Facilitated transport of methotrexate polyglutamates into lysosomes derived from S180 cells. Further characterization and evidence for a simple mobile carrier system with broad specificity for homo- or heteropeptides bearing a C-terminal glutamyl moiety.

Studies are presented further characterizing a facilitative system transporting methotrexate (MTX) polyglutamates into lysosomes derived from S180 cells. Initial influx of [3H]MTX + G1 (MTX with 1 additional glutamyl residue) exhibited a slightly alkaline pH optimum (pH 7.7) and was moderately temperature-dependent (Q10 27-37 degrees C = 3.1 +/- 0.1). An analysis of the kinetics of intralysosomal accumulation of [3H]MTX + G1 showed saturation kinetics for initial influx, but linear kinetics for the steady-state level of exchangeable [3H]MTX + G1 at different external concentrations of [3H]MTX + G1. In addition, the system exhibited substantial directional asymmetry with respect to the interaction with MTX + G1 during influx and efflux. Accelerated homo- and heteroexchange diffusion was demonstrated for influx of [3H]MTX + G1, while decelerated homoexchange diffusion was demonstrated for efflux of [3H]MTX + G1 following trans-positioning of MTX + G1 or glutamyl-gamma-glutamate in the opposite compartment. These observations were consistent with a single mobile carrier system mediating influx and efflux of this polyglutamate. Based upon an analysis of competitive interactions with [3H] MTX + G1, this system displayed specificity for MTX-gamma-glutamates, folyl-gamma-polyglutamates, alpha- or gamma-glutamyl peptides and heteropeptides bearing a C-terminal gamma-glutamate but not for MTX or glutamate, themselves. Among polyglutamates, gamma-glutamyl chain length was not a significant factor for transport except in the case of MTX polyglutamates. Overall, our results appear to delineate in the lysosomal membrane a simple mobile carrier system with broad specificity for folyl- or non-folyl-bearing peptides responsible for the transport of MTX polyglutamates.     

Metabolic turnover of methotrexate polyglutamates in lysosomes derived from S180 cells. Definition of a two-step process limited by mediated lysosomal permeation of polyglutamates and activating reduced sulfhydryl compounds.

Transport and metabolic turnover of methotrexate (MTX) polyglutamates were examined in lysosomes derived from S180 cells. These studies extend prior work from this laboratory (Barrueco, J. R., and Sirotnak, F. M. (1991) J. Biol. Chem 266, 11732-11737) which described basic properties of a facilitative transport system in lysosomes capable of mediating intralysosomal accumulation of MTX polyglutamates. In the present report, we show that the rate of turnover of MTX polyglutamates in lysosomes, which releases MTX in the extralysosomal space, is limited by the extent of mediated intralysosomal accumulation of the polyglutamate and reduced sulfhydryls that activate the enzyme folylpolyglutamate hydrolase. Evidence is presented that cysteine functions as the naturally occurring reduced sulfhydryl compound in lysosomes being equipotent to 2-mercaptoethanol as an activator of folylpolyglutamate hydrolase. Folylpolyglutamate hydrolase in permeabilized lysosomes from S180 cells exhibited a low pH optimum characteristic of a lysosomal enzyme, was activated at concentrations of reduced sulfhydryl at 0.1 mM and above, and exhibited Km values in the range of 0.2-3 microM that decreased with increase in polyglutamate chain length. Values for Km for MTX polyglutamates of folylpolyglutamate hydrolase activity were 100-200-fold lower than values for Km or Ki for facilitated intralysosomal transport, whereas capacities for both processes were similar. This relationship between the kinetic properties of each process ensures efficient hydrolysis of MTX polyglutamates within the lysosome.     

Oligosaccharide transport: pumping waste from the ER into lysosomes.

N-glycans play important roles during the folding and secretion of glycoproteins. Surprisingly, during the N-glycosylation of glycoproteins, considerable amounts of unconjugated polymannose-type oligosaccharides ('free OS') are generated. Although free oligosaccharides have no known function in mammalian cells, a sophisticated cellular machinery enables them to be cleared from the endoplasmic reticulum (ER) into the cytosol and then re-enter the endomembrane system at the level of the lysosome. One possible function of this pathway is to stop free OS from interfering with the carbohydrate-dependent aspects of glycoprotein folding and transport along the secretory pathway.     

The permeability of rat liver lysosomes to sugars. Evidence for carrier-mediated facilitated diffusion.

1. By the osmotic-protection method, the penetration of sugars through the rat liver lysosomal membranes was studied with a view of determining whether sugar uptake was by facilitated diffusion. 2. The following criteria for this type of transport were established: sugar specificity, the order of uptake being 2-deoxy-D-glucose less than D-glucose less than D-mannose less than D-galactose less than D-ribose less than 2-deoxy-D-ribose; stereospecificity, the uptake of L-glucose and L-ribose being 50% slower than their D-stereoisomers; inhibition by 1 MM-phlorrhizin and 1 M-cytochalastin B; competition between sugars for uptake, and a Q10 (rate difference over a 10 degrees C temperature range) for uptake of approx. 2.8. 3. It is proposed that sugar uptake into lysosomes from rat liver is by facilitated diffusion.     

Effect of varying polyglutamate chain length on the structure and stability of ferricytochrome c

The effect of varying polyglutamate chain length on local and global stability of horse heart ferricytochrome c was studied using scanning calorimetry and spectroscopy methods. Spectral data indicate that polyglutamate chain lengths equal or greater than eight monomer units significantly change the apparent pK(a) for the alkaline transition of cytochrome c. The change in pK(a) is comparable to the value when cytochrome c is complexed with cytochrome bc(1). Glutamate and diglutamate do not significantly alter the temperature transition for cleavage of the Met(80)-heme iron bond of cytochrome c. At low ionic strength, polyglutamates consisting of eight or more glutamate monomers increase midpoint of the temperature transition from 57.3+/-0.2 to 66.9+/-0.2 degrees C. On the other hand, the denaturation temperature of cytochrome c decreases from 85.2+/-0.2 to 68.8+/-0.2 degrees C in the presence of polyglutamates with number of glutamate monomers n >or approximately equal 8. The rate constant for cyanide binding to the heme iron of cytochrome c of cytochrome c-polyglutamate complex also decreases by approximately 42.5% with n>or approximately equal 8. The binding constant for the binding of octaglutamate (m.w. approximately 1000) to cyt c was found to be 1.15 x 10(5) M(-1) at pH 8.0 and low ionic strength. The results indicate that the polyglutamate (n>or approximately equal 8) is able to increase the stability of the methionine sulfur-heme iron bond of cytochrome c in spite of structural differences that weaken the overall stability of the cyt c at neutral and slightly alkaline pH.     

Renaturation of formiminotransferase-cyclodeaminase from guanidine hydrochloride. Quaternary structure requirements for the activities and polyglutamate specificity

Formiminotransferase-cyclodeaminase denatured in 6 M guanidine hydrochloride (Gdn.HCl) refolds and reassembles to the native octameric structure upon dilution into buffer. Both enzymic activities are recovered to greater than 90%, and the renatured enzyme "channels" the formiminotetrahydropteroylpentaglutamate intermediate. Under conditions where the two activities are recovered simultaneously, the rate-limiting step in reactivation is first order with respect to protein, with k = 1.9 X 10(-5) s-1 at 22 degrees C and delta E approximately equal to 15 kcal mol-1. In the presence of 1.5 M urea, renaturation is arrested at the level of dimers having only transferase activity. Subsequent dialysis to remove the urea leads to recovery of deaminase activity and formation of octamer. Kinetic studies with mono- and pentaglutamate derivatives of the folate substrates demonstrated that native and renatured enzyme as well as deaminase-active dimers [Findlay, W. A., & MacKenzie, R. E (1987) Biochemistry 26, 1948-1954] have much higher affinity for polyglutamate substrates, while the transferase-active dimers do not. These results indicate that the transferase activity is associated with one type of subunit-subunit interaction in the native tetramer of dimers and that the polyglutamate binding site and the deaminase activity are associated with the other interface. A dimeric transferase-active fragment generated by limited proteolysis of the native enzyme can also be renatured from 6 M Gdn.HCl, confirming that it is an independently folding domain capable of reforming one type of subunit interaction.     

Circumvention of the methotrexate transport system by methotrexate-phosphatidylethanolamine derivatives: effect of fatty acid chain length

Methotrexate has been conjugated (amide bond) via either the alpha or gamma, or both alpha and gamma, glutamyl carboxyl groups to the amino function of dihexanoylphosphatidylethanolamine (C6C6PE) and 1-tetradecanoyl-2-hexanoylphosphatidylethanolamine (C14C6PE). These phospholipid prodrugs (either free or incorporated into liposomes) were compared with the corresponding ditetradecanoylphosphatidylethanolamine (C14C14PE) conjugates, some of whose properties have been described previously, for their ability to inhibit the proliferation of human leukemic cells (CEM/O) or cells derived therefrom (CEM/MTX) that are resistant to methotrexate because of a defective drug transport system. Regardless of chain length, the gamma conjugates were more effective than either the alpha or the alpha, gamma conjugates, in inhibiting growth of the parent cells, confirming initial experiments with mouse cells. Chain length had, however, a pronounced influence on the capacity of the various gamma derivatives to circumvent the transport defect. For example, CEM/MTX cells were 120-fold less susceptible than CEM/O cells to inhibition by either methotrexate or methotrexate-gamma-C6C6PE, whereas both cell lines were equally sensitive to methotrexate-gamma-C14C14PE. Although less potent than either of the foregoing, methotrexate-gamma-C14C6PE could partially by-pass the defective transport system. These results suggest that methotrexate-gamma-PE derivatives with appropriate acyl residues might be useful probes to investigate the mechanism by which phospholipids in general are able to traverse cell membranes.     

Evidence for glucocorticoid transport into AtT-20/D-1 cells.

Glucocorticoid uptake by AtT-20/D-1 mouse pituitary adenocarcinoma cells grown in tissue culture was examined. The binding of triamcinolone acetonide, a potent synthetic glucocorticoid, by intact cells and by cell cytosol was studied at both 4 and 25 degrees. Specific binding of [3H]triamcinolone acetonide by intact cells was markedly different from cell-free cytosol binding at 4 degrees. Intact cells bound a relatively small amount of labeled steroid within 2 min, after which no further binding was observed. In contrast, the receptor in a cell-free cytosol preparation was capable of binding steroid progressively at 4 degrees, indicating that the limited binding by intact cells was not a consequence of receptor characteristics. At 25 degrees, uptake by intact cells and cytosol was nearly identical and appeared to be limited only by the binding kinetics of the cytosol receptor. Estradiol-17 beta, a nonglucocorticoid steroid, was not bound by the AtT-20/D-1 cell at 4 degrees. Triamcinolone was not bound significantly at 4 or 25 degrees by an adrenal carcinoma cell that does not appear to be a glucocorticoid target cell. An Arrhenius plot of cell steroid uptake vs. the reciprocal of absolute temperature revealed an abrupt change in slope at 16 degrees, which is compatible with the temperature-dependent mechanism involved in glucocortidoid uptake being associated with lipid constituents of the cell membrane. These data suggest that glucocorticoid uptake by this target cell involves a mechanism of specific, temperature-dependent transport through the cell membrane.     

Cytosol-to-lysosome transport of free polymannose-type oligosaccharides. Kinetic and specificity studies using rat liver lysosomes

In hepatocellular carcinoma HepG2 cells, free polymannose-type oligosaccharides appearing in the cytosol during the biosynthesis and quality control of glycoproteins are rapidly translocated into lysosomes by an as yet poorly defined process (Saint-Pol, A., Bauvy, C., Codogno, P., and Moore, S. E. H. (1997) J. Cell Biol. 136, 45-59). Here, we demonstrate an ATP-dependent association of [2-3H]mannose-labeled Man5GlcNAc with isolated rat liver lysosomes. This association was only observed in the presence of swainsonine, a mannosidase inhibitor, which was required for the protection of sedimentable, but not nonsedimentable, Man5GlcNAc from degradation, indicating that oligosaccharides were transported into lysosomes. Saturable high affinity transport (Kuptake, 22.3 microM, Vmax, 7.1 fmol/min/unit of beta-hexosaminidase) was dependent upon the hydrolysis of ATP but independent of vacuolar H+/ATPase activity. Transport was inhibited strongly by NEM and weakly by vanadate but not by sodium azide, and, in addition, the sugar transport inhibitors phloretin, phloridzin, and cytochalasin B were without effect on transport. Oligosaccharide import did not show absolute specificity but was selective toward partially demannosylated and dephosphorylated oligosaccharides, and, furthermore, inhibition studies revealed that the free reducing GlcNAc residue of the oligosaccharide was of critical importance for its interaction with the transporter. These results demonstrate the presence of a novel lysosomal free oligosaccharide transporter that must work in concert with cytosolic hydrolases in order to clear the cytosol of endoplasmic reticulum-generated free oligosaccharides.     

Complex formation between ribosomal protein S1, oligo-and polynucleotides: chain length dependence and base specificity.

In order to examine the nature of the complex formation between the ribosomal protein S1 and nucleic acids three methods were used: Inhibition of the reaction of n-ethyl[2.3 14C]-maleimide with S1 by the addition of oligonucleotides; adsorption of the complexes to nitrocellulose filters; and equilibrium dialysis. The complex formation is Mg2+ dependent at low salt concentrations and becomes Mg2+ independent at an ionic strength greater than 90 mM. Oligouridylates of increasing chain length reach an optimal KA of 3-3-10(7) M-1 at a chain length of n=13-14. Protein S1 contains one binding site for long chain oligouridylates, such as U12, and the standard-free-energy change on binding caused by one Pu increment is 0.41 kcal/mol, when n varies between five and fourteen. Complex formation is insensitive to the capacity of the homopolynucleotide bases to form hydrogen bonds. Homopolynuceotides, however, showing a Tm less than 250 in the buffer system used show an increased affinity for S1 compared to poly(A) and poly(C) (Tm greater than 40 degrees). The data are discussed with respect to the proposed binding of protein S1 to the 3-terminal end of the 16S RNA.     

Purification and properties of a pig heart thiolase with broad chain length specificity and comparison of thiolases from pig heart and Escherichia coli.

A thiolase (acetyl CoA acyltransferase, EC 2.3-1.16) which acts on substrates of various chain lengths (thiolase I) has been purified from pig heart muscle 366-fold to near homogeneity as judged by gel electrophoresis. Its molecular weight was estimated to be 200,000 in the absence and 46,000 in the presence of sodium dodecyl sulfate. Kinetic measurements with acetoacetyl coenzyme A, 3-ketohexanoyl-CoA, 3-ketooctanoyl-CoA, and 3-ketodecanoyl-CoA yielded apparent Km values of 16, 8.3, 2.4, and 1.8 micron, respectively, whereas apparent Vmax values of 65 to 69 mumol/min/mg were obtained with all substrates except for acetoacetyl-CoA, with which a value of 26.5 mumol/min/mg was observed. Antibodies prepared against this thiolase were used to demonstrate that thiolase I and acetoacetyl-CoA thilase (thiolase II) from pig heart mitochondria are immunologically unrelated. The antibodies cross-reacted, however, with thiolase I from beef heart. Kinetic constants (Km, Vmax) were also determined for thiolases I and II from Escherichia coli, as were the native and subunit molecular weights of E. coli thiolase II. Although the E. coli thiolases were found to be immunologically distinct from the pig heart enzymes, their physical and kinetic properties are strikingly similar to those of the heart thiolases. In view of this finding and in view of the known physiological functions of the E. coli thiolases, it is proposed that thiolase I from pig heart is only involved in fatty acid metabolism, whereas thiolase II functions solely in ketone body degradation.     

Nature of the facilitated messenger ribonucleic acid transport from isolated nuclei.

Cytoplasmic macromolecules were previously identified which regulate both qualitatively and quantitatively the release of messenger-like RNA from isolated nuclei. These macromolecules are now shown to be denatured at 45-50 degrees C and their synthesis is sensitive to pactamycin or cycloheximide. The putative regulatory proteins are essentially quantitatively precipitated with high specificity from the cytosol by streptomycin at a concentration 10-fold higher than that used to precipitate RNA. The nuclear concentration-dependence of RNA transport from successive samples of nuclei strongly suggests that the regulatory factors are recycled. Quantitative changes in the sequences transported at various dilutions of the cytosol suggest that not all the different classes of the putative regulatory macromolecules are present in an effective concentration at any one dilution.     

Binding properties of the 5-methyltetrahydrofolate/methotrexate transport system in L1210 cells

A binding component with a high affinity for 5-methyltetrahydrofolate (K_D = 0.11μm) is present on the external surface of L1210 cells. The amount of binder (1 pmol/mg protein) corresponds to 8 × 10⁴ sites per cell. The participation of this component in the high-affinity 5-methyltetrahydrofolate/methotrexate transport system is supported by similarities in the K_D values for 5-methyltetrahydrofolate and methotrexate binding and the K_t values of these compounds for transport. Relative affinities for other folate substrates (aminopterin, 5-formyltetrahydrofolate, and folate) and various competitive inhibitors (thiamine pyrophosphate, ADP, AMP, arsenate, and phosphate) are also similar for both the binding component and the transport system. The measured binding activity does not represent low-temperature transport of substrate into cells, since it is readily saturable with time and is eliminated by either washing the cells with buffer or by the addition of excess unlabeled substrate.     

Immunoglobulin alpha heavy chain derived from human epithelial cancer cells promotes the access of S phase and growth of cancer cells

It is generally believed that under normal conditions only B lymphocytes express immunoglobulin. Interestingly, our previous work demonstrated that epithelial cancer tissues and cancer cell lines also express Ig alpha heavy chain. So we further analyzed the potential function of cancer-derived Ig alpha heavy chain. Here we show that blockade of cancer-derived Ig alpha suppressed the growth and viability of cancer cells. And cancer-derived Ig alpha promotes the malignant proliferation ability of cancer cells. Furthermore, we demonstrated that Ig alpha protein increases the access percentage of S phase from the early mitosis of synchronized cancer cells. Our findings support the important role of cancer-derived Ig alpha as a growth promoter of cancer cells, and reveal a novel molecular mechanism for growth and proliferation of cancer cells.     

Substrate and inhibitor specificity of monocarboxylate transport into heart cells and erythrocytes. Further evidence for the existence of two distinct carriers.

A range of short-chain aliphatic monocarboxylates, both unsubstituted and substituted with hydroxy, chloro and keto groups, were shown to inhibit transport of L-lactate and pyruvate into both guinea-pig cardiac myocytes and rat erythrocytes. The carrier of heart cells exhibited a higher affinity (approx. 10-fold) for most of the monocarboxylates than did the erythrocyte carrier. A notable exception was L-lactate, whose Km for both carriers was similar. The K1 values of the two carriers for inhibitors such as phenylpyruvate and alpha-cyanocinnamate derivatives were also different. The high affinity of the heart cell carrier for ketone bodies and acetate may be physiologically important, since these substrates are used as fuels by the heart.     

Translocation of sugars into rat liver lysosomes. Evidence against a common carrier for D-glucose and D-ribose.

A conflict exists in the literature concerning the mode of translocation of D-glucose and D-ribose across the lysosome membrane. The more rapid net uptake of ribose, when measured by the osmotic-protection technique, has been attributed either to its smaller size and lower hydrogen-bonding capacity, or to a lower affinity for a transport system shared by both sugars. The latency of acid beta-hexosaminidase in isolated rat liver lysosomes was measured after preincubation for periods up to 1 h in various solutions containing glucose and/or ribose, and in some cases sucrose. After confirmation of the superior osmotic protection afforded by glucose (than by ribose), it was shown that a solution 0.125 M in both glucose and ribose provided protection intermediate between that given by 0.25 M-glucose and that given by 0.25 M-ribose. This result is inconsistent with the common-carrier hypothesis.     

Assembly and transport properties of invariant chain trimers and HLA-DR-invariant chain complexes.

The MHC class II-associated invariant chain behaves as a resident endoplasmic reticulum protein in the absence of class II molecules. In humans, two predominant forms exist; one, p35, differs from the other, p33, by an N-terminal cytoplasmic extension of 16 amino acids that contains a strong endoplasmic reticulum-retention signal. Here we show that one mechanism for retention of p33 is its association with p35 in mixed invariant chain trimers. However, even for p33 homotrimers transport from the endoplasmic reticulum is inefficient. In an MHC class II-positive B cell line, the formation of invariant chain trimers is rapid and is the first intermediate in the assembly of a nine-chain alpha beta-invariant chain complex. With time, three higher molecular weight complexes are progressively formed. These correspond to an invariant chain trimer with one alpha beta dimer, two alpha beta dimers, and three alpha beta dimers, respectively. No free alpha beta dimers are detectable early in biosynthesis. However, beginning at 2 h of chase, alpha beta dimers begin to appear concomitant with the disappearance of the completely assembled alpha beta-invariant chain complex. This conversion is virtually complete by 4 h, and presumably reflects the proteolytic degradation of the invariant chain component of the alpha beta-invariant chain complex and the generation of endosomal alpha beta dimers capable of binding antigenic peptides.