Human Cytosolic and Mitochondrial Folylpolyglutamate Synthetase Are Electrophoretically Distinct

Abstract

Folylpolyglutamate synthetase (FPGS) activity in CCRF-CEM human leukemia cells was found in the cytosolic (Ϸ67% of total) and mitochondrial (Ϸ22%) fractions. A polyclonal antipeptide antibody (430Ab) to human FPGS specifically recognized distinct immunoreactive bands (Ϸ60 kDa) present in each subcellular fraction. Human cytosolic FPGS (hcFPGS) migrated more rapidly than mitochondrial FPGS (hmFPGS); their estimated difference in molecular mass was 1 kDa. The human K562 acute nonlymphocytic leukemia and the A253 and FaDu head and neck cancer cell lines also expressed the two FPGS isoforms, and the ratio of hcFPGS to hmFPGS protein in each cell line was similar. Since K562 and A253 cells are intrinsically resistant to pulse methotrexate (MTX) exposure relative to CCRF-CEM and FaDu cells, respectively, because of decreased MTX polyglutamate synthesis (despite having similar levels of total FPGS activity expression), these data suggest that the natural difference in drug sensitivity cannot be explained by compartmentalization of FPGS activity. Higher expression of hmFPGS relative to hcFPGS was observed in some sublines of CCRF-CEM with acquired MTX resistance suggesting that differential expression of the hmFPGS isoform may contribute to MTX resistance caused by decreased FPGS activity.It has been known for years that mitochondria have their own folate-dependent enzymes and folylpolyglutamate pool, but it has not been established how that pool is acquired or maintained (reviewed in Refs. 1-3). A central enzyme in establishing and maintaining folylpolyglutamate pools in whole cells is folylpolyglutamate synthetase (FPGS) 1 (4,5). Early studies indicated that FPGS activity was present in both cytosol and mitochondria (6, 7), suggesting that folate monoglutamates were transport forms and that the essential (8) folate polyglutamate forms were then synthesized independently in each compartment. Studies with isolated mitochondria have confirmed that folate monoglutamates are transported by a carrier-mediated facilitated-diffusion mechanism (9); if folate polyglutamates pass the mitochondrial membrane, they do so slowly, since the cytosolic and mitochondrial folate pools are not in equilibrium (2, 10). More detailed studies later showed the presence of FPGS activity in Chinese hamster ovary (CHO) cell cytosol and mitochondria (2). The function of FPGS within the two compartments has recently been studied. Shane and co-workers (2, 11-13) studied CHO AUXB1 cells (which lack expression of both cFPGS and mFPGS) transfected with Esherichia coli or human FPGS that was expressed in either or both compartments. In cells expressing cytosolic FPGS activity, mitochondrial folates were absent, and cells were auxotrophic for glycine and methionine (12). However, cells expressing activity only in mitochondria also contained cytosolic folates, although the levels were low, and as a consequence, folate metabolism was not optimal. The results indicate that both FPGS isoforms must be expressed to establish normal folate metabolism and opti...

Forced Expression of Survivin-2B Abrogates Mitotic Cells and Induces Mitochondria-dependent Apoptosis by Blockade of Tubulin Polymerization and Modulation of Bcl-2, Bax, and Survivin

“…Cytoplasmic and mitochondrial subcellular fractions were isolated as previously described (23). Resultant supernatants (cytosolic fraction) and pellets (mitochondrial fraction) were analyzed for survivin-2B expression by Western blot analysis.…”

5-Amino-4-Imidazolecarboxamide Riboside Potentiates Both Transport of Reduced Folates and Antifolates by the Human Reduced Folate Carrier and Their Subsequent Metabolism

“…Subsequent steps were done at 4jC. Cells were homogenized as described (22), except that hypotonic buffer at 20-fold the packed cell volume was used, and the resulting homogenate was centrifuged for 6 minutes at 1,000 Â g to yield a nuclear pellet and post-nuclear supernatant. Based on controls, >91% of cells are lysed during homogenization.…”

“…The resulting supernatant was centrifuged at 100,000 Â g (60 minutes) to yield the 100,000 Â g supernatant (cytosol). The 100,000 Â g pellet (microsomes) and combined mitochondria-lysosome pellet were each washed in isotonic buffer (22) and recentrifuged. Pellets were solubilized and quantitated as described above.…”

The Reduced Folate Carrier (RFC) Is Cytotoxic to Cells under Conditions of Severe Folate Deprivation

“…Therefore, folate deficiency may impair the de novo biosynthesis of purines and thymidylate and thereby disrupt DNA and RNA metabolism, homocysteine remethylation, methionine biosynthesis, and subsequent formation of S-adenosylmethionine, the universal methyl donor, which in turn may lead to the impairment of methylation reactions (1)(2)(3)(4)(5)(6). Based on their key role in cellular metabolism, folate cofactors are efficiently retained in cells via polyglutamylation, an ATPdependent reaction in which up to 9 eq of glutamate units are added to the ␥-carboxyl residue of folate cofactors (8,9) (see Fig. 1A).…”

“…1A). Whereas this reaction is catalyzed by the enzyme folylpoly-␥-glutamate synthetase (9), the enzyme ␥-glutamyl hydrolase (GGH) 2 catalyzes the hydrolysis of these terminal ␥-glutamyl residues from polyglutamylated folates (10). Importantly the long chain (n Ͼ 3) folate polyglutamylate derivatives can no longer be extruded via ATP-dependent efflux transporters such as the multidrug resistance proteins (MRPs/ABCCs) (11,12) and breast cancer resistance protein (BCRP/ABCG2) (13) as well as through the reduced folate carrier (RFC), a bidirectional folate transporter (14).…”