Distribution of CTP:Phosphocholine Cytidylyltransferase (CCT) Isoforms

CTP:phosphocholine cytidylyltransferase is a major regulator of phosphatidylcholine biosynthesis. A single isoform, CCTα, has been studied extensively and a second isoform, CCTβ, was recently identified. We identify and characterize a third cDNA, CCTβ2, that differs from CCTβ1 at the carboxyl-terminal end and is predicted to arise as a splice variant of the CCTβ gene. Like CCTα, CCTβ2 is heavily phosphorylated in vivo, in contrast to CCTβ1. CCTβ1 and CCTβ2 mRNAs were differentially expressed by the human tissues examined, whereas CCTα was more uniformly represented. Using isoform-specific antibodies, both CCTβ1 and CCTβ2 localized to the endoplasmic reticulum of cells, in contrast to CCTα which resided in the nucleus in addition to associating with the endoplasmic reticulum. CCTβ2 protein has enzymatic activity in vitro and was able to complement the temperature-sensitive cytidylyltransferase defect in CHO58 cells, just as CCTα and CCTβ1 supporting proliferation at the nonpermissive conditions. Overexpression experiments did not reveal discrete physiological functions for the three isoforms that catalyze the same biochemical reaction; however, the differential cellular localization and tissue-specific distribution suggest that CCTβ1 and CCTβ2 may play a role that is distinct from ubiquitously expressed CCTα.

PtdCho 1 is the major membrane phospholipid in higher eukaryotes and is also secreted by particular tissues for important extracellular tasks. For example, it is a significant component of lung surfactant, serum lipoproteins, and bile. CCT is a key regulator of PtdCho biosynthesis (1) and membraneprotein interaction is one important mechanism that governs cellular CCT activity (1,2). Recently a second isoform, CCT␤, was discovered which is encoded by a second gene (3). CCT␣ and CCT␤ have nearly identical amino acid sequences in the catalytic domain which extends approximately from residues 72 to 233 in both proteins, and also near identity in the membrane-interaction domain which extends approximately from residues 256 to 288. Both isoforms are dependent on interaction with phospholipids for catalytic activity (3)(4)(5)(6)(7)(8)(9), as would be predicted from the high degree of identity in the membraneinteraction domains. These domains are characterized by three 11-residue amphipathic repeats that form ␣-helices upon association with phospholipid regulators (10 -13).
The amino terminus of CCT␤ bears no resemblance to the amino terminus of CCT␣ and does not include a nuclear localization sequence as was identified in the CCT␣ protein (14,15). CCT␣ has been localized predominantly in the nucleus but the physiological significance of the nuclear localization of CCT␣ remains unclear. CCT␤ protein was localized outside the cell nucleus by indirect immunofluorescent microscopy (3). CCT␤ consists of 330 amino acids, in contrast with the 367 residues of CCT␣, and lacks most of the carboxyl-terminal phosphorylation domain that is found in the CCT␣ protein (9,16). Phosphorylation of CCT␣ interferes with the lipid stimulation of enzyme activity in vitro (17) and correlates with a reduction of PtdCho biosynthesis in vivo (18 -24). Despite the differences at the amino and carboxyl termini of the proteins, both CCT␣ and CCT␤ exhibit high activity when overexpressed in COS-7 cells (3,9,25,26) resulting in accumulation of cellular CDP-choline and increased radiolabeling of PtdCho (3,27).
In this work we identify a third isoform of CCT, called CCT␤2, which is a splice variant of CCT␤. CCT␤2 encodes a 369-amino acid protein which is identical to the CCT␤1 isoform described previously from amino acids 1 to 320. However, CCT␤2 also has a carboxyl-terminal sequence that resembles the phosphorylation domain of CCT␣. The existence of two distinct CCT genes and two CCT␤ splice variants raises the possibility of regulation of CCT activity at the level of gene expression as well as subcellular localization and phosphorylation (3). Thus, we investigated the expression of the CCT isoforms in human tissues, determine whether CCT␤2 has a phosphorylated carboxyl-terminal domain, and whether these structural differences alter the cellular localization of or the ability of CCT isoforms to complement defective CCT activity in vivo (28).
Antibodies-Anti-CCT␣ rabbit polyclonal antiserum was raised against a synthetic peptide (MDAQSSAKVNSRKRRKE) corresponding to the first 17 amino acids of CCT␣. Anti-CCT␤ antibody (B1 epitope) was a rabbit polyclonal antiserum raised against a peptide (MEE-IEHTCPQPRL) corresponding to amino acids 27-39 of CCT␤1 and CCT␤2. The anti-CCT␤ antibody (B2 epitope) was a rabbit polyclonal antiserum raised against a synthetic peptide (TTDAESETGIPKSL-SNEP) corresponding to amino acids 5-22 of CCT␤1 and CCT␤2. Anti-CCT␤2 antibody (B3 epitope) was a rabbit polyclonal antiserum raised against a synthetic peptide (PPSSPKAASRSISSMSEGD) corresponding to amino acids 347-365 of CCT␤2. Resequencing of the CCT␤2 clone identified that the correct residue at position 10 of the B3 peptide is an alanine instead of an arginine. The B1 and B2 epitope antibodies recognized both CCT␤1 and CCT␤2 whereas the B3 epitope antibody recognized only CCT␤2. Peptides and peptide antigens were prepared by the Molecular Resource Center of St. Jude Children's Research Hospital. The B1 and B2 antigens were prepared by coupling each peptide to keyhole limpet hemocyanin via an additional cysteine at the carboxyl terminus of the peptide whereas the B3 antigen was coupled at the amino terminus. Immunization of rabbits and collection of antiserum was performed by Rockland, Inc., according to their standard schedule. Antisera were purified by affinity chromatography on Affi-Gel 10 cross-linked to the peptide as described previously (3).
Isolation of the CCT␤2 cDNA and Construction of Expression Plasmids-The EST data base was searched using the published CCT␤ sequence (GenBank TM /EBI Data Bank accession number AF052510). A clone from human brain was identified (GenBank TM accession number AA683266) and purchased from Genome Systems. The cDNA sequence was determined on both strands using primers that flanked the multiple cloning sites and internal primers that were synthesized to ensure a complete read on both strands. A second EST clone from human testis was identified (GenBank TM accession number AI041180) and purchased from Research Genetics. The cDNA sequence of the second clone was also determined. Clone AA683266 was subcloned into pcDNA3 using BamHI and XhoI (pAL1). pcDNA3 has an SspI site approximately 1 kilobase from the 5Ј end of the T7 promoter and pAL1 retains the SspI site of CCT␤. The cDNA encoding CCT␤1 in pcDNA3 (pPJ34) (3) was also digested with SspI. The approximately 1.1-kilobase fragment derived from plasmid pPJ34 was ligated to the 5.5-kilobase fragment of pAL1 to generate pAL2.
Construction of the CCT␤1(M27A) Mutant-The M27A point mutation was constructed using overlap extension PCR with the CCT␤1 cDNA as template in pBlueScript SK Ϫ and using the primers: M13 reverse: 5Ј-CAGGAAACAGCTATGACC-3Ј, M27A forward: 5Ј-CAGAA-ACCGCGGAGGAAATAGAGC-3Ј, M27A reverse: 5Ј-ATTTCCTCCGCG-GTTTCTGAG-3Ј, and SnaB1 reverse: 5Ј-AGGGAGCATCTCTGATAAC-TTCGTC-3Ј. Primers M27A forward and M27A reverse replace the ATG codon for methionine 27 with GCG encoding alanine. In the first round of PCR the pairs of primers M13rev-M27Arev and M27Afor-SnaB1rev generated products of 280 and 381 bp, respectively. 10 ng of these products were gel purified and used as template for the second round of PCR with primers M13rev and SnaB1rev. The 642-bp product was cloned into pCR2.1 (plasmid pPJ76) and sequenced to verify that it had the desired mutation. The BamHI-SnaBI fragment of pPJ76 was ligated into the CCT␤1 cDNA replacing the BamHI-SnaBI fragment of AA382871. The mutated CCT␤1 cDNA was subcloned into pcDNA3 using BamHI-XhoI.
CCT Assay-CCT activity was determined essentially as described previously (3). The standard assay contained 150 mM bis-Tris-HCl, pH 6.5, 10 mM MgCl 2 , 4 mM CTP, 64 M lipid activator (PtdCho:oleic acid, 1:1), 1 mM phospho[ 14 C]choline (specific activity 4.5 mCi/mmol) in a final volume of 50 l. The reaction mixture was incubated at 37°C for 10 min. The reaction was stopped by the addition of 5 l of 0.5 M Na 3 EDTA, and the tubes were vortexed and placed on ice. Next, 40 l of each sample was spotted on preadsorbent Silica Gel G thin layer plates, which were developed in 2% ammonium hydroxide, 95% ethanol (1:1, v/v). CDP-[ 14 C]choline was identified by comigration with a standard, scraped from the plate, and quantified by liquid scintillation counting. Protein was determined according to the Bradford method (29).
Transfection Experiments-COS-7 cells were grown in 100-mm dishes to 80% confluency in Dulbecco's modified Eagle's medium sup-plemented with 10% fetal bovine serum and 1% glutamine. CHO58 cells were grown in 100-mm dishes at 33°C in Ham's F-12 medium supplemented as above. Transfections using LipofectAMINE reagent were performed according to the manufacturer's instructions. Briefly, 10 g of plasmid and 60 l of LipofectAMINE reagent were diluted separately into 0.8 ml of serum-free medium. The two solutions were combined and incubated at room temperature for 45 min. Next, 6.4 ml of serum-free medium was added to each tube and the diluted solution was overlaid onto cells that had been previously rinsed with serum-free medium. The cells and reagents were incubated at 37°C for 5 h, and then 8 ml of growth medium containing twice the normal amount of serum was added. The medium was replaced 24 h after the start of the transfection procedure. COS-7 cells were incubated for an additional 24 h at 37°C and then harvested for analysis. CHO58 cells were transferred to 40°C. After incubation for an additional 72 h at the restrictive temperature, CHO58 cells were washed twice with 10 ml of phosphate-buffered saline, cells were fixed by incubation for 5 min in CH 3  Metabolic Labeling-COS-7 cells were grown in 100-mm dishes and transfected with 10 g of vector expressing CCT␤1, CCT␤2, or a pcDNA3 control vector without a cDNA insert. Cells were washed with PBS 48 h after transfection and fresh medium was added containing 1.6 mCi/dish of [ 32 P]orthophosphate. Cells were incubated for 60 min and then immunoprecipitated (see below).
RNA Analysis-RT-PCR was performed using human poly(A) ϩ RNA that was purchased from Invitrogen, Inc., or using poly(A) ϩ RNA isolated from HeLa cells. The cDNA cycle kit (Invitrogen) was used to synthesize the first strand of cDNA following manufacturer's recommended procedure. Poly(A) ϩ RNA (1 g) from each source was used in each 20 l of reaction with random and oligo(dT) primers. The two tubes, where the reverse transcriptase reaction was performed, were combined and 5 l of the first strand cDNA synthesis mixture was used for PCR amplification of CCT sequences. The forward primer for detection of CCT␣ expression was 5Ј-GAAGGTGGAGGAAAAAAG-3Ј corresponding to 795-812 bp of the CCT␣ cDNA sequence, and the reverse primer was 5Ј-ACAGAAAGGGAGGACAG-3Ј corresponding to 1123-1159 bp of the CCT␣ cDNA sequence. The forward primer for CCT␤ was 5Ј-CAAGTGGACAAAATGAAGG-3Ј corresponding to 733-751 bp of the CCT␤ cDNA sequence and the reverse primer was 5Ј-CTAGAAGTCT-CTGCACCTCG-3Ј corresponding to 1299 -1238 bp of the CCT␤2 sequence or 974 -993 bp of the CCT␤1 sequence. The PCR was performed in 50-l reaction volume with 35 thermocycles at 94°C for 1 min, 56°C for 2 min, and 72°C for 2 min. The PCR products were separated by agarose gel electrophoresis.
Transcription-translation Analysis-Plasmid DNA was isolated, transcribed, translated, and labeled with [ 35 S]methionine using the Promega T7-coupled transcription/translation kit according to the manufacturer's instructions. The labeled proteins were analyzed by SDS-gel electrophoresis and visualized by autoradiography.
Immunoblots and Immunoprecipitation-Cell lysates (50 g of protein) were separated by SDS-gel electrophoresis on 12% polyacrylamide gels and transferred by electroblotting onto nitrocellulose membranes. Immunoblotting was performed by incubation of the membranes with purified anti-CCT␣ (1:2000 dilution), purified anti-CCT␤1 (B2 epitope) (1:2000 dilution), or purified anti-CCT␤2 (B3 epitope) (1:2000 dilution) as primary antibody. The Amersham Pharmacia Biotech ECL Western blotting reagents and protocol were used to identify the immunoreactive proteins. For immunoprecipitations, cells were washed twice with PBS and lysed in the culture dish with lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 2% aprotinin, 5 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 50 mM sodium fluoride, 100 M Na 3 VO 4 ) for 30 min in 4°C with gentle agitation. Cell lysates and debris were scraped from the dish and centrifuged for 10 min at 10,000 ϫ g at 4°C. Lysate supernatants were incubated for 1 h with 8 g of anti-CCT␤ (B2 epitope) purified antibody at 4°C and then with the protein A-Sepha-rose beads pre-equilibrated in lysis buffer for 1 h at 4°C. The beads were collected and washed thoroughly. Immune complexes were disrupted by addition of Laemmli buffer and heated in boiling water for 3 min. Proteins were separated by SDS-gel electrophoresis and phosphoproteins were detected by autoradiography.
Fluorescent Labeling of Affinity Purified Antibodies-The antibodies were labeled according to the instructions provided with Molecular Probes' FluoReporter TM labeling kits. Briefly, 200 l of the 1-2 mg/ml antibody in PBS was combined with 20 ml of 1 M sodium bicarbonate, pH 8.0. An appropriate amount of 5 mg/ml reactive dye solution in Me 2 SO was added to the mixture. The amount of dye was calculated according to the following formula: l of dye stock solution ϭ (mg/ml protein ϫ 0.2 ml ϫ MW reactive dye ϫ 200 ϫ MR)/MW protein . Where 200 is a unit conversion factor, and MR is the molar ratio of dye to protein in the reaction mixture. The reaction was stirred in the dark for 1 h and stopped by the addition of 5.5 l of hydroxylamine provided with the kit and additional stirring for 15 min. Labeled antibodies were purified using spin columns provided with the kit. The degree of labeling was determined by measuring protein and dye concentrations (extinction coefficients provided by Molecular Probes) in a spectrophotometer and calculating protein/dye ratio. Typical labeling reaction resulted in 5-10 molecules of dye per one bivalent antibody molecule.
Direct Immunofluorescence Experiments-BAC1.2F5 cells (32) and HeLa cells (33) were cultured as described previously. Cells were grown in 4-chamber LabTek TM II Chamber Slides. The cells were rinsed twice with PBS, fixed, and permeabilized. Six different fixation and permeabilization procedures were investigated to evaluate the reproducibility of staining patterns with Oregon Green TM -labeled anti-CCT␣ antibodies. The procedure of choice entailed fixation in 3.7% formaldehyde for 20 min at 25°C followed by washing with PBS and permeabilization with 0.2% Triton X-100 for 10 min at 25°C. The other five methods that were tested included: 1) fixation in 3.6% formaldehyde for 10 min at 25°C and permeabilization with methanol:acetone (1:1) for 5 min at 25°C (15); 2) fixation in 3.7% formaldehyde for 20 min and permeabilization with cold acetone for 20 min at Ϫ20°C (3); 3) fixation and permeabilization in cold methanol for 6 min at Ϫ20°C; 4) fixation and permeabilization in methanol:acetone (1:1) for 20 min at 4°C; 5) fixation and permeabilization with 70% ethanol in 50 mM glycine for 15 min at Ϫ20°C. All of the procedures, except the last one, resulted in the distribution of anti-CCT␣ staining in both nuclear and cytoplasmic compartments. Using the last fixation/permeabilization procedure, anti-CCT␣ was found in the cytoplasmic compartment only. After fixation and permeabilization, the cells were subjected to 3 ϫ 5-min washes with PBS containing 1% dry milk and nonspecific binding was blocked with PBS with 1% dry milk for 1 h. Cells were then washed 3 ϫ 5 min with PBS and treated with 1:50 dilution in PBS of the appropriate antibody. For antibody specificity controls, CCT antibodies were incubated for 1 h in the cold room rotator with a 20-fold molar excess of peptide before application to the cells. The antibody treatment was followed by 5 ϫ 10-min washes with PBS with continuous shaking at 25°C. For the colocalization studies, cells were treated with 50 g/ml concanavalin A conjugates, 50 g/ml wheat germ agglutinin conjugates, or a 1:100 dilution of fluorophore-labeled anti-protein disulfide isomerase for 1 h at 25°C after the treatment with the anti-CCT antibody and washed an additional 5 ϫ 5 min with PBS with shaking. Slides were mounted with Prolong TM antifade in the mounting medium and covered with coverslips. For the localization of the nucleus, cells were treated with 1 g/ml Hoechst 33258 dye.
Fluorescent antibodies and conjugates were visualized using a Leica DM IRBE laser scanning confocal microscope equipped with the TCS-NT scanning laser. The pictures were taken using Leica TCS-NT computer software. For high-resolution pictures, the images were digitally zoomed to bring a single cell into the field of view. Oregon Green TM 488 fluorophore was visualized using an argon-ion laser and a fluorescein isothiocyanate filter set (488, 514 nm); Texas Red TM was visualized with the krypton-ion laser using a tetramethylrhodamine isothiocyanate filter set (568, 647 nm); colocalization studies were conducted with argon and krypton lasers and double fluorescein isothiocyanate/tetramethylrhodamine isothiocyanate filter sets. Hoechst 33258 dye was visualized with the UV laser and 352/461 nm filter set.

RESULTS
Identification and Sequence of the CCT␤2 cDNA-Two human cDNA clones similar to CCT␤1 were identified (Gen-Bank TM accession numbers AA683266 and AI041180) using a BLAST search of the public expressed sequence-tagged data base of the National Center for Biotechnology Information. The DNA sequences were verified/corrected and completed, and analysis of the sequence information revealed the existence of a unique CCT␤ mRNA, called CCT␤2, that was identical to CCT␤1 at the 5Ј end of the open reading frame but was predicted to encode a protein with a very different carboxyl terminus (Fig. 1). The cDNA sequence of clone AI041180 included both carboxyl termini representing the two variants of CCT␤ with two in-frame stop codons to terminate translation, as well as the entire 5Ј coding sequence. These data indicated that two transcripts were expressed from the same gene and also indicated that the exon encoding the ␤2 carboxyl terminus precedes the one encoding the ␤1 terminus in the genomic structure. The sequence analysis indicated that CCT␤2 is a splice variant of CCT␤1. The cDNA for CCT␤2 was assembled to exclude the possibility of expression of CCT␤1, and subcloned into the expression vector pcDNA3.
CCT Isoform Protein Sequence Comparison-The predicted amino acid sequence of CCT␤2 was aligned with the sequences of CCT␣ and CCT␤1 (Fig. 2). The predicted CCT␤2 protein had 369 amino acids and was identical to CCT␤1 from amino acids 1 to 320. After residue 320 there were 39 additional amino acids, including two groups of 5 and 4 amino acids (SSPTR, residues 321-325, and RSPS, residues 328 -331), respectively, which were identical to sequences in CCT␣ and missing from CCT␤1. The carboxyl terminus of CCT␤2 had 21 potential phosphorylation sites after position 310, including 19 serines and 2 threonine residues. As shown in Fig. 2 only 9 serines and 1 threonine of CCT␤2 align with the corresponding residues of CCT␣.
Phosphorylation of CCT␤ Isoforms-The existence of 21 potential serine and threonine phosphorylation sites in the predicted carboxyl-terminal domain of CCT␤2 suggested that this enzyme was phosphorylated similar to the modification of CCT␣ protein. This point was tested by transfecting COS-7 cells with CCT␤1, CCT␤2, or a vector control and followed labeling 48 h later with [ 32 P]orthophosphate (160 Ci/ml) for 60 min. Both CCT␤ isoforms were immunoprecipitated with the amino-terminal anti-CCT␤ antibody (B2 epitope), fractionated by SDS-PAGE, and the radiolabeled proteins were visualized by autoradiography. CCT␤2 was highly phosphorylated (Fig. 3) confirming the prediction made from the analysis of the primary structure of its carboxyl terminus. CCT␤1 was also phosphorylated, although to a signficantly lesser extent as was predicted from the fact that CCT␤1 had only 3 potential phosphorylation sites after amino acid 310. These data are consistent with the idea that the carboxyl-terminal domains of CCT␤1 and CCT␤2 were the exclusive sites of phosphorylation, as was shown with CCT␣ (9,16).
Expression and Amino-terminal Modification of CCT␤ Isoforms-In our previous report (3) describing CCT␤1 we suggested that the CCT␤1 protein was modified when expressed in COS-7 cells. Two immunoreactive proteins with apparent molecular masses of approximately 40 or 35 kDa were identified using anti-CCT␤ (epitope B1) following transfection of COS-7 cells with CCT␤1 cDNA. The faster migrating protein species co-migrated with the major [ 35 S]methionine-labeled product of an in vitro transcription/translation reaction using the CCT␤1 cDNA as template. We proposed that the slower migrating CCT␤1 form identified in COS-7 cells may result from posttranslational modification. We therefore attempted to obtain evidence of possible glycosylation, acylation or ubiquitination of the CCT␤1 protein, however, our efforts to identify the biochemical nature of the putative modification were unsuccessful. Experiments with a different lot of the commercial preparation of reticulocyte lysate also yielded two CCT␤1 translation products in vitro (Fig. 4), rather than the single product that was originally described (3), and addition of microsomes to the lysate did not alter the relative amounts of the two radiolabeled protein products. Therefore, we tested the hypothesis that the faster migrating protein produced in the in vitro transcription at translation originated from an alternative translational initiation at methionine 27, since this second predicted methionine in the CCT␤ open reading frame was within a favorable Kozak consensus context (34). Methionine 27 was changed to alanine by mutagenesis of the CCT␤1 cDNA and the derived expression construct, plasmid pPJ82, was used as a template for in vitro expression. CCT␤1(M27A) protein migrated at the same position as the "modified" CCT␤1 expressed in cells (Fig. 4). These data indicated that the initiation site in vivo was the first methionine in the open reading frame and also identified the correct mobility in SDS-PAGE for the full-length protein. On the other hand, expression of CCT␤1[⌬1 -26] in which the first 26 amino acids were deleted from the amino terminus yielded a protein that co-migrated with the "unmodified" or fastermigrating CCT␤1 expressed in cells (Fig. 4), and the major band produced by the in vitro transcription/translation system. These data showed the correct mobility for a protein that was 26 amino acids smaller corresponding to a CCT␤1 protein initiating at methionine 27 in the open reading frame. Thus, CCT␤ was not post-translationally modified at the amino terminus when expressed in a cellular context and the differences in the in vivo and in vitro results was due to the artifactual initiation at an alternative methionine in the in vitro experiments.
Tissue-specific Expression of CCT Isoforms-The tissue distribution and indication of the relative abundance of the CCT mRNAs was addressed by RT-PCR in a series of human tissues. The forward primer for the detection of both CCT␤ isoforms was complementary to sequence within the 5Ј coding region of CCT␤ and a sequence-specific reverse primer corresponded to the 3Ј ends of each of the two coding sequences for CCT␤1 and CCT␤2. The anticipated size of the CCT␤1 product was 256 bp whereas the CCT␤2 product was predicted to be 586 bp. The CCT␣ primers were predicted to yield a PCR product 345 bp long. We incubated the CCT␤ primers with the CCT␣ purified cDNA as template, and conversely, the CCT␣ primers were incubated with the CCT␤2 purified cDNA as template to verify the specificity of the primers under the thermocycling conditions. In both cases no DNA products were detectable (data not shown).
The data indicated that CCT␣ was expressed in all tissues approximately at the same levels (Fig. 5). In contrast, the expression of the CCT␤ isoforms differed among the tissues tested. Both isoforms of CCT␤ were expressed in brain, with CCT␤2 being predominant. Liver also expressed both isoforms, with CCT␤1 giving a stronger signal. Placental tissue contained CCT␤1 transcripts with no detectable signal for CCT␤2. On the other hand, CCT␤2 was the predominant isoform expressed in HeLa cells whereas lower amounts of CCT␤1 were detected. An interesting variation in the development of lung tissue was suggested in that CCT␣ and both CCT␤ isoforms were expressed in fetal lung whereas mRNA from adult lung did not yield a signal for CCT␤ and only CCT␣ was expressed. These data are consistent with the results from Post's lab (35) where cDNAs encoding only CCT␣ were cloned from an adult lung library.
Subcellular Localization of CCT␤ Isoforms-We have developed two new antibodies, anti-CCT␤ (B2 epitope) and anti-CCT␤2 (B3 epitope), to study the subcellular localization of the CCT isoforms. Immunoblotting data were obtained to confirm the specificity of the antibodies, following expression in COS-7 cells (Fig. 6). The anti-CCT␤ antibody (B2 epitope) was directed against the amino terminus of CCT␤ and recognized both the CCT␤1 and CCT␤2 isoforms on immunoblots as predicted. The anti-CCT␤2 antibody (B3 epitope) was directed against the unique carboxyl terminus of CCT␤2 and reacted only with the CCT␤2 isoform. The anti-CCT␣ antibody reacted only with the CCT␣ isoform.
CCT␣ overexpressed in CHO58 cells was found to be localized mainly in the cell nucleus using a specific peptide antibody directed against the amino terminus of the protein (15, 36). On the other hand, CCT␣ was also found to be both a nuclear and cytoplasmic protein in primary hepatocytes using an antibody   that recognized the membrane interaction domain of CCT␣ and that could potentially cross-react with CCT␤ (37). In our previous report (3) we showed that CCT␤1 was an extranuclear protein using a specific anti-CCT␤ amino-terminal antibody (B1 epitope). The above studies utilized conventional immunofluorescent microscopy to visualize the CCT proteins. In the present report, we used confocal microscopy to investigate the cellular localization of the ␤ isoforms in more detail. Confocal microscopy was advantageous because it detected proteins at the same focal plane, thoroughly increasing the resolution of cellular structures compared with previously used techniques. We also coupled the fluorescent dyes directly to the affinitypurified primary antibodies at a high molar ratio (5-10 mol of dye/mol of bivalent antibody), thus increasing the sensitivity of detection of endogenously expressed protein. We compared the distribution of CCT␤2 in cells with the distribution of the two other CCT isoforms, CCT␣ and CCT␤1, using direct immunofluorescence microscopy with confocal imaging and affinitypurified isoform-specific antibodies.
All of the CCT antipeptide antibodies used were raised in rabbits and direct coupling of different dyes to the antibodies also allowed the co-visualization of the CCT isoforms in the same in situ context. Fixation and permeabilization conditions were optimized as described under "Experimental Procedures" and the conditions (3.7% formaldehyde, 0.2% Triton) were chosen on the basis of reproducibility, consistency with the other methods, and preservation of morphology. An antibody dilution series was performed following each coupling reaction and cellular fluorescence patterns were recorded using antibody preparations at as high a dilution as possible to minimize possible nonspecific detection of unrelated proteins. The specificity of the fluorescent signal in cells was confirmed by preincubation of the antibodies with the corresponding peptide epitopes (Fig.  7). Multiple CCT␤ antibodies (B1, B2, and B3 epitopes) were used to confirm the results for CCT␤. The CCT␣ proteins from both human and rodent species are known to be identical at the amino terminus (38 -41) and the mouse and human CCT␤ proteins are also identical at the amino terminus. 2 The CCT␣ and -␤ isoforms were visualized in several different cell types, including BAC1.2F5 murine macrophage cells, HeLa human carcinoma cells, and the CHO58 hamster ovary cells (Fig. 8A). In all three cell lines, CCT␣ protein was largely found in the nucleus but a significant signal was also detected outside of the nucleus. The extranuclear CCT␣ co-localized with concanavalin A (Fig. 8B), an agglutinin with a high affinity for mannose residues and a marker for the ER (42), as determined by computer-mediated overlay of the two distinct fluorescent images. Since there was only one antibody specific for CCT␣, a cDNA encoding an FLAG epitope-tagged CCT␣ was also transfected into CHO58 cells and localized with anti-FLAG antibody (M2 antibody) to confirm results obtained with the anti-CCT␣ antibody and ensure that an unrelated protein did not possess the same peptide epitope (Fig. 8C). CCT␤2 protein was also found in the three cell lines and was situated outside of the nucleus (Fig. 8A). Antibodies that recognized both CCT␤ isoforms (B1 and B2 epitopes) and those that were specific for CCT␤2 co-localized not only with concanavalin A but also with anti-protein disulfide isomerase (Fig. 9), another marker protein for the ER organelle (43). Antibodies for CCT␤ and CCT␤2 also co-localized with each other (Fig. 10). Neither the CCT␣ nor CCT␤ antibodies associated to a high degree with the Golgi bodies as determined by co-staining with fluorescently tagged wheat germ agglutinin (data not shown), a marker for the Golgi organelle (44). These data support the conclusion that CCT␣ was found both in the nucleus and associated with the ER and that the CCT␤ isoforms were associated with the ER. Direct evidence of CCT␤2 expression and ER association was obtained with this approach but the specific occurrence of CCT␤1 could not be determined with these immunological reagents.
Overexpression of CCT␤2 in COS-7 Cells-The similarities among amino acid sequences of CCT␤2, CCT␤1, and CCT␣ suggested that CCT␤2 would also exhibit CCT enzyme activity. Transfection of COS-7 cells with plasmid pAL2 containing the CCT␤2 cDNA resulted in significantly increased CCT enzyme specific activity (Fig. 10), from 2.5 to 27 nmol/min/mg, in the crude cell lysates. Overexpression of CCT␤2 activity also resulted in an increased incorporation of [methyl-3 H]choline into cellular CDP-choline, PtdCho, and glycerophosphocholine (data not shown) comparable to the levels of radioactive metabolites following overexpression of CCT␤1 in COS-7 cells (3).
CCT␤ Rescued CHO58 Cells-The CHO58 cell line is conditionally defective for CCT activity (28) and cannot synthesize sufficient PtdCho to support growth at 40°C. Transfection of CHO58 cells with a CCT␣ cDNA complements the defective CCT activity and the overexpression of the ␣ isoform supported the proliferation of CHO58 cell colonies after shifting the cultures to the restrictive temperature (45). The differences in primary structure and subcellular localization between CCT␣ and CCT␤2 raised a question as to whether CCT␤2 had a cellular function similar to that of CCT␣. To address this issue, CHO58 cells were transiently transfected with cDNAs encoding CCT␤1, CCT␤2, and CCT␣ as a positive control, or vector alone as a negative control (Fig. 11). After 72 h at 40°C, 7 colonies remained, in the control dishes transfected with vector alone, indicating that reversion of the background genetic phenotype did not occur under these experimental conditions. In contrast, the dishes transfected with CCT␣, CCT␤1, or CCT␤2 cDNAs, hundreds of colonies were evident, indicating that overexpression of any of the CCT isoforms could complement the CCT defect in the CHO58 cells. These data suggest that CCT␣, CCT␤1, and CCT␤2 perform equivalent biochemical functions.  (1-3). Also, both the ␣ (6) and ␤ isoforms are inhibited by antineoplastic phospholipids as would be predicted from the similarity of their primary sequences and the metabolic redistribution of the PtdCho precursors in drug-treated cells that express all isoforms (i.e. BAC1.2F5 (6) and HeLa cells (33)). CCT␤1 and CCT␤2 likely arise from alternate splicing of the CCT␤ mRNA which results in the production of two mRNAs that encode proteins that differ only at their carboxyl terminus. CCT␤1 is a protein of 330 amino acids whereas CCT␤2 has 369 amino acids. The additional 39 carboxyl-terminal residues in CCT␤2 closely resembles the carboxyl-terminal phosphorylation domain of CCT␣. Within this domain, CCT␤2 has 22 potential phosphorylation sites (19 serines and 2 threonines) compared with the 13 serine residues known to be phosphorylated in the carboxyl-terminal domain of CCT␣ (16). Accordingly, CCT␤2, like CCT␣, is extensively phosphorylated in vivo. CCT␤1 lacks the numerous phosphorylation sites present in CCT␣ and CCT␤2 and is phosphorylated to a minor extent in vivo indicating that CCT␤1 may not be subject to regulation by protein kinases. After the splice junction at amino acid 323, CCT␤2 has two regions of five (SSPTR) and four (RSPS) residues identical to sequences known to be phosphorylated in CCT␣ (16). Also, CCT␤2 contains a unique sequence, SSPTRSRSPSRSP, containing the RSPXR motif similar to the one found in neurofilament H (KSPXK) that specifies phosphorylation by cyclin-dependent kinase 5 (46). Phosphorylation attenuates CCT␣ biochemical activity by interfering with lipid stimulation (17) and unphosphorylated CCT␣ exhibits a higher degree of membrane association in cells (24). Phos-phorylation is predicted to exert the same regulatory influence on CCT␤2 as on CCT␣.
CCT␤1 was proposed to be post-translationally modified following overexpression in COS-7 cells, resulting in slower migration during SDS-PAGE (3). Truncation of the amino-terminal 26 amino acids resulted in a protein that co-migrated slightly faster than the full-length product of in vitro transcription/translation of the CCT␤1 cDNA (3). Examination of new data that was obtained during comparison with the most recently discovered isoform, CCT␤2, revealed that the faster migrating product of the in vitro transcription/translation was an artifact where translation was initiating at Met-27. CCT␤1 and CCT␤2 proteins initiated at Met-1 when expressed in vivo as demonstrated by interaction with the antibody specific for residues 5 through 22 (B2 epitope) and confirmed by co-migration with the M27A mutant (Fig. 4).
CCT␤1 and CCT␤2 are distinguished from CCT␣ by their selective localization to the ER suggesting that the ␤ isoform plays a special role in PtdCho metabolism in the ER compartment. Our use of laser-scanning confocal microscopy and direct labeling of the primary antibodies with fluorescent tags resulted in improved resolution of cellular structures and a more sensitive detection of endogenous CCT isoforms than in previous studies. Computer overlays of the immunofluorescent images confirm that both CCT␤ isoforms colocalize with each other and with ER-specific markers. Although, significant amounts of CCT␣ are found distributed throughout the interphase nucleus, except the nucleolus, CCT␣ also colocalizes with ER-specific markers. These results are in general agreement with the available information from other laboratories. An investigation of the cellular distribution of CCT␣ in hepatocytes using immunoelectron microscopy and an antibody that was potentially cross-reactive with CCT␤ localized CCT␣ to both the nuclear and extranuclear compartments (37). CCT␣ was assigned to both the cytoplasm and the ER, however, membranous structures were not distinct in the images used as evidence for cytoplasmic localization, probably due to the limitations of the fixation procedure. It is clear from our images ( Fig. 8)   cytoplasmic fluorescence in some of their images, the bright nuclei coupled with the inability of this technique to examine thin sections through the cells may have obscured extranuclear CCT␣. This may be particularly relevant in their experiments using CHO58 cells to localize overexpressed CCT␣ (15,36,40) since it is possible that the number of CCT sites on the ER is limiting and that supraphysiological concentrations of CCT␣ accumulate in the nucleus. Nuclear CCT␣ is the likely source of the soluble CCT pool defined by subcellular fractionation and digitonin permeabilization experiments (1) since the remaining CCT staining in cells is associated with membrane systems. CCT is primarily regulated by the membrane lipid environment (1, 2) and localization of CCT to the ER places the enzyme in a prime position to respond to changes in the bulk membrane environment to maintain homeostasis.
All CCT isoforms have the same biochemical function and accelerate PtdCho synthesis when overexpressed in cells (Fig.  10) (3, 26). Also, CCT␣, CCT␤1, or CCT␤2 can supply the enzymatic activity necessary to support the growth of a cell line conditionally defective in CCT activity. We detected both CCT␣ and CCT␤ proteins in the mutant CHO58 cell line (Fig. 8A) indicating that both proteins may be conditionally defective to obtain the temperature-sensitive defect in PtdCho biosynthesis (28). The ability of either CCT␣ or CCT␤ to complement the temperature-sensitive phenotype suggests that reversion at either the CCT␣ or CCT␤ genetic locus could give rise to a temperature-resistant derivative cell line (48). However, any conclusions reached on the basis of expression studies in CHO58 cells must be tempered by the understanding that overexpression studies are a very blunt experimental tool to address the functionality of isoforms or mutants. Catalytically compromised or mislocalized proteins can complement mutant phenotypes if the defective proteins are expressed at a high enough level. Supraphysiological concentrations of CCT swamp the cells with CDP-Cho and trigger a compensatory response to the overproduction of PtdCho by enhancing degradation (3,26,27). Thus, enforced CCT␤ may complement a CCT␣ function(s) by swamping the cells with CDP-Cho. Also, CCT␣ proteins defective in catalytic activity, regulatory function, or cellular localization may complement the CHO58 cells when overexpressed, whereas they may not be able to sustain growth if present as a single copy. The definition of the specific functions of the CCT isoforms and the importance of nuclear versus ER localization await more detailed genetic experiments.
A specific cellular function for CCT␤ remains speculative, although analysis of the tissue-specific distribution of CCT␤ isoforms does suggest some hypotheses. CCT␤2 could play an important role in neuronal development and function since the brain has the highest levels of CCT␤2 expression (Fig. 5) and PtdCho biosynthesis is critical to axons (49). The finding of CCT␤ associated with the ER suggests that this isoform may be involved in tissues that secrete PtdCho. For example, CCT␤1 is highly expressed in placenta (Fig. 5) and may play a role in PtdCho bioysnthesis in this lipogenic tissue which secretes and supplies phospholipid to a developing embryo (50). Liver and fetal lung also express both CCT␤ isoforms, but CCT␤1 is predominant (Fig. 5). The absence of CCT␤ expression in adult lung (Fig. 5) does not fit with this hypothesis since PtdCho biosynthesis plays an important role in surfactant secretion and pulmonary function. A precise determination of a function for CCT␤ will require the analysis of genetically engineered animals that do not express this isoform.