Modification of Rab5 with a photoactivatable analog of geranylgeranyl diphosphate.

A photoprobe analog of geranylgeranyl diphosphate (2-diazo-3,3,3-trifluoropropionyloxy-farnesyl diphosphate or DATFP-FPP) inhibits mevalonate-dependent prenylation of in vitro translated Rab5 in rabbit reticulocyte lysate, suggesting that it competes for lipid binding to the Rab geranylgeranyl transferase. Modification of Rab5 with DATFP-FPP, demonstrated by gel mobility shift and Triton X-114 phase separation experiments, confirms that the enzyme uses the analog as a substrate. The sedimentation of DATFP-modified Rab5 as a larger mass complex on sucrose density gradients indicates that it binds to other factors in rabbit reticulocyte lysate. Most importantly, DATFP-Rab5 cross-links to these soluble factors upon exposure to UV light. Immunoprecipitation with antibodies raised against proteins known to interact with Rab5 reveals that the cross-linked complexes contain Rab escort protein and GDI-1. DATFP-Rab5 also associates with membranes in a guanosine-5'-O-(3-thiotriphosphate)-stimulated manner. However, although prenylated Rab5 can be cross-linked to two unknown membrane-associated factors by the chemical cross-linker disuccinimidyl suberate, these proteins fail to be UV cross-linked to membrane-bound DATFP-Rab5. These results strongly suggest that membrane-associated factors bind Rab5 through protein-protein interactions rather than protein-prenyl interactions. The modification of Rab5 with DATFP-FPP establishes a novel photoaffinity technique for the characterization of prenyl-binding sites.

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.

Expression of stimulator of Fe transport is not enhanced in Hfe knockout mice.

Hfe knockout (-/-) mice recapitulate many of the biochemical abnormalities of hereditary hemochromatosis (HH), but the molecular mechanisms involved in the etiology of iron overload in HH remain poorly understood. It was found previously that livers of patients with HH contained 5-fold higher SFT (stimulator of Fe transport) mRNA levels relative to subjects without HH. Because this observation suggests a possible role for SFT in HH, we investigated SFT mRNA expression in Hfe(-/-) mice. The 4- and 10-wk-old Hfe(-/-) mice do not have elevated levels of hepatic SFT transcripts relative to age-matched Hfe(+/+) mice, despite having 2.2- and 3.3-fold greater hepatic nonheme iron concentrations, respectively. Northern blot analyses of various mouse tissues revealed that SFT is widely expressed. The novel observation that SFT transcripts are abundant in brain prompted a comparison of SFT transcript levels and nonheme iron levels in the brains of Hfe(+/+) and Hfe(-/-) mice. Neither SFT mRNA levels nor nonheme iron levels differed between groups. Further comparisons of Hfe(-/-) and Hfe(+/+) mouse tissues revealed no significant differences in SFT mRNA levels in duodenum, the site of increased iron absorption in HH. Important distinctions between Hfe(-/-) mice and HH patients include not only differences in the relative rate and magnitude of iron loading but also the lack of fibrosis and phlebotomy treatment in the knockout animals.

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.

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.

Studies on the inhibition of endosome fusion by GTPgammaS-bound ARF.

Using a cell free assay, we have previously shown that ARF is not required for endosome fusion but that inhibition of fusion by GTPgammaS is dependent on a cytosolic pool of ARFs. Since ARF is proposed to function in intracellular membrane traffic by promoting vesicle biogenesis, and components of clathrin- and COP-coated vesicles have been localized on endosomal structures, we investigated whether ARF-mediated inhibition of early endosome fusion involves the recruitment or irreversible association of these proteins onto endosomal membranes. We now report that depletion of components of clathrin coated vesicles (clathrin, AP-1 and AP-2) or COPI vesicles (beta COP) does not affect the capacity of GTPgammaS-activated ARF to inhibit endosome fusion. Inhibition of fusion by activated ARF is also independent of endosomal acidification since assays performed in the presence of the vacuolar ATPase inhibitor bafilomycin A1 are equally sensitive to GTPgammaS-bound ARF. Finally, in contrast to reported effects on lysosomes, we demonstrate that ARF-GTPgammaS does not induce endosomal lysis. These combined data argue that sequestration of known coat proteins to membranes by activated ARF is not involved in the inhibition of early endosome fusion and that its capacity to inhibit fusion involves other specific interactions with the endosome surface. These results contrast with the mechanistic action of ARF on intra-Golgi transport and nuclear envelope assembly.

Immunological analysis of beta-thalassemic mouse intestinal proteins reveals up-regulation of sucrase-isomaltase in response to iron overload.

Maintenance of iron homeostasis must balance the demand for iron due to heme synthesis, which is driven by hematopoiesis, and the restricted intestinal uptake of iron, which otherwise limits absorption of this toxic element. The consequences of perturbed iron homeostasis are witnessed in inherited forms of beta-thalassemia in which erythroid hyperplasia results in enhanced intestinal iron absorption despite tissue iron overload. To gain a better understanding of intestinal factors that are induced when iron homeostasis is disrupted, a panel of monoclonal antibodies that recognize intestinal microvillous membrane proteins of the beta-thalassemic Hbbd(th3)/Hbbd(th3) mouse was established. The monoclonal antibodies were screened by differential Western blotting against normal and beta-thalassemic mouse intestine to identify antigens modulated in the disease state. Here we report the initial characterization of one immunoreactive species that is up-regulated in beta-thalassemic mouse intestine and the tentative identification of this antigen as sucrase-isomaltase. Studies in Caco-2 cells revealed the rather unexpected finding that expression of this intestinal hydrolase is increased in response to iron toxicity.

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.

Expression of SFT (stimulator of Fe transport) is enhanced by iron chelation in HeLa cells and by hemochromatosis in liver.

SFT (stimulator of Fe transport) is a novel transport protein that has been found to facilitate uptake of iron presented to cells as either Fe(II) or Fe(III). When HeLa cells are exposed to the iron chelator desferrioxamine, levels of SFT mRNA increase in an actinomycin D-sensitive manner. In contrast, cells exposed to high levels of iron down-regulate SFT expression in a time-dependent and reversible fashion. Thus, homeostatic regulation of SFT expression not only ensures that sufficient levels of iron are maintained but also limits excessive assimilation to prevent potentially harmful effects of this toxic metal. The unexpected observation that SFT transcript levels are up-regulated in hemochromatosis patients therefore suggests that enhanced SFT expression contributes to the etiology of this iron overload disorder.

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.

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.

Metabolic depletion inhibits the uptake of nontransferrin-bound iron by K562 cells.

The effect of metabolic inhibitors on nontransferrin bound iron transport by K562 cells was investigated. Incubation with 1 microM rotenone, 10 microM antimycin, or 0.5 mM 2,4-dinitrophenol effectively reduced ATP levels by approximately 50%. Both the rate and extent of Fe+3 uptake were impaired in ATP-depleted cells, which display a reduced Vmax for uptake. K562 cell ferrireductase activity was also lowered by metabolic inhibitors, suggesting that the apparent energy requirements for transport reside in the reduction of Fe+3 to Fe+2. However, ATP depletion was found to inhibit the rate and extent of Fe+2 uptake as well. Thus, the transbilayer passage of Fe+2 and/or Fe+3 appears to be an energy-requiring process. These features possibly reflect properties of the transport mechanism associated with a recently identified K562 cell transport protein, called SFT for "Stimulator of Fe Transport," since exogenous expression of its activity is also affected by ATP depletion.

Characterization and chromosomal mapping of the human gene for SFT, a stimulator of Fe transport.

Hemochromatosis is the most common genetic disorder known in man and results in progressive tissue deposition of iron leading to cirrhosis of the liver, hepatic carcinoma, congestive heart failure, endocrinopathies, and premature death. SFT (stimulator of Fe transport) is a newly discovered transport protein that facilitates uptake of iron. Recent studies have demonstrated that although SFT expression is reciprocally regulated in response to cellular iron levels, it is aberrantly upregulated in the liver of hemochromatosis patients, indicating that enhanced SFT expression contributes to the etiology of this disease. Here we report the molecular cloning and characterization of the human gene for SFT. FISH analysis maps the SFT gene to human chromosome 10q21. PCR analysis indicates 1000 nucleotides of intervening intron sequence near the 3' end of the coding region for SFT. Based on DNA sequence analysis of the additional 5' untranslated region obtained from the genomic clone, SFT lacks known metal-regulated transcriptional or translational control elements. These studies provide the basis for future elucidation of the mechanisms that control SFT expression in order to discover how this regulation is lost in hemochromatosis.

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.

Rab11 is associated with transferrin-containing recycling compartments in K562 cells.

3'RACE PCR was used to survey Rab transcripts synthesized by the human hematopoietic K562 cell line. Among the identified GTP-binding proteins, Rab11 was discovered. This result was unexpected since Rab11 previously had been found associated with polarized and secretory cells. Rab11 mRNA was abundant compared to that for other Rabs in K562 cells; protein levels represented 0.05-0.1% of total membrane protein. Localization of Rab11 using confocal immunofluoresence microscopy revealed extensive overlap with transferrin in recycling and/or exocytic compartments and suggests that Rab11 in non-polarized and non-secretory cells may play a role in the trafficking and recycling of internalized proteins.

Synthesis and effect of nonhydrolyzable xanthosine triphosphate derivatives on prenylation of Rab5D136N.

A novel and convenient method for nucleoside triphosphate synthesis was applied to the preparation of potentially nonhydrolyzable xanthosine triphosphate derivatives. The N-methylimidazolide of xanthosine 5'-monophosphate reacted rapidly with methylenediphosphonic acid and imidodiphosphonic acid to give xanthosine 5'-(beta, gamma-methylene)triphosphate and xanthosine 5'-(beta, gamma-imido)triphosphate, respectively, in good yields. Both compounds inhibited the xanthosine-diphosphate-dependent prenylation of a mutant of Rab5, Rab5D136N, the nucleotide specificity of which had been converted from GTP to xanthosine triphosphate. The results indicate that xanthosine 5'-(beta, gamma-methylene)triphosphate and xanthosine 5'-(beta, gamma-imido)triphosphate bound to the mutant protein with similar affinities and were not hydrolyzed under the assay conditions. These novel derivatives may be useful tools for the study of the role of individual GTPases mutated to xanthosine triphosphate specificity in the background of other GTP-binding proteins.

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.

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.