Further deletions from either end of this sequence greatly reduced the nuclear localization of beta-galactosidase. Deletions of amino acids within the nuclear localization signal or of the entire signal disrupted CT nuclear localization, but CT was not completely excluded from the nucleus. Clones of stable transfectants of the nuclear localization signal-deficient CT expressed in Chinese hamster ovary (CHO) 58 cells, which is temperature-sensitive for growth and CT activity, were isolated and characterized. The deletion mutants were active under the same conditions as the wild-type enzyme. Despite the difference in subcellular location from wild-type CT, the nuclear localization mutants were fully able to complement the CT-deficient cell line CHO 58 for both growth and choline incorporation into phosphatidylcholine at the nonpermissive temperature. The mobility of the mutant enzymes on SDS gels was altered relative to the mobility of wild-type CT; however, the extent of phosphorylation of the mutant enzymes was decreased only slightly. Thus, the distribution of CT in both cytoplasm and nucleus, rather than exclusively nucleus, has little effect on the ability of CT to function in growing CHO cells.

INTRODUCTION

Phosphatidylcholine (PC) (^1)is the principal phospholipid of mammalian cell membranes. It is also an abundant component of pulmonary surfactant and serum lipoproteins. In addition to these major structural roles, PC hydrolysis products, including diacylglycerol, phosphatidate, and arachidonate have been identified as lipid second messengers in signal transduction(1, 2) . CTP:phosphocholine cytidylyltransferase (CT) is a key regulatory enzyme for phosphatidylcholine synthesis in mammalian cells(3) . CT is an ambiquitous protein existing in both soluble and particulate forms. The particulate form is generally considered to be the more active form while the soluble enzyme appears to serve as a relatively inactive reservoir in vivo. The soluble CT can be converted into the more active, particulate form under a variety of conditions, including treatment of CHO-K1 cells with phospholipase C (4, 5) and stimulation of Hela cells with oleate(6) . Moreover, soluble CT is highly phosphorylated while the particulate enzyme is less phosphorylated(4, 6) . Thus, translocation and activation of CT are accompanied by dephosphorylation(4, 6) . Recently, Jackowski (7) has shown that phosphorylation of CT is regulated during the cell cycle. To understand the mechanism of CT regulation, it is important to understand the subcellular localization of both the soluble and particulate forms of CT and the means by which CT is transported to its locations. Early cell fractionation experiments suggested that the membranous form of CT is associated with the endoplasmic reticulum (8, 9) or Golgi apparatus(10, 11) , and it has long been assumed that the soluble form is cytoplasmic. Using immunofluorescence staining as well as biochemical studies, however, we found that soluble CT is nuclear (12) . Translocation of CT from the nuclear matrix to the nuclear envelope was observed upon activation by treatment with oleate or phospholipase C(5, 12, 13) . This is a surprising observation because the enzyme catalyzing the final step in the CDP-choline pathway, choline phosphotransferase, is in the endoplasmic reticulum(14) , as are many other enzymes of lipid biosynthesis. It is not obvious why an enzyme regulating phosphatidylcholine biosynthesis should be located in the nucleus. In order to study the physiological function of the nuclear localization of CT, we have identified the nuclear localization signal of CT by deletion analysis as well as by constructing chimeric fusions of CT fragments to beta-galactosidase. Stably transfected cells expressing mutant forms of CT deficient in the nuclear localization signal were also isolated and characterized.

EXPERIMENTAL PROCEDURES

Cell Culture and Transfections

CHO-K1 and CHO 58 (15) cells were grown in F-12 medium (Sigma) supplemented with 10% fetal bovine serum (Whittaker). For transient transfection assays, cells were plated at 4 times 10^4 cells/well on glass coverslips in a 24-well dish for immunofluorescence or 4 times 10^5 cells in a 60-mm culture dish for immunoblotting. The cells were grown at 34 °C for 1 day, then washed twice with calcium- and magnesium-free phosphate-buffered saline (CMF-PBS) (137 mM sodium chloride, 1.5 mM potassium monobasic phosphate, 6.6 mM sodium dibasic phosphate, 2.7 mM potassium chloride). Plasmid DNA was transfected into CHO 58 cells using the Lipofectin method (Life Technologies, Inc.). For 60-mm dishes, 10 µg of plasmid DNA and 40 µg of Lipofectin were used in 1.5 ml of Opti-MEM medium (Life Technologies, Inc.), and for 24-well dishes, 1.0 µg of DNA and 2.5 µg of Lipofectin were used in 1.0 ml of Opti-MEM/well. The transfections were stopped 6 h after transfection by adding an equal volume of F-12 medium supplemented with 20% fetal bovine serum. The next day, the medium was replaced with F-12 plus 10% fetal bovine serum, and cells were allowed to grow for another day before analysis. For stable transfection, cells were transfected the same way as for transient transfections except that 30 µg of DNA, 3 µg of pKOneo(16) , and 40 µg of Lipofectin in 8 ml of Opti-MEM were used in a 100-mm culture dish. Cells were then grown for 2 days in F-12 medium supplemented with 10% fetal bovine serum. The cells were then split 1:15 and cultured in four 100-mm culture dishes in the same medium plus 20 mM Hepes and 0.8 mg/ml G418 (Life Technologies, Inc.) for 2 weeks. The individual clones were isolated and screened with indirect immunofluorescence, as described below, to determine which clones were expressing exogenous CT. Positive clones were picked, re-cloned, and subjected to a second screen to obtain pure clones.

Plasmid Construction

Deletion mutagenesis was performed according to the method developed by Eckstein et al.(17) with the oligonucleotide-directed in vitro mutagenesis system, version 2.1 (Amersham Corp.). The parent plasmid used for mutagenesis was pCMV4CT (the same as pCMV4RCCT in (18) ), in which the cDNA for rat liver CT is under the control of the cytomegalovirus immediate early promoter. The 1.2-kilobase HindIII/XbaI fragment which contained the entire CT gene was subcloned into M13mp18 to produce M13mp18CT. Single-stranded DNA was obtained by transformation of M13mp18CT to Escherichia coli TG-1 cells. The sequences of the two oligonucleotides for N1 and N2 deletions are 5`-GTTCAGCTAAAGTCAATTCAGAGGTACCTGGCCCTAAT-3` (DeltaN1) and 5`-ACTTGCAAGAACGAGTTGATGATGTGGAGGAAAAGTCGA-3` (DeltaN2), respectively. The double mutations (DeltaN1N2) were made using the DeltaN2 oligonucleotide and M13mp18CTDeltaN1 as the template. All mutant constructs were sequenced to confirm that the appropriate mutations had been obtained. The entire mutant CT sequences were then subcloned into the HindIII/XbaI site of pCMV4 (19) to produce pCMV4CTDeltaN1, pCMV4CTDeltaN2, and pCMV4CTDeltaN1N2, respectively. pCMV4CTDelta2-32 was obtained by polymerase chain reaction (PCR) with pCMV4CT as template and 5`-GGAATTCAGATCTATGAAAGTGCAGCGCTGTGCA-3` and 5`-AGATCTAGATTATTAGTCCTCTTCATCCTC-3` as primers. The PCR product was then subcloned into the BglII/XbaI site of the pCMV4 vector to produce pCMV4CTDelta2-32. The plasmids were purified using the QIAGEN plasmid purification kit and transfected into CHO 58 cells(18) .

For construction of CT(x)-lacZ fusions, the E. coli lacZ gene, starting from codon 7, was produced by PCR amplification with pcD-SRalpha 296-HMGal (kindly provided by Dr. Robert D. Simoni) as template and 5`-GAAGATCTGCCATGGATATCAAGCTTGAGCTCTGCAGAATTCCACTGGCCGTCGTTTTA-3` and 5`-GCTCTAGATTATTATTTTTGACACC-3` as primers. The PCR product was subcloned into the BglII/XbaI site of the pCMV4 vector to produce pCMV4-lacZ. The CT portion of different CT(x)-lacZ fusions were produced by PCR with the NH(2)-terminal primer 5`-CGCGGATCCAGATCTATGGATGCACAGAGTTCA3` and different COOH-terminal primers followed by a HindIII restriction site. The primers were as follows: pCMV4CT-lacZ: 5`-CGGGATCCAAGCTTAGGGCCAGGTACCTCTT-3`; pCMV4CT-lacZ: 5`-CCGGATCCAAGCTTTGTTGCTCCATTAGGGC-3`; pCMVCT-lacZ: 5`-CCGGATCCAAGCTTATCTTCCTCTGTTGCT-3`; pCMV4CT-lacZ: 5`CGGGATCCAAGCTTGGAAGGAATTCCATC-3`; pCMV4CT-lacZ: 5`-CCCAAGCTTCCTGCAGGCTTCTTCCA-3`; pCMV4CT-lacZ: 5`-CGGGATCCAAGCTTCCCTGCCGAAGAGTAG-3`; pCMV4CT-lacZ: 5`-CGGGATCCAAGCTTCACAAATTCTTTCGACT-3`; pCMV4CT-lacZ: 5`-AGCGAGGATGAAGAGGACGAGCAGAAGCTTATCAGCGAGGAGGACCTCTAGAGTCGACTCGAG-3`. The PCR products were then digested with BglII and HindIII and subcloned into the BglII/HindIII site of pCMV4-lacZ in frame to produce pCMV4CT(x)-lacZ, where times is the last codon of the CT fragment at each construct. The junction of each CT(x)-lacZ fusion was sequenced.

Plasmid pCMV4CTDeltaN1-lacZ was obtained by PCR amplification with pCMV4CTDeltaN1 as template and 5`-CGCGGATCCAGATCTATGGATGCACAGAGTTCA-3` and 5`-CGGGATCCAAGCTTGGAAGGAATTCCATC-3` as primers. The PCR product was then subcloned into the BglII/HindIII site of pCMV4lacZ to produce pCMV4CTDeltaN1-lacZ. pCMV4CTDelta2-11-lacZ was produced by PCR using pCMV4CT-lacZ as template and 5`-CGCGGATCCAGATCTATGAGGAGGAGGAGGAAAGAGG-3` and 5`-GCTCTAGATTATTATTTTTGACACC-3` as primers. pCMV4N1-lacZ was obtained by PCR with pCMV4-lacZ as template and 5`-CGCGGATCCAGATCTATGAGGAAGAGGAGGAAAGATATCAAGCTTGAGCT-3` and 5`-GCTCTAGATTATTATTTTTGACACC-3` as primers. pCMV4CTDelta2-7-lacZ was produced by PCR with pCMV4CT-lacZ as template and 5`-GGAATTCAGATCTATGAAAGTCAATTCAAGGAAG-3` and 5`-GCTCTAGATTATTATTTTTGACACC-3` as primers. The three previous PCR products were subcloned into the BglII/XbaI sites of pCMV4 to produce pCMV4CTDelta2-11-lacZ, pCMV4N1-lacZ, and pCMV4CTDelta2-7-lacZ, respectively. The CT portions of all of these constructs were sequenced to confirm that there were no undesired mutations. All the plasmids were purified using QIAGEN plasmid purification kit and transfected into CHO 58 cells.

Antibodies

The N antibody against a peptide corresponding to the NH(2)-terminal 17 residues of rat liver CT was affinity-purified as described elsewhere(5) . The sequences of the first 18 residues of rat liver CT and CHO-K1 CT are identical(18, 20) .

To prepare the rabbit anti-rat liver CT antibody, CT was purified as described (21) from insect cells infected for 72 h with a baculovirus clone containing the cDNA for rat liver CT, and a polyclonal antibody to the recombinant CT was raised in rabbits. To obtain CT-specific antibodies from the serum of immunized rabbits we first isolated the IgG fraction by passage of the serum over protein A-agarose. Immune serum was added to protein A-agarose which had previously been equilibrated in 0.15 M phosphate containing 0.9% NaCl and 0.3% bovine serum albumin, pH 8.2 and incubated with gentle rocking for 30-60 min at room temperature. The slurry was then poured into a column (1 times 5 cm) and washed with approximately 10 volumes of phosphate buffer. Antibodies were eluted with 5 ml of 0.1 M glycine-HCl, pH 2.3 into 3 ml of 3.0 M Tris-HCl containing 0.1% bovine serum albumin, pH 8.6. The IgG fraction was then dialyzed overnight against 4 liters of PBS, lyophilized, and resuspended in 1.0 ml of deionized water.

A CT affinity column was constructed by binding approximately 500 µg of purified recombinant CT to CNBr-activated Sepharose (Pharmacia Biotech Inc.), following the manufacturer's instructions. The column was stored in PBS containing 0.2% azide. The purified IgG fraction was incubated at room temperature for 2 h with gentle rocking with CT-Sepharose which had been previously equilibrated with phosphate buffer as above. The slurry was returned to the column and CT-specific antibody was eluted, dialyzed and lyophilized as described above. The CT-Sepharose was regenerated by washing with 2-3 volumes of 6 M guanidine HCl followed by 10 volumes of PBS containing 0.2% azide.

Mouse anti-E. coli beta-galactosidase monoclonal antibody was purchased from Promega Biotech Inc. FITC-conjugated goat anti-rabbit IgG(H+L) antibody and FITC-conjugated horse anti-mouse IgG(H+L) antibody were from Vector Laboratories. Horseradish peroxidase-conjugated goat anti-rabbit IgG(H+L) was from Life Technologies, Inc.

Indirect Immunofluorescence

Cells were plated on coverslips in 24-well culture dishes and transfected as described above. Two days after stopping the transfection, cells were washed twice with cold PBS and then fixed and immunostained as described(12) . For N antibody, a 1:200 dilution of affinity-purified antibody was used. For CT antibody, a 1:100 dilution of affinity-purified antibody was used. For mouse anti-beta-galactosidase antibody, a 1:200 dilution was used. The samples were analyzed using either a Zeiss Axioskop MC100 microscope in Dr. Jack Dixon's laboratory or a Bio-Rad MRC600 confocal microscope in the Cell Biology Laboratory (University of Michigan).

Immunoblotting

Cell fractionation with digitonin was performed as described previously(6) . The samples were immediately mixed with six-fold concentrated SDS gel sample buffer (22) to preserve the phosphorylation state. Equal amounts of total protein or equal amounts of CT protein were loaded and separated by 10% SDS-PAGE. Conditions for immunoblotting were as described(6) . Both the first and the second antibodies were diluted 1:3000. The blot was detected by the enhanced chemiluminescence method (Amersham Corp.).

Choline Uptake

Cells were plated at 4 times 10^5 cells per 60-mm dish in F-12 medium supplemented with 10% fetal bovine serum and grown for 1 day at 34 °C, then 24 h at 40 °C. The cells were washed twice with cold CMF-PBS and incubated with 1 mCi/ml of [methyl-^3H]choline in F-12 medium supplemented with 10% fetal bovine serum for 30 min to 2 h at 40 °C. Cells were washed twice with cold CMF-PBS and harvested in 1 ml of water. Lipids were extracted by the Bligh-Dyer method(23) , and the radioactivity in the lipid phase was determined in a Beckman LS1707 scintillation counter.

CT Assay

Cells were fractionated into soluble and particulate fractions by addition of digitonin buffer(6) . CT activity in the presence or absence of lipids was measured as described by Weinhold and Feldman(24) . ADP (6 mM) was added in both soluble and particulate fractions in the assay to prevent CTP hydrolysis in crude cell extracts. Samples of 25 and 50 µl were used for enzyme assay at 37 °C for 1 h, which is within the linear range for this assay.

Protein Determination

Protein levels were determined by the Bradford (25) method using bovine serum albumin as the standard.

In Vivo P Labeling and Immunoprecipitation

Cells were plated at 2 times 10^5 cells/60-mm dish for 3 days. The cells were then washed with phosphate-free Dulbecco's modified Eagle's medium (Sigma) twice and then cultured with 0.2 mCi of carrier-free P(i) (ICN) in 1.5 ml of phosphate-free Dulbecco's modified Eagle's medium for 3 h at 34 °C. The cells were then washed with cold CMF-PBS twice and harvested in 0.5 ml of digitonin buffer with 1% Nonidet P-40 and 5 µg/ml leupeptin, 200 µM benzamidine, 5 µg/ml antipain, 5 µg/ml chymostatin, 10 µg/ml pepstatin, and 100 µM phenylmethylsulfonyl fluoride.

Immunoprecipitation was performed as previously described (6) except 5 µl of affinity-purified CT antibody were used as the first antibody. The immunoprecipitation sample were resolved on SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and subjected to autoradiography. The relative amount of phosphorylation was quantitated with a PhosphorImager from Molecular Dynamics. The same samples were then probed with CT antibody as the first antibody and alkaline phosphatase-conjugated goat anti-rabbit IgG as the second antibody. After the alkaline phosphatase reaction was run, the membrane was made transparent with ethylene glycol and the relative amount of CT protein was quantitated with a Pharmacia LKB Ultrascan XL. The level of phosphorylation was then normalized to the amount of CT protein.

RESULTS

A Necessary Nuclear Localization Signal in Rat CT

Most nuclear localization signals consist of a segment of basic amino acid residues(26, 27) . Sequence analysis of rat liver CT revealed two such segments that could possibly function as nuclear localization signals (Fig. 1). The first segment, RKRRK, referred to as N1, is similar to the nuclear localization signal of SV40 large T antigen(28) . The N1 sequence is conserved among rat(20) , CHO K1(18), and mouse (29) CTs. The second basic residue-rich sequence, KVKKKVK, referred to as N2, resembles a bipartite nuclear localization sequence (26) in that the dibasic sequence KK is upstream of N2. The N2 sequence is also conserved among rat, mouse and CHO-K1 CT sequences. To determine if these segments actually function as nuclear localization signals in CT, we used site-directed mutagenesis to delete each segment alone (DeltaN1 and DeltaN2) and also made the double mutant (DeltaN1N2). Plasmids containing wild-type CT or deletion mutants of CT were transfected into CHO 58 cells, which have a low background of CT protein(12, 15, 18) . The expression of CT was assayed by immunoblotting. Because the N1 sequence is part of the peptide used for raising our anti-amino-terminal peptide antibody(20) , this antibody did not effectively detect the DeltaN1 or DeltaN1N2 mutants of CT (Fig. 2). We then used CT antibody, which was prepared against the entire recombinant rat liver CT. This antibody detected the DeltaN1 and DeltaN1N2 mutants as well as wild-type CT and the DeltaN2 mutant (Fig. 2). The CT antibody was then used for detecting the subcellular localization of the putative nuclear localization mutants expressed in CHO strain 58 cells, which are deficient in CT(15, 18) . As expected, wild-type rat liver CT expressed in CHO 58 was located in the nucleus (Fig. 3). Deleting the N1 sequence disrupted CT localization, rendering CT both cytoplasmic and nuclear. The N2 deletion had no apparent effect on CT nuclear localization because the DeltaN2 mutant was entirely nuclear and the DeltaN1N2 double mutant was found in both the nucleus and cytoplasm, as was the DeltaN1 mutant. (Although the location of CT in theDeltaN1N2-expressing cells shown in Fig. 3looks more cytoplasmic than in the DeltaN1 cells, there was considerable cell-to-cell variation in the level of cytoplasmic versus nuclear CT in these cells (see below).) These results suggest that the N1 sequence, but not the N2 sequence, is an essential signal to ensure the exclusive nuclear location of CT.

Figure 1: Construction of two potential NLS mutants. Sequence analysis of rat liver CT (4) revealed two basic motifs of amino acids which could be potential nuclear localization signals (black bars). The first one is close to the NH(2) terminus of CT(N1). The second one is in the COOH-terminal one-third of CT(N2). Deletion mutants of either one or both of the two putative nuclear localization signals were constructed by site-directed mutagenesis and subcloned into mammalian expression vector pCMV4.


Figure 2: Immunoblot of wild-type and deletion mutants of rat liver CT. Plasmids of wild-type and deletion mutants of CT were transfected into CHO 58 cells in a 60-mm tissue culture dish. Two days after the transfection was stopped, the cells were separated into soluble (S) and particulate (P) fractions in digitonin buffer. Equal amounts of protein were loaded on each lane for the immunoblot. A, 1:3000 dilution of affinity-purified N-antibody; B, 1:1000 dilution of affinity-purified CT-antibody. Lane 1, wild-type rat liver CT; lane 2, N1 deletion of rat liver CT; lane 3, N2 deletion of rat liver CT; lane 4, N1N2 double deletion of rat liver CT.


Figure 3: Subcellular localizations of CT nuclear localization signal mutants. CHO 58 cells were transfected with the CT expression plasmids, and the location of the transiently expressed proteins was determined by indirect immunofluorescence with N-antibody for wild-type CT and CT-antibody for the deletion mutants of CT. A and B, wild-type rat liver CT (pCMV4CT). A is stained with DAPI for DNA and B is immunofluorescence. C and E, N2 deletion of CT (pCMV4CTDeltaN2). C is phase contrast and E is immunofluorescence; D, immunofluorescence of N1 deletion of CT (pCMV4CTDeltaN1); F, Immunofluorescence of N1N2 double deletion (pCMV4CTDeltaN1N2).


An NH(2)-terminal CT Sequence Sufficient for Nuclear Targeting

To determine which segment of CT could direct a cytoplasmic protein to the nucleus, we fused various segments of CT to the amino terminus of the E. coli beta-galactosidase, which is cytoplasmic when expressed in mammalian cells ( Fig. 4and Fig. 5). The location of the fusion protein was determined with an anti-beta-galactosidase antibody. The N1 sequence alone was not a sufficient signal to direct beta-galactosidase to the nucleus (Fig. 4I). When the entire CT sequence was fused to beta-galactosidase, the resulting chimeric protein was exclusively nuclear (Fig. 4C). We then deleted CT progressively toward the NH(2) terminus and found that the first 28 residues of CT were sufficient to direct beta-galactosidase completely to the nucleus in the majority (64%) of the cells examined. The first 25 residues of CT could direct beta-galactosidase to the nucleus in only a small percentage (20%) of the cells. The first 21 residues of CT, however, could not render beta-galactosidase nuclear. Thus, the first 28 residues of CT including residues 22-28 were important to observe substantial direction of beta-galactosidase to the nucleus.

Figure 4: Representative subcellular locations of CT(X)-lacZ fusions. Cells were transfected with pCMV4-lacZ (A and B); pCMV4CT-lacZ (C); pCMV4CT-lacZ (D); pCMV4CTDeltaN1-lacZ (E); pCMV4CT-lacZ (F); pCMV4CTDelta2-7-lacZ (G); pCMV4CT-lacZ (H); pCMV4N1-lacZ (I).


Figure 5: Summary of subcellular locations of beta-galactosidase protein fused to CT. CHO 58 cells were transfected with indicated pCMV4CT(X)-lacZ plasmid. Two days after the transfection was stopped, cells were fixed with 3% formaldehyde followed by methanol:acetone (1:1). Subcellular locations were detected with monoclonal antibody to beta-galactosidase (1:200) and FITC-conjugated rabbit anti-mouse second antibody. Open bar, CT; slashed bar, lacZ (not proportional to scale); black bar, N1.


We then began deletion from the NH(2) terminus using the first 28 residues of CT as the starting point to determine which residues within this portion of the sequence are sufficient for nuclear targeting. Deletion of residues 2 though 7 had no significant effect on nuclear localization (Fig. 4G). Deletion of residues 2 though 11 eliminated the ability of the CT sequence to direct beta-galactosidase to the nucleus (Fig. 5). The smallest segment of CT that is sufficient for nuclear targeting was thus identified as ^8KVNSRKRRKEASSPNGATEED. It is interesting to note that although the deletion of the 5-residue N1 sequence did not exclude CT from the nucleus (Fig. 3), an N1 deletion in a CT fusion protein containing the first 32 residues of CT resulted in the exclusion of beta-galactosidase from the nucleus (Fig. 5E). The N1 sequence, therefore, is necessary, but not sufficient, for targeting beta-galactosidase to the nucleus.

Characterization of Stable Transfectants

To study the effect of altered subcellular location of CT on its biochemical and physiological function, stable cell lines expressing CT mutants deficient in the nuclear targeting signal were isolated. In CTDeltaN1, only the 5-residue N1 sequence was deleted, and in CTDelta2-32, the deletion included an entire segment sufficient for nuclear localization of beta-galactosidase. Characterization of the several stable clones for each mutation revealed that CT in all clones was both cytoplasmic and nuclear. The relative levels of nuclear and cytoplasmic CT, however, varied from clone to clone (Fig. 6). For example, CTDeltaN1 in some clones appeared to be mostly cytoplasmic (Fig. 6A) while in other clones it was evenly distributed between the nucleus and the cytoplasm (Fig. 6B). The same phenomenon was observed for CTDelta2-32 (Fig. 6, C and D). The reason for the clonal variation in the location of mutant CT is not clear.

Figure 6: Confocal indirect immunofluorescence of CTDeltaN1 and CTDelta2-32 clones. Cells were fixed and detected the same as in Fig. 4except 1:100 dilution of CT-antibody was used as first antibody. Immunofluoresence was then detected with a confocal microscope with a 0.5-nm slice at about 12-nm thickness from the bottom of the cells. CTDeltaN1 clone 3-5 (A); CTDeltaN1 clone 28-3 (B); CTDelta2-32 clone 3-5 (C); CTDelta2-32 clone 10-3 (D).


CHO 58, the host cell line used for these transfections, is both deficient in and temperature sensitive for CT(15) . The cells can grow at 34 °C but not at 40 °C, and expression of wild-type rat liver CT in these cells allows them to grow at 40 °C(18) . Growth rates at 40 °C of CHO 58 cells expressing CTDeltaN1 or CTDelta 2-32 were no different from those of cells expressing wild-type CT, indicating that the mutant CTs were fully capable of complementing the mutant phenotype.

PC Metabolism

To understand the significance of the nuclear location of CT, it was of interest to determine the effect of the altered location of CT on its ability to participate in the CDP-choline pathway. The NH(2)-terminal deletions apparently did not affect enzymatic activity, because cells expressing CTDelta2-32 or CTDeltaN1, either in transient assays or as stable cell lines, had as much CT activity as cells expressing wild-type rat CT (data not shown). To test the ability of the mutants to function in phosphatidylcholine biosynthesis, the rate of incorporation of [^3H]choline into phosphatidylcholine was measured for CHO 58 cells expressing wild-type CT (WT-4) as well as two stable clones each expressing CTDeltaN1 and CTDelta2-32. Since CT is a rate-limiting step for phosphatidylcholine biosynthesis under these conditions, the rate of choline incorporation into phosphatidylcholine is a reflection of CT activity in vivo. The ability of untransfected CHO 58 cells to synthesize phosphatidylcholine is virtually eliminated at 40 °C (Fig. 7) due to its temperature-sensitive mutant CT (15) . Each mutant clone was able to overcome the defect in phosphatidylcholine biosynthesis as well as the wild-type clone (Fig. 7).

Figure 7: Choline incorporation into lipids. Cells were plated at 4 times 10^5 cells per 60-mm dish and grown for 1 day at 34 °C and then 40 °C for 24 h. Cells were then labeled with 1 µCi/ml [methyl-^3H]choline for 0.5-2 h. Lipids were extracted by the Bligh-Dyer method. CHO 58 cells (open circle), rat liver wild-type CT stable transfection (open square), CTDeltaN1 clone 3-5 (open diamond), CTDeltaN1 clone 28-3 (closed square), CTDelta2-32 clone 3-5 (closed circle), CTDelta2-32 clone 10-1 (closed diamond).


Phosphorylation Pattern of Nuclear Localization Mutants

Differences in the phosphorylation state of CT can be monitored on Western blots(4, 6) . The highly phosphorylated, soluble form migrates more slowly as two or three bands while the dephosphorylated, membrane-associated form migrates more rapidly as a single band. To determine the effect of mutations in the nuclear localization sequence on mobility in SDS-PAGE, Western blots of extracts from the stably-transfected cell lines were performed (Fig. 8). As expected, CTDelta2-32 migrated faster on SDS-PAGE than CTDeltaN1 since it is 26 residues shorter than CTDeltaN1. Comparison of wild-type CT with two clones each of CTDeltaN1 and CTDelta2-32 indicated that the mutant CT enzymes migrated predominantly as single bands, and faster than wild-type CT (Fig. 8). The two clones of each mutant were selected because the CT localization in one clone was more cytoplasmic and in the other was more nuclear. In spite of this apparent difference in the degree of cytoplasmic association, the mobilities of CT expressed in the two clones were identical and significantly different from the wild type. Although the mobilities of the CT mutants resembled that of membrane-associated CT, the amount of membrane-associated CT in the cells expressing the mutant CT constructs was not greater than in the cells expressing wild-type CT.

Figure 8: Immunoblot of stable transfection clones of CTDeltaN1 and CTDelta2-32. Clones of stable transfection of wild-type rat liver CT as well as CTDeltaN1 and CTDelta2-32 were isolated as described. Cells were plated at 4 times 10^5 cells/60-mm tissue culture dish and grown for 2 days at 34 °C. The cells were then washed twice with CMF-PBS and harvested in digitonin buffer as soluble (S) and particulate (P) fractions. Lane 1, wild-type rat liver CT; lane 2, N1 deletion of rat liver CT clone 3-5; lane 3, N1 deletion of rat liver CT clone 28-3; lane 4, CTDelta2-32 deletion of rat liver CT clone 3-5; lane 5, CTDelta2-32 deletion of rat liver CT clone 10-1. The particulate fraction of CTDelta2-32 deletion clone 10-1 (not shown) was the same as the particulate fractions in lanes 1-4.


To analyze further the CT phosphorylation pattern, CT was immunoprecipitated from cells labeled in vivo with P(i). The CT protein levels were determined by immunoblot analysis and phosphorylation levels were detected by autoradiography (Fig. 9). Although there was much more fast-migrating CT in the deletion mutants than in wild-type CT, the extent of phosphorylation of the mutants was, on average, only 20% lower than that of wild-type CT (Table 1). Furthermore, the two-dimensional tryptic phosphopeptide maps of the mutants were similar to that of the wild-type CT (not shown).

Figure 9: Phosphorylation of rat liver wild-type and nuclear localization signal deletion mutants of CT. Cells were labeled and samples were immunoprecipitated as described under ``Experimental Procedures.'' The precipitated samples were then separated by SDS-PAGE and subjected to immunoblotting with CT antibody (A) and autoradiography (B). Lane 1, wild-type rat liver CT; lane 2, CTDelta2-32 clone 3-5; lane 3, CTDelta2-32 clone 10-1; lane 4, CTDeltaN1 clone 3-5; lane 5, CTDeltaN1 clone 28-3.


DISCUSSION

The results reported in this study identify residues 8-28 of CT as a nuclear localization signal. This sequence represents a minimum sequence to direct a non-nuclear protein into the nucleus. Deletion of several residues at either end of this sequence greatly diminished nuclear targeting. Within this sequence is found a more classic nuclear targeting sequence, RKRRK. Although similar short, basic sequences are capable of directing some proteins to the nucleus(26, 27) , the N1 sequence was not sufficient for nuclear targeting of beta-galactosidase.

Deletion of the N1 sequence from a nuclear-targeted fusion protein rendered beta-galactosidase completely cytoplasmic. In contrast, deletion of N1 or even the more extensive region of residues 2-32 from CT rendered it both cytoplasmic and nuclear. There are several possible reasons for the continued presence of the CT mutants in the nucleus. First, CT might be interacting with another nuclear protein and could be transported into the nucleus while attached to the other protein. For example, deletion of the nuclear localization signal of the product of the retinoblastoma gene does not exclude this nuclear protein from the nucleus(30) . Additional mutations, however, in a region involved in protein:protein interaction result in exclusion from the nucleus (30) . It has been shown that CT interacts with a 110-kDa protein(31) , but the nature and the region of the interaction are not clear. It is possible that CT is brought into the nucleus though interaction with this or another protein. Second, CT might have an additional nuclear localization signal. Polyoma large T antigen (32) and yeast ribosomal protein L29 (33) each have two nuclear localization signals. Mutation of either one individually impairs but does not eliminate the ability of the protein to enter the nucleus; mutations of both result in an exclusively cytoplasmic location. While it is not likely that the N2 region serves as an additional signal, there may be a sequence in CT that is not a conventional, basic residue-rich nuclear localization sequence. Third, rat liver CT expressed in the transfected cells may interact with the endogenous CT in CHO 58 cells. CT is known to be a dimer(34, 35) . The CT in CHO 58 cells is apparently less stable than wild-type CT. At the permissive temperature (34 °C), the mutant cell lines contain approximately 5% of the CT activity found in wild-type CHO K1 cells, while at the non-permissive temperature (40 °C) the activity drops to less that 1%(36) . The decreased activity is accompanied by decreased protein levels(18) . Expression of exogenous rat liver CT in CHO 58 cells could result in heterodimer formation between rat liver CT and CHO 58 CT. If rat liver CT could stabilize CHO 58 CT in the heterodimer, then it is possible that the total CT level in the nucleus could be substantial. An argument against this possibility, however, is that immunoblot analysis of extracts from CTDelta2-32 cells did not reveal a significant increase in endogenous CHO 58 CT levels.

The altered subcellular location of CT resulting from deletions of the nuclear targeting signal has no effect on the ability of exogenous CT to complement the defective CHO 58 CT. Since the product of the CT-catalyzed reaction, CDP-choline, is soluble, it should be able to diffuse to the site of choline phosphotransferase whether CT is nuclear or cytoplasmic. While the deletions of the nuclear localization signal had no apparent effect on phosphatidylcholine biosynthesis, they affected the mobility of CT on SDS gels as detected by Western blots. The faster mobility of CT in both mutants suggested a lower level of phosphorylation in the mutants, but a quantitative estimation of the degree of phosphorylation indicated only a 20% reduction. Furthermore, there was not a dramatic change in the pattern of phosphorylation as detected on two-dimensional phosphopeptide maps. Thus it appears that the altered location of CT in the mutants does not subject CT to abnormal phosphorylation or phosphatase activity.

While it is clear that a substantial change in the location of the enzyme has no dramatic effect on phosphatidylcholine biosynthesis, the apparent presence of some CT in the nucleus in the mutant cell lines precludes a definitive conclusion at this time as to the importance of CT location for phosphatidylcholine biosynthesis. In fact, given the low level of CT in CHO strain 58, which functions normally at 34 °C, it is likely that the level of nuclear CT in the nuclear localization mutants is sufficient to carry out any functions that must be performed in the nucleus. Further experiments to determine the necessity of the nuclear location of CT must await either the identification of the region of CT responsible for its continued nuclear location in the mutants or an alternative means of anchoring CT in the cytoplasm.

FOOTNOTES

*
This work was supported by American Cancer Society Grant BE126. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

(^1)
The abbreviations used are: PC, phosphatidylcholine; CT, CTP:phosphocholine cytidylyltransferase; CHO, Chinese hamster ovary; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; CMF, calcium and magnesium free; FITC, fluorescein isothiocyanate; PAGE, polyacrylamide gel electrophoresis.

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