Functional expression cloning and characterization of SFT, a stimulator of Fe transport.

A stimulator of Fe transport (SFT) was identified by functional expression cloning in Xenopus oocytes. SFT-mediated transport has properties defined for transferrin-independent Fe uptake, but its cytolocalization in recycling endosomes and the observed stimulation of transferrin-bound Fe assimilation indicate a key role in intracellular Fe membrane transport as well. SFT has six predicted transmembranous domains and a functionally important RExxE motif that resembles domains involved in yeast Fe transport and Fe-binding by ferritin L-chains. The observation that SFT oligomerizes, along with other structural and mechanistic features, suggests it may be a member of either the ATP-binding cassette or cation diffusion facilitator families. The 3' untranslated region of SFT contains a translation inhibitory element and inhibition of SFT expression in Xenopus oocytes was found to be relieved by coinjection of transcripts from other defined cDNAs that are also described in this report. SFT is the first component of the mammalian Fe membrane transport machinery to be identified.

Properties of Rab5 N-terminal domain dictate prenylation of C-terminal cysteines.

Rab5 is a Ras-related GTP-binding protein that is post-translationally modified by prenylation. We report here that an N-terminal domain contained within the first 22 amino acids of Rab5 is critical for efficient geranylgeranylation of the protein's C-terminal cysteines. This domain is immediately upstream from the "phosphate binding loop" common to all GTP-binding proteins and contains a highly conserved sequence recognized among members of the Rab family, referred to here as the YXYLFK motif. A truncation mutant that lacks this domain (Rab5(23-215) fails to become prenylated. However, a chimeric peptide with the conserved motif replacing cognate Rab5 sequence (MAYDYLFKRab5(23-215) does become post-translationally modified, demonstrating that the presence of this simple six amino acid N-terminal element enables prenylation at Rab5's C-terminus. H-Ras/Rab5 chimeras that include the conserved YXYLFK motif at the N-terminus do not become prenylated, indicating that, while this element may be necessary for prenylation of Rab proteins, it alone is not sufficient to confer properties to a heterologous protein to enable substrate recognition by the Rab geranylgeranyl transferase. Deletion analysis and studies of point mutants further reveal that the lysine residue of the YXYLFK motif is an absolute requirement to enable geranylgeranylation of Rab proteins. Functional studies support the idea that this domain is not required for guanine nucleotide binding since prenylation-defective mutants still bind GDP and are protected from protease digestion in the presence of GTP gamma S. We conclude that the mechanism of Rab geranylgeranylation involves key elements of the protein's tertiary structure including a conserved N-terminal amino acid motif (YXYLFK) that incorporates a critical lysine residue.

Wortmannin alters the transferrin receptor endocytic pathway in vivo and in vitro.

Treatment with the phosphatidylinositol 3-kinase inhibitor wortmannin promotes approximately 30% decrease in the steady-state number of cell-surface transferrin receptors. This effect is rapid and dose dependent, with maximal down-regulation elicited with 30 min of treatment and with an IC50 approximately 25 nM wortmannin. Wortmannin-treated cells display an increased endocytic rate constant for transferrin internalization and decreased exocytic rate constants for transferrin recycling. In addition to these effects in vivo, wortmannin is a potent inhibitor (IC50 approximately 15 nM) of a cell-free assay that detects the delivery of endocytosed probes into a common compartment. Inhibition of the in vitro assay involves the inactivation of a membrane-associated factor that can be recruited onto the surface of vesicles from the cytosol. Its effects on the cell-free assay suggest that wortmannin inhibits receptor sorting and/or vesicle budding required for delivery of endocytosed material to "mixing" endosomes. This idea is consistent with morphological changes induced by wortmannin, which include the formation of enlarged transferrin-containing structures and the disruption of the perinuclear endosomal compartment. However, the differential effects of wortmannin, specifically increased transferrin receptor internalization and inhibition of receptor recycling, implicate a role for phosphatidylinositol 3-kinase activity in multiple sorting events in the transferrin receptor's membrane traffic pathway.

Iron metabolism.

The understanding of iron metabolism at the molecular level has been enormously expanded in recent years by new findings about the functioning of transferrin, the transferrin receptor and ferritin. Other recent developments include the discovery of the hemochromatosis gene HFE, identification of previously unknown proteins involved in iron transport, divalent metal transporter 1 and stimulator of Fe transport, and expanded insights into the regulation and expression of proteins involved in iron metabolism. Interactions among principal participants in iron transport have been uncovered, although the complexity of such interactions is still incompletely understood. Correlated efforts involving techniques and concepts of crystallography, spectroscopy and molecular biology applied to cellular processes have been, and should continue to be, particularly revealing.

Chemistry and biology of eukaryotic iron metabolism.

With rare exceptions, virtually all studied organisms from Archaea to man are dependent on iron for survival. Despite the ubiquitous distribution and abundance of iron in the biosphere, iron-dependent life must contend with the paradoxical hazards of iron deficiency and iron overload, each with its serious or fatal consequences. Homeostatic mechanisms regulating the absorption, transport, storage and mobilization of cellular iron are therefore of critical importance in iron metabolism, and a rich biology and chemistry underlie all of these mechanisms. A coherent understanding of that biology and chemistry is now rapidly emerging. In this review we will emphasize discoveries of the past decade, which have brought a revolution to the understanding of the molecular events in iron metabolism. Of central importance has been the discovery of new proteins carrying out functions previously suspected but not understood or, more interestingly, unsuspected and surprising. Parallel discoveries have delineated regulatory mechanisms controlling the expression of proteins long known--the transferrin receptor and ferritin--as well as proteins new to the scene of iron metabolism and its homeostatic control. These proteins include the iron regulatory proteins (IRPs 1 and 2), a variety of ferrireductases in yeast an mammalian cells, membrane transporters (DMT1 and ferroportin 1), a multicopper ferroxidase involved in iron export from cells (hephaestin), and regulators of mitochondrial iron balance (frataxin and MFT). Experimental models, making use of organisms from yeast through the zebrafish to rodents have asserted their power in elucidating normal iron metabolism, as well as its genetic disorders and their underlying molecular defects. Iron absorption, previously poorly understood, is now a fruitful subject for research and well on its way to detailed elucidation. The long-sought hemochromatosis gene has been found, and active research is underway to determine how its aberrant functioning results in disease that is easily controlled but lethal when untreated. A surprising connection between iron metabolism and Friedreich's ataxia has been uncovered. It is no exaggeration to say that the new understanding of iron metabolism in health and disease has been explosive, and that what is past is likely to be prologue to what is ahead.

Molecular mechanisms of iron transport.

The homeostasis of iron transport and absorption represents a critical mechanism to maintain the necessary physiologic balance of this essential nutrient. Recent advances in our understanding of the molecular mechanisms involved in the transport of iron and its membrane translocation are reviewed. Translational and transcriptional control mechanisms that have been characterized to play a role in iron homeostasis are also discussed.

An integrin-mobilferrin iron transport pathway in intestine and hematopoietic cells.

A pathway of iron transport not involving transferrin or transferrin receptors has been described as functioning in iron uptake into intestinal cells and in a hematopoietic cell line. Evidence was presented to show that it is mediated by a transmembrane protein (integrin) and a cytosolic protein (mobilferrin).

Biochemistry of iron uptake.

Recent information gained from genetic and biochemical studies of iron transport in yeast, coupled with the identification of specific mutations causing iron uptake disorders in mice and man, has provided new clues about the mechanisms involved in iron uptake. This article summarizes these discoveries and discusses their impact on our current understanding of the biochemistry of iron uptake.

Iron transport.

Iron homeostasis is maintained by regulating its absorption: Under conditions of deficiency, assimilation is enhanced but iron uptake is otherwise limited to prevent toxicity due to overload. Iron deficiency remains the most important micronutrient deficiency worldwide, but increasing awareness of the genetic basis for iron-loading diseases points to iron overload as a major public health issue as well. Recent identification of mutant alleles causing iron uptake disorders in mice and humans provides new insights into the mechanisms involved in iron transport and its regulation. This article summarizes these discoveries and discusses their impact on our current understanding of iron transport and its regulation.

Brefeldin A down-regulates the transferrin receptor in K562 cells.

The fungal metabolite brefeldin A (BFA) induces profound alterations in the morphology of intracellular organelles. Although BFA promotes the formation of extensive tubular endosomal domains, our understanding of the effects of the antibiotic on vesicle traffic events associated with endocytosis is limited. Thus, alterations in the transferrin (Tf) receptor's endocytic/recycling pathway upon treatment of human erythroleukemia K562 cells with BFA were studied as a pharmacological response. Treatment of K562 cells with BFA caused a down-regulation in the number of cell surface Tf receptors. This effect is highly reminiscent of the well-known action of phorbol 12-myristate 13-acetate (PMA) on Tf receptor traffic in K562 cells. However, our results demonstrate that these two agents down-regulate the Tf receptor via different mechanisms. The effects of BFA and PMA were additive when K562 cells were incubated with both together. Using the In/Sur method, the endocytic rate constant for Tf internalization was determined and PMA was found to greatly enhance ke, from 0.28 min-1 to 0.43 min-1, while BFA had little effect (Ke = 0.20 min-1). In contrast, BFA-treatment alters the exocytic rate constant for return of internalized receptors to the cell surface, with the largest effect exerted on a 'slow-release', monensin-sensitive, compartment. The sum of the endocytic and exocytic kinetic data support a model in which BFA and PMA down-regulate the Tf receptor in K562 cells by mechanistically distinct actions, with BFA targeting exocytic monensin-sensitive intracellular compartments and PMA acting to exert a profound influence on elements of receptor internalization.

Inhibition of in vitro endosomal vesicle fusion activity by aminoglycoside antibiotics.

The effects of two aminoglycoside antibiotics, neomycin and Geneticin, on the endocytic pathway were studied using a cell-free assay that reconstitutes endosome-endosome fusion. Both drugs inhibit the rate and extent of endosome fusion in a dose-dependent manner with IC50 values of approximately 45 microM and approximately 1 mM, respectively. Because the IC50 for neomycin falls within the range of affinities reported for its binding to acidic phospholipids, notably phosphatidylinositol 4,5-bisphosphate (PIP2), these data suggest that negatively charged lipids are required for endosome fusion. A role for negatively charged lipids in membrane traffic has been postulated to involve the activity of a PIP2-dependent phospholipase D (PLD) stimulated by the GTP-binding protein ADP-ribosylation factor (ARF). Although neomycin blocks endosome fusion at a stage of the in vitro reaction that is temporally related to steps inhibited by cytosolic ARFs when they bind guanosine-5'-gamma-thiophosphate (GTPgammaS), these inhibitors appear to act in a synergistic manner. This idea is confirmed by the fact that addition of a PIP2-independent PLD does not suppress neomycin inhibition of endosome fusion; moreover, in vitro fusion activity is not affected by the pleckstrin homology domain of phosphoinositide-specific phospholipase C delta1, which binds to acidic phospholipids, particularly PIP2, with high affinity. Thus, although aminoglycoside-sensitive elements of endosome fusion are required at mechanistic stages that are also blocked by GTPgammaS-bound ARF, these effects are unrelated to inhibition of the PIP2-dependent PLD activity stimulated by this GTP-binding protein. These results argue that there are additional mechanistic roles for acidic phospholipids in the endosomal pathway.

GEF-mediated GDP/GTP exchange by monomeric GTPases: a regulatory role for Mg2+?

GTPases share highly conserved guanine nucleotide-binding domains and fulfill diverse functions through a common molecular switch. An inactive GDP-bound protein is turned on by a guanine nucleotide exchange factor (GEF) that catalyzes exchange of GTP for GDP, but unfortunately little is known about the mechanism of GEF action. A common mechanism for GDP/GTP exchange can be envisioned wherein GEFs activate monomeric GTPases through transient disruption of Mg2+ coordination in the nucleotide-binding pocket while stabilizing a nucleotide-free (and cation-free) conformation. After guanine nucleotide exchange, Mg2+ coordination is restored to complete the conformational switch to the active GTP-bound state. Evidence in the literature highlighting an important regulatory role for Mg2+ in the mechanism of GEF-mediated GDP/GTP exchange by monomeric GTPases is summarized.

Structural and functional analysis of SFT, a stimulator of Fe Transport.

Previous studies demonstrated that SFT (Stimulator of Fe Transport) facilitates both transferrin and nontransferrin-bound iron uptake in HeLa cells (Yu, J., and Wessling-Resnick, M. (1998) J. Biol. Chem. 273, 6909-6915). To further characterize the structure and function of SFT, we studied this human factor in rodent BHK cells. Kyte-Doolittle analysis suggests that SFT has six transmembrane-spanning segments. This transport protein also displays an REXXE motif resembling domains involved in iron binding by ferritin and in iron uptake mediated by the yeast transporter Ftr1. Using N- and C-terminal epitope tags, we have identified that modification of either protein terminus does not interfere with SFT function in nontransferrin-bound iron uptake. The N- and C-terminal domains are intracellularly disposed since antibodies against these epitopes fail to recognize expressed proteins unless BHK cells are solubilized with detergents. To define the topology of two large extramembranous loop domains, anti-peptide antibodies were employed; anti-loop 4 antibodies show no immunoreactivity unless cells are permeabilized but anti-loop 5 antibodies recognize and bind surface SFT. Thus, loop 4 must be intracellular while loop 5 is extracellular. These topological studies situate the putative iron-binding REXXE domain on the cytosolic face of the plasma membrane. However, 55Fe-binding studies reveal that the ability of SFT to bind and mediate transport of extracellular iron is defective in mutants with Glu --> Ala conversions in this motif. Curiously, we also find that depletion of intracellular iron by desferrioxamine impairs SFT transport and iron-binding functions. These observations lead to the speculation that the REXXE motif may play an important role in regulating SFT activity through interaction with intracellular iron and demonstrate that iron transport mediated by SFT is itself an iron-dependent process.

Characterization of transferrin-independent iron transport in K562 cells. Unique properties provide evidence for multiple pathways of iron uptake.

The present study characterizes the transport of nontransferrin (non-Tf) iron by K562 cells. Accumulation of radiolabel by cells incubated with 55Fe-nitrilotriacetate (NTA) is a saturable process that is time and temperature dependent (Ea approximately 20 kcal/mol). Initial rate analysis of iron influx yields values of Vmax = 855 fmol/min/10(6) cells and apparent Km = 0.54 microM. NHCL4 and chloroquine, agents that block cellular acquisition of iron from Tf, do not interfere with assimilation from FeNTA, demonstrating that uptake is truly independent of the Tf-mediated pathway. Furthermore, the inactivation of this transport mechanism by limited proteolytic digestion on ice indicates that specific cell surface proteins are involved. The extent of radiolabel incorporation into heme and ferritin is the same regardless of whether K562 cells acquire iron from 55FeNTA via the cell surface mechanism or from 55Fe-Tf via receptor-mediated endocytosis. Unlike other Tf-independent iron transport pathways that have been described, the K562 cell transport mechanism is not inhibited by divalent cations such as Ni2+, Co2+, or Mn2+. Uptake from 55FeNTA can be blocked by Cu2+ but at concentrations > 1500-fold molar excess. However, Cd2+ is a fairly specific inhibitor of 55Fe uptake by K562 cells (IC50 approximately 50 microM). Additionally, the K562 cell transport mechanism is not Ca2+ dependent and does not appear to be regulated by extracellular iron salts, in contrast to features noted for non-Tf iron uptake by fibroblasts (Sturrock, A., Alexander, J., Lamb, J., Craven, C. M., and Kaplan, J. (1991) J. Biol. Chem. 265, 3139-3145; Kaplan, J., Jordan, I., and Sturrock, A. (1991) J. Biol. Chem. 266, 2997-3004). These unique characteristics of the K562 cell uptake mechanism suggest that multiple transport systems function in Tf-independent iron assimilation.

Influence of copper depletion on iron uptake mediated by SFT, a stimulator of Fe transport.

We recently identified a novel factor involved in cellular iron assimilation called SFT or Stimulator of Fe Transport (Gutierrez, J. A., Yu, J., Rivera, S., and Wessling-Resnick, M. (1997) J. Cell Biol. 149, 895-905). When stably expressed in HeLa cells, SFT was found to stimulate the uptake of both transferrin- and nontransferrin-bound Fe (iron). Assimilation of nontransferrin-bound Fe by HeLa cells stably expressing SFT was time- and temperature-dependent; both the rate and extent of uptake was enhanced relative to the activity of control nontransfected cells. Although the apparent Km for Fe uptake was unaffected by expression of SFT (5.6 versus 5.1 microM measured for control), the Vmax of transport was increased from 7.0 to 14.7 pmol/min/mg protein. Transport mediated by SFT was inhibitable by diethylenetriaminepentaacetic acid and ferrozine, Fe3+- and Fe2+-specific chelators. Because cellular copper status is known to influence Fe assimilation, we investigated the effects of Cu (copper) depletion on SFT function. After 4 days of culture in Cu-deficient media, HeLa cell Cu,Zn superoxide dismutase activity was reduced by more than 60%. Both control cells and cells stably expressing SFT displayed reduced Fe uptake as well; levels of transferrin-mediated import fell by approximately 80%, whereas levels of nontransferrin-bound Fe uptake were approximately 50% that of Cu-replete cells. The failure of SFT expression to stimulate Fe uptake above basal levels in Cu-depleted cells suggests a critical role for Cu in SFT function. A current model for both transferrin- and nontransferrin-bound Fe uptake involves the function of a ferrireductase that acts to reduce Fe3+ to Fe2+, with subsequent transport of the divalent cation across the membrane bilayer. SFT expression did not enhance levels of HeLa cell surface reductase activity; however, Cu depletion was found to reduce endogenous activity by 60%, suggesting impaired ferrireductase function may account for the influence of Cu depletion on SFT-mediated Fe uptake. To test this hypothesis, the ability of SFT to directly mediate Fe2+ import was examined. Although expression of SFT enhanced Fe2+ uptake by HeLa cells, Cu depletion did not significantly reduce this activity. Thus, we conclude that a ferrireductase activity is required for SFT function in Fe3+ transport and that Cu depletion reduces cellular iron assimilation by affecting this activity.

Fe-nitrilotriacetic acid--binding proteins associated with rat liver plasma membranes.

The uptake of nontransferrin-bound iron by hepatocytes is known to occur and may contribute to the deposition of iron and resulting injury during hemochromatosis. To examine the proteins that may function in the transport of nontransferrin-bound iron, the properties of FeNTA-binding to rat liver basolateral plasma membranes were characterized. The binding of 55FeNTA to purified liver basolateral plasma membranes was measured using a simple centrifugation assay. The binding activity could be solubilized with 0.1% octylglucoside; apparent molecular weight Mapp approximately 210 kd for the binding complex was determined by gel filtration chromatography. Immobilized metal affinity chromatography was used to further purify binding protein(s) from rat liver plasma membranes and at least six polypeptides were identified by silver staining. If associated in a stoichiometric complex, the molecular mass of these proteins would predict a size of approximately 227 kd in fairly close agreement with the gel filtration experiments. The characterization of FeNTA-binding proteins associated with basolateral membranes is the first step towards understanding elements responsible for the uptake of nontransferrin-bound iron by the liver.

Allosteric behavior in transducin activation mediated by rhodopsin. Initial rate analysis of guanine nucleotide exchange.

Photolyzed rhodopsin acts in a catalytic manner to mediate the exchange of GTP for GDP bound to transducin. We have analyzed the steady-state kinetics of this activation process in order to determine the molecular mechanism of interactions between rhodopsin, transducin, and guanine nucleotides. Initial velocities (Vo) of the exchange reaction catalyzed by rhodopsin were measured for various transducin concentrations at several fixed levels of the GTP analog, [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S). The initial rate data analysis rigorously demonstrates that rhodopsin mediates the activation of transducin by a double-displacement catalytic mechanism. The Michaelis-Menten curves determined as a function of [transducin] reveal remarkable allosteric behavior; analysis of this data yields a Hill coefficient of 2. Lineweaver-Burk plots of Vo-1 versus [transducin]-1 display curvilinearity indicative of positive cooperativity and a series of parallel lines are generated by plotting Vo-1 as a function of [transducin]-2. The plots of Vo-1 versus [GTP gamma S]-1 show no evidence of allosterism and are a parallel series. Furthermore, the allosteric behavior observed in the activation of transducin is also witnessed in the rhodopsin-catalyzed guanine nucleotide exchange of the G protein's purified alpha subunit in the absence of the beta X gamma subunit complex. The latter observation implies that the molecular basis for allosterism in the activation process resides in the interactions between the photoreceptor and transducin's alpha subunit.

Kinetic and hydrodynamic properties of transducin: comparison of physical and structural parameters for GTP-binding regulatory proteins.

Transducin is a member of the family of GTP-binding regulatory proteins that interact with cell surface receptors and that include Gs, Gi, and Go. Kinetic and physical properties of purified bovine transducin were characterized by the following results: (1) Initial rate analysis demonstrates a dissociative-type mechanism for the guanine nucleotide exchange process of transducin in the absence of rhodopsin. A second-order rate constant of kf = (1.7-2.7) X 10(-7) M-1 s-1 was determined for this reaction. (2) Equilibrium binding measurements indicated a Kd of 0.05-0.10 microM for guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding to transducin. (3) Neither the rate nor the extent of GTP gamma S binding was affected in the presence of up to 50 mM Mg2+, as compared to values obtained in the presence of excess ethylenediaminetetraacetic acid. (4) Sucrose density gradient ultracentrifugation gave S20,w values for transducin, its alpha subunit, and its beta gamma subunit complex of 4.23 +/- 0.25, 3.42 +/- 0.37, and 4.04 +/- 0.2, respectively. (5) Incubation of transducin in the presence of up to 20 mM Mg2+ did not alter its sedimentation behavior; however, the presence of guanine nucleotides did produce a shift in transducin's migration in the sucrose gradient. (6) Gel filtration over Sephacryl S-300 indicated that transducin elutes at a Stokes radius of 37.5 A and that transducin's alpha subunit displays a Stokes radius of 24 A. (7) A molecular mass of 68 kDa for transducin is derived from the determined hydrodynamic parameters. These results are compared with properties known for other G proteins, and functional differences between transducin and Gs, Gi, and Go are proposed in relation to the proteins' primary sequences.

Transducin interactions with rhodopsin. Evidence for positive cooperative behavior.

Transducin and rhodopsin belong to a family of guanine nucleotide-binding (G) protein-coupled receptor systems that provide signal transduction mechanisms resulting in numerous metabolic responses in a variety of cell types. A simple, direct binding assay has been developed to investigate the molecular interactions between transducin and rhodopsin. The binding curves generated by these studies are sigmoidal, indicating an allosteric response. The Scatchard plots of this data display an asymptotic, bell-shaped character representative of the positive cooperative behavior. A Hill coefficient, nH = 1.92, was determined and found to be in close agreement with previous kinetic studies of allosterism described for rhodopsin's catalytic mechanism (Wessling-Resnick, M., and Johnson G.L. (1987) J. Biol. Chem. 262, 3697-3705). The value for Kd app was determined to be 0.05 microM. Bmax values obtained from the binding studies suggest that oligomeric complexes of rhodopsin may be involved in interactions with transducin to form multiple high affinity binding sites for the G protein. The positive cooperative behavior demonstrated in this investigation can provide insight into the molecular basis for regulation of other G protein-coupled receptor systems.

The sorting and segregation mechanism of the endocytic pathway is functional in a cell-free system.

A cell-free system which reconstitutes early stages of receptor-mediated endocytosis has been developed, based on detection of the association between avidin-beta-galactosidase (Av beta Gal) and biotin-transferrin (B-Tf). Initially, Av beta Gal (a fluid-phase marker) and B-Tf (receptor-bound) are internalized and delivered to a common endosomal compartment in vivo and in vitro. Subsequently, these two probes enter divergent intracellular pathways: Av beta Gal is sorted from the endosome and directed for delivery to lysosomes, whereas B-Tf is segregated away from the fluid-phase marker, remaining bound to the transferrin receptor for return to the cell surface. Using the avidin-biotin association reaction to monitor the co-localization of these two probes, we have been able to reconstruct this sorting and segregation process in a cell-free system. The in vitro reaction is time-, temperature-, and ATP-dependent, and is not affected by NH4Cl; cell-free segregation of the two probes is also sensitive to N-ethylmaleimide. As these characteristics are also properties of in vitro endocytic vesicle fusion, it is likely that the latter event is a prerequisite for the sorting and segregation process. Both the in vivo and in vitro sorting of Av beta Gal and B-Tf to their respective and distinct destinations can be followed by subcellular fractionation on Percoll gradients. Our observations provide the first evidence that the cellular mechanism to identify, sort, and sequester endocytosed material can be reconstituted in a cell-free system.