Download Intracellular metal transport proteins

RIKEN Review No. 35 (May, 2001): Focused on New Trends in Bio-Trace Elements Research
Intracellular metal transport proteins
Makoto Hiromura and Hiromu Sakurai
Department of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University
Metal ions are essential cofactors for functional expressions of many proteins in living organisms. In cells, several
trace elements are needed to activate and stabilize enzymes, such as superoxide dismutase, metalloproteases, protein
kinases, and transcriptional factors containing zinc finger proteins. Recently, intracellular metal trafficking proteins,
that carry metal ions to specific target proteins, have been identified in various organisms including mammalians.
Such proteins are thus called metallochaperones. This review focuses on the structural and functional characteristics
of several metal transport systems, such as those of copper, iron, zinc, manganese, magnesium, and nickel.
Abbreviations: Ctr or COPT, copper transporter; Atx1p,
antioxidant protein 1; Hah1p, human ATX1 homolog; CopZ
or CCH, copper chaperone; CCS, copper chaperone for
SOD; Cox17p, cytochrome oxidase 17 protein; CopA, CopB,
Ccc2, ATP7A or ATP7B, copper-cation P-type ATPase;
RAN1, responsive-to-antagonist 1; PAA1, putative metal
transporting P-type ATPase; DMT1, divalent metal transporter 1; Nramp, natural-resistance-associated macrophage
protein; Fre, ferrireductase; Fet, multicopper oxidase (Fe
transporter); Ftr1p, high-affinity ferric ion transport protein;
IRT, iron-regulated transporter; ZRT, zinc-regulated transporter; ZIP, ZRT-, and IRT-like protein; Smf, manganese
transporter; PMR, P-type ATPase manganese transporter;
Alr or Mgt, magnesium transporter; Nik or Nic, nickel transporter; GST, glutathione S-transferase; M, methionine; C,
cysteine; T, threonine; K, lysine; G, glycine; and X, any
amino acids.
Table 1. Homologous proteins that affect metal ion metabolism in bacteria, yeast, plant, and human.
Copper transport proteins
The systems consisting of intracellular copper chaperones
were identified in bacteria, yeast, plants, and mammals (Table 1).1) As shown in Fig. 1, the copper chaperones consisting of three independent pathways in the cells deliver copper ions to specific target proteins. The first protein is a
Atx1p family protein, which targets to copper-cation P-type
ATPase in trans-Golgi. This protein has a copper-binding
motif, MTCXXC, in the N terminus and a lysine-rich motif, KTGK, in the C terminus. The second protein is a CCS
(copper chaperone for SOD) family protein, which has three
domains, namely, domain I, domain II, and domain III. Domain I and domain III have metal-binding motifs, namely,
MXCXXC and CXC, respectively. Among these three domains, domain III is essential to the transport of copper ions
to Cu/Zn SOD. The third protein is a Cox17 family protein,
which delivers copper ions to cytochrome c oxidase in mitochondria.
Recently, cDNAs of Atx1p and CCS homolog proteins were
cloned from rat. 2,3) The rat ATX1 homolog protein named
Rah1p was found to have both copper-binding and lysinerich motifs. This protein shows 35% and 89% identities in
deduced amino acid sequence with Atx1p and Hah1p (human
ATX1 homolog), respectively. Thus, Rah1p was investigated
whether it is complementary to a null atx1 mutant strain.
The null atx1 mutant strain was defective at the high-affinity
Fig. 1. Human metal transport machinery.
site for uptake of ferrous ions by Fet3p and thus required an
iron-supplemented medium for growth. Moreover, Rah1pexpressing-strains grow in an iron-depleted medium. 2) From
these observations, Rah1p was concluded to be an intracel-
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lular copper transport protein, which delivers copper ions to
the copper-cation P-type ATPases.
The rat copper chaperone for SOD (rCCS) has a copperbinding motif (MXCXXC) in domain I, a homolog of Cu/Zn
SOD in domain II, and an active site (CXC) in domain III.
The amino acid sequence of rCCS shows 27, 85, and 94% identities with those of three CCSs, namely, yCCS (yeast CCS),
hCCS (human CCS), and mCCS (mouse CCS), respectively.
Interestingly, domain I of rCCS shows 33% identity in amino
acid sequence with Rah1p, and domain I of yCCS was found
to have a very similar structure to that of Atx1p. The amino
acid sequence of domain II of rCCS shows 50% identity with
that of Cu/Zn SOD. This domain was confirmed to bind with
Cu/Zn SOD, using a GST column binding assay. From these
experiments, domain II of rCCS was determined to be necessary for the binding of rCCS with Cu/Zn SOD, but both
MXCXXC and CXC motifs are not essential for this binding.3)
Iron transport proteins
Iron transport proteins were identified in various organisms 1 )
(Table 1). DMT1 (divalent metal transporter 1) cloned from
rat, is a member of Nramp2 (natural-resistance-associated
macrophage protein) family and its cDNA encodes 562 amino
acids with 12 putative membrane domains. DMT1, which is a
proton-coupled iron transporter protein, mediates the active
transport of iron, but incorporates other divalent metal ions,
such as Zn2+ , Mn2+ , Co2+ , Cd2+ , Cu2+ , Ni2+ , and Pb2+ ,
in the proximal duodenum. Ferroportin1 was cloned from
zebrafish, mouse, and human. This protein has 570 amino
acids with 10 putative membrane domains and its mRNA
was found to be expressed at the basolateral surface of the
duodenal enterocyte. Thus, ferroportin1 was proposed to be
involved in the transport of iron from the basolateral surface
to the circulatory system.
Saccharomyces cerevisae has a multiple-iron transport protein. Fre1p (Ferrireductase1) identified as a membrane protein, reduced ferric ions to ferrous ions, which were in turn
transferred from Fre1p to Fet3p on the cell surface. Fet3p is a
multicopper oxidase that displays high functional homology
to ceruloplasmin. This protein reoxidizes ferrous ions to ferric ions and transfers them to Ftr1p (high-affinity ferric iron
transport protein). Ftr1p is also a membrane protein, and
is involved in the uptake of ferric ions in the cell. The iron
transport system of the Fet3p and Ftr1p is specific for ferric
ions and functions when environmental iron is deficient.
IRT (iron-regulated transporter) was identified in Arabidopsis thaliana as a ferrous transport protein. This protein is a
cation transporter, which incorporates several metal ions in
the cell.
Zinc transport proteins
The zinc transport protein is a ZIP (ZRT- and IRT-like) family protein (Table 1). ZRT (zinc-regulated transporter) is a
zinc transporter in yeast. 4) ZIP family has amino acid sequence homology in yeast, plants, and mammals. The hu-
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man zinc transport proteins are known as hZIP, hZIP2, and
hZIP3. hZIP2 may transport not only zinc ions but also
other metal ions in the cell. However, the function of hZIP2,
which specifically incorporates zinc ions in the cell, was inhibited by the presence of other metal ions such as Fe2+ , Co2+ ,
Cd2+ , Cu2+ , and Mn2+ . Moreover, the zinc-uptake activity
of hZIP2 is energy-independent.
S. cerevisiae has three zinc transporters, namely ZRT1,
ZRT2, and ZRT3. When both ZRT1 and ZRT2 act synergetically, it was suggested that zinc uptake by the proteins was
regulated by different systems. These three transporters have
their own unique characteristics. ZRT1 is a high functional
zinc transporter (Km = 10 nM Zn2+ ) and is active in zincdeficient cells. On the other hand, ZRT2 is a low functional
zinc transporter (Km = 100 nM Zn2+ ) and is detectable in
zinc-replete cells. Finally, ZRT3, which is localized in the vacuolar membrane, was suggested to control a system of zinc
influx and efflux transporters in the vacuole.
ZIP1, ZIP2, and ZIP3 in plants have different time-,
temperature-, and concentration-dependent zinc-uptake activities. On the other hand, ZIP4 was also identified as a ZIP
family protein, however, when ZIP4 was expressed, there was
no uptake of zinc ions in the cells. ZIP4 has a chloroplasttargeting sequence and therefore, this protein may be expressed in the chloroplast of plant cells.
Other metal transport proteins (Table 1)
Manganese
Smf1 and Smf2, identified as manganese transport proteins
in S. cerevisiae, were proposed to function as high- and
low-affinity transporters for manganese uptake, respectively.
PMR1, which is a member of the P-type ATPase family and
is localized in the Golgi apparatus, accumulates elevated levels of intracellular manganese ions. This protein is involved
in the homeostasis of manganese ions in yeast cells. Nramp1
and Nramp2 in mammals have high homologies in amino acid
sequence to Smf1 and Smf2, respectively. Nramp2 was found
to complement the function of the Smf yeast mutant, in contrast, Nramp1 could not complement this function. Thus,
Nramp2 was suggested to be involved in the uptake of manganese ions in the cell.
Magnesium
Magnesium transport proteins were characterized in Gramnegative bacteria.5) Two magnesium transporters, MgtA and
MgtB, are members of the P-type ATPases that are localized
in the periplasmic membrane and mediate magnesium ion
influx. These transporters were found to be related to the
roles of calcium ATPases in eukaryotes. PhoP and PhoQ are
also magnesium-regulated proteins in bacteria. PhoQ, which
functions as a membrane sensor kinase of magnesium ions,
is present as an inactive form when bound with magnesium
ions. However, when magnesium ions dissociate from PhoQ,
this protein is activated, which in turn phosphorylates PhoP.
Therefore, PhoQ is called a magnesium-dependent transcriptional factor.
Nickel
Nickel transport proteins activate some nickel-dependent en-
zymes such as urease, hydrogenase, and carbon monoxide dehydrogenase.6) Highly specific nickel transporters were identified as five genes in the Nik ABCDE operon in Escherichia
coli under anaerobic conditions. Nik A is a nickel-binding
protein, which is localized in the periplasmic membrane. Nik
B and NikC are integral membrane subunits, and NikD and
NikE are nucleotide-binding proteins. NixA was cloned as
a nickel permease in Helicobacter pylori. The expression of
this protein in E. coli allowed nickel transport and increased
urease activity. UreE, the urease accessory protein in Klebsiella aerogenes, specifically delivers nickel ions to the urease
apoprotein, then the urease is activated. Therefore, UreE is
a nickel chaperone protein, which targets urease. Nic1p was
identified as the firstly reported eukaryotic nickel permease as
well as a homolog protein of the bacterial nickel transporter
in Schizosaccharomyces pombe. This protein has four characteristic amino acids that are conserved in the bacteria nickel
transporter. Two motifs are located within the transmembrane segment and other motifs are shown to be essential for
transport activity. Nic1p is a high-affinity nickel transporter
and its function is inhibited by cobalt ions.
Conclusions
Several metal ions are essential for all living organisms, and
some metal ions activate many enzymes and maintain the
homeostasis of the cells. In this review, the recently reported
metal transport proteins are summarized. Metal transport
proteins exist widely in both prokaryotes and eukaryotes and
many transport proteins are identified. Metal transport proteins recognize specific metal ions. Membrane-type transporters carry metal ions from outside of the cell to inside,
then the intracellular metal chaperones transport the metal
ions to the specific targeted proteins.
Recently, it has been reported that some diseases are caused
by abnormal metabolisms of certain metal ions in cells and
tissues. For example, Menkes and Wilson’s diseases, that are
hereditary disorders of copper metabolism, were found to be
caused by mutated ATP7A and ATP7B genes that encode
copper-cation P-type ATPases. Excess iron in cells is related
to the pathologies of hereditary hemochromatosis, Parkinson’s disease, and the neurological disease Friedreich ataxia.
When manganese ions are present at abnormal levels in cells,
pediatric neurological disorders develop. On the basis of the
numerous observations reported, metal transport proteins are
important in protecting living organisms from metabolic disorders that in turn develop into diseases and in maintaining
the metal ion homeostasis in them.
References
1) D. Radisky and J. Kaplan: J. Biol. Chem. 274, 4481 (1999).
2) M. Hiromura and H. Sakurai: Biochem. Biophys. Res. Commun. 265, 509 (1999).
3) M. Hiromura et al.: Biochem. Biophys. Res. Commun. 275,
394 (2000).
4) M.-L. Gueriot: Biochim. Biophys. Acta 1465, 190 (2000).
5) M. Beth et al.: J. Biol. Inorg. Chem. 4, 523 (1999).
6) R.-P. Hausinger: J. Biol. Inorg. Chem. 2, 279 (1997).
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