A Formiminotransferase Cyclodeaminase Isoform Is Localized to the Golgi Complex and Can Mediate Interaction of Trans-Golgi Network-derived Vesicles with Microtubules*

Dagmar HennigDagger §, Suzie J. Scales, Anne MoreauDagger , Laura L. Murleyparallel , Jan De MeyDagger , and Thomas E. Kreis**

From the Dagger  Department of Supramolecular and Cell Biology, Institut Jacques Monod, 2 Place Jussieu, 75251 Paris, France,  Department of Cell Biology, University of Geneva, Sciences III, 30 Quai Ernest-Ansermet, 1211 Geneva 4, Switzerland, and parallel  Department of Biochemistry, McGill University, Montréal, Québec H3G 1Y6, Canada

    ABSTRACT
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A protein of 60 kDa (p60) has been identified using a quantitative in vitro vesicle-microtubule binding assay. Purified p60 induces co-sedimentation with microtubules of trans-Golgi network-derived vesicles isolated from polarized, perforated Madin-Darby canine kidney cells. Sequencing of the cDNA coding for this protein revealed that it is the chicken homologue of formiminotransferase cyclodeaminase (FTCD), a liver-specific enzyme involved in the histidine degradation pathway. Purified p60 from chicken liver has formiminotransferase activity, confirming that it is FTCD or an isoform of this enzyme. Isoforms of FTCD were identified in chicken hepatoma and HeLa cells, and immunolocalize to the region of the Golgi complex and vesicular structures in its vicinity. Furthermore, 58K, a previously identified microtubule-binding Golgi protein from rat liver (Bloom, G. S., and Brashear, T. A. (1989) J. Biol. Chem. 264, 16083-16092), is identical to FTCD. Both proteins co-purify with microtubules and co-localize with membranes of the Golgi complex. The capacity of FTCD to bind both to microtubules and Golgi-derived membranes may suggest that this protein, or one of its isoforms, might have in addition to its enzymatic activity, a second physiological function in mediating interaction of Golgi-derived membranes with microtubules.

    INTRODUCTION
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Interphase microtubules and their associated motor proteins direct intracellular movements of essentially all cytoplasmic membrane-bounded organelles, and they are thus also responsible for the correct spatial arrangement of these organelles in a cell (1). Transport of trans-Golgi network derived vesicles (TGVs)1 in polarized epithelial Madin-Darby canine kidney (MDCK) cells to the apical and basolateral membrane occurs in two classes of vesicles and depends on microtubule-based motor proteins (2). In contrast to the kinesin-dependent transport of basolaterally targeted vesicles, the delivery of apically destined vesicles to the plasma membrane requires kinesin and cytoplasmic dynein. The directionality of these motors correlates well with the topology of microtubules in polarized MDCK cells, arranged mainly apico-basolaterally, with their minus ends near the cell apex and their plus ends toward the basal cytoplasm (3). In addition, motor protein independent binding of TGVs to microtubules occurs in vitro and depends on two different protein activities, suggesting that these interactions of apical and basolateral TGVs require specific proteins (4). These data reflect the complex mechanisms of the exocytic transport machinery between the trans-Golgi network and the cell surface in epithelial cells (5). Accessory factors have been identified which can improve the efficiencies of cytoplasmic dynein- and kinesin-dependent movements (6, 7). Of these, the dynactin complex is clearly the best characterized (8). Recently, a new family of proteins mediating interaction of membranous organelles with microtubules has been discovered using a number of different in vitro assays. These cytoplasmic linker proteins (CLIPs) appear to be involved in the initial docking of membranes to microtubules preceding the motor-dependent movement (9).

We have used the previously established in vitro microtubule-binding assay (4) for further characterizing the specific interaction of MDCK TGVs with microtubules at the molecular level and have identified a 60-kDa chicken liver microtubule-binding protein (MBP). Cloning and sequencing of the cDNA coding for p60 revealed that it is the chicken homologue of formiminotransferase cyclodeaminase (FTCD) (10), a liver-specific enzyme, and 58K, a rat liver enzyme described in the accompanying paper (56).2 FTCD was originally identified and characterized in pig liver (11) and is involved in the histidine degradation pathway. A potential dual physiological function of FTCD, or one of its isoforms, will be discussed.

    EXPERIMENTAL PROCEDURES
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Cell Culture-- MDCK II cells (12) and HeLa spinner cells (9) were cultured as described previously. Adherent HeLa cells were grown in minimal Eagle's medium supplemented with 2 mM glutamine, 5% fetal calf serum, and 1% nonessential amino acids. DU249 chicken hepatoma cells (13) were grown in RPMI 1640 supplemented with 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 5% FCS. All cell cultures were grown with 5% CO2 in a humidified 37 °C incubator. Media and reagents for cell culture were purchased from Life Technologies, Inc. (Eggenstein, FRG or Cergy, France) unless stated otherwise.

Immunofluorescence-- Immunofluorescence was essentially performed as described previously (14). DU249 chicken hepatoma cells grown on glass coverslips were rinsed three times in PHE10M (60 mM K-PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgOAc, pH 6.9) at 37 °C, prior to fixation for 20 min at room temperature with 3% paraformaldehyde (Merck, Darmstadt, FRG) in PHE10M, pH 7.5. They were then permeabilized in 0.1% Triton X-100 in PBS with or without 0.05% SDS for 4 min at room temperature. HeLa cells were fixed and extracted for 4 min at -20 °C in methanol. The following secondary antibodies were used: Cy3-labeled anti-rabbit (Sigma) and fluorescein-labeled anti-mouse (Jackson Immunoresearch Co., West Grove, PA) for the DU249 cells; and rhodamine-labeled anti-rabbit and fluorescein-labeled anti-mouse for HeLa cells (15).

DU249 cells were visualized with a multimode videomicroscope including a modified Zeiss Axiovert 135. Images were recorded with a cooled CCD camera (Photometrics CH250), controlled by an Apple Macintosh Quadra 950. Image processing was achieved using Adobe Photoshop software, and photographs were printed on a Kodak ColorEase PS printer. Visualization of HeLa cells was done with a Zeiss inverted fluorescence microscope (Axiovert TV10) and a cooled CCD camera (Photometrics CH250), controlled by an Apple Macintosh 840a. Images of HeLa cells were processed using IPLab Spectrum V2.3 software (Signal Analytics Corp., Vienna, VA) and printed on a Slidewriter IS200 (Focus Graphics, Foster City, CA).

Antibodies-- The following antibodies were used: mAbs against rat liver 58K (anti-58K-2, -4, -7, -9, and -12) (16), mAb SUK4 against kinesin (17), mAb 100/3 against gamma -Adaptin (18), maD against beta -COP (19), mAb 2D6 (20), and a rabbit polyclonal antibody against porcine FTCD (from Dr. MacKenzie, McGill University, Montréal, who also generously provided us with purified recombinant FTCD).

Antibodies against synthetic peptides of chicken p60 ("LVEC," aa 4-17; "KKVQ," aa 217-228; "IIEY," aa 321-333; "KLPK," aa 424-438; and "KQGS," aa 526-541) were raised in rabbits or rats essentially as described earlier (14). For immunization peptides were either synthesized on an 8-branched polylysine core (21) or coupled to keyhole limpet hemocyanin as a carrier. Peptide antibodies were affinity-purified on the corresponding peptides coupled to BSA on CNBr-Sepharose beads (Pharmacia LKB GmbH, Freiburg, FRG). 1 mg of peptide was incubated with 1 mg of BSA and 8 mg of 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide (Sigma) in a final volume of 1 ml (in H2O) for 1 h at room tempeature on a rotating wheel. After dialysis against 0.2 M NaHCO3, 0.5 M NaCl pH 8.5, the peptide/BSA solution was coupled to CNBr-activated Sepharose beads, and antibodies were affinity purified. Polyclonal antibodies were also raised in rabbits against the p60 band from chicken liver excised from preparative 10% SDS-PAGE. The gel piece was washed with PBS, homogenized, and emulsified in an equal volume of complete (first immunization) or incomplete (first/second boosts) Freund's adjuvant (Life Technologies, Inc.), or diluted in PBS (for further boosts). Sera (no. 148) were affinity-purified on immunoblots with purified chicken liver or recombinant p60 (15). Polyclonal antibodies were also raised in rabbits against purified bacterially expressed poly-his-tagged full-length p60 (no. N1) or a 153-aa N-terminal fragment of p60 (no. 568) according to the same immunization protocol and affinity purified as indicated above (14). All these antibodies raised against p60 reacted with the antigen by immunoblotting.

Organelle-Microtubule Binding Assay-- The in vitro binding assay was performed as described (4), except that [35S]methionine-labeled, flotation-purified vesicles were used (obtained from ~0.25-0.5 × 106 pulse-labeled, filter-grown MDCK II cells). The amount of radioactivity in the supernatant and pellet fractions was quantified by liquid scintillation counting, using Ready Safe scintillation mixture (Beckman Instruments GmbH, München, FRG). The percentage of binding was calculated using the formula Rp/(Rp + Rs), where Rp and Rs denote the amount of radioactivity in the pellet and supernatant, respectively. The total recovery of radioactivity (Rp + Rs) was in the range of 80-90% of the total input radioactivity.

Tubulin was prepared from bovine brain and polymerized with taxol (referred to as "microtubules" in this work) as described previsously (15). Microtubules were stored in liquid nitrogen for up to 6 months.

Pulse-labeling of filter-grown MDCK II cells was performed according to previously described procedures (12). MDCK II cells from a confluent 75-cm2 flask were seeded in 20 ml of growth medium onto one 100-mm diameter, 0.4-µm pore size, premounted polycarbonate filter (Costar, Cambridge, MA) and grown for 3 days in a special holder in a Petri dish containing 140 ml of growth medium. For pulse-labeling, cells were rinsed twice with PBS containing 0.9 mM CaCl2 and 0.5 mM MgCl2 (PBS+). The basal side of the filter was then placed onto 2 ml of "pulse-labeling medium" (methionine-free minimal Eagle's medium containing 2.2 g/liter NaHCO3, 0.2% BSA, 10 mM HEPES, pH 7.3, 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin) containing 500 µCi of [35S]methionine (Amersham Buchler, Braunschweig, FRG) on Parafilm in a humid chamber and covered with 2.5 ml of the same medium lacking radioactivity. Labeling was stopped after 90 min by washing and incubating the cells for 2 h at 20 °C in "20 °C medium" (methionine-free minimal Eagle's medium containing 0.35 g/liter NaHCO3, 0.2% BSA, 10 mM HEPES, pH 7.3, 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin) supplemented with 150 µg/ml unlabeled methionine (10-fold excess) and 20 µg/ml cycloheximide. TGVs were then isolated and purified on discontinuous equilibrium density gradients as described (22). This flotation-purified vesicle fraction was then either used immediately as the "TGVs" in the in vitro organelle-microtubule binding assays or frozen in aliquots in liquid nitrogen and stored at -80 °C.

Cytosol from HeLa spinner and MDCK II cells was prepared as described (4). For heat treatment, cytosol was incubated for 3 min at 95 °C, cooled on ice and cleared by centrifugation at 10,000 × g for 10 min at 4 °C. To cleave cytoplasmic dynein, HeLa cytosol was treated with UV light in the presence of vanadate and ATP (23, 24). HeLa cytosol was immunodepleted of kinesin by incubation with Sepharose beads carrying the mAb SUK4. Immunodepletion of CLIP-170 from HeLa cytosol was performed with mAb 2D6 as described previously (25).

Purification of p60 from Liver-- Livers (~70 g) from four freshly sacrificed chickens were minced on ice with scalpels and homogenized with a Polytron tissue grinder (Kinematica, Lucerne, Switzerland) three times, 20 s each, with 20-s intervals in 1.1 volume of ice-cold homogenization buffer (20 mM K-HEPES, 0.3 M sucrose, pH 7.0) supplemented with 1 mM dithiothreitol and protease inhibitors (1 mM phenylmethylsulfonyl fluoride and 1 µg/ml chymotrypsin, leupeptin, and pepstatin A; Sigma, Deisenhofen, FRG). All subsequent steps were performed at 0-4 °C unless otherwise specified. A post-nuclear supernatant, prepared by centrifugation of the homogenate at 17,000 × g for 15 min, was filtered through glass wool by suction and recentrifuged at 180,000 × g for 90 min to obtain a liver cytosol fraction (yield ~50 ml at ~30 mg protein/ml). This cytosol fraction was diluted 3-fold to a final concentration of 10 mM K-HEPES, 0.1 M sucrose, pH 6.8, and incubated for 30-40 min with 0.5 volume of S-Sepharose cation-exchange beads (Pharmacia LKB GmbH, Freiburg, FRG) preequilibrated in 10 mM K-HEPES, pH 6.8. The beads were sedimented at 1000 × g for 2 min, and the supernatant was further clarified by centrifugation at 12,000 × g for 10 min. The resulting supernatant was adjusted to pH 8.0 by addition of 0.01 volume of 1 M Tris and loaded onto an EMD-DEAE anion-exchange column (Merck, Darmstadt, FRG) containing 1 volume of packed beads preequilibrated with buffer A (10 mM Tris-Hepes, 6.5 mM HEPES, pH 8.0). Fractions of 2.5 ml eluted from the column with an increasing salt gradient of buffer B (1 M KCl in buffer A) on a FPLC system (Pharmacia LKB GmbH, Freiburg, FRG) were analyzed in vitro in the organelle-microtubule binding assay. The fractions containing the peak of activity (from ~400 to 450 mM KCl) were pooled and dialyzed against PB buffer (0.1 M PIPES-KOH, 1 mM EGTA, 1 mM MgSO4, pH 6.9). To these pooled and dialyzed EMD-DEAE fractions (~1 mg/ml) microtubules were added to a final concentration of 0.5 mg/ml. Taxol (20 µM) was present in this and all subsequent steps. After 15 min of incubation at 25 °C, this solution was centrifuged at 25 °C at 46,000 × g for 30 min through a 0.3 volume cushion of 30% sucrose in PB buffer containing Taxol. The pellet was resuspended in 1/12 the original volume of PB buffer containing 0.8 M KCl and Taxol, incubated for 15 min at 25 °C, and spun as above without a cushion. The supernatant containing p60 was dialyzed against PB buffer, aliquots (at ~0.5 mg/ml) were frozen in liquid nitrogen and stored at -80 °C. Taking into account all volume changes during the purification of p60, the equivalent volume of the final p60 fraction was about one-fourth that of the initial cytosol. Preparation of p60 from turkey liver was carried out according to the same protocol.

Protein Sequencing-- Approximately 1 nmol (~85 µg) of purified chicken liver p60 was run on preparative 6-12% SDS-PAGE. Trypsin digestion of the Coomassie Blue-stained p60 band and sequencing of the resulting purified tryptic peptides were performed as described (26, 27).

cDNA Cloning and Sequencing-- Six degenerate oligonucleotide hybridization probes with a length of 23 bases were synthesized from the following peptide sequences: NFSEGCN(K) (amino acids 10-17, oligonucleotides 1a, b); FILEEEH(K) (amino acids 292-299, oligonucleotides 2a, b); and VQAGQED(K) (amino acids 326-333, oligonucleotide 3). The sequences of the oligonucleotides were: 1a, 5'-AA(CT)TT(CT)TC(ACGT)GA(AG)GG(ACGT)TG(CT)AA(CT)AA-3'; 1b, 5'-AA(CT)TT(CT)AG(CT)GA(AG)GG(ACGT)TG(CT)AA(CT)AA-3'; 2a, 5'-TT(CT)AT(ACT)CT(ACGT)GA(AG)GA(AG)GA(AG)CA(CT)AA-3'; 2b, 5'-TT(CT)AT(ACT)TT(AG)GA(AG)GA(AG)GA(AG)CA(CT)AA-3'; and 3, 5'-GT(ACGT)CA(AG)GC(ACGT)GG(ACGT)CA(AG)GA(AG)GA(CT)AA-3'.

All nucleotides in parentheses were included at that position. Oligonucleotides 1a, 1b, 2a, 2b, and 3 were 512, 256, 384, 192, and 1024-fold degenerates, respectively. Approximately 1,400,000 phage plaques of an oligo(dT)-primed chicken liver cDNA library made in the lambda ZAPII vector (Stratagene, La Jolla, CA) were analyzed in the first round of screening. Briefly, oligonucleotides 1a, 1b, 2a, and 2b were 32P-labeled with T4 polynucleotide kinase and [gamma -32P]ATP (Amersham Buchler, Braunschweig, FRG). Plating of phages and preparation of duplicate nitrocellulose filters (Schleicher & Schüll, Dassel, FRG) were performed using standard techniques (28). After baking for 2 h at 80 °C, nitrocellulose filters were washed four times, 15 min each, in 3× SSC, 0.1% SDS at room temperature, 1.5 h in 3× SSC, 0.1% SDS at 65 °C, then in 6× SSC for 10 min at room temperature to remove bacterial debris. Prehybridization was done overnight at 42 °C in 6× SSC, 5× Denhardt's solution, 0.05% sodium pyrophosphate, 100 µg/ml herring sperm DNA, and 0.5% SDS. Hybridization was carried out for 24 h at 39 °C in 6× SSC, 1× Denhardt's solution, 0.05% sodium pyrophosphate, and 100 µg/ml yeast tRNA supplemented with a mixture of 32P-kinase-labeled oligonucleotides 1a, 1b, 2a, and 2b at 1.1 × 106 cpm/ml. Filters were washed four times, 15 min each, at room temperature in filter wash buffer (6× SSC, 0.05% sodium pyrophosphate), followed by three successive washes for 30 min each at 40, 44, and 48 °C, then 15 min at 50 °C, before being exposed wet against x-ray film (X-Omat AR films, Eastman Kodak Co.) for 2 days at -70 °C. Filters were re-wetted in 6× SSC, washed for 30 min at 54 °C in filter wash buffer, and reexposed for 1.5 days at -70 °C. 21 positive phage plaques were identified in the first screening, which were verified and further isolated in second and third rounds of screening. These screening cycles were performed as described above, except that triplicate nitrocellulose filters were prepared and hybridized separately with 32P-kinase-labeled oligonucleotides 1a + b, 2a + b, and 3 at 40 °C for 19 h at 1 × 106 cpm/ml. Filters were washed as described for the first screening, except that the washing procedure was stopped at 48 °C for filters hybridized with oligonucleotides 2a + b, at 50 °C with oligonucleotides 1a + b and at 54 °C with oligonucleotide 3. 11 clones remained which hybridized with all three oligonucleotides.

Clones obtained from the lambda ZAPII library were excised as Bluescript KS plasmids with helper phage according to the manufacturer's instructions. DNA isolation from transformed bacteria, and restriction map analyses of the clones were carried out using standard methods (28). Restriction enzymes and other molecular biology reagents were purchased from Boehringer (Mannheim, FRG), unless stated otherwise. Clones were also analyzed by dideoxy sequencing of their 5'- and 3'-terminal ends, using plasmid DNA purified on Qiagen columns (Diagen GmbH, Düsseldorf, FRG). Sequencing was performed with alpha -35S-labeled dATP (Amersham Buchler, Braunschweig, FRG) and a T7 sequencing kit (Pharmacia LKB GmbH, Freiburg, FRG). Of the 11 clones analyzed, clone 9 was selected for further characterization, since it had the correct size expected for the cDNA of a 60-kDa protein and contained the putative ATG initiation codon. Other clones were identical, smaller, or extended clone 9 to the 3' end. Unidirectional deletion clones of clone 9 were made, using a nested deletion kit (Pharmacia LKB GmbH) with exonuclease III (Freiburg, FRG) and sequenced as described above. The sequence of the opposite strand of clone 9 was determined by dideoxynucleotide sequencing with fluorescent synthetic oligonucleotides as primers and the EMBL sequencing device. Using these two strategies, overlapping identical sequences on both strands were obtained for the entire cDNA. Sequence data were compiled and analyzed using the University of Wisconsin GCG programs (Madison, WI) (29).

Sequence Analysis-- Molecular weight, amino acid composition, and secondary structure prediction of p60 were established using the GCG programs PEPTIDESORT and PEPTIDE-STRUCTURE (31). FASTA (30) and BLAST (31) were used to search the EMBL/GenBankTM nucleotide data library (release 53) and the SwissProt protein data library (release 35) for sequence homologies. Potential sites for post-translational modifications were identified by searching the PROSITE data library (32) with the program SCRUTINEER (33). Comparison of p60 with itself, the so far characterized MBPs and porcine FTCD was performed using the Interactive Sensitive Sequence Comparison program ISSC (34). The window length for these comparisons ranged from 5 to 35, in steps of 2, at a stringency of 40.

In Vitro Transcription and Translation-- The different Bluescript templates were linearized at convenient 3'-restriction sites and RNAs were produced by in vitro transcription using an in vitro transcription kit with T3 polymerase (Promega, Madison, WI) according to the manufacturer's instructions. Translation of the RNAs was carried out in rabbit reticulocyte lysate (Promega) with [35S]methionine for 60 min at 30 °C (28). Translation products were analyzed by SDS-PAGE and fluorography.

The reaction mixture of [35S]methionine-labeled p60, in vitro translated from clone 9, was clarified at 15,000 × g for 15 min at 4 °C, and the supernatant was used for immunoprecipitation. Briefly, the supernatant was diluted 1:20 with TNT buffer (50 mM Tris-Cl, pH 7.4, 100 mM NaCl, 0.5% Triton X-100) and incubated with 0.01 volume of anti-p60 in the presence of hemoglobin (0.5 mg/ml final concentration). After 12-16 h, 30 µl of protein A-Sepharose (a 50% v/v slurry in TNT; Pharmacia LKB GmbH, Freiburg, FRG) was added for 2 h. All incubations were at 4 °C with gentle mixing. The beads were then washed 3 × 10 min in TNT buffer, and rinsed once in water. Protein bound to the beads was solubilized in 50 µl of gel sample buffer (35) and analyzed by SDS-PAGE.

Cytosol for FTCD Enzyme Assays-- For total cell extract and cytosol preparation, confluent chicken hepatoma (DU249) and adherent HeLa cells were washed twice with ice-cold PBS and then once with ice-cold homogenization buffer Kpi (0.1 M potassium phosphate, pH 7.3; 20% glycerol; 0.02% TX-100; 35 mM 2-mercaptoethanol; 1 mM benzamidine; 1 mM phenylmethylsulfonyl fluoride), as used for the purification of recombinant FTCD (10). All steps were carried out at 0-4 °C. Cells were scraped in Kpi (1.5 ml per 100-mm Petri dish) and sedimented at 300 × g for 2 min. The pellet of cells from 10 Petri dishes was resuspended in 500 µl of Kpi. DU249 cells were Dounce-homogenized, 8-10 times; HeLa cells were broken by four cycles of dounce homogenization (20 strokes) followed by 20 passages through a 22-gauge needle. Aliquots of this total cell extract were snap frozen in liquid nitrogen and stored at -80 °C or incubated with 1 volume of 95 °C preheated 2× gel sample buffer (35) for 5 min at 95 °C and analyzed by SDS-PAGE. The rest of the homogenate was spun at 17,000 × g for 15 min to pellet nuclei and cell debris, and the post-nuclear supernatant was further clarified at 180,000 × g for 90 min to obtain the cytosolic fraction. Aliquots of this cytosol were frozen in liquid nitrogen and stored at -80 °C. Cytosol from rat liver was prepared as described above for chicken liver, except that the homogenization buffer was PB containing 1 mM dithiothreitol and protease inhibitors. A sample for SDS-PAGE of total cell extract of chicken or rat liver was taken immediately after homogenization of the tissues.

Formiminotransferase assays were performed as described previously (36, 37). Protein concentrations were determined according to the method of Bradford (38), using BSA as the standard.

Gel Electrophoresis and Immunoblotting-- Gel electrophoresis of proteins on SDS-PAGE, silver staining of gels, and immunoblotting were performed as described previously (17). Two-dimensional gel electrophoresis using the mini-gel system of Bio-Rad Laboratories GmbH (München, FRG) was carried out as described elewhere (39). For the analysis of in vitro translation products, gels were stained with Coomassie Blue, destained, washed in water for 30 min, treated with 1 M sodium salicylate for 30 min, vacuum-dried, and exposed to preflashed x-ray films (X-Omat AR films; Kodak) at -70 °C. Immunoblotting followed by complete protein staining with AuroDye (Amersham, Les Ulis, France) was as described previously (40), except that alkaline phosphatase-labeled antibodies were used.

    RESULTS
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Identification of p60 with an in Vitro TGV-Microtubule Binding Assay-- We have previously shown that binding of C6NBD-ceramide-labeled TGVs from MDCK II cells to microtubules depends on vesicular membrane proteins and cytosolic factors (4). For convenience of identification of the cytosolic proteins involved in binding TGVs to microtubules, we have modified this assay by using radioactivity instead of C6NBD-ceramide for labeling the vesicles. These radioactively labeled purified TGVs had two advantages: (i) purified by flotation to their equilibrium density, residual cytosolic components and membrane contaminants were effectively removed (22), and (ii) quantitation of TGVs co-sedimenting with microtubules was rapid and reproducible. In the presence of HeLa cytosol, about 35% of the [35S]methionine-labeled TGVs co-sedimented with microtubules, compared with only about 3% in the absence of cytosol, and heat-inactivated cytosol was unable to stimulate binding (Fig. 1). Inactivation of cytoplasmic dynein by UV treatment or immunodepletion of kinesin with mAb SUK4 also did not interfere with the normal binding of TGVs to microtubules. Thus, modifying the labeling procedure of TGVs appeared not to alter their performance. In addition, immunodepletion with mAb 2D6 of CLIP-170, a protein mediating endosome-microtubule interactions (25), left the TGV-microtubule binding activity of HeLa cytosol unchanged. Taken together, these data suggest that cytosolic factors other than CLIP-170, kinesin, or cytoplasmic dynein are involved in the binding of TGVs to microtubules.


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Fig. 1.   Cytosol-dependent binding of TGVs to microtubules is independent of kinesin, cytoplasmic dynein, and CLIP-170. The in vitro binding assay was performed with [35S]methionine-labeled TGVs from MDCK II cells as described under "Experimental Procedures." The vesicle fraction was incubated with microtubules in the absence of cytosol (lane 1) and in the presence of 3 mg/ml untreated (lane 3) or an equivalent volume of pretreated HeLa cytosol (lanes 2 and 4-6) and separated by centrifugation through 30% sucrose. Binding was determined by measuring radioactivity in the pellet and supernatant fractions as described under "Experimental Procedures." HeLa cytosol was heat-treated at 95 °C for 3 min (heat), treated with UV light in the presence of vanadate and ATP (UV+), or preincubated with Sepharose beads coupled to mAbs directed against kinesin (SUK4) or CLIP-170 (2D6).

The following purification procedure (described in detail under "Experimental Procedures") was used for identifying candidate proteins involved in the interaction of TGVs with microtubules. Chicken liver cytosol was used, since it is fully active in the binding assay and can easily be prepared in sufficient amounts and at high protein concentration. Enrichment of activity was monitored using the TGV-microtubule binding assay and SDS-PAGE (Fig. 2). In the first step, cytosol (lane 1) was incubated with S-Sepharose; the supernatant had essentially the same activity in the assay as the starting material (lane 2) and was used for further purification. Interestingly, however, a peak of activity, albeit smaller than in the initial cytosol, could be eluted from the cation-exchange beads using a salt gradient (data not shown). Since we used a floatation-purified vesicle fraction which contains at least two classes of TGVs (22), it is conceivable that the two active fractions may correspond to the different factors responsible for the interaction of apically and basolaterally targeted vesicles with microtubules. In a second step, the S-Sepharose supernatant was fractionated by EMD-DEAE anion-exchange chromatography using a continuous KCl salt gradient. The activity of the EMD-DEAE peak fraction was comparable to that of the original cytosol (lane 3). In the final step we selected from this EMD-DEAE peak fraction proteins with an affinity for microtubules, since this should be one of the characteristics of a protein mediating TGV-microtubule interactions. In fact, MBPs prepared from chicken or turkey liver have TGV-microtubule linking activity in vitro (not shown). Microtubule affinity purification of potential TGV-CLIPs was performed by addition of Taxol-stabilized microtubules to the EMD-DEAE peak fraction and subsequent sedimentation of microtubules and MBPs. Microtubule-bound proteins were eluted with high salt concentrations, and this fraction was highly enriched in a protein of 60 kDa (p60; lane 5) and active in the in vitro binding assay. In contrast, the supernatant after microtubule-MBP sedimentation was depleted of p60 and could no longer mediate TGV-microtubule interactions (lane 4). The p60 fraction also contained tubulin (most likely a contaminant from the "microtubule-affinity" step), and several other minor polypeptides. Since none of these polypeptides were stoichiometric with p60, we assume that they are contaminants. These data suggest that p60 can mediate TGV-microtubule interactions. When compared with the initial cytosol (100% activity), an equivalent volume (see "Experimental Procedures" for calculations) of enriched p60 had ~60% linker activity (not shown), whereas two volumes of p60 showed ~123% linker activity (lane 5). This reduced binding activity of the final p60 fraction may be due to deterioration of the protein during the purification procedure. On the other hand, the elevated plateau for p60 mediating TGV-microtubule interactions may be due to removal of potential inhibitory factors. Essentially the same results were obtained when turkey liver p60 was used.


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Fig. 2.   A 60-kDa protein is involved in binding TGVs to microtubules in vitro. The purification of p60 (described in detail under "Experimental Procedures") used chicken liver cytosol as starting material (lane 1). The unbound fraction after the cation-exchange chromatographic step (lane 2) and the pooled fractions obtained by chromatography on EMD-DEAE-cellulose (lane 3) are shown. Microtubule-affinity purification of the EMD-DEAE peak fractions yielded a supernatant depleted of microtubule-binding proteins (lane 4), and a microtubule pellet from which p60 was eluted (lane 5). The different fractions resulting from the purification procedure were analyzed by SDS-PAGE (A) and in the in vitro binding assay (B). The assay was performed in the presence of an equivalent (lanes 1-4) or 2-fold equivalent volume (lane 5) of the cytosolic fractions as described in the legend to Fig. 1. Molecular masses are indicated (×103 kDa).

cDNA Cloning of p60-- A chicken liver cDNA library was screened with three oligonucleotides derived from partial protein sequences obtained by tryptic digestion of gel-purified p60. Eleven phage clones hybridizing with all three oligonucleotides were isolated, and one (clone 9), which had the right size and contained a putative ATG start codon, was sequenced. Although the N terminus of p60 could not be directly sequenced, this ATG is probably the start codon, as it is near a consensus Kozak (41) sequence, AGGACCATGGGCC(A/G)CCATGG (41), and no other start codons were found upstream in any of the eleven clones. Clone 9 revealed an open reading frame of 1623 base pairs (terminated by a stop codon at position 1654), encoding a protein of 541 aa with a calculated molecular weight of 59,100 (Fig. 3A). All six tryptic peptides are present in this sequence (underlined in Fig. 3A), each preceded by the expected lysine or arginine. A 19-base pair poly(A) tail was identified at the 3' terminus of the cDNA, preceded 23 base pairs upstream in the noncoding region by the sequence CATAAA, which represents a slight variant of the polyadenylation signal AAUAAA (42).


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Fig. 3.   Analysis of the cDNA sequence of p60. A, the complete cDNA and deduced amino acid sequence of p60 is shown. Underlined regions indicate matches of tryptic peptide sequences of chicken liver p60 with the cDNA-deduced p60 protein sequence. Five peptides used for raising antibodies are represented by dotted lines; antibodies were raised in rabbits (dots) or in rat (double-under lined region). The asterisk denotes the stop codon. The sequence data are available from EMBL/GenBankTM under accession no. AJ224473. B, p60 is almost idenical to FTCD. Comparison of the p60 with FTCD (accession no. L16507) (12) using an ISSC homology search matrix (36) shows structural identity of the two proteins. Comparison of p60 with itself using the same method gives an almost identical pattern (not shown).

Several other lines of evidence confirmed that clone 9 encodes full-size p60. First, the major polypeptide produced by in vitro transcription/translation of clone 9 cDNA, and which can be immunoprecipitated by specific antibodies against p60, co-migrates with purified chicken liver p60 (Fig. 4, D and A, respectively). In addition, a subset of polypeptides with slightly different pI values and which overlapped with polypeptides present in the p60 fraction from chicken or turkey was also found (Fig. 4). Thus it appears that different isoforms or post-translational modifications of p60 exist and that some of the putative post-translational modifications of p60 (see below) also occur in reticulocyte lysate on the clone 9 polypeptide. Second, the calculated pI of 6.13 for clone 9 is consistent with the pI of ~6 determined for the major form of chicken liver p60 (Fig. 4). Third, antibodies raised against five different peptides derived from the clone 9 cDNA sequence (see double and dotted lines in Fig. 3A) all recognize chicken liver p60 by immunoblotting, a reaction which can be competed with the corresponding peptides (data not shown). Furthermore, chicken liver p60 was also recognized by immunoblotting with antibodies raised against bacterially expressed clone 9 polypeptide. We therefore conclude that clone 9 cDNA encodes full-length p60.


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Fig. 4.   Comparison of in vitro translated p60 with purified p60 from chicken liver. [35S]Methionine-labeled p60, obtained by in vitro transcription/translation of the full-length cDNA of clone 9, was immunoprecipitated with antibody no. 148 raised against native purified p60 and subjected together with chicken liver p60 in one sample to 2D gel electrophoresis. After Coomassie Blue staining, which revealed only chicken liver p60 (A), the same gel was subjected to autoradiography to identify the in vitro translated p60 (D). Both proteins migrated at the same position in the gel. The purified p60 from chicken (B) or turkey (C) liver and in vitro translated p60 (E) were separated on 2D gels and visualized by silver staining (B and C) or autoradiography (E), which revealed a major form (2) and four isoforms (1, 3, 4, and 5) of the protein. Molecular masses are indicated (×103).

A search of the EMBL/GenBankTM nucleotide and the SwissProt protein data libraries with FASTA (30) and BLAST (31) revealed very strong homology of p60 with FTCD (accession no. L16507) (10). The significance of this homology was confirmed by using ISSC (34). Chicken p60 and pig FTCD were alignable over their entire amino acid sequence (Fig. 3B) with a similarity of 80.2% and an identity of 68.4%. Thus most likely, p60 is the chicken homologue of FTCD. Several unpublished human EST sequences putatively encoding FTCD were likewise alignable with p60 (not shown), and the only other significant homology (31%) was with a putative serine cycle enzyme (accession no. Q49135) (43). There is no obvious yeast homologue of p60, consistent with the observed lack of FTCD activity in yeast (44). We also performed pairwise comparison of p60 with the so far characterized microtubule-associated proteins and MBPs using ISSC and found no homologies. Finally, we could not identify internal repeats in the p60 sequence.

Using the program "peptide structure" (29), we examined the secondary structure and charge display of p60. p60 has no hydrophobic sequence long enough to span a lipid bilayer. The lack of obvious alpha -helical stretches, beta -sheet domains, or clusters of charged amino acids suggests that p60 is a globular, hydrophilic protein. In fact, p60 structure may thus resemble that of FTCD, which has been shown to be composed of eight identical subunits forming a planar ring-shaped structure (36, 45), but is more aptly described as a tetramer of dimers (37). Indeed, preliminary experiments indicate that p60 may sediment as an oligomer (data not shown). Several potential phosphorylation sites, including consensus sequences for casein kinase II and protein kinase C, were identified in the p60 sequence by searching the PROSITE data library (32). We also identified consensus sequences for glycosylation, myristoylation, amidation, farnesylation and a peroxisomal signal sequence, but do not yet know if any of these modifications occur on p60. The different putative isoforms of p60 identified by 2D SDS-PAGE do suggest that p60 is post-translationally modified. No known consensus nucleotide-binding motifs were found.

Finally, comparison of partial amino acid sequences of rat 58K (16) revealed that five out of eight peptides have more than 66% identity with the corresponding p60 amino acid sequence (56), strongly suggesting that p60 and 58K are homologous proteins.

Localization of p60 in Tissue Culture Cells-- A battery of specific antibodies was raised against p60 for further characterization of the protein, including five peptide antibodies (for the peptides, see Fig. 3A), one against the purified chicken liver protein, as well as against recombinant full-length and N-terminal 153 aa of p60. All these antibodies react with chicken liver cytosolic p60 by immunoblotting. p60 was localized by immunofluorescence in chicken hepatoma (DU249; Fig. 5) and HeLa cells (Fig. 6). Labeling was preferentially over the Golgi region and on a few vesicular structures randomly distributed throughout the cytoplasm. Double immunofluorescence labeling with anti-p60 and mAb 58K-9 revealed essentially identical patterns (Fig. 5, A and B, and Fig. 6, A and B). Double immunofluorescence labeling with anti-p60 and mAb 100/3 against gamma -adaptin, a component of the Golgi adaptor complex (AP-1), indicated, however, that the vesicular structures containing p60 and AP-1 can be clearly distinguished (Fig. 5, C and D, and Fig. 6, C and D), suggesting that p60 is not associated with Golgi derived clathrin-coated vesicles. Furthermore, little overlap of p60 could be detected with coatomer-coated vesicles (see arrows in Fig. 5, E and F) visualized with mAb maD against beta -COP (Fig. 5, E and F, and Fig. 6, E and F). Unfortunately, affinity-purified antibodies against FTCD, which recognize their antigen by immunoblotting both in DU249 and HeLa cells, do not label these cells by immunofluorescence. These results clearly establish that p60, as has been previously shown for 58K (18), is localized to the region of the Golgi complex, apparently on Golgi membranes as well as some potentially Golgi complex-derived vesicular structures. This distribution of p60 is consistent with its putative role in mediating interaction of Golgi-derived vesicles with microtubules.


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Fig. 5.   Immunofluorescence localization of a p60 isoform in DU249 cells. Chicken hepatoma cells (DU249) were fixed with paraformaldehyde and double-labeled for conventional fluorescence microscopy with anti-KKVQ against p60 (A, C, and E), and monoclonal antibodies against 58K (B), gamma -adaptin (D), and beta -COP (F), respectively. As secondary antibodies Cy3- or fluorescein-conjugated anti-rabbit and anti-mouse, respectively, were used. The p60 isoform was specifically associated with the Golgi complex and vesicular structures radiating from it. The specific labeling for p60 in the Golgi region (A) was nearly identical to the compact Golgi staining using anti-58K (B). Although the Golgi complex was stained by both anti-p60 (C) and anti-gamma -adaptin (D), no vesicles containing p60 labeled for gamma -adaptin. Vesicular labeling for the p60-isoform (E) and beta -COP (F) was clearly different (arrowheads in E and F), although there was some overlap (arrows in E and F). Note that when cells were permeabilized with Triton X-100 without SDS, vesicles stained by anti-p60 are more prominent and dispersed throughout the cell (E), whereas a similar, but more diffuse vesicular labeling for p60 was obtained when cells were treated with Triton X-100 and SDS (A and C). Bar = 10 µm.


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Fig. 6.   p60 co-localizes with 58K, rather than with beta -COP or gamma -adaptin in HeLa cells. HeLa cells were fixed with methanol and double-labeled for conventional fluorescence microscopy with anti-p60, no. 148 (A, C, and E), and mAbs against 58K (B), gamma -adaptin (AP-1, D), and beta -COP (F), respectively. As secondary antibodies rhodamine- or fluorescein-conjugated anti-rabbit and anti-mouse, respectively, were used. The p60 isoform was specifically associated with the Golgi complex and vesicular structures in its vicinity. Specific labeling for the p60 isoform in the Golgi region (A) was nearly identical to the Golgi staining using the anti-58K (B). None of the AP-1 positive vesiclular structures outside of the strongly labeled Golgi area (D) contained p60 (C). Vesicular labeling for p60 (E) and beta -COP (F) was also clearly different, although there was some overlap. Bar = 10 µm.

Identification of Isoforms of p60 in Nonliver Cells-- A 60-kDa band was detected by immunoblotting of total extracts of chicken embryos or adult liver, primary hepatocytes prepared from 17-day-old chicken embryos, or DU249 and HeLa cells with antibodies against p60 (data not shown). To better identify the two-dimensional gel immunoblotting signal, immunodetection was followed by colloidal gold staining of the complete electropherogram on the same membrane. Purified chicken and turkey liver p60 were also analyzed by 2D gel electrophoresis (Fig. 4, B and C). We identified a major protein of 60 kDa with a pI of ~6, as well as at least four isoforms of p60 with different pI values. These polypeptides could represent post-translationally modified forms of p60, the nature of which is currently unknown. Immunoblotting of 2D gels of purified chicken liver p60 added to DU249 and HeLa cytosol with antibodies against p60 revealed several isoforms of this protein. The p60 proteins from DU249 and HeLa cytosol show a difference in pI to chicken liver p60 of ~1.5 and 2.0 units, respectively, and migrate faster than their liver relative (data not shown). This is probably indicative of p60 isoforms, although in HeLa cells it could also be due to species specific differences.

Two-dimensional gel immunoblotting was also used to compare p60 and 58K from chicken and rat liver cytosol, respectively (Fig. 7). A well characterized mAb against rat 58K (58K-9), which was also used for the immunofluorescence studies, did not recognize the chicken homologue by immunoblotting (Fig. 7C), while it labeled two major immunoreactive spots in rat liver cytosol at ~55 and 58 kDa with a pI similar to that of p60 (Fig. 7F). The polypeptide at 55 kDa (arrowhead) is probably a degradation product of the 58K protein.3 Three additional minor polypeptides were identified with this mAb, differing from 58K (and 55K) in their pI, which may represent modified isoforms of 58K. Interestingly, the two major spots (58K and 55K) were also detected by the antibody against the KKVQ peptide of chicken p60 (Fig. 7E). The exact position of the immunodetected spots within the total protein pattern was determined by total protein staining on the same membrane (Fig. 7, A and D). In fact, eight out of the 11 antibodies against p60 recognize 58K in rat liver cytosol, and conversely two out of five mAbs against 58K recognize p60 in chicken liver cytosol (data not shown). Taken together, these results suggest that chicken p60 and the 58K from rat liver share similar, but not identical, antigenic sites and are therefore most likely closely related proteins.


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Fig. 7.   p60/58K isoforms detected by specific antibodies in chicken and rat liver cytosol. Cytosol from chicken (A-C) or rat (D-F) liver was separated by 2D gel electrophoresis and subjected to immunoblotting using affinity-purified anti-KKVQ, antibodies raised against a p60 peptide (B and E) and anti-58K, mAb no. 9 (C and F). Complete protein staining with AuroDye (A and D) was subsequently carried out on the anti-KKVQ membranes (from B and E). p60 is indicated by a long arrow (A and B), the major form of 58K by a short arrow (D-F), and three minor isoforms of 58K by vertical bars (F). 55K, a shorter form of 58K, is shown by a arrowhead (D, E, and F). The position of actin is marked by the asterisk. Molecular masses are indicated (×103).

We also analyzed the immunological relationship between chicken p60 and porcine FTCD. A polyclonal antibody against FTCD reacts by immunoblotting with p60 in chicken liver cytosol (data not shown). Furthermore, eight out of the 11 antibodies against p60 recognized purified recombinant porcine FTCD by immunoblotting (data not shown), confirming the significant homology between p60 and FTCD. To further characterize the relationship between p60 isoforms and FTCD, we assayed their formiminotransferase activity. Purified chicken liver p60 has a specific activity of 10.5 µmol/min/mg (Table I), about 25% of that observed for the purified porcine liver FTCD (36, 46). The 75% loss of activity is probably due to our purification procedure for p60, which was not optimized for preservation of its enzymatic activity. Formiminotransferase activity was also found in chicken liver total extract and was slightly enriched in chicken liver cytosol. The chicken liver total extract was 6-7-fold less active than that of the corresponding fraction from porcine liver (data not shown), although we used the same buffer conditions for preparation of chicken liver extracts as for the purification of porcine FTCD (10). In contrast, no formiminotransferase activity was detectable in total DU249 or HeLa cell extracts or cytosols prepared under the same conditions. However, estimation by immunoblotting of the relative expression levels of p60 in DU249 and HeLa cells revealed that it was reduced 80-100-fold compared with chicken liver (data not shown). The assay may thus not have been sensitive enough to detect the cellular forms of the enzyme. Taken together, our results demonstrate that an isoform of p60 is expressed in nonliver cells which shows a similar, but not identical, electrophoretic mobility on 2D gels compared with liver p60 and crossreacts with some, but not all, of the antibodies raised against the liver p60 protein.

                              
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Table I
Formiminotransferase enzyme assays

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Using an in vitro assay for binding of TGVs to microtubules we have identified a 60-kDa protein, p60. We have purified, cloned, and sequenced p60, and found that it is most likely the chicken homologue of 58K, a previously identified Golgi-associated MBP from rat liver (16), and porcine FTCD (10), a folate-dependent liver-specific enzyme implicated in the histidine degradation pathway (11). Purified chicken liver p60 has significant formiminotransferase activity and chicken liver extracts show deaminase activity (data not shown). By immunofluorescence microscopy it has been established that p60/58K/FTCD is located on the Golgi complex and cytoplasmic vesicular membranes (56).

How can p60/58K/FTCD show microtubule binding properties? We consider two possibilities. It is possible that p60 can oligomerize into a protein complex sufficiently large to co-sediment with microtubules. FTCD is known to assemble into an octameric protein complex of identical subunits arranged as a circular tetramer of dimers (36, 37, 45), channelling polyglutamylated folate between its two types of catalytic sites (47, 48). Protein complexes within that range of molecular mass (~500 kDa), including coatomer, may be found under certain conditions in microtubule pellets.4 We have so far not found conditions under which in vitro translated p60 co-sediments with microtubules, suggesting that either p60 does not interact with microtubules, or that the polypeptide has not folded or assembled correctly under these conditions or that additional post-translational modifications are required for its proper function. Alternatively, since each FTCD octamer has four high affinity polyglutamate binding sites (49), and since pig FTCD is normally enriched in a final step on a polyglutamate column (10), p60 may bind to the polyglutamate on microtubules. Brain microtubules (used in this study for assaying in vitro TGV-microtubule binding) are highly polyglutamylated (50). It has been shown that each subunit of the octameric FTCD consists of an N-terminal transferase and a C-terminal deaminase active site, separated by a short linker sequence (51). Both domains self-dimerize, confirming that FTCD octamers include two different types of subunit interfaces which are most likely maintained for expression of both activities (52, 53). It has been suggested that the polyglutamate-binding site resides at the subunit interface formed between the deaminase domains (51). Since chicken liver p60 has formiminotransferase activity, it may have similar quaternary structure to porcine FTCD, and its deaminase subunits might thus bind to polyglutamated tubulin.

Interestingly, different isoforms of p60 have been detected. In vitro translation of p60 results in several, most likely post-translationally modified, polypeptides and different isoforms are also present in liver extracts, as well as in cytosol from different tissue culture cells, including DU249 and HeLa. The proteins from chicken DU249 cells and liver have a different electrophoretic mobility and pI. We have not been able to measure formiminotransferase activity in DU249 or HeLa cells. While this lack of measurable activity may be due to the low expression of the p60 in these cell lines, it is also possible that these isoforms of FTCD may have lost their enzymatic activity and exert another cellular function. Formiminotransferase activity has been found predominantly in liver and to a lesser degree in kidney, but not in epidermis, skeletal muscle or brain in mammals (11, 54). The liver-specific expression of FTCD has been confirmed by Northern blotting of different mouse tissues with an FTCD specific porcine cDNA probe (10). Formiminotransferase activity is reduced in hepatomas to less than 10% of the levels found in normal liver (55).

The reason for why p60 mediates binding of TGVs to microtubules in vitro is unclear. It is conceivable that the homo-octameric protein complex may, despite its overall negative pI and binding to an anion exchange column, have sufficiently positively charged domains to mediate an interaction with potentially negatively charged TGVs. Concomitant interaction of p60 with polyglutamated tubulin would thus enable the protein complex to bridge microtubules and TGVs. Alternatively, we have established that p60 (like 58K) is a protein localized to membranes of the Golgi complex (see also Bloom and Brashear (16)), as well as cytoplasmic vesicles which may be derived from the TGN. Thus, if these proteins do indeed have an affinity for microtubules (e.g. via polyglutamate residues), they should, as a consequence, lead to Golgi membrane/TGV-microtubule interactions and therefore also be capable of linking these membranes to microtubules in vitro. Thus, while it is likely that p60 is not the specific CLIP mediating TGV-microtubule interaction that we sought for, this possibility should nevertheless not be completely dismissed.

    ACKNOWLEDGEMENTS

We thank Roland Kellner and Tony Houthaeve for skilful assistance with protein sequencing and for synthesis of peptides. We also thank Brigitte Joggerst-Thomalla for excellent technical assistance. We acknowledge many helpful and stimulating discussions with George Bloom, and our colleagues at the EMBL in Heidelberg (particularly Kai Simons, Mark Bennett, and Paul Duprée) and at the Institut Jacques Monod. Taxol was obtained from Dr. N. Lomax (Department of Health and Human Services, National Institutes of Health, Bethesda, MD). We are grateful to Evelyre Martinez and Micheline Vautravers for help with the photographic figures and also thank Dr. Jean-Claude Courvalin for giving us DU249 chicken hepatoma cells, Dr. R. E. MacKenzie for the recombinant FTCD and antibodies against it, Drs. George Bloom for the antibodies against 58K, Ernst Ungewickell for the antibodies against gamma -adaptin, and Jonathan Scholey for the antibodies against kinesin.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by a long-term EMBO fellowship and by the CNRS.

** Supported by the Fonds Nationale Suisse and the Canton de Genève. To whom correspondence should be addressed. Tel./Fax: 41-22-702-6747; E-mail: Thomas.Kreis{at}cellbio.unige.ch.

1 The abbrevations used are: TGV, trans-Golgi network-derived vesicle; BSA, bovine serum albumin; CLIP, cytoplasmic linker protein; FTCD, formiminotransferase cyclodeaminase; mAb, monoclonal antibody; MDCK, Madin-Darby canine kidney; MBP, microtubule-binding protein; PBS, phosphate-buffered saline; PIPES, piperazine-N',N'-bis(2-ethanesulfonic acid); PAGE, polyacrylamide gel electrophoresis; 2D, two-dimensional.

2 In the accompanying paper (56), Bashour and Bloom report that 58K, a microtubule-binding protein, is a FTCD.

3 G. Bloom, personal communication

4 S. Martín, F. Perez, and T. E. Kreis, unpublished data.

    REFERENCES.
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Goodson, H. V., Valetti, C., and Kreis, T. E. (1997) Curr. Opin. Cell Biol. 9, 18-28[CrossRef][Medline] [Order article via Infotrieve]
  2. Lafont, F., Burkhardt, J. K., and Simons, K. (1994) Nature 372, 801-803[Medline] [Order article via Infotrieve]
  3. Bacallao, B., Antony, C., Dotti, C., Karsenti, E., Stelzer, E. H. K., and Simons, K. (1989) J. Cell Biol. 109, 2817-2832[Abstract]
  4. van der Sluijs, P., Bennett, M. K., Antony, C., Simons, K., and Kreis, T. E. (1990) J. Cell Sci. 95, 545-553[Abstract]
  5. Keller, P., and Simons, K. (1997) J. Cell Sci. 110, 3001-3009[Abstract/Free Full Text]
  6. Schroer, T. (1996) Semin. Cell Dev. Biol. 7, 321-328[CrossRef]
  7. Sheetz, M. P., and Yu, H. (1996) Semin. Cell Dev. Biol. 7, 329-334[CrossRef]
  8. Allan, V. (1996) Curr. Biol. 6, 630-633[Medline] [Order article via Infotrieve]
  9. Rickard, J. E., and Kreis, T. E. (1996) J. Cell Biol. 6, 178-183
  10. Murley, L. L., Mejia, N. R., and MacKenzie, R. E. (1993) J. Biol. Chem. 268, 22820-22824[Abstract/Free Full Text]
  11. Tabor, H., and Wyngarden, L. (1959) J. Biol. Chem. 234, 1830-1846[Free Full Text]
  12. Bennett, M. K., Wandinger-Ness, A., and Simons, K. (1988) EMBO J. 7, 4075-4085[Abstract]
  13. Langlois, A. J., Lapis, K., Ishizaki, R., Beard, J. W., and Bolognesi, D. P. (1974) Cancer Res. 34, 1457-1464[Medline] [Order article via Infotrieve]
  14. Kreis, T. E. (1986) EMBO J. 5, 931-941[Abstract]
  15. Rickard, J. E., and Kreis, T. E. (1990) J. Cell Biol. 110, 1623-1633[Abstract]
  16. Bloom, G. S., and Brashear, T. A. (1989) J. Biol. Chem. 264, 16083-16092[Abstract/Free Full Text]
  17. Ingold, A. L., Cohn, S. A., and Scholey, J. M. (1988) J. Cell Biol. 107, 2657-2667[Abstract]
  18. Ahle, S., Mann, A., Eichelsbacher, U., and Ungewickell, E. (1988) EMBO J. 7, 919-929[Abstract]
  19. Pepperkok, R., Scheel, J., Horstmann, H., Hauri, H.-P., Griffiths, G., and Kreis, T. E. (1993) Cell 74, 71-82[Medline] [Order article via Infotrieve]
  20. Rickard, J. E., and Kreis, T. E. (1991) J. Biol. Chem. 266, 17597-17605[Abstract/Free Full Text]
  21. Tam, J. P. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5409-5413[Abstract]
  22. Wandinger-Ness, A., Bennett, M. K., Antony, C., and Simons, K. (1990) J. Cell Biol. 111, 987-1000[Abstract]
  23. Gibbons, I. R., Lee-Eiford, A., Mocz, G., Phillipson, C. A., Tang, W. J., and Gibbons, B. H. (1987) J. Biol. Chem. 262, 2780-2786[Abstract/Free Full Text]
  24. Scheel, J., and Kreis, T. E. (1991) J. Biol. Chem. 266, 18141-18148[Abstract/Free Full Text]
  25. Pierre, P., Scheel, J., Rickard, J. E., and Kreis, T. E. (1992) Cell 70, 887-900[Medline] [Order article via Infotrieve]
  26. Gausepohl, H., Trosin, M., and Frank, R. (1986) in Advanced Methods in Protein Microsequence Analysis (Wittmann-Liebold, B., Salnikow, J., and Erdmann, V. A., eds), pp. 149-160, Springer Verlag, Berlin
  27. Kurzchalia, T. V., Dupree, P., Parton, R. G., Kellner, R., Virta, H., Lehnert, M., and Simons, K. (1992) J. Cell. Biol. 118, 1003-10014[Abstract]
  28. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  29. Devereux, J., Haebeli, P., and Smithies, O. (1984) Nucleic Acids Res. 12, 387-395[Abstract]
  30. Pearson, W. R., and Lipman, D. J. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 2444-2448[Abstract]
  31. 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]
  32. Bairoch, A. (1989) in EMBL Biocomputing Technical Document 4, European Molecular Biology Laboratory, Heidelberg, Germany
  33. Sibbald, P. R., and Argos, P. (1990) Comput. Appl. Biosci. 6, 279-288[Abstract]
  34. Rechid, R., Vingron, M., and Argos, P. (1989) Comput. Appl. Biosci. 5, 107-1013[Abstract]
  35. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
  36. Drury, E. J., Bazar, L. S., and MacKenzie, R. E. (1975) Arch. Biochem. Biophys. 169, 662-668[Medline] [Order article via Infotrieve]
  37. MacKenzie, R. E. (1980) Methods Enzymol. 66, 626-630[Medline] [Order article via Infotrieve]
  38. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
  39. Shestakova, E., Vandekerckhove, J., and De Mey, J. R. (1998) Eur. J. Cell Biol., in press
  40. Daneels, G., Moeremans, M., De Raeymaeker, M., and De Mey, J. (1986) J. Immunol. Methods 89, 89-91[Medline] [Order article via Infotrieve]
  41. Kozak, M. (1987) Nucleic Acids Res. 15, 8125-8143[Abstract]
  42. Nevius, J. R. (1983) Annu. Rev. Biochem. 52, 441-446[Medline] [Order article via Infotrieve]
  43. Chistoserdova, L. V., and Lidstrom, M. E. (1994) J. Bacteriol. 176, 7398-7407[Abstract]
  44. Shane, B., and Stokstad, E. L. R. (1984) in Folates and Pterins: Chemistry and Biochemistry of Folates (Blakey, R. L., and Benkovic, S. J., eds), Vol. 1, pp. 433-455, John Wiley & Sons, New York
  45. Beaudet, R., and MacKenzie, R. E. (1976) Biochim. Biophys. Acta 453, 151-161[Medline] [Order article via Infotrieve]
  46. Findlay, W. A., Zarkadas, C. G., and MacKenzie, R. E. (1989) Biochim. Biophys. Acta 999, 52-57[Medline] [Order article via Infotrieve]
  47. MacKenzie, R. E. (1979) Dev. Biochem. 4, 443-446
  48. MacKenzie, R. E., and Baugh, C. M. (1980) Biochim. Biophys. Acta 611, 187-195[Medline] [Order article via Infotrieve]
  49. Paquin, J., Baugh, C. M., and MacKenzie, R. E. (1985) J. Biol. Chem. 260, 14925-14931[Abstract/Free Full Text]
  50. Edde, B., Rossier, J., Le Caer, J. P., Desbruyeres, E., Gros, F., and Denoulet, P. (1990) Science 247, 83-85[Medline] [Order article via Infotrieve]
  51. Murley, L. L., and MacKenzie, R. E. (1995) Biochemistry 34, 10358-10364[Medline] [Order article via Infotrieve]
  52. Findlay, W. A., and MacKenzie, R. E. (1987) Biochemistry 26, 1948-1954[Medline] [Order article via Infotrieve]
  53. Findlay, W. A., and MacKenzie, R. E. (1988) Biochemistry 27, 3404-3408[Medline] [Order article via Infotrieve]
  54. McClain, L. D., Carl, G. F., and Bridgers, W. F. (1975) J. Neurochem. 24, 719-722[Medline] [Order article via Infotrieve]
  55. Jackson, R. C., and Niethammer, D. (1979) Dev. Biochem. 4, 665-670
  56. Bashour, A.-M., and Bloom, G. S. (1998) J. Biol. Chem. 273, 19612-19617[Abstract/Free Full Text]


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