1 Department of Biochemistry and Molecular Biology, Royal Free and University College Medical School, London NW3 2PF, 2 Metalloprotein Research Group, The Randall Centre for Molecular Mechanisms of Cell Function, New Hunt's House, King's College London, Guy's Campus, London SE1 1UL and 3 Department of Crystallography, Birkbeck College, Malet Street, London WC1E 7HX, UK
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Abstract |
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Keywords: ceruloplasmin/ferroxidase/hephaestin/homology modelling
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Introduction |
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Hephaestin is homologous to ceruloplasmin (Cp), a member of the family of `blue' copper oxidases that includes ascorbate oxidase (Messerschmidt, 1992) and laccase (Messerschmidt and Huber, 1990
). Copper oxidases are expressed by a wide variety of organisms and perform a disparate number of functions. Members of this family form a nexus between copper and iron metabolism in both yeast and metazoa. For example, the trinuclear copper-containing oxidase fet3 from yeast Saccharomyces cerevisiae is a structural and functional homologue of Cp and has been shown to be a mandatory component for influx of iron by the Ftr1 transporter (Askwith et al., 1994
; Stearmen et al., 1996).
Ceruloplasmin is the principal copper-binding protein of the plasma, containing ~95% of total plasma copper, and contains at least six copper atoms per molecule (Zaitseva et al., 1996). Ceruloplasmin has been implicated in the efflux of iron from the tissues into the plasma. The evidence for this comes from the autosomal recessive disorder aceruloplasminaemia, caused by a genetic defect in the gene encoding Cp and the murine model for aceruloplasminaemia. Aceruloplasminaemic patients cannot mobilize iron from their tissues (Harris et al., 1995
; Yoshida et al., 1995
) and suffer marked iron elevation in the liver, pancreas and brain (Morita et al., 1995
) that can lead to diabetes mellitus and neurological symptoms (Klomp and Gitlin, 1996
). In Cp/ mice cellular iron uptake was normal; however, there was severe impairment of iron efflux from reticuloendothelial cells and hepatocytes (Harris et al., 1999
). These observations indicate that Cp plays an essential role in oxidizing Fe2+ to Fe3+ for efficient uptake of iron by transferrin (Tf). The ferroxidase activity of the Cp would require a ligand binding site and putative iron-binding residues (E272, E935, H940 and D1025) have been identified in the X-ray structure of ceruloplasmin (Lindley et al., 1997
).
The biochemical activity of Heph has yet to be elucidated but the extensive sequence similarity between the ecto-domain of hephaestin and ceruloplasmin (~50% sequence identity) suggests that they share the same fold and further implies that it may function as a ferroxidase. Natural mutations in Cp and hephaestin genes result in different phenotypes probably reflecting their distinct site-specific roles.
Vulpe and co-workers used radiation hybridization to locate the hHeph gene, mapping it to within 14.55 cR of DXS1194 in Xq11-q12 (lod score = 7.81), a region with homology by synteny to the sla region (Vulpe et al., 1999). The availability of the human genome sequence has presented the opportunity to locate and characterize the human homologue of the murine hephaestin gene.
In this paper, we have assembled the genomic and cDNA structure of the human hephaestin (hHeph) gene from sequences in publicly accessible databases. Furthermore, we have built a three-dimensional homology model of the putative copper oxidase domain using the coordinates from the structure of human ceruloplasmin (Zaitseva et al., 1996) as a template with a view to understanding the ferroxidase activity of hephaestin. The model of this novel protein supports the role of hephaestin as a membrane-tethered ferroxidase.
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Materials and methods |
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Initial searches were performed with the BLAST algorithm (Altschul et al., 1997) using the murine hephaestin protein and nucleotide sequences (Accession No. AF082567). Searches were performed on the OWL database (http://www.bioinf.man.ac.uk/dbbrowser/OWL/OWL.html) and from the Chromosome X genomic DNA sequence available from Sanger Centre at http://www.sanger.ac.uk/HGP/ChrX.html. The human hephaestin sequence (AJ296162) was assembled and aligned using the Omega 2.0 suite of sequence analysis software (Oxford Molecular, Oxford, UK). Following assembly and identification of the hHeph ORF, the physiochemical parameters were calculated by ProtParam tool at http://www.expassy.ch/tool/protparam.html.
Molecular modelling
Comparative modelling of human hephaestin (AJ296162) was carried out with the Modeler 4.0 package using hCp as the template structure (Sali and Blundell, 1993). This used a distance restraint algorithm with spatial restraints extracted from alignments of target sequences with the template structure. Model consistency and viability were assessed by use of the structure validity software Procheck (Laskowsky et al., 1993
), WHAT IF available on-line (http://biotech.ebi.ac.uk:8400/chk/whatif/index.html) (Vriend, 1990
). Pairwise sequence alignment between the structural template, human ceruloplasmin (hCp) (PDB code 1KCW) and human hephaestin was calculated using a protein identity matrix in Clustal X with a gap penalty of 6 and a gap extension of 1.65 (Higgins and Sharp, 1988
). The initial sequence alignment between hCp and hHeph was iteratively modified to optimize the ProsaII energy profile of the corresponding model obtained by Modeler until no further improvement could be achieved. The final alignment accounts for 47% sequence identity between hCp and hHeph.
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Results |
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The mouse hephaestin protein sequence was used in a BLAST search (Altschul et al., 1997) of the OWL database, which revealed two overlapping human sequences. One sequence (Accession No. AB014598) had been reported as a cDNA from the human brain for an unidentified protein (Ishikawa et al., 1998
). The other sequence (Accession No. AL030998) had been identified as a putative ceruloplasmin and factor V and VIII homologue as part of the annotation of the human genome sequence project. There was complete identity within the overlap of 586 amino acid residues. Together, the two sequences accounted for the 3291 nucleotides corresponding to the amino acid residues (601157) of the murine hephaestin sequence. The remainder of the 5' end of the human hephaestin gene was assembled from BLAST searches of the human X chromosome genome sequence using the mouse hephaestin sequence as a probe. This search also confirmed the cDNA sequence that had been obtained from the two earlier clones.
The structure of human hephaestin gene
The human hephaestin gene comprises 20 exons and spans ~100 kB. In comparison, the hCp gene has 19 exons spanning across ~45 kB (Daimon et al., 1995). It has two transcripts of 3.7 and 4.2 kB, which arise from alternative polyadenylation sites in the 3' UTR. In this study we characterized the 713 bp of the 3' UTR of human hephaestin gene and the polyadenylation sequence [AA(T/U)AAA] remains to be determined. The first 45 bp of the 3'UTR of human and mouse hephaestin are found to be 66% homologous whilst hHeph and hCp share only 15% sequence identity. It has been suggested that the 3' UTR of mRNA encoding structurally homologous proteins can be markedly conserved among species (Yaffe et al., 1985
).
Figure 1 shows the deduced ORF of the complete human hephaestin and the 5' and 3' UTRs. The positions of the 20 exons are also indicated and the putative signal peptide sequence (Nielsen et al., 1997
) is highlighted. The start codon follows a sequence that is consistent with Kozak's rules (Kozak, 1996
).
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The alignment of the deduced amino acid sequence of human hephaestin (hHeph) with the murine homologue (mHeph) and human (hCp) and mouse ceruloplasmin (mCp) is shown in Figure 2. The complete hHeph cDNA encodes a protein of 1158 amino acids that has ~85% sequence identity at both the protein and the nucleotide level with the corresponding mouse protein. hHeph and mHeph have ~50% identity with human and mouse ceruloplasmin. Immediately after the start codon, hHeph has a short putative N-terminal signal peptide (von Heijne, 1983
, 1985
). Analysis, using neural network-based methods for signal peptidase cleavage sites, indicates that the most likely cleavage site for the human hephaestin sequence is between positions 23 and 24 (TDG-AT) (Nielsen et al., 1997
).
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Sequence alignment of both the human and mouse hephaestin with ceruloplasmin shows that hephaestin contains an additional 86 amino acid residues at the C-terminus that are highly conserved (Figure 2). This region includes the predicted transmembrane (TM) domain [residues 11031123, human numbering (Klein et al., 1999
)], which has a type Nt topology (Hartmann et al., 1989
). The region in hHeph also contains three characteristic membrane targeting signals (S/T-X-L/V) recognized by proteins containing a PDZ domain (Saras and Heldin, 1996
). One of these motifs (S1145IL) would be a candidate for PDZ proteins, which play a role in targeting/sorting membrane proteins in the polarized epithelial cells (Kaech et al., 1998
; Perego et al., 1999
). The murine hephaestin sequence also shows conservation of the S1145IL motif.
The D1148DSFKL1153L motif in the C-terminus of hHeph protein may play a role in the sorting of hephaestin. It also has the characteristic acidic amino acids (Asp: 11481149), 45 residues upstream of the di-leucine motif that are significant for protein trafficking (Pond et al., 1995; Geisler et al., 1998
). A glutamate/di-leucine motif towards the C-terminus is required for the correct expression of vasopressin V2 receptor (Schulein et al., 1998
). Di-leucine residues can also play a significant role in trafficking of proteins from the trans-Golgi apparatus to a late endosomal/lysosomal compartment (Letourneur and Klausner, 1992
; Bremnes et al., 1994
; Ogata and Fukuda, 1994
). In addition, Valdenaire et al. have shown that di-leucine-based motifs account for different subcellular localization of the isoforms for the human endothelial converting enzyme (ECE-1) (Valdenaire et al., 1999
). Interaction of the above-mentioned motifs with cellular proteins is likely to play a role in the subcellular localization of hephaestin. In a recent study, hephaestin has been localized to the supranuclear region of the duodenal endothelial cells (Frazer et al., 2001
).
Tissue distribution of hephaestin
Murine hephaestin was cloned from the duodenum (Vulpe et al., 1999) and the cDNA for part of the human hephaestin sequence was isolated from the brain (AB014598). Searches of the murine and human expressed sequence tags (EST) databases indicate widespread tissue distribution for the hephaestin. At present human Heph ESTs available in dbEST at NCBI server indicate that Heph is expressed in the breast (AW601924), colon (AW361871) and bone trabecular cells (AI752764). Vulpe and co-workers have also reported a human EST (W46354) from senescent fibroblast (Vulpe et al., 1999
). A search with human hHeph also revealed a bovine EST (AW654997) from pooled tissue from lymph node, ovary, fat, hypothalamus and pituitary with 90% sequence identity. A number of murine ESTs with over 99% sequence identity have been identified: from ES neural cells (AW244344), lymph (AA390074) and foetus (AI529524).
Homology model of human hephaestin
In the absence of structural data for hephaestin, a comparative model for human hephaestin was built using the known crystal structure of human ceruloplasmin (Zaitseva et al., 1996) in view of the high degree of sequence similarity (47% sequence identity) between human hephaestin and ceruloplasmin. Figure 3a
d show the overall structure of the human hephaestin protein. The protein comprises three major domains, which share extensive internal sequence homology (3540%) analogous to ceruloplasmin (Ortel et al., 1984
) and to the three homologous (A-type) domains in factor V and VIII (Church et al., 1984
). Moreover, examination of the hHeph model reveals that the structural features important for Cp function are likely to be conserved in hHeph. These include histidine residues involved in copper binding, the putative iron-binding residues and cysteine residues involved in disulphide bond formation (Figures 2, 3g and 3h
).
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Ceruloplasmin contains at least six copper atoms. Three of these occupy mononuclear centres in domains 2, 4 and 6 and the remaining three form a trinuclear cluster sited at the interface between domain 1 and 6 (Zaitseva et al., 1996). The hephaestin model shows that the geometry for the copper centres is maintained. The putative ligands involved in binding the active site copper atoms are shown in Figure 3g and h
. In hephaestin, like ceruloplasmin, the domain 4 and 6 mononuclear coppers have a typical type I blue copper environment with two histidine ligands, a methionine and a cysteine. However, the mononuclear copper site in domain 2 is structurally different from the mononuclear sites in domain 4 and 6 as it lacks a methionine ligand (Figure 2
). The trinuclear copper cluster in hephaestin is coordinated by four pairs of histidine residues. Two pairs, H126, H128 and H186, H188 are donated by domain 1. A proline and a serine residue separate the components of each pair, respectively. Domain 6 donates the remaining pairs H1003, H1005 and H1045, H1047, separated by phenylalanine and cysteine residues, respectively. The C1046 coordinates the mononuclear copper in domain 6. Like ceruloplasmin, two of the copper atoms in the trinuclear cluster are liganded by three histidines (H128, H186 and H1047; H188, H1005 and H1047), whilst the copper further away from the domain 6 copper atom (a type II copper in ceruloplasmin) is bound by only two histidine residues (H126 and H1003); in all cases the pairs of histidines bridge two copper atoms.
Iron binding site
Crystallographic data from ceruloplasmin suggest that iron binding involves the residues E272, E935, H940 and D1025 (Lindley et al., 1997). Also, structural information on the yeast homologue Fet3 derived from homology modelling based on the ascorbate oxidase backbone revealed E185, Y354 and D409 as potential iron ligands (Bonaccorsi di Patti et al., 1999
). The putative iron-binding site in Cp has also been identified on the hephaestin model (Figure 3h
). The homologous residues in hephaestin coordinating the iron were identified as E300, E960, H965 and D1050. The charge distribution over the top surface of the human hephaestin is shown in Figure 3e and f
. A series of negatively charged residues around this region were also identified (D259, 302, 998, 955, 1023) in human hephaestin. These aspartate residues could provide an electrostatic pathway for ferric (Fe3+) ions when released from the putative active site.
Potential glycosylation sites in hephaestin
The hHeph protein contains six potential N-glycosylation motifs at positions 164, 244, 588, 714, 829 and 931 and several putative attachment sites for O-glycosylation at Thr174, -520, -528 and -846 and Ser145 and -1070. Although analysis of the hCp sequence shows potential glycosylation sites at positions 119, 208, 339, 378, 569, 743, 907. Putnam and co-workers have shown that N-linked carbohydrate moieties are only present at positions 119, 339, 378 and 743 (Takahashi et al., 1984). Sequence alignment (Figure 2
) of hephaestin and ceruloplasmin reveals that none of the N-linked glycosylated sites in Cp are conserved in either the human or murine hephaestin. However, the non-glycosylated residues (Asn: 208, 569, 907) in hCp are conserved in hephaestin. Analysis of the hHeph model shows that residues 164, 588, 714 and 829 are good candidates for N-linked glycosylation. Residues 145, 528 and 846 may have possible O-linked sugars present whereas residues 244 and 931 and Thr174 and -520 are partially exposed.
Cysteine location
The mature hCp has 14 cysteine residues, of which 10 are involved in disulphide bond formation. Three residues (C319, C680, C1021, hCp numbering) are involved in type I copper binding (Figure 2). The remaining free cysteine residue (C221), in domain 2, is present on a surface-exposed region. Comparison of the hephaestin model with the ceruloplasmin structure model shows that cysteine residues involved in the formation of disulphide bonds [Cys: domain 1 (180 + 206), domain 2 (285 + 366), domain 3 (534 + 560), domain 4 (637 + 718) and domain 5 (877 + 903)] are conserved with respect to ceruloplasmin. The three copper-binding residues (Cys: 334, 686 and 1033) are also conserved, as is the free cysteine (C221, hCp numbering) in hHeph (C249). In addition, both human and mouse heph have a cysteine residue at position 747 (hHeph numbering), which is absent in the Cp protein. Murine hephaestin also features an additional cysteine residue at position 1114 in the putative trans-membrane domain in the C-terminus. Examination of the three-dimensional model structure of hHeph reveals that the free C249 and C747 are located too far apart (41.5 Å) to form a disulphide bridge.
The sla mutation
The homology model has enabled us to designate the sla mutation on the human hephaestin structure and offer an explanation for the absence or limited efflux of iron in the mutant protein. In sla mice the 155 kDa band expected for wild-type hephaestin is absent and a smaller band of 110 kDa is present, indicating that the mice synthesize a truncated form of the protein (Frazer et al. 1999). The sla mutation has been identified as a deletion of 582 nucleotides (an omission of 194 residues) towards the N-terminus of the murine hepheastin, resulting in an in-frame translation of a truncated protein (Vulpe et al., 1999
). The region deleted by the sla mutation is depicted in magenta on the ribbon diagram in Figure 3c
and includes residues equivalent to 508694 in the human hephaestin sequence, which would be lacking in the mutated sla protein. The type 1 copper and the labile copper in domain 3 and 4 are depicted in yellow and blue, respectively.
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Discussion |
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The full-length human gene shows striking conservation (~85%) with the murine homologue at both the nucleotide and the protein level. Furthermore, the extensive homology with ceruloplasmin (~50%) gives confidence in the hephaestin model presented in this paper. The model shows that key features (histidine residues involved in type I, II and III copper binding, methionine residues involved in type I copper coordination, putative iron-binding site and the cysteine residues involved in disulphide bonds) are all preserved with respect to ceruloplasmin. An explanation for the putative ferroxidase activity of the hephaestin protein with respect to iron absorption is explored. In addition, a structural explanation for the sla mutation with respect to ferroxidase activity is proferred.
The human hephaestin sequence was in part determined from a cDNA clone from the brain (AB014598) (Ishikawa et al., 1998). It is already known that a glycosylphosphatidylinositol (GPI)-anchored form of ceruloplasmin is expressed by astrocytes in the central nervous system (Patel and David, 1997
; Patel et al., 2000
). At present, it is unclear why two membrane-bound copper oxidases are present in the brain. It may be that they have different substrates or activities. However, it is also possible that the two proteins are trafficked to different cellular compartments. The GPI anchor has previously been identified as an apical target for various cell types (Lisanti et al., 1991
). The C-terminal domain in hephaestin, that includes the predicted transmembrane (TM) domain [residues 11031123, human numbering (Klein et al., 1999
)], has a type Nt topology (Hartmann et al., 1989
). This concurs with the predicted signal sequence (Nielsen et al., 1997
) which emerges at the extreme N-terminus and would translocate the copper oxidase domain of hephaestin into the lumen of the endoplasmic reticulum where it could be transported to an intracellular vesicle or to the basolateral cell surface. The conserved di-leucine motifs and the PDZ domain in the predicted cytoplasmic C-terminus may be necessary for trafficking hephaestin correctly (Figure 2
). These motifs are also present in the recently identified iron transporter, the IREG1 protein (McKie et al., 2000
) which has been proposed to act in concert with hephaestin in the release of iron from the basolateral membrane in the enterocyte. However, there is no report of hephaestin being present at the basolateral surface. Anderson and colleagues, using an antibody raised against a peptide to the C-terminus, have localized hephaestin to the supranuclear region in the mature villus enterocytes of the duodenum (Frazer et al., 2001
).
The extensive sequence similarity between the ecto-domain of hephaestin with ceruloplasmin (~50% sequence identity) suggests that the two proteins share the same ß-barrel fold and further infers that hephaestin may function as a ferroxidase. The iron-binding site and ferroxidase activity of ceruloplasmin are vital for iron homeostasis. The three-dimensional structure of ceruloplasmin in relation to metal binding has been investigated by Lindley and colleagues (Lindley et al., 1997). The residues implicated in iron binding in ceruloplasmin, as well as their relative positions, are conserved in the human hephaestin sequence. Moreover, Bonaccorsi di Patti and co-workers have shown that E185 and Y354 in the yeast multicopper oxidase Fet3, analogous to residues E272 and H940 in Cp, are important structural determinants for ferroxidase activity (Bonaccorsi di Patti et al., 2000
). E185 and Y354 in Fet3 are analogous to E300 and H965, respectively, in the human hephaestin and have been modelled in the ferroxidase center. Figure 3e and f
show the electrostatic potential around the hephaestin protein. The series of negatively charged aspartate residues (D259, 302, 998, 955, 1023) in the vicinity of the putative iron-binding site could provide an electrostatic guidance for ferric (Fe3+) ions when they are released from the putative active site.
It is well established that ceruloplasmin (and by analogy, therefore, hephaestin) is involved in oxidation of ferrous (Fe2+) to ferric (Fe3+) prior to its binding by transferrin. Whether this process requires direct interaction between hephaestin and transferrin at the basolateral membrane of the duodenum (site of iron efflux) is under investigation. Currently there is no firm evidence to suggest that ceruloplasmin or hephaestin interacts with Tf, but an interaction between lactoferrin, a member of the transferrin family, and Cp at physiological pH has been demonstrated (Zakharova et al., 2000). The negatively charged region on Cp formed by loops in the first and second strands in the six domains (Zaitseva et al., 1996
), and therefore by analogy in hephaestin (Figure 3a
c), may, however, offer a potential binding site. A complementary positively charged surface may dock on to this inter-domain groove postulated as the holding site for iron and sequester iron prior to transporting it to sites of storage and utilization. The idea is especially attractive as unbound ferric (Fe3+) is able to participate in the Fenton and HaberWeiss type reactions and generate hazardous hydroxyl free radicals. The basolateral iron transporter IREG1 may also play a pivotal role in this interaction.
One of the interesting consequences of modelling hephaestin is that we can map the sla mutation on the hephaestin structure (Figure 3c). It is clear from Figure 3c
that the mutation results in a loss of a substantial portion of the secondary structure of domains 3 and 4, analogous to ceruloplasmin. It is unlikely, therefore, that these domains are folded in the sla product. Also, the binding sites for type I copper associated with these domains will have been lost, with concomitant loss of ferroxidase activity. In addition, the intimate nature of the domaindomain interactions in the Cp fold suggests that the structural stability of the remaining domains is also likely to be compromised, resulting in a substantial fraction of unfolded, inactive protein. It is unlikely, therefore, that any truncated protein could mediate the release of iron from the enterocyte or its oxidation prior to uptake by transferrin. The impairment of the ferroxidase activity is consistent with the gradual accumulation of intracellular iron in the enterocyte leading to microcytic anaemia as evidenced in the sla mice.
Since the proliferation of the genomic data, comparative modelling has an increasingly important role in predicting the fold of proteins provided that homology with known templates can be reliably identified. We have described here an application of this approach for building a model of the copper oxidase ecto-domain and the putative iron-binding sites of human hephaestin. The model can be tested and refined as biophysical data becomes available for hephaestin.
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Notes |
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Acknowledgments |
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Received June 26, 2001; revised November 15, 2001; accepted December 7, 2001.