scholarly journals Characterization of a cysteine-less human reduced folate carrier: localization of a substrate-binding domain by cysteine-scanning mutagenesis and cysteine accessibility methods

2003 ◽  
Vol 374 (1) ◽  
pp. 27-36 ◽  
Author(s):  
Wei CAO ◽  
Larry H. MATHERLY
Keyword(s):  
Amino Acids ◽  
Mammalian Cells ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Substrate Binding ◽  
Chinese Hamster ◽  
Water Soluble ◽  
Cysteine Residues ◽  
Reduced Folate Carrier ◽  
Cysteine Scanning Mutagenesis

The human reduced folate carrier (hRFC) mediates the transport of reduced folates and classical anti-folates into mammalian cells. Whereas the functionally important domains in hRFC are poorly characterized, previous studies with anti-folate-resistant cells suggest critical roles for transmembrane domain (TMD) 1 and residues (Gly44, Glu45, Ser46 and Ile48) in or flanking this region. An hRFC mutant devoid of cysteine residues was prepared by deleting the C-terminal 56 amino acids, including four cysteine residues, and mutagenizing the remaining cysteine residues to serine residues. A fully functional cysteine-less hRFC protein was expressed in transport-impaired MtxRIIOuaR2-4 Chinese-hamster ovary cells. To explore the role of residues in or flanking TMD1 in transport, all 24 amino acids from Trp25 to Ile48 of hRFC were mutated individually to cysteine residues, and the mutant hRFCs were transfected into MtxRIIOuaR2-4 cells. All of the 24 cysteine mutants were expressed and, with the exception of R42C (Arg42→Cys), were capable of mediating methotrexate uptake above the low level in MtxRIIOuaR2-4 cells. We found that by treating the transfected cells with the small, water-soluble, thiol-reactive anionic reagent, sodium (2-sulphonatoethyl) methanethiosulphonate, methotrexate transport by several of the cysteine-substituted hRFC mutants was significantly inhibited, including Q40C, G44C, E45C and I48C. Sodium (2-sulphonatoethyl) methanethiosulphonate transport inhibition of the Q40C, G44C and I48C mutants was protected by leucovorin [(6R,S)-5-formyltetrahydrofolate], indicating that these residues lie at or near a substrate-binding site. Using surface-labelling reagents [N-biotinylaminoethyl methanethiosulphonate and 3-(N-maleimidylpropionyl)biocytin, combined with 4-acetamido-4′-maleimidylstilbene-2,2′-disulphonic acid] with cysteine mutants from positions 37–48, the extracellular TMD1 boundary was found to lie between residues 39 and 40, and amino acids 44–46 and 48 were localized to the TMD1 exofacial loop. Collectively, our results imply that amino acids 40, 44, 48 and, possibly, 42 serve important roles in hRFC transport, albeit not as structural components of the putative transmembrane channel for folate substrates.

scholarly journals Localization of a Substrate Binding Domain of the Human Reduced Folate Carrier to Transmembrane Domain 11 by Radioaffinity Labeling and Cysteine-substituted Accessibility Methods

2005 ◽  
Vol 280 (43) ◽  
pp. 36206-36213 ◽  
Author(s):  
Zhanjun Hou ◽  
Sarah E. Stapels ◽  
Christina L. Haska ◽  
Larry H. Matherly
Keyword(s):  
Hela Cells ◽  
Mammalian Cells ◽  
Transmembrane Domain ◽  
Substrate Binding ◽  
Binding Domain ◽  
Reduced Folate Carrier ◽  
Western Blots ◽  
Transmembrane Channel ◽  
Functional Components ◽  
Substrate Binding Domain

The human reduced folate carrier (hRFC) mediates the membrane transport of reduced folates and classical anti-folates into mammalian cells. RFC is characterized by 12 transmembrane domains (TMDs), internally oriented N and C termini, and a large central linker connecting TMDs 1–6 and 7–12. By co-expression and N-hydroxysuccinimide methotrexate (Mtx) radioaffinity labeling of hRFC TMD 1–6 and TMD 7–12 half-molecules, combined with endoproteinase GluC digestion, a substrate binding domain was previously localized to within TMDs 8–12 (Witt, T. L., Stapels, S. E., and Matherly, L. H. (2004) J. Biol. Chem. 279, 46755–46763). In this report, this region was further refined to TMDs 11–12 by digestion with 2-nitro-5-thiocyanatobenzoic acid. A transportcompetent cysteine-less hRFC was used as a template to prepare single cysteine-replacement mutant constructs in which each residue from Glu-394 to Asp-420 of TMD 11 and Tyr-435 to His-457 of TMD 12 was replaced individually by a cysteine. The mutant constructs were transfected into hRFC-null HeLa cells. Most of the 50 single cysteine-substituted constructs were expressed at high levels on Western blots. With the exception of G401C hRFC, all mutants were active for Mtx transport. Treatment with sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) had no effect on hRFC activity for all of the cysteine mutants within TMD 12 and for the majority of the cysteine mutants within TMD 11. However, MTSES inhibited Mtx uptake by the T404C, A407C, T408C, T412C, F416C, I417C, V418C, and S419C mutants by 25–65%. Losses of activity by MTSES treatment for T404C, A407C, T412C, and I417C hRFCs were appreciably reversed in the presence of excess leucovorin, a hRFC substrate. Our results strongly suggest that residues within TMD 11 are likely critical structural and/or functional components of the putative hRFC transmembrane channel for anionic folate and anti-folate substrates.

scholarly journals Structural requirements for palmitoylation of surfactant protein C precursor

2002 ◽  
Vol 361 (3) ◽  
pp. 663-671 ◽  
Author(s):  
Anja ten BRINKE ◽  
Arie B. VAANDRAGER ◽  
Henk P. HAAGSMAN ◽  
Anja N. J. A. RIDDER ◽  
Lambert M. G. van GOLDE ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein C ◽  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Chinese Hamster Ovary Cells ◽  
Membrane Surface ◽  
Chinese Hamster ◽  
Surfactant Protein ◽  
Structural Requirements ◽  
Surfactant Protein C

Pulmonary surfactant protein C (SP-C) propeptide (proSP-C) is a type II transmembrane protein that is palmitoylated on two cysteines adjacent to its transmembrane domain. To study the structural requirements for palmitoylation of proSP-C, His-tagged human proSP-C and mutant forms were expressed in Chinese hamster ovary cells and analysed by metabolic labelling with [3H]palmitate. Mutations were made in the amino acid sequence representing mature SP-C, as deletion of the N- and C-terminal propeptide parts showed that this sequence by itself could already be palmitoylated. Substitution of the transmembrane domain by an artificial transmembrane domain had no effect on palmitoylation. However, an inverse correlation was found between palmitoylation of proSP-C and the number of amino acids present between the cysteines and the transmembrane domain. Moreover, substitution by alanines of amino acids localized on the N-terminal side of the cysteines had drastic effects on palmitoylation, probably as a result of the removal of hydrophobic amino acids. These data, together with the observation that substitution by alanines of the amino acids localized between the cysteines and the transmembrane domain had no effect on palmitoylation, suggest that the palmitoylation of proSP-C depends not on specific sequence motifs, but more on the probability that the cysteine is in the vicinity of the membrane surface. This is probably determined not only by the number of amino acids between the cysteines and the transmembrane domain, but also by the hydrophobic interaction of the N-terminus with the membrane. This may also be the case for the palmitoylation of other transmembrane proteins.

Interplay between insulin and nutrients in the regulation of translation factors

2001 ◽  
Vol 29 (4) ◽  
pp. 541-547 ◽  
Author(s):  
C. G. Proud ◽  
X. Wang ◽  
J. V. Patel ◽  
L. E. Campbell ◽  
M. Kleijn ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Mammalian Target Of Rapamycin ◽  
Chinese Hamster ◽  
Elongation Factor ◽  
Nucleotide Exchange ◽  
S6 Kinase ◽  
P70 S6 Kinase ◽  
Eef2 Kinase

Protein synthesis in mammalian cells is regulated through alterations in the states of phosphorylation of eukaryotic initiation factors and elongation factors (eIFs and eEFs respectively) and of other regulatory proteins. This modulates their activities or their abilities to interact with one another. Insulin activates several of these proteins including the following: the guanine-nucleotide exchange factor eIF2B; the eIF4F complex, which (through eIF4E) interacts with the cap of the mRNA; p70 S6 kinase; and elongation factor eEF2, which mediates the translocation step of elongation. Control of the last three of these is linked to mTOR (mammalian target of rapamycin). In Chinese hamster ovary cells, regulation of all these proteins by insulin is modulated by the presence of amino acids and/or glucose in the medium. For example, p70 S6 kinase activity declines in the absence of amino acids and cannot be stimulated by insulin under this condition. The readdition of amino acids, especially leucine, restores activity and sensitivity to insulin. With eIF2B and eEF2, both amino acids and glucose must be provided for insulin to regulate their activities. In contrast, insulin-stimulation of the formation of eIF4F complexes requires glucose but not amino acids. Glucose metabolism is required for this permissive effect. Our recent studies have also identified the mechanism by which mTOR signalling regulates the phosphorylation of eEF2. eEF2 kinase is phosphorylated by p70 S6 kinase at Ser-366; this results in the inactivation of eEF2 kinase, especially at low (micromolar) Ca concentrations.

scholarly journals Analysis of the membrane topology for transmembrane domains 7-12 of the human reduced folate carrier by scanning cysteine accessibility methods

2004 ◽  
Vol 378 (1) ◽  
pp. 201-206 ◽  
Author(s):  
Wei CAO ◽  
Larry H. MATHERLY
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Transmembrane Domains ◽  
Membrane Topology ◽  
Cysteine Residues ◽  
Reduced Folate Carrier ◽  
Cell Permeabilization ◽  
Topology Structure ◽  
Ovary Cells ◽  
Pre Treatment

The hRFC (human reduced folate carrier) is the major membrane transporter for both reduced folates and antifolates in human tissues and tumours. The primary amino acid sequence of hRFC predicts a membrane topology involving 12 TMDs (transmembrane domains) with cytosolic oriented N- and C-termini, and a large internal loop connecting TMDs 6 and 7. Previous studies using haemagglutinin epitope insertion and scanning glycosylation mutagenesis methods verified portions of the predicted topology model, including TMDs 1–8 and the N- and C-termini of hRFC. However, the topology structure for TMDs 9–12 remains controversial. To further determine the membrane topology of the hRFC protein, single cysteine residues were introduced into the predicted extracellular or cytoplasmic loops of a fully functional cysteine-less hRFC expressed in transport impaired MtxRIIOuaR2-4 Chinese hamster ovary cells. The membrane orientations of the substituted cysteines were determined by treatments with the thiol reagents 3-(N-maleimidylpropionyl)-biocytin (biotin maleimide) and 4-acetamido-4´maleimidylstilbene-2,2´-disulphonic acid (stilbenedisulphonate maleimide; SM) or N-ethylmaleimide, combined with the cell-permeabilizing reagent SLO (streptolysin O). We found that cysteine residues placed in the predicted extracellular loops between TMDs 7 and 8 (position 301), 9 and 10 (360), and 11 and 12 (429) could be biotinylated with 200 µM biotin maleimide, and labelling could be blocked with SM. However, biotinylation of cysteines placed in the predicted intracellular loops between TMDs 8 and 9 (position 332) and TMDs 10 and 11 (position 388) was only detected after cell permeabilization with SLO and was abolished by pre-treatment with N-ethylmaleimide. These results strongly support a 12-TMD topology structure for the hRFC protein.

Recombinant Human Soluble Thrombomodulin Delivers Bounded Thrombin to Antithrombin III: Thrombomodulin Associates with Free Thrombin and Is Recycled to Activate Protein C

1993 ◽  
Vol 70 (03) ◽  
pp. 418-422 ◽  
Author(s):  
Masaharu Aritomi ◽  
Naoko Watanabe ◽  
Rika Ohishi ◽  
Komakazu Gomi ◽  
Takao Kiyota ◽  
...  
Keyword(s):  
Protein C ◽  
Antithrombin Iii ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Time Lag ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Soluble Thrombomodulin ◽  
Simulation Based ◽  
Recombinant Human Soluble Thrombomodulin

SummaryRecombinant human soluble thrombomodulin (rhs-TM), having no transmembrane domain or chondroitin sulfate, was expressed in Chinese hamster ovary cells. Interactions between rhs-TM, thrombin (Th), protein C (PC) and antithrombin III (ATIII) were studied. Equilibrium between rhs-TM and Th had no detectable time lag in clotting inhibition (K d = 26 nM) or PC activation (K d = 22 nM), while ATIII inhibited Th at a bimolecular rate constant = 5,200 M-1s-1 (K d <0.2 nM). A mixture of ATIII, Th and rhs-TM showed that ATIII reacted with Th slower than rhs-TM, whose presence did not affect the reaction between ATIII and Th. In a mixture of rhs-TM, ATIII and PC, the repeated addition of Th caused the repeated activation of PC; which was consistent with the Simulation based on the assumption that rhs-TM is recycled as a Th cofactor. From these results, we concluded that upon inhibition of the rhs-TM-Th complex by ATIII, rhs-TM is released to recombine with free Th and begins to activate PC, while the Th-ATIII complex does not affect rhs-TM-Th equilibrium.

Thermotropic lipid and protein transitions in Chinese hamster lung cell membranes: relationship to hyperthermic cell killing

1983 ◽  
Vol 61 (6) ◽  
pp. 421-427 ◽  
Author(s):  
James R. Lepock ◽  
Kwan-Hon Cheng ◽  
Hisham Al-Qysi ◽  
Jack Kruuv
Keyword(s):  
Mammalian Cells ◽  
Plasma Membranes ◽  
Chinese Hamster ◽  
Cell Killing ◽  
Water Soluble ◽  
Growth Data ◽  
Protein Fluorescence ◽  
Polar Groups ◽  
Spin Resonance ◽  
Intrinsic Protein Fluorescence

Exposure of mammalian cells to hyperthermic temperatures (ca. 41–45 °C) appears to act as a direct or triggering effect to produce some later response such as cell death, thermotolerance, or heat-shock protein synthesis. The high activation energy of cell killing indicates that the direct effect of hyperthermia might be a thermotropic transition in some cellular component, for this particular response. Both hyperthermic survival and growth data imply that the temperature for the onset of hyperthermic cell killing is 40–41.5 °C for Chinese hamster lung V79 cells. Studies using the electron spin resonance label 2,2-dimethyl-5-dodecyl-5-methyloxazolidine-N-oxide and the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene show the existence of lipid transitions at approximately 7–8 and 23–36 °C (or a broad transition between these temperatures) in mitochondria and whole cell homogenates, that correlate well with changes in growth and hypothermic killing. No lipid transition was detected near 40–41.5 °C that could correlate with hyperthermic killing in either mitochondrial or plasma membranes, but measurements of intrinsic protein fluorescence and protein fluorophore to trans-paranaric acid energy transfer demonstrate the existence of an irreversible transition in protein structure or arrangement above ca. 40 °C in both mitochondrial and plasma membranes. This transition is due to protein rearrangement and (or) unfolding such that there is increased exposure of protein tryptophan and tyrosine residues to polar groups and to paranaric acid. The strength of the transition implies that a significant fraction of total membrane protein is involved in this transition, which may be analogous to the heat-induced denaturation of water-soluble proteins. This alteration in membrane structure above ca. 40 °C could cause many of the observed changes in plasma membrane and mitochondrial function, which may further be involved in cellular responses to hyperthermia.

scholarly journals Molecular cloning of the cDNA for a mutant mouse ribonucleotide reductase M1 that produces a dominant mutator phenotype in mammalian cells.

1988 ◽  
Vol 8 (7) ◽  
pp. 2698-2704 ◽  
Author(s):  
I W Caras ◽  
D W Martin
Keyword(s):  
Ribonucleotide Reductase ◽  
Mammalian Cells ◽  
Mutant Mouse ◽  
Chinese Hamster Ovary Cells ◽  
Spontaneous Mutation ◽  
Single Point ◽  
Chinese Hamster ◽  
Fold Increase ◽  
Single Point Mutation ◽  
Mutator Phenotype

Mammalian ribonucleotide reductase is regulated by the binding of dATP and other nucleotide effectors to allosteric sites on subunit M1. Using mRNA from a mutant mouse T-lymphoma (S49) cell line, we have isolated a cDNA which encodes an altered, dATP feedback-resistant subunit M1. The mutant cDNA contains a single point mutation (a G-to-A transition) at codon 57, converting aspartic acid to asparagine. Proof that this mutation is responsible for the phenotype of dATP feedback resistance is provided by the following evidence. (i) The mutation was detected only in mutant S49 cells containing dATP feedback-resistant ribonucleotide reductase and not in wild-type or other mutant S49 cells. (ii) Transfection of Chinese hamster ovary cells with an expression plasmid containing the mutant M1 cDNA resulted in the production of dATP feedback-resistant ribonucleotide reductase. Transfected CHO cells expressing the mutant M1 cDNA exhibited a 15- to 25-fold increase in the frequency of spontaneous mutation to 6-thioguanine resistance, confirming that dATP feedback-resistant ribonucleotide reductase produces a mutator phenotype in mammalian cells. The availability of a cDNA which encodes dATP feedback-resistant subunit M1 thus provides a means of manipulating by transfection the frequency of spontaneous mutation in mammalian cells.

scholarly journals Replacement of both tryptophan residues at 388 and 412 completely abolished cytochalasin B photolabelling of the GLUT1 glucose transporter

1994 ◽  
Vol 302 (2) ◽  
pp. 355-361 ◽  
Author(s):  
K Inukai ◽  
T Asano ◽  
H Katagiri ◽  
M Anai ◽  
M Funaki ◽  
...  
Keyword(s):  
Glucose Transporter ◽  
Cytochalasin B ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Site Directed Mutagenesis ◽  
Transport Activity ◽  
Wild Type ◽  
In The Wild ◽  
Glut1 Glucose Transporter

A mutated GLUT1 glucose transporter, a Trp-388, 412 mutant whose tryptophans 388 and 412 were both replaced by leucines, was constructed by site-directed mutagenesis and expressed in Chinese hamster ovary cells. Glucose transport activity was decreased to approx. 30% in the Trp-388, 412 mutant compared with that in the wild type, a similar decrease in transport activity had been observed previously in the Trp-388 mutant and the Trp-412 mutant which had leucine at 388 and 412 respectively. Cytochalasin B labelling of the Trp-388 mutant was only decreased rather than abolished, a result similar to that obtained previously for the Trp-412 mutant. Cytochalasin B labelling was finally abolished completely in the Trp-388, 412 mutant, while cytochalasin B binding to this mutant was decreased to approx. 30% of that of the wild-type GLUT1 at the concentration used for photolabelling. This level of binding is thought to be adequate to detect labelling, assuming that the labelling efficiency of these transporters is similar. These findings suggest that cytochalasin B binds to the transmembrane domain of the glucose transporter in the vicinity of helix 10-11, and is inserted covalently by photoactivation at either the 388 or the 412 site.

scholarly journals Cell cycle-dependent effects on deoxyribonucleotide and DNA labeling by nucleoside precursors in mammalian cells.

1987 ◽  
Vol 7 (1) ◽  
pp. 532-534 ◽  
Author(s):  
J M Leeds ◽  
C K Mathews
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
G1 Phase ◽  
Ovary Cells ◽  
Dna Labeling ◽  
Metabolic Pool ◽  
Specific Activities ◽  
Cycle Dependent

dCTP pools equilibrated to equivalent specific activities in Chinese hamster ovary cells or in nuclei after incubation of cells with radiolabeled nucleosides, indicating that dCTP in nuclei does not constitute a distinct metabolic pool. In the G1 phase, [5-3H]deoxycytidine labeled dCTP to unexpectedly high specific activities. This may explain reports of replication-excluded DNA precursor pools.

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