Reconstitution of a surface transferrin binding complex in insect form Trypanosoma brucei.

In the bloodstream of the mammalian host, Trypanosoma brucei takes up host transferrin by means of a high-affinity uptake system, presumably a transferrin receptor. Transferrin-binding activity is seen in the flagellar pocket and is absent in insect form trypanosomes. By transfection we have reconstituted a transferrin-binding complex in insect form trypanosomes. Formation of this complex requires the products of two genes that are part of a variant surface glycoprotein expression site, expression site-associated gene (ESAG) 6 (encoding a protein with GPI-anchor) and ESAG 7 (encoding a protein without any obvious membrane attachment). This complex can be precipitated by transferrin-Sepharose and by an antibody directed only against the ESAG 6 protein. Transfection of ESAG 6 or 7 alone did not result in transferrin binding. In the transfected trypanosomes, the products of ESAG 6 alone and the combination of ESAG 6 and 7 did not exclusively localize to the flagellar pocket, but were present all over the surface of the trypanosome. The reconstituted transferrin-binding complex also did not result in the uptake of transferrin. Additional proteins present in bloodstream trypanosomes, but not in sufficient amounts in insect form trypanosomes, may therefore be required for the correct routing of the transferrin-binding complex to the flagellar pocket, and for its rapid internalization after ligand binding.     

Sequence requirements for trafficking of the CRAM transmembrane protein to the flagellar pocket of African trypanosomes

CRAM is a cysteine-rich acidic transmembrane protein, highly expressed in the procyclic form of Trypanosoma brucei. Cell surface expression of CRAM is restricted to the flagellar pocket of trypanosomes, the only place where receptor mediated endocytosis takes place in the parasite. CRAM can function as a receptor and was hypothesized to be a lipoprotein receptor of trypanosomes. We study mechanisms involved in the presentation and routing of CRAM to the flagellar pocket of insect- and bloodstream-form trypanosomes. By deletional mutagenesis, we found that deleting up to four amino acids from the C terminus of CRAM did not affect the localization of CRAM at the flagellar pocket. Shortening the CRAM protein by 8 and 19 amino acids from the C terminus resulted in the distribution of the CRAM protein in the endoplasmic reticulum (ER) (the CRAM protein is no longer uniquely sequestered at the flagellar pocket). This result indicates that the truncation of the CRAM C terminus affected the transport efficiency of CRAM from the ER to the flagellar pocket. However, when CRAM was truncated between 29 and 40 amino acids from the C terminus, CRAM was not only distributed in the ER but also located to the flagellar pocket and spread to the cell surface and the flagellum. Replacing the CRAM transmembrane domain with the invariant surface glycoprotein 65-derived transmembrane region did not affect the flagellar pocket location of CRAM. These results indicate that the CRAM cytoplasmic extension may exhibit two functional domains: one domain near the C terminus is important for efficient export of CRAM from the ER, while the second domain is of importance for confining CRAM to the flagellar pocket membrane.     

Regulation of protein trafficking by glycosylphosphatidylinositol valence in African trypanosomes

The structure, biosynthesis, and attachment of glycosylphosphatidylinositol (GPI) anchors were all first determined for the variant surface glycoprotein (VSG) of African trypanosomes, and all of the basic aspects of this work have been shown to be universal in eukaryotic organisms. However, the role of GPI anchors in protein trafficking within trypanosomes has lagged behind the more standard eukaryotic model systems such as yeast and polarized epithelial cells. Trypanosomes are also highly polarized cells in which all endocytosis and exocytosis intersect at a discrete domain of the plasma membrane, the flagellar pocket. Within these convergent pathways trafficking of GPI anchored proteins correlates strongly with valence: homodimeric VSG with two GPIs is stably incorporated into the cell surface coat, heterodimeric transferrin receptor with a single GPI is found in the flagellar pocket and is slowly delivered to the lysosome for degradation, and recombinant GPI minus VSG reporters are rapidly degraded in the lysosome. Here we summarize recent data confirming this correlation using a tool kit of recombinant GPI anchored reporters, including a reporter designed to be conditionally modulated between a GPI valence of one and two.     

Clathrin-dependent targeting of receptors to the flagellar pocket of procyclic-form Trypanosoma brucei

In trypanosomatids, endocytosis and exocytosis occur exclusively at the flagellar pocket, which represents about 0.43% of the pellicle membrane and is a deep invagination of the plasma membrane where the flagellum extends from the cell. Receptor molecules are selectively retained at the flagellar pocket. We studied the function of clathrin heavy chain (TbCLH) in the trafficking of the flagellar pocket receptors in Trypanosoma brucei by using the double-stranded RNA interference approach. It appears that TbCLH is essential for the survival of both the procyclic form and the bloodstream form of T. brucei, even though structures resembling large coated endocytic vesicles are absent in procyclic-form trypanosomes. Down-regulation of TbCLH by RNA interference (RNAi) for 24 h rapidly and drastically reduced the uptake of macromolecules via receptor-mediated endocytosis in procyclic-form trypanosomes. This result suggested the importance of TbCLH in receptor-mediated endocytosis of the procyclic-form trypanosome, in which the formation of large coated endocytic vesicles may not be required. Surprisingly, induction of TbCLH RNAi in the procyclic T. brucei for a period of 48 h prohibited the export of the flagellar pocket-associated transmembrane receptor CRAM from the endoplasmic reticulum to the flagellar pocket, while trafficking of the glycosylphosphatidylinositol-anchored procyclin coat was not significantly affected. After 72 h of induction of TbCLH RNAi, procyclics exhibited morphological changes to an apolar round shape without a distinct structure of the flagellar pocket and flagellum. Although trypanosomes, like other eukaryotes, use similar organelles and machinery for protein sorting and transport, our studies reveal a novel role for clathrin in the secretory pathway of trypanosomes. We speculate that the clathrin-dependent trafficking of proteins to the flagellar pocket may be essential for the biogenesis and maintenance of the flagellar pocket in trypanosomes.     

Trypanosoma rhodesiense: antigenicity and immunogenicity of flagellar pocket membrane components

A low density membrane fraction, isolated from the bloodstream stage of Trypanosoma rhodesiense and enriched in flagellar pocket membrane, was characterized with regard to antigenicity using antibodies raised against purified flagellar pocket membrane. Mild trypsinolysis of flagellar pocket membrane released two small peptides (Mr = 13-16 X 10(3)) separated by chromatofocusing (pI = 6.8 and 5.8) that were antigenic as monitored by fused rocket immunoelectrophoresis. Both of these antigenic peptides were enriched in relative fluorescence when flagellar pocket membrane was prepared from surface labeled (fluorescamine-beta-cyclodextrin) trypanosomes, indicating that cleaved peptides were on the external (luminal) side of the flagellar pocket membrane. More extensive release of fluorescamine labeled flagellar pocket membrane components was affected using mild detergent treatment (0.15% Zwittergent 3-12/0.4% Triton X-100), crossed immunoelectrophoresis separating two prominent antigens was more pronounced after incubation of flagellar pocket membrane with either porcine pancreas phospholipase A2 or umbilical cord sphingomyelinase. The use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent electroblotting to nitrocellulose also revealed two principal flagellar pocket membrane antigens (Mr approximately 60 and 66 X 10(3)), the latter showing greater release after exposure to sphingomyelinase or phospholipase, compared to mild detergent or 50 mM acetate, pH 5.0. Both antigens were glycoprotein as judged by electroblotting and the use of concanavalin A conjugated horseradish peroxidase as probe. Neither flagellar pocket membrane antigen was found to react with monoclonal antibodies prepared against T. rhodesiense variable surface antigen. The use of flagellar pocket membrane in the presence of Freund's complete adjuvant was found to protect mice against challenge infections with either the CP344 clone or uncloned CT Well-come isolate of T. rhodesiense.     

Rab11 mediates selective recycling and endocytic trafficking in Trypanosoma brucei

Trypanosoma brucei possesses a streamlined secretory system that guarantees efficient delivery to the cell surface of the critical glycosyl‐phosphatidylinositol (GPI)‐anchored virulence factors, variant surface glycoprotein (VSG) and transferrin receptor (TfR). Both are thought to be constitutively endocytosed and returned to the flagellar pocket via TbRab11+ recycling endosomes. We use conditional knockdown with established reporters to investigate the role of TbRab11 in specific endomembrane trafficking pathways in bloodstream trypanosomes. TbRab11 is essential. Ablation has a modest negative effect on general endocytosis, but does not affect turnover, steady state levels or surface localization of TfR. Nor are biosynthetic delivery to the cell surface and recycling of VSG affected. TbRab11 depletion also causes increased shedding of VSG into the media by formation of nanotubes and extracellular vesicles. In contrast to GPI‐anchored cargo, TbRab11 depletion reduces recycling of the transmembrane invariant surface protein, ISG65, leading to increased lysosomal turnover. Thus, TbRab11 plays a critical role in recycling of transmembrane, but not GPI‐anchored surface proteins. We proposed a two‐step model for VSG turnover involving release of VSG‐containing vesicles followed by GPI hydrolysis. Collectively, our results indicate a critical role of TbRab11 in the homeostatic maintenance of the secretory/endocytic system of bloodstream T. brucei.      

Characterization of glyceraldehyde-3-phosphate dehydrogenase as a novel transferrin receptor

A majority of cells obtain of transferrin (Tf) bound iron via transferrin receptor 1 (TfR1) or by transferrin receptor 2 (TfR2) in hepatocytes. Our study establishes that cells are capable of acquiring transferrin iron by an alternate pathway via GAPDH.These findings demonstrate that upon iron depletion, GAPDH functions as a preferred receptor for transferrin rather than TfR1 in some but not all cell types. We utilized CHO-TRVb cells that do not express TfR1 or TfR2 as a model system. A knockdown of GAPDH in these cells resulted in a decrease of not only transferrin binding but also associated iron uptake. The current study also demonstrates that, unlike TfR1 and TfR2 which are localized to a specific membrane fraction, GAPDH is located in both the detergent soluble and lipid raft fractions of the cell membrane. Further, transferrin uptake by GAPDH occurs by more than one mechanism namely clathrin mediated endocytosis, lipid raft endocytosis and macropinocytosis. By determining the kinetics of this pathway it appears that GAPDH-Tf uptake is a low affinity, high capacity, recycling pathway wherein transferrin is catabolised. Our findings provide an explanation for the detailed role of GAPDH mediated transferrin uptake as an alternate route by which cells acquire iron.     

Identification of the segments of the mouse transferrin receptor 1 required for mouse mammary tumor virus infection

Most enveloped viruses enter cells through binding of virion surface envelope proteins to receptors found on the plasma membrane of the cell. The beta retrovirus mouse mammary tumor virus (MMTV) uses transferrin receptor 1 (TfR1) to enter cells in a pH-dependent mechanism, probably co-trafficking with TfR1 to an acidic compartment where virus entry occurs. We have shown here that, although mouse and rat TfR1 function as entry receptors, cat, dog, hamster, or human TfR1s do not support MMTV infection. We also demonstrated that MMTV entry is independent of transferrin, iron, and the TfR1 cofactor hereditary hematochromatosis HFE protein. Using chimeric mouse/human hybrid TfR1 constructs, we determined the site of interaction with MMTV and found that it maps to two segments physically disparate from the TfR and HFE binding sites. Thus, MMTV has apparently evolved to enter cells independently of the iron status of the host.     

Endocytosis, membrane recycling and sorting of GPI-anchored proteins: Trypanosoma brucei as a model system

In the flagellated protozoon Trypanosoma brucei, endo- and exocytosis are restricted to a small area of the plasma membrane, the flagellar pocket. All endosomal compartments and the single Golgi complex are located within the posterior part of the cell between the flagellar pocket and the nucleus. The use of reverse genetic tools, including RNA interference, in combination with quantitative 3D-fluorescence and electron microscopic techniques has provided an insight into endosomal membrane traffic, which occurs at a very high rate and appears to exhibit a lower level of complexity than in mammalian cells. The flagellate is an excellent model system for studies on endocytosis, sorting and recycling of glycosylphosphatidylinositol-anchored glycoproteins, because 10(7) molecules of the variant surface glycoprotein form a dense coat at the cell's surface. Because the endocytic rate varies widely at different stages in the parasite's life cycle, trypanosomes may be used for investigating developmental aspects of their endocytic system.     

The extracellular regions of PSMA and the transferrin receptor contain an aminopeptidase domain: implications for drug design

The Aeromonas proteolytica aminopeptidase (AMP), Pseudomonas sp. (RS-16) carboxypeptidase G2 (CPG2), and Streptomyces griseus aminopeptidase (SGAP) are zinc dependent proteolytic enzymes with cocatalytic zinc ion centers and a conserved aminopeptidase fold. A BLAST search with the sequence of the solved AMP structure indicated that a similar domain could be found in prostate-specific membrane antigen (PSMA) and the transferrin receptor (TfR). When the PSMA or TfR sequence was input into the THREADER program, the top structural matches were SGAP and AMP confirming that these are structurally conserved domains. Optimal sequence alignment of PSMA and TfR using the known three-dimensional structures of AMP, CPG2, and SGAP shows that the critical amino acids involved in forming the catalytic pocket are conserved in PSMA but absent in the TfR. The specificity pocket in AMP is formed from four aromatic side chains and the equivalent region in CPG2/PSMA has a changed sequence pattern. Since CPG2 and PSMA are folate hydrolases, the changed specificity pocket leaves space to accommodate the large pteroate moiety of folic acid. In contrast, no enzyme function has been ascribed to the TfR.     

HFE association with transferrin receptor 2 increases cellular uptake of transferrin-bound iron

Mutations in either HFE or transferrin receptor 2 (TfR2) cause decreased expression of the iron regulatory hormone hepcidin and hemochromatosis. HFE and TfR2 were recently discovered to form a stable complex at the cell membrane when co-expressed in heterologous cell lines. We analyzed the functional consequences of the co-expression of these proteins using transfected TRVb cells, a Chinese hamster ovary derived cell line without endogenous HFE or transferrin receptor. The co-expression of TfR2 in TRVb cells expressing HFE led to accelerated HFE biosynthesis and late-Golgi maturation, suggesting interaction prior to cell surface localization. The co-expression of HFE in cells expressing TfR2 led to increased affinity for diferric transferrin, increased transferrin-dependent iron uptake, and relative resistance to iron chelation. These observations indicate that HFE influences the functional properties of TfR2, and suggests a model in which the interaction of these proteins might influence signal transduction to hepcidin.     

A conserved flagellar pocket exposed high mannose moiety is used by African trypanosomes as a host cytokine binding molecule

Trypanosomes use antigenic variation of their variant-specific surface glycoprotein (VSG) coat as defense against the host immune system. However, in order to sustain their growth, they need to expose conserved epitopes, allowing host macromolecule binding and receptor-mediated endocytosis. Here we show that Trypanosoma brucei uses the conserved chitobiose-oligomannose (GlcNAc(2)-Man(5-9)) moieties of its VSG as a binding ligand for tumor necrosis factor (TNF), a host cytokine with lectin-like properties. As endocytosis in trypanosomes is restricted to the flagellar pocket, we show that soluble flagellar pocket extracts, and in particular soluble VSG, inhibit the binding of (125)I-TNF to trypanosomes. The interaction between TNF and VSG is confirmed by affinity chromatography, biosensor, and dot-blot affinity measurements, and soluble VSG inhibition of TNF-mediated trypanolysis. In all approaches, removal of N-linked carbohydrates abrogates the TNF-VSG interaction. In addition, synthetic high mannose oligosaccharides can block TNF-VSG interactions, and a VSG glycopeptide carrying the GlcNAc(2)-Man(5-9) moiety is shown to inhibit TNF-mediated trypanosome killing in mixed parasite/macrophage cell cultures. Together, these results support the observation that TNF plays a role in growth control of trypanosomes and, moreover, suggest that, by the use of conserved VSG carbohydrates as lectin-binding epitopes, trypanosomes can limit the necessity to express large numbers of invariant surface exposed receptors.     

Oligomerized transferrin receptors are selectively retained by a lumenal sorting signal in a long-lived endocytic recycling compartment

Cross-linking of surface receptors results in altered receptor trafficking in the endocytic system. To better understand the cellular and molecular mechanisms by which receptor cross-linking affects the intracellular trafficking of both ligand and receptor, we studied the intracellular trafficking of the transferrin receptor (TfR) bound to multivalent-transferrin (Tf10) which was prepared by chemical cross- linking of transferrin (Tf). Tf10 was internalized about two times slower than Tf and was retained four times longer than Tf, without being degraded in CHO cells. The intracellular localization of Tf10 was investigated using fluorescence and electron microscopy. Tf10 was not delivered to the lysosomal pathway followed by low density lipoprotein but remained accessible to Tf in the pericentriolar endocytic recycling compartment for at least 60 min. The retained Tf10 was TfR-associated as demonstrated by a reduction in surface TfR number when cells were incubated with Tf10. The presence of Tf10 within the recycling compartment did not affect trafficking of subsequently endocytosed Tf. Retention of Tf10 within the recycling compartment did not require the cytoplasmic domain of the TfR since Tf10 exited cells with the same rate when bound to the wild-type TfR or a mutated receptor with only four amino acids in the cytoplasmic tail. Thus, cross-linking of surface receptors by a multivalent ligand acts as a lumenal retention signal within the recycling compartment. The data presented here show that the recycling compartment labeled by Tf10 is a long-lived organelle along the early endosome recycling pathway that remains fusion accessible to subsequently endocytosed Tf.     

Factors affecting the level and localization of the transferrin receptor in Trypanosoma brucei

Transfer of bloodstream-form Trypanosoma brucei variant 221a from calf serum to dog serum-based medium induces acute iron starvation, as the transferrin receptor (Tf-R) of variant 221a binds dog Tf poorly. We show here that transfer to dog serum induces a 3-5-fold increase in Tf-R mRNA and protein within one doubling time (8 h). Because iron stores are still high 8 h after transfer, we infer that the signal for Tf-R overproduction is the decreased availability of cytosolic iron when cellular iron import drops. Up to 30% of the extra Tf-R spills out of the flagellar pocket onto the pellicular surface. Because the 5-fold increase in Tf-R is accompanied by a 5-fold increase in bovine Tf uptake, the up-regulation of Tf-R levels in response to Tf starvation helps the trypanosome to compete for limiting amounts of Tf. We noted that Tf-R levels also vary in calf serum medium. Cells in dense cultures contain up to 5-fold more Tf-R mRNA and protein than in dilute cultures. Only one-tenth of the extra Tf-R reaches the pellicular surface. The increase cannot be explained by a lack of Tf or to cell density sensing but is due to pericellular hypoxia. Our results show that bloodstream-form trypanosomes can regulate the expression of the two Tf-R subunit genes and the localization of their gene products in a flexible manner. This flexibility is made possible by the promoter-proximal position of the two genes in the variant surface glycoprotein expression site.     

Molecular Evolution of the Transferrin Receptor/Glutamate Carboxypeptidase II Family

The transferrin receptor family is represented by at least seven different homologous proteins in primates. Transferrin receptor (TfR1) is a type II membrane glycoprotein that, as a cell surface homodimer, binds iron-loaded transferrin as part of the process of iron transfer and uptake. Other family members include transferrin receptor 2 (TfR2), glutamate carboxypeptidase II (GCP2 or PSMA), N-acetylated α-linked acidic dipeptidase-like protein (NLDL), N-acetylated α-linked acidic dipeptidase 2 (NAALAD2), and prostate-specific membrane antigen-like protein (PMSAL/GCPIII). We compared 86 different sequences from 24 different species, from mammals to fungi. Through this comparison, we have identified several highly conserved residues specific to each family not previously associated with clinical mutations. The evolutionary history of the TfR/GCP2 family shows repeated episodes of duplications consistent with recent theories that nondispensable, slowly evolving genes are more likely to form multiple gene families. [Reviewing Editor: Dr. Gail Simmons]     

Pumping iron: the strange partnership of the hemochromatosis protein, a class I MHC homolog, with the transferrin receptor

People suffering from hereditary hemochromatosis (HH) can not regulate the uptake of iron properly and gradually accumulate iron in their body over their lifetime. The protein involved in HH, HFE, has been recently identified as a class I major histocompatibility complex (MHC) homolog. The wild-type HFE associates and co-traffics with the transferrin receptor (TfR). The mutation responsible for 83% of HH (C260Y) results in the failure of HFE to form a critical disulfide bond, bind β₂ microglobulin, bind TfR, and traffic to the cell surface. In non-polarized cells, the partnership of HFE and TfR results in decreased iron uptake into cells. The mechanism whereby a class I MHC homolog modifies the function of a membrane receptor and how this dynamic complex of molecules regulates iron transport across intestinal epithelial cells is the subject of this review.     

Endocytosis by African trypanosomes. I. Three-dimensional structure of the endocytic organelles in Trypanosoma brucei and T. congolense

African trypanosomes multiply rapidly during the course of infection obtaining nutrients from the host blood and other body fluids. The organelles involved in endocytosis were revealed ultrastructurally using horseradish peroxidase (HRP) and colloidal gold coupled to bovine transferrin (Au-Tf) or bovine serum albumin (Au-BSA). At 0 degree C the markers bound to the cell surface and neither entered the flagellar pocket nor were internalized. Upon warming to 37 degrees C, the markers were found in the flagellar pocket and appeared to enter all the intracellular endocytic organelles within 5 min. Serial sectioning of resin-embedded cells was employed to obtain pseudo three-dimensional views of these organelles. The organelles involved were of three types: (1) small vesicles and cisternae (20-25 nm in diameter), (2) large tubular networks (200 nm diameter) similar to endosomes of mammalian cells, and (3) large lysosome-like vesicles. These organelles were located between the flagellar pocket and the nucleus and were also associated with one face of the Golgi apparatus. In pulse-chase experiments HRP was not detected in intracellular organelles after 410 min but Au-Tf was seen in residual bodies. No exocytosis of Au-Tf from the flagellar pocket was observed. The data suggests that the processes of endocytosis in these parasitic protozoa may be similar to the endocytic processes found in mammalian cells.     

A differential role for actin during the life cycle of Trypanosoma brucei

Actin is expressed at similar levels but in different locations in bloodstream and procyclic forms of Trypanosoma brucei. In bloodstream forms actin colocalizes with the highly polarized endocytic pathway, whereas in procyclic forms it is distributed throughout the cell. RNA interference demonstrated that in bloodstream forms, actin is an essential protein. Depletion of actin resulted in a rapid arrest of cell division, termination of vesicular traffic from the flagellar pocket membrane leading to gross enlargement of the pocket, loss of endocytic activity and eventually cell death. These results indicate that actin is required for the formation of coated vesicles from the flagellar pocket membrane, which is the first step in the endocytic pathway. Although loss of actin in procyclic cells did not affect growth, the trans region of the Golgi became distorted and enlarged and appeared to give rise to a heterogeneous population of vesicles. However, the flagellar pocket was not affected. These findings suggest that trypanosomes have different functional requirements for actin during the bloodstream and procyclic phases of the life cycle.     

Endocytosis of a glycosylphosphatidylinositol-anchored protein via clathrin-coated vesicles,sorting by default in endosomes,and exocytosis via RAB11-positive carriers

Recently, proteins linked to glycosylphosphatidylinositol (GPI) residues have received considerable attention both for their association with lipid microdomains and for their specific transport between cellular membranes. Basic features of trafficking of GPI-anchored proteins or glycolipids may be explored in flagellated protozoan parasites, which offer the advantage that their surface is dominated by these components. In Trypanosoma brucei, the GPI-anchored variant surface glycoprotein (VSG) is efficiently sorted at multiple intracellular levels, leading to a 50-fold higher membrane concentration at the cell surface compared with the endoplasmic reticulum. We have studied the membrane and VSG flow at an invagination of the plasma membrane, the flagellar pocket, the sole region for endo- and exocytosis in this organism. VSG enters trypanosomes in large clathrin-coated vesicles (135 nm in diameter), which deliver their cargo to endosomes. In the lumen of cisternal endosomes, VSG is concentrated by default, because a distinct class of small clathrin-coated vesicles (50-60 nm in diameter) budding from the cisternae is depleted in VSG. TbRAB11-positive cisternal endosomes, containing VSG, fragment by an unknown process giving rise to intensely TbRAB11- as well as VSG-positive, disk-like carriers (154 nm in diameter, 34 nm in thickness), which are shown to fuse with the flagellar pocket membrane, thereby recycling VSG back to the cell surface.     

Defective trafficking and localization of mutated transferrin receptor 2: implications for type 3 hereditary hemochromatosis

Transferrin receptor 2 (TfR2), a homologue of transferrin receptor 1 (TfR1), is a key molecule involved in the regulation of iron homeostasis. Mutations in TfR2 result in iron overload with similar features to HFE-associated hereditary hemochromatosis. The precise role of TfR2 in iron metabolism and the functional consequences of disease-causing mutations have not been fully determined. We have expressed wild-type and various mutant forms of TfR2 that are associated with human disease in a mouse liver cell line. Intracellular and surface analysis shows that all the TfR2 mutations analyzed cause the intracellular retention of the protein in the endoplasmic reticulum, whereas the wild-type protein is expressed in endocytic structures and at the cell surface. Our results indicate that the majority of mutations that cause type 3 hereditary hemochromatosis are a consequence of the defective localization of the protein.