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58K, a Microtubule-binding Golgi Protein, Is a Formiminotransferase Cyclodeaminase*

  • Anne-Marie Bashour
    Footnotes
    Affiliations
    Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas 75235
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  • George S. Bloom
    Correspondence
    To whom correspondence should be addressed: Dept. of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235. Tel.: 214-648-7680; Fax: 214-648-9160;
    Affiliations
    Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas 75235
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  • Author Footnotes
    * This work was supported by grants from the American Cancer Society (CB-58E) and the Robert A. Welch Foundation (I-1236) (to G.S.B.).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.
    ‡ Submitted to partially fulfill the requirements for a doctorate of philosophy at the University of Texas Southwestern Medical Center.
Open AccessPublished:July 31, 1998DOI:https://doi.org/10.1074/jbc.273.31.19612
      58K was previously identified as a rat liver protein that binds microtubules in vitro and is associated with the cytoplasmic surface of the Golgi apparatus in vivo(Bloom, G. S., and Brashear, T. A. (1989) J. Biol. Chem. 264, 16083–16092). We now report that 58K is a formiminotransferase cyclodeaminase (FTCD), a bifunctional enzyme that catalyzes two consecutive steps in the modification of tetrahydrofolate to 5,10-methenyl tetrahydrofolate. Comparative immunoblotting using several monoclonal antibodies made against 58K and a polyclonal antibody made against a chicken liver protein (p60) with similar properties (Hennig, D., Scales, S. J., Moreau, A., Murley, L. L., De Mey, J., and Kreis, T. E. (1998) J. Biol. Chem. 273, 19602–19611) demonstrated precise co-purification of protein recognized by all antibodies through multiple fractionation steps, including gel filtration and ion exchange chromatography, and sucrose gradient ultracentrifugation. Eight peptides derived from 58K showed high sequence identity to amino acid sequences predicted by full length cDNA for p60 and porcine liver FTCD. Furthermore, purified 58K was associated with formiminotransferase and cyclodeaminase activities. Based on these collective results, 58K was concluded to be a rat liver version of FTCD. Microtubules assembled from brain tubulin, but not from liver tubulin, were able to bind rat liver FTCD. Binding to brain microtubules is suspected to occur via polyglutamates that are added post-translationally to tubulin in brain, which was shown to contain very low levels of FTCD, but not to tubulin in liver, which was determined to be the richest tissue source, by far, of FTCD. The physiological significance of the microtubule binding activity of FTCD is thus called into question, but an association of FTCD with the Golgi apparatus has now been established.
      A powerful approach for identifying factors that function in concert with microtubules (MTs)
      The abbreviations used are: MT, microtubule; FTCD, formiminotransferase cyclodeaminase; MAP, microtubule-associated protein; PAGE, polyacrylamide gel electrophoresis; THF, tetrahydrofolate; PIPES, 1,4-piperazinediethanesulfonic acid.
      1The abbreviations used are: MT, microtubule; FTCD, formiminotransferase cyclodeaminase; MAP, microtubule-associated protein; PAGE, polyacrylamide gel electrophoresis; THF, tetrahydrofolate; PIPES, 1,4-piperazinediethanesulfonic acid.
      has been to seek proteins that bind to MTs in preparations of cytosol. This strategy was originally applied to mammalian brain cytosol, leading to the discovery of numerous MT-associated proteins (MAPs) (
      • Bloom G.S.
      • Luca F.C.
      • Vallee R.B.
      ,
      • Bloom G.S.
      • Schoenfeld T.A.
      • Vallee R.B.
      ,
      • Sloboda R.D.
      • Rudolph S.A.
      • Rosenbaum J.L.
      • Greengard P.
      ,
      • Weingarten M.D.
      • Lockwood A.H.
      • Hwo S.-Y.
      • Kirschner M.W.
      ), which are structural components of MTs and regulate their assembly. Modifications of this general approach also led to the discovery of brain versions of kinesin (
      • Brady S.T.
      ,
      • Vale R.D.
      • Reese T.S.
      • Sheetz M.P.
      ) and cytoplasmic dynein (
      • Paschal B.M.
      • Shpetner H.S.
      • Vallee R.B.
      ), the first two MT motor proteins to have been found outside of cilia and flagella.
      To broaden the search for MAPs, molecular motors and other factors that may interact with MTs, several groups have made use of cytosol isolated from pure cell populations or non-neural tissues as potential sources of new MT-binding proteins (
      • Bloom G.S.
      • Luca F.C.
      • Vallee R.B.
      ,
      • Vallee R.B.
      • Bloom G.S.
      ,
      • Scholey J.M.
      • Neighbors B.
      • McIntosh J.R.
      • Salmon E.D.
      ,
      • Collins C.A.
      • Vallee R.B.
      ,
      • Kotani S.
      • Murofushi H.
      • Maekawa S.
      • Sato C.
      • Sakai H.
      ,
      • Kotani S.
      • Murofushi H.
      • Maekawa S.
      • Aizawa H.
      • Sakai H.
      ,
      • Olmsted J.B.
      • Lyon H.D.
      ,
      • Bulinski J.C.
      • Borisy G.G.
      ). In one such case, our laboratory discovered a novel MT-binding protein, 58K, in preparations of rat liver cytosol (
      • Bloom G.S.
      • Brashear T.A.
      ). Curiously, 58K was localized by immunofluorescence to the Golgi apparatus in cultured hepatoma cells and was present on purified liver Golgi membranes as a peripheral membrane protein (
      • Bloom G.S.
      • Brashear T.A.
      ). 58K was also detected by both Western blotting
      A.-M. Bashour and G. S. Bloom, unpublished results.
      2A.-M. Bashour and G. S. Bloom, unpublished results.
      and immunofluorescence microscopy (
      • Ktistakis N.T.
      • Roth M.G.
      • Bloom G.S.
      ,
      • Donaldson J.G.
      • Lippincott-Schwartz J.
      • Bloom G.S.
      • Kreis T.E.
      • Klausner R.D.
      ) in a wide variety of cultured mammalian cell types. As was found for hepatoma cells, immunofluorescence labeling of other cell types with anti-58K antibodies yielded bright staining of the Golgi complex.
      Although the localization of a MAP-like protein on Golgi membranes was initially surprising, it was not unprecedented (
      • Allan V.J.
      • Kreis T.E.
      ) and offered potential insight into the intriguing relationship between MTs and the Golgi complex in interphase cells. The Golgi complex in nondividing cells typically comprises an interconnected network of stacked cisternae (
      • Rambourg A.
      • Clermont Y.
      • Marrano A.
      ), which are located either near the nucleus (
      • Louvard D.
      • Reggio H.
      • Warren G.
      ), or in some types of polarized epithelial cells, adjacent to the apical surface (
      • Ihrke G.
      • Neufeld E.B.
      • Meads T.
      • Shanks M.R.
      • Cassio D.
      • Laurent M.
      • Schroer T.A.
      • Pagano R.E.
      • Hubbard A.L.
      ,
      • Fawcett D.W.
      ). Exposure of interphase cells to MT depolymerizing drugs, such as colcemid or nocodazole, causes the Golgi apparatus to break apart into scores of fragments that are widely distributed throughout the cytoplasm (
      • Rogalski A.A.
      • Singer S.J.
      ,
      • Moskalewski S.
      • Thyberg J.
      • Lohmander S.
      • Friberg U.
      ). Similar effects on the Golgi apparatus have been observed in cells exposed to Taxol (
      • Rogalski A.A.
      • Singer S.J.
      ,
      • Wehland J.
      • Henkart M.
      • Klausner R.
      • Sandoval I.V.
      ), a drug which stimulates MT assembly, and as a by-product, disrupts the normal organization of MTs. It is evident, therefore, that the structural integrity and normal localization of the Golgi apparatus in interphase cells depends upon the presence and normal organization of MTs. At the time that 58K was initially characterized, the dependence of the Golgi on MTs was well known, but poorly understood mechanistically. We therefore decided to investigate the hypothesis that 58K anchors Golgi membranes to MTs and helps to maintain the structural unity of the Golgi and its proper positioning within the cell (
      • Bloom G.S.
      • Brashear T.A.
      ).
      New evidence derived from biochemical, immunological, molecular biological, and enzymological studies indicates that 58K is a version of formiminotransferase cyclodeaminase (FTCD), a bifunctional enzyme which catalyzes two consecutive steps in the modification of tetrahydrofolate (THF) to 5,10-methenyl-THF (
      • Tabor H.
      • Wyngarden L.
      ,
      • Drury E.J.
      • Bazar L.S.
      • MacKenzie R.E.
      ). We thus refer to 58K as rat liver FTCD. MTs assembled from brain tubulin, but not from liver tubulin, were able to bind avidly to rat liver FTCD, although Western blotting of rat tissue extracts revealed that FTCD is far more abundant in liver than in all other tissues examined, including brain. Considering that FTCD is virtually liver-specific, but does not bind to liver MTs, the ability of FTCD to bind brain MTs is unlikely to be physiologically relevant. Although the hypothesis that 58K (FTCD) links Golgi membranes to MTs in vivo must now be regarded with skepticism, an unexpected association of FTCD with the Golgi apparatus has been revealed. It is noteworthy that analogous results were obtained independently in another laboratory for a chicken liver version of FTCD, and are reported in Henning et al. (
      • Hennig D.
      • Scales S.J.
      • Moreau A.
      • Murley L.L.
      • De Mey J.
      • Kreis T.E.
      ).

      DISCUSSION

      Based on several criteria, 58K has been shown to be an FTCD. By immunoblotting, antibodies made against chicken (Fig. 1) or porcine (not shown) liver FTCD labeled protein that co-migrated with 58K in SDS-PAGE, and precisely co-fractionated with 58K during its purification from rat liver cytosol. In addition, several monoclonal anti-58K antibodies reacted with purified native or recombinant porcine liver FTCD (not shown). High sequence identity was observed between eight peptide fragments of 58K, and the full-length sequences of porcine and chicken liver FTCD (Fig. 2). Finally, both enzyme activities known to be associated with porcine liver FTCD, formiminotransferase and cyclodeaminase (
      • Tabor H.
      • Wyngarden L.
      ,
      • Drury E.J.
      • Bazar L.S.
      • MacKenzie R.E.
      ), were also associated with purified 58K. Until more is known about the structure of FTCD genes and flanking regions of genomic DNA, it will not be possible to judge whether the rat, chicken, and porcine proteins discussed here represent species-specific products of equivalent, as opposed to closely related, genes. Likewise, ambiguities in the amino acid sequence of rat FTCD at several positions (see Fig. 2) raise the possibility that multiple FTCD isoforms are encoded by multiple genes within a single animal species. Regardless, in light of the new evidence about rat liver 58K, its classification as an FTCD seems amply justified.
      Although 58K was associated with formiminotransferase activity, the measured activity was only ∼20% of that reported for recombinant porcine FTCD (
      • Murley L.L.
      • Mejia N.R.
      • MacKenzie R.E.
      ). The following considerations, individually or collectively, might account for this discrepancy. First, no effort was made to optimize recovery of the enzyme activities of 58K, which was purified by a method (see “Experimental Procedures”) that was radically different than the procedure used to purify recombinant porcine FTCD (
      • Murley L.L.
      • Mejia N.R.
      • MacKenzie R.E.
      ). Next, isoelectric focusing gel electrophoresis resolved purified 58K into eight closely spaced variants that collectively were recognized by antibodies to rat 58K, chicken p60, and porcine FTCD (data not shown). The electrophoretic diversity of 58K could reflect the presence of multiple, distinct translation products, post-tranlsational modifications, or both, although no evidence that might discriminate among these possibilities is presently available. Regardless, by comparison to recombinant porcine FTCD, the purified 58K that was used for enzyme assays was very diverse in molecular terms, and by extension, perhaps in enzymatic properties as well. Finally, species-specific differences in enzymatic efficiency might explain, at least in part, the low formiminotransferase activity of rat liver 58K relative to that of recombinant porcine FTCD.
      Before 58K was shown to be an FTCD, studies of the protein demonstrated that it could bind MTs in vitro, was localized by immunofluorescence to the Golgi apparatus of cultured cells, and was a cytoplasmically oriented, peripheral membrane protein of isolated Golgi membranes (
      • Bloom G.S.
      • Brashear T.A.
      ,
      • Ktistakis N.T.
      • Roth M.G.
      • Bloom G.S.
      ,
      • Donaldson J.G.
      • Lippincott-Schwartz J.
      • Bloom G.S.
      • Kreis T.E.
      • Klausner R.D.
      ). In light of the ability of 58K to bind MTs and Golgi membranes, and the fact that a properly localized, structurally intact Golgi requires MTs to be present and normally organized (
      • Rogalski A.A.
      • Singer S.J.
      ,
      • Moskalewski S.
      • Thyberg J.
      • Lohmander S.
      • Friberg U.
      ,
      • Wehland J.
      • Henkart M.
      • Klausner R.
      • Sandoval I.V.
      ), we postulated that 58K anchors Golgi membranes to MTs (
      • Bloom G.S.
      • Brashear T.A.
      ). New evidence presented here, however, strongly suggests that 58K, now realized to be an FTCD, does not cross-link Golgi membranes to MTsin vivo.
      The pertinent evidence is that FTCD binds to MTs assembled from brain, but not liver tubulin (Fig. 3). Thus, FTCD is unlikely to bind MTs in the only tissue in which it is abundant, namely liver (Fig. 4). There is one obvious difference between brain and liver tubulin that might account for the preferential binding of FTCD to brain MTs. Located near the C termini of both α-tubulins and β-tubulins are glutamic acid residues to which polyglutamic acid side chains of variable length can be added post-translationally in vivo (
      • Wolff A.
      • de Néchaud B.
      • Chillet D.
      • Mazarguil H.
      • Desbruyères E.
      • Audebert S.
      • Eddé B.
      • Gros F.
      • Denoulet P.
      ,
      • Eddé B.
      • Rossier J.
      • Le Caer J.-P.
      • Desbruyères E.
      • Gros F.
      • Denoulet P.
      ). In brain, approximately half of the α-tubulin (
      • Eddé B.
      • Rossier J.
      • Le Caer J.-P.
      • Desbruyères E.
      • Gros F.
      • Denoulet P.
      ), and a lesser, but substantial, amount of the β-tubulin (
      • Wolff A.
      • de Néchaud B.
      • Chillet D.
      • Mazarguil H.
      • Desbruyères E.
      • Audebert S.
      • Eddé B.
      • Gros F.
      • Denoulet P.
      ) are polyglutamylated. In other tissues, including liver, polyglutamylated tubulin has also been detected, but at dramatically lower levels and only on β-tubulins (
      • Wolff A.
      • de Néchaud B.
      • Chillet D.
      • Mazarguil H.
      • Desbruyères E.
      • Audebert S.
      • Eddé B.
      • Gros F.
      • Denoulet P.
      ).
      Why might FTCD preferentially bind MTs assembled from polyglutamylated, as compared with unmodified tubulin? FTCD is a homooctomeric enzyme (
      • Beaudet R.
      • MacKenzie R.E.
      ), and each molecule comprises 4 binding sites for the polyglutamate moiety of THF (
      • Paquin J.
      • Baugh C.M.
      • MacKenzie R.E.
      ). Indeed, affinity chromatography using a polyglutamate-Affi-Gel column has been used as a final enrichment step for purification of recombinant porcine liver FTCD (
      • Murley L.L.
      • Mejia N.R.
      • MacKenzie R.E.
      ). We postulate, therefore, that the polyglutamates which are uniquely abundant on brain tubulin enable MTs assembled from brain, but not liver, tubulin to bind avidly to FTCD. The formal proof of this hypothesis ultimately must rest on MT binding studies using tubulin whose polyglutamylation state can be altered experimentally. Unfortunately, such experiments are not presently possible, because none of the enzymes responsible for post-translationally adding glutamates to tubulin or subsequently removing them are reported to have been discovered.
      It may seem puzzling that FTCD appears by immunofluorescence microscopy to be localized predominantly on the Golgi and post-Golgi vesicles (
      • Bloom G.S.
      • Brashear T.A.
      ,
      • Hennig D.
      • Scales S.J.
      • Moreau A.
      • Murley L.L.
      • De Mey J.
      • Kreis T.E.
      ,
      • Ktistakis N.T.
      • Roth M.G.
      • Bloom G.S.
      ,
      • Donaldson J.G.
      • Lippincott-Schwartz J.
      • Bloom G.S.
      • Kreis T.E.
      • Klausner R.D.
      ), but is routinely purified from cytosol, rather than from membranes (
      • Bloom G.S.
      • Brashear T.A.
      ,
      • Hennig D.
      • Scales S.J.
      • Moreau A.
      • Murley L.L.
      • De Mey J.
      • Kreis T.E.
      ,
      • Tabor H.
      • Wyngarden L.
      ,
      • Drury E.J.
      • Bazar L.S.
      • MacKenzie R.E.
      ). One potential explanation for this apparent anomaly is that both soluble and membrane-associated pools of FTCD exist, but FTCD is far more concentrated on membranes than in the soluble phase of cytoplasm. Bearing in mind that immunofluorescence reports relative concentrations of accessible epitopes, it is possible that the majority of FTCD is soluble, but at a concentration near or below the level of detectability by immunofluorescence. Furthermore, because FTCD is a cytoplasmically oriented peripheral membrane protein that binds membranes by relatively weak ionic interactions (
      • Bloom G.S.
      • Brashear T.A.
      ,
      • Hennig D.
      • Scales S.J.
      • Moreau A.
      • Murley L.L.
      • De Mey J.
      • Kreis T.E.
      ), it might be easily solubilized from membrane surfaces by the homogenization and extraction procedures that routinely are used to prepare cytosol as a first step in the purification of FTCD. An analogous situation exists for the MT motor protein, kinesin. Immunofluorescence and other in situ localization methods indicate that ∼30–90% of the cellular pool of kinesin is associated with membrane surfaces in vivo (
      • Pfister K.K.
      • Wagner M.C.
      • Stenoien D.A.
      • Brady S.T.
      • Bloom G.S.
      ,
      • Hirokawa N.
      • Sato-Yoshitake R.
      • Kobayashi N.
      • Pfister K.K.
      • Bloom G.S.
      • Brady S.T.
      ,
      • Hollenbeck P.J.
      ,
      • Elluru R.
      • Bloom G.S.
      • Brady S.T.
      ), even though kinesin is purified from cytosol (
      • Vale R.D.
      • Reese T.S.
      • Sheetz M.P.
      ).
      While emphasizing that FTCD is unlikely to bind MTs in vivo, the results shown here and in a companion study about chicken FTCD (
      • Hennig D.
      • Scales S.J.
      • Moreau A.
      • Murley L.L.
      • De Mey J.
      • Kreis T.E.
      ) also raise the question of why FTCD is associated with the Golgi. FTCD catalyzes two consecutive reactions: transfer of a single carbon from formiminoglutamate to THF, followed by conversion of the product, 5-formimino-THF, to 5,10-methynyl-THF plus ammonia (
      • Tabor H.
      • Wyngarden L.
      ). Several metabolically relevant purposes are served by these reactions (
      • Voet D.
      • Voet J.G.
      ). First, formiminoglutamate is a product of the histidine metabolism pathway, and its modification by FTCD completes the multistep conversion of histidine to glutamic acid. Next, 5,10-methynyl-THF, the end product of FTCD catalysis, can be converted directly to active coenzymes, such as 5-formyl-THF and 10-formyl-THF. Finally, 5,10-methynyl-THF also lies directly in the pathways for synthesis of several nucleotides and amino acids, as well as other compounds, including S-adenosylmethionine and formylmethionine tRNA.
      The two reactions catalyzed by FTCD have classically been considered to occur in vivo within the soluble phase of cytoplasm. The finding that FTCD is localized, at least in part, on the Golgi apparatus and post-Golgi vesicles (
      • Bloom G.S.
      • Brashear T.A.
      ,
      • Hennig D.
      • Scales S.J.
      • Moreau A.
      • Murley L.L.
      • De Mey J.
      • Kreis T.E.
      ) raises the possibility that its site of action is actually on membrane surfaces. Liver apparently contains the highest level of folates of all tissues (
      • Zamierowski M.
      • Wagner C.
      ), and acquires its folates from plasma as complexes of folate binding protein and 5-methyl-THF (
      • Zamierowski M.
      • Wagner C.
      ,
      • Weitman S.
      • Anderson R.G.W.
      • Kamen B.A.
      ). Folate binding protein is a soluble, desialylated version of the membrane-associated, glycosylphosphatidylinositol-anchored folate receptor, and like other asialoglycoproteins, is believed to enter cells by receptor-mediated endocytosis through coated pits (
      • Goldstein J.L.
      • Brown M.S.
      • Anderson R.G.W.
      • Russell D.W.
      • Schneider W.J.
      ). After 5-methyl-THF enters hepatocytes, it transits to the cytoplasm from the luminal space of membrane-bounded compartments, presumably late endosomes and lysosomes. Because these late endocytotic compartments are often localized very close to the Golgi complex, concentrating FTCD on the outer surface of the Golgi may enhance the rate of productive collisions between FTCD and 5-methyl-THF. An appealing feature of this model is that it does not exclude the possibility that a soluble pool of FTCD is also capable of modifying 5-methyl-THF.

      ACKNOWLEDGEMENTS

      We thank Drs. Clive Slaughter and Kim Orth, and Carolyn Moomaw for sequencing the rat liver FTCD peptides; Drs. Thomas Kreis of l'Université de Genève, and Dagmar Hennig and Jan De Mey of Institut Jacques Monod for sharing their unpublished data and anti-chicken FTCD antibodies with us; Drs. Laura Lea Murley and Robert MacKenzie of McGill University for providing us with native and recombinant porcine liver FTCD, and a polyclonal antibody to porcine liver FTCD, and for performing formiminotransferase and cyclodeaminase assays on purified rat liver 58K; Dr. Tony Frankfurter of the University of Virginia for supplying purified rat liver tubulin; and Aaron Fullerton and Dr. Jean-Marie Sontag of the Bloom laboratory for technical assistance.

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