Export of mRNA from microinjected nuclei of Xenopus laevis oocytes.

Export of mRNA from the nucleus to the cytoplasm was studied in mature Xenopus laevis oocytes. In vitro transcribed, capped 32P-labeled mRNA was microinjected into nuclei, and its appearance in the cytoplasm measured by counting radioactivity or by RNA extraction and gel electrophoresis. Both for a 5.0-kb transferrin receptor mRNA and a 2.0-kb 4F2 antigen heavy chain mRNA we found saturable transport with an apparent Km of 3.6 x 10(8) molecules per oocyte nucleus. Under non-saturating conditions the half-time for mRNA export from the nucleus was approximately 2 min at 20 degrees C. At higher concentrations of injected mRNA this half-time was prolonged, and the maximal transport rate was reached at approximately 1.6 x 10(8) molecules/min. mRNA transport showed properties of an energy-dependent mechanism, since it was inhibited at 4 degrees C or by ATP depletion. Co-injection of the cap dinucleotide m7GpppG blocked the export effectively, suggesting a role for the cap in this process. The export was also inhibited by the pre-injection of wheat germ agglutinin. The effect of the lectin was specific and abolished by co-injection of N-acetylglucosamine. Finally, we found significant competitive inhibition in mRNA export by the presence of tRNA. Our results suggest that mRNA transport is a facilitated process which may share common steps with tRNA transport. Preliminary gel retardation experiments show that injected mRNA associates with endogenous nuclear proteins and suggest an exchange of some of the bound components during the transport to the cytoplasm.

Luminal heterodimeric amino acid transporter defective in cystinuria.

Mutations of the glycoprotein rBAT cause cystinuria type I, an autosomal recessive failure of dibasic amino acid transport (b(0,+) type) across luminal membranes of intestine and kidney cells. Here we identify the permease-like protein b(0,+)AT as the catalytic subunit that associates by a disulfide bond with rBAT to form a hetero-oligomeric b(0,+) amino acid transporter complex. We demonstrate its b(0,+)-type amino acid transport kinetics using a heterodimeric fusion construct and show its luminal brush border localization in kidney proximal tubule. These biochemical, transport, and localization characteristics as well as the chromosomal localization on 19q support the notion that the b(0,+)AT protein is the product of the gene defective in non-type I cystinuria.

Crystallization and preliminary X-ray diffraction data for the aconitase form of human iron-regulatory protein 1.

Iron-regulatory proteins (IRPs) 1 and 2 are closely related molecules involved in animal iron metabolism. Both proteins can bind to specific mRNA regions called iron-responsive elements and thereby control the expression of proteins involved in the uptake, storage and utilization of iron. In iron-replete cells, IRP1, but not IRP2, binds a [4Fe-4S] cluster and functions as a cytoplasmic aconitase, with simultaneous loss of its RNA-binding ability. Whereas IRP2 is known to be involved in Fe homeostasis, the role of IRP1 is less clear; it may provide a link between citrate and iron metabolisms and be involved in oxidative stress response. Here, two crystal forms of the aconitase version of recombinant human IRP1 are reported. An X-ray fluorescence measurement performed on a gold-derivative crystal showed the unexpected presence of zinc, in addition to gold and iron. Both native and MAD X-ray data at the Au, Fe and Zn absorption edges have been collected from these crystals.

A novel interferon-gamma regulated human melanoma-associated antigen, gp33-38, defined by monoclonal antibody Me14-D12. II. Molecular cloning of a genomic probe.

The human Me14-D12 antigen is a cell surface glycoprotein regulated by interferon-gamma (IFN-gamma) on tumor cell lines of neuroectodermal origin. It consists of two non-convalently linked subunits with apparent mol. wt sizes of 33,000 and 38,000. Here we describe the molecular cloning of a genomic probe for the Me14-D12 gene using the gene transfer approach. Mouse Ltk- cells were stably cotransfected with human genomic DNA and the Herpes Simplex virus thymidine kinase (TK) gene. Primary and secondary transfectants expressing the Me14-D12 antigen were isolated after selection in HAT medium by repeated sorting on a fluorescence activated cell sorter (FACS). A recombinant phage harboring a 14.3 kb insert of human DNA was isolated from a genomic library made from a positive secondary transfectant cell line. A specific probe derived from the phage DNA insert allowed the identification of two mRNAs of 3.5 kb and 2.2 kb in primary and secondary L cell transfectants, as well as in human melanoma cell lines expressing the Me14-D12 antigen. The regulation of Me14-D12 antigen by INF-gamma was retained in the L cell transfectants and could be detected both at the level of protein and mRNA expression.

Interaction between iron-regulatory proteins and their RNA target sequences, iron-responsive elements.

In this chapter, we have focused on the biochemistry of IRP-1 and the features which distinguish it from the related RNA-binding protein, IRP-2. IRP-1 is the cytoplasmic isoform of the enzyme aconitase, and, depending on iron status, may switch between enzymatic and RNA-binding activities. IRP-1 and IRP-2 are trans-acting regulators of mRNAs involved in iron uptake, storage and utilisation. The finding of an IRE in the citric acid cycle enzymes, mitochondrial aconitase and succinate dehydrogenase, suggests that the IRPs may also influence cellular energy production. These two proteins appear to bind RNAs with different but overlapping specificity, suggesting that they may regulate the stability or translation of as yet undefined mRNA targets, possibly extending their regulatory function beyond that of iron homeostasis. The interaction between the IRPs and the IRE represents one of the best characterised model systems for posttranscriptional gene control, and given that each IRP can also recognise its own unique set of RNAs, the search for new in vivo mRNA targets is expected to provide yet more surprises and insights into the fate of cytoplasmic mRNAs.

Iron and gene expression: molecular mechanisms regulating cellular iron homeostasis.

In recent years, specific post-transcriptional mechanisms in the cytoplasm of vertebrate cells have been elucidated that directly affect the stability and translation of mRNAs coding for central proteins in iron metabolism. This review shall focus primarily on these mechanisms. Other levels of control, either affecting gene transcription and/ or related to the function of iron-capturing substances and transmembrane transport, are also likely to exist and to influence the iron balance and utilization. They are, however, much less clear.

Monoclonal antibodies recognizing the secreted and membrane domains of the IgA dimer receptor.

The receptor that mediates the specific uptake and intracellular transport of dimeric immunoglobulin A (IgA dimer) in mucosal and glandular epithelia is identical with a transmembrane precursor of secreted secretory component. During transport, the IgA dimer receptor (membrane SC) is cleaved into two domains, a membrane anchorage peptide and secreted secretory component. We have produced monoclonal antibodies with distinct specificity against both domains of the rabbit IgA dimer receptor. Two mouse hybridoma lines were obtained by fusion of SP2/0 myeloma cells with spleen cells from mice immunized with purified receptor from rabbit liver and by screening of culture supernatants in an immunoprecipitation assay with radiolabeled receptor. One antibody, designated anti-SC 303, reacts both with membrane and secreted SC and is therefore directed to a determinant on the secreted domain of the IgA dimer receptor. The other antibody, anti-SC 166, unable to interact with secreted SC, recognizes the membrane domain of the receptor. We discussed the unique precursor relationship between a cell-surface receptor and a secreted protein and its implications in the IgA dimer transport system.

Coordination of cellular iron metabolism by post-transcriptional gene regulation.

Maintenance of cellular iron homeostasis demands the coordination of iron uptake, intracellular storage, and utilization. Recent investigations suggest that a single genetic regulatory system orchestrates the expression of proteins with central importance for all three aspects of cellular iron metabolism at the level of mRNA stability and translation. Two components of this regulatory system have been defined: a cis-acting mRNA sequence/structure motif called "iron-responsive element" (IRE) and a specific trans-acting cytoplasmic binding protein, here referred to as "IRE-binding protein" (IRE-BP). As an early event in the regulatory cascade, cellular iron deprivation induces the IRE-binding activity of IRE-BP, whereas binding activity is reduced in iron-replete cells. IRE-BP is highly homologous to the iron-sulphur (Fe-S) protein aconitase which strongly suggests that IRE-BP is an Fe-S protein itself. Control over IRE-BP activity by the cellular iron status is exerted post-translationally and likely involves changes between (4Fe-4S) and (3Fe-4S) states of the postulated IRE-BP Fe-S cluster. In addition, post-translational regulation of IRE-BP activity via heme has been proposed. Subsequent to its activation, IRE-BP binds with high affinity to single IREs contained in the 5' untranslated regions (UTRs) of ferritin and erythroid 5-aminolevulinic acid synthase (eALAS) mRNAs. The binding represses translation of these proteins involved in iron storage and utilization, respectively. In contrast, iron uptake is largely regulated via multiple IREs in the 3' UTR of transferrin receptor (TfR) mRNA. TfR-IREs are required for the iron-sensitive control of TfR mRNA stability. IRE-BP binding stabilizes TfR gene transcripts against as yet undefined ribonucleases. As a result of these regulatory interactions, iron starvation induces the expression of TfR, thereby increasing iron uptake, and represses the synthesis of proteins involved in iron storage and utilization. As cellular iron levels rise, the homeostatic balance is maintained by lowering iron uptake and increasing iron storage in ferritin.

The inability of cells to grow in low iron correlates with increasing activity of their iron regulatory protein (IRP).

We studied the factors that determine the differing growth requirements of low-iron-tolerant (LIT) versus high-iron-dependent (HID) cells for extracellular nontransferrin iron. The growth of LIT cells HeLa and THP-1, when transferred from transferrin (5 micrograms/ml) medium into low-iron (5 microM ferric citrate) medium, was not significantly affected while HID cells Jiyoye and K562 showed nearly no growth. HeLa and THP-1 cells, as well as Jiyoye and K562 cells, do not produce transferrin in sufficient amounts to support their growth in low-iron medium. Surprisingly, similar rates of iron uptake in low-iron medium (0.033 and 0.032 nmol Fe/min and 10(6) cells) were found for LIT cells HeLa and HID cells K562. Furthermore, the intracellular iron level (4.64 nmol/10(6) cells) of HeLa cells grown in low-iron medium was much higher than iron levels (0.15 or 0.20 nmol/10(6) cells) of HeLa or K562 cells grown in transferrin medium. We demonstrated that the activity (ratio activated/total) of the iron regulatory protein (IRP) in HID cells Jiyoye and K562 increased more than twofold (from 0.32 to 0.79 and from 0.47 to 1.12, respectively) within 48 h after their transfer into low-iron medium. In the case of LIT cells HeLa and THP-1, IRP activity stayed at similar or slightly decreased levels (0.86-0.73 and 0.58-0.55, respectively). Addition of iron chelator deferoxamine (50 microM, i.e., about half-maximal growth-inhibitory dose) resulted in significantly increased activity of IRP also in HeLa and THP-1 cells. We hypothesize that the relatively higher bioavailability of nontransferrin iron in LIT cells, over that in HID cells, determines the differing responses observed under low-iron conditions.

Transferrin receptor expression is controlled differently by transferrin-bound and non-transferrin iron in human cells.

We studied the effects of iron supplied as transferrin-bound iron and iron supplied as non-transferrin iron on transferrin receptor expression by human cell lines. Defined conditions of iron supply were represented by (i) 5 microg/ml of iron-saturated transferrin (transferrin medium) and by (ii) 500 microM ferric citrate (ferric citrate medium). Transferrin receptor expression of studied cell lines (HeLa, K562, Jiyoye) grown as long-term cultures in transferrin medium was somewhat higher (up to 137% of the mean fluorescence intensity) than in ferric citrate medium. The receptor expression corresponded with cellular iron regulatory protein (IRP) activity (ratio activated/total), which was also higher in transferrin medium (0.69-0.84) than in ferric citrate medium (0.33-0.60). However, unexpectedly much higher (about 65-135-fold) cellular iron levels were found in ferric citrate medium (13.9-14.9 nmol/10(6) cells) than in transferrin medium (0.11-0.21 nmol/10(6) cells). In contrast to the iron levels, cellular ferritin levels of the cells in ferric citrate medium (38.3-130 ng/10(6) cells) were only about 2-7-fold higher than in transferrin medium (6.8-61.5 ng/10(6) cells). We suggest that iron supplied as non-transferrin iron (ferric citrate) is apparently less available for the control of transferrin receptor expression via IRP activity than iron supplied as transferrin.

The transferrin receptor: a key function in iron metabolism.

Iron is essential to cell proliferation. Its uptake by cells requires specific binding of the major serum iron-transport protein, transferrin, to cell surface transferrin receptors, followed by endocytosis of the receptor-ligand complexes and release of iron from endosomal vesicles to the cytoplasm. The structural and functional aspects of this pathway are reviewed. Intracellular iron either serves as a substrate for the biosynthesis of haem and iron-containing proteins or is stored in ferritin deposits. Recent studies are presented which establish that iron plays an important role in the maintenance of its own homeostasis by regulating coordinately the expression of both the transferrin receptor and ferritin. The elucidation of these regulatory mechanisms may become important to the understanding of certain disorders in iron-metabolism.

Molecular regulation of iron proteins.

Cellular iron metabolism comprises pathways of iron-protein synthesis and degradation, iron uptake via transferrin receptor (TfR) or release to the extracellular space, as well as iron deposition into ferritin and remobilization from such stores. Different cell types, depending on their rate of proliferation and/or specific functions, show strong variations in these pathways and have to control their iron metabolism to cope with individual functions. Studies with cultured cells have revealed a specific cytoplasmic protein, called 'iron regulatory protein' (IRP) (previously known as IRE-BP or IRF), that plays a key role in iron homoeostasis by regulating coordinately the synthesis of TfR, ferritin, and erythroid 5-aminolevulinate synthase (eALAS). Present in all tissues analysed, IRP is identical with the [4Fe-4S] cluster containing cytoplasmic aconitase. Under conditions of iron chelation, IRP is an apo-protein which binds with high affinity to specific RNA stem-loop elements (IREs) located 5' of the initiation codon in ferritin and eALAS mRNA, and 3' in the untranslated region of TfR mRNA. At 5' sites IRF blocks mRNA translation, whereas 3' it inhibits TfR mRNA degradation. Both effects compensate for low intracellular iron concentrations. Under high iron conditions, IRP is converted to the holo-protein and dissociates from mRNA. This reverses the control towards less iron uptake and more iron storage. Iron can therefore be considered as a feedback regulator of its own metabolism. It has recently become evident that nitric oxide, produced by macrophages and other cell types in response to interferon-gamma, induces the IRE-binding activity of IRF. Moreover measurements of the RNA-binding activity of IRP in tissue extracts may provide valuable information on iron availability.

Noncoding 3' sequences of the transferrin receptor gene are required for mRNA regulation by iron.

The cell-surface receptor for transferrin mediates cellular uptake of iron from serum. Transferrin receptor protein and mRNA levels are increased in cells treated with iron chelating agents, and are decreased by treatment with iron salts or hemin. Here we report that expression of human transferrin receptor cDNA constructions in stably transfected mouse fibroblasts is regulated both by the iron chelator, desferrioxamine, and by hemin. We found that sequences within the 3' noncoding region are required for the iron-dependent feed-back regulation of receptor expression, whereas the presence of the transferrin receptor promoter region is not necessary. Regulation by iron is observed when transcription is initiated at either the SV-40 early promoter or the transferrin receptor promoter, but deletion of a 2.3 kb fragment within the 2.6 kb 3' noncoding region of the cDNA abolishes regulation and increases the constitutive level of receptor expression. Furthermore, the 3' deletion does not affect the decrease in receptors which is observed in response to growth arrest, indicating that transferrin receptor expression is controlled by at least two distinct mechanisms.

A stem-loop in the 3' untranslated region mediates iron-dependent regulation of transferrin receptor mRNA stability in the cytoplasm.

Expression of the human transferrin receptor (hTR) and its mRNA is strongly induced by iron deprivation. By measuring transcription elongation rates, levels of hTR-specific nuclear RNA, and mRNA half-lives, we found this regulation to occur posttranscriptionally in the cytoplasm. Analysis of hTR cDNA mutants with deletions in the 3' untranslated region revealed the existence of two distinct domains, both of which are essential for regulation in mouse L cells. The regulated phenotype correlates with the presence of a stem-loop structure predicted by a computer algorithm. Expression of point and deletion mutants affecting the stem-loop confirmed the requirement for this secondary structure in regulation. The 3' untranslated region of hTR cDNA was sufficient to confer iron-dependent regulation on another gene.

Role of secretory component, a secreted glycoprotein, in the specific uptake of IgA dimer by epithelial cells.

The binding of secretory component (SC) to epithelial cells and its role in the specific uptake of immunoglobulin A (IgA) dimer has been studied in rabbit mammary gland and liver. SC, Mr approximately 80,000, secreted by epithelial cells of the mammary gland was found associated with the cell surface of mammary cells in intact tissue. Dispersed mammary cells and plasma membrane-enriched fractions obtained from mammary glands of midpregnant rabbits bound 125I-labeled SC in a saturable time- and temperature-dependent process. The association rate followed a second order reversible reaction (k+1 approximately equal to 2.7 x 10(6) M-1 min-1 at 4 degrees C) and equilibrium was reached in about 4 h at 4 degrees C. The dissociation rate for membranes was first order (k-1 approximately equal to 1.7 x 10(-2) min-1 at 4 degrees C), whereas displacement from cells was incomplete. The apparent affinity constant was similar for membranes and cells (Ka approximately equal to 5 x 10(8) M-1) with one class of binding sites. The number of binding sites varied from one animal to another (260 to 7,000 sites/mammary cell) in relation to endogenous occupancy by SC, which was assessed by immunocytochemistry and complement-mediated cytotoxicity. Rabbit liver and heart membranes did not bind SC, and serum proteins present in rabbit milk failed to interact with mammary cells or membranes. Mammary membranes or cells and liver membranes bound 125I-labeled IgA dimer in a saturable, reversible time- and temperature-dependent process. Association and dissociation rate constants at 4 degrees C (k+1 approximately equal to 5 x 10(6) M-1 min-1 and k-1 approximately equal to 5 x 10(-3) min-1, respectively) and the apparent affinity constant (Ka approximately equal to 10(9) M-1) were similar for liver and mammary membranes; these parameters differed, however, from those reported for free SC-IgA dimer interaction. The binding capacity of membranes for IgA dimer was directly related to the amount of free SC bound to membranes. Interaction of IgA dimer with mammary or liver membranes or cells was abrogated by excess of free SC and was prevented by preincubation of membranes or cells with Fab antibody fragments directed against SC. These data indicate that the first step in the translocation process of polymeric immunoglobulins across epithelia consists of binding of SC to the surface of epithelial cells which in turn acts as a receptor for the specific uptake of IgA dimer.

Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress.

As an essential nutrient and a potential toxin, iron poses an exquisite regulatory problem in biology and medicine. At the cellular level, the basic molecular framework for the regulation of iron uptake, storage, and utilization has been defined. Two cytoplasmic RNA-binding proteins, iron-regulatory protein-1 (IRP-1) and IRP-2, respond to changes in cellular iron availability and coordinate the expression of mRNAs that harbor IRP-binding sites, iron-responsive elements (IREs). Nitric oxide (NO) and oxidative stress in the form of H2O2 also signal to IRPs and thereby influence cellular iron metabolism. The recent discovery of two IRE-regulated mRNAs encoding enzymes of the mitochondrial citric acid cycle may represent the beginnings of elucidating regulatory coupling between iron and energy metabolism. In addition to providing insights into the regulation of iron metabolism and its connections with other cellular pathways, the IRE/IRP system has emerged as a prime example for the understanding of translational regulation and mRNA stability control. Finally, IRP-1 has highlighted an unexpected role for iron sulfur clusters as post-translational regulatory switches.

Interaction of rabbit secretory component with rabbit IgA dimer.

Secretory component (SC), a glycoprotein with an apparent molecular weight of approximately 80,000, has been isolated from rabbit milk and found to be heterogenous in size and charge. Functionally intact IgA dimer has been dissociated from milk secretory IgA using a chaotropic agent and further purified to homogeneity. The interaction between SC and IgA dimer is a reversible time- and temperature-dependent process. At 23 degrees C, the association rate constant (2.4 x 10(5) M-1 min-1) and the dissociation rate constant (1.8 x 10(-3) min-1) have been measured independently and the affinity constant based on these rates (1.3 x 10(8) M-1) is similar to that calculated from Scatchard plots (1.9 x 10(8) M-1). One class of binding sites has been estimated from Scatchard plots in spite of the observed heterogeneity of SC. The interaction is tighter at low temperatures because the decrease in dissociation rate is greater than the decrease in association rate. The thermodynamic calculations reveal a delta G of -11.0 kcal . mol-1, a delta H of -8.9 kcal . mol-1 and a delta S of +7.0 cal. mol-1 degree-1. The pH range over which interaction occurs is rather large (5 to 8) with no significant differences in apparent Ka.