From the 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
Department of Biochemistry, McGill University, Montréal,
Québec H3G 1Y6, Canada
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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.
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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.
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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).
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 -Adaptin (18), maD against
-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).
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 atPurification 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 [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.
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.
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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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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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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 -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
-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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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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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 -adaptin, and Jonathan Scholey for the antibodies against
kinesin.
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FOOTNOTES |
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* 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.
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REFERENCES. |
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