From the Division of Immunology and Cell Biology, Department of Pathology, School of Medicine, University of Utah, Salt Lake City, Utah 84132
Transition metals are essential for the function
of many proteins, either by facilitating redox reactions or by
stabilizing protein structure. To satisfy requirements for these
metals, cells have numerous mechanisms for the solubilization and
uptake of metals from the extracellular environment. Cells must,
however, simultaneously protect themselves from the hazards inherent in the versatility of these metals, as the facile redox reactions can
produce toxic free radicals if cytosolic metal concentrations are not
carefully regulated. Numerous human disorders of metal homeostasis have
been identified. Excess iron uptake has been implicated in the
pathologies of hereditary hemochromatosis, Parkinson's disease, and
the neurological disease Friedreich ataxia. Menkes' syndrome and
Wilson's disease result from aberrant copper homeostasis. Excess
manganese supplements have been implicated in pediatric neurological
disorders. The yeast Saccharomyces cerevisiae has proven a powerful
model for investigation of the metal uptake machinery. Extensive
investigations using this single cell organism have demonstrated
parallels between the mechanisms by which this simple eucaryote obtains
different transition metals from the extracellular environment. Metal
transport systems have been found to consist of both low and high
affinity transporters (Fig. 1). High
affinity transporters are selective for their target metals and are
tightly regulated according to metal need. Low affinity transporters
are less responsive to metal need and are somewhat less selective for
metals transported. This system of dual uptake allows the maintenance
of metal homeostasis in conditions of either metal limitation or
excess.
Iron is the most versatile transition metal in biological redox
reactions. Variation in the environment of the iron-binding site can
effect a 1000-mV difference in redox potential (1), and iron is a
component of numerous cellular redox reactions. The high affinity of
iron for oxygen has also made iron the active site in heme, which is
commonly involved in oxygen binding and oxygen-based enzymatic
reactions. The same properties of facile electron transport, however,
make iron potentially toxic, as free iron generates toxic superoxide
anion and hydroxyl radicals in the presence of oxygen. Cells face the
conundrum of accumulating an essential but relatively toxic metal by
tightly regulating the concentration of free cytosolic iron. Higher
eucaryotes regulate cytosolic free iron by controlling both the amount
of iron uptake mediated by the transferrin receptor and the amount of
iron storage in ferritin. These processes are inversely regulated by
the iron regulatory proteins (cf. Ref. 2 for review).
To date, there is no compelling data that yeast cells contain a
ferritin homologue. Some experiments have suggested that iron is stored
in the vacuole (3, 4), although alternative explanations for these
experiments have also been proposed (5). Regulation of iron
status in yeast, therefore, is mediated primarily by regulating
plasma membrane iron transport.
Yeast have multiple iron transport systems, all of which appear to
require ferrous iron as a substrate. This form of iron, however, is not
usually present under aerobic conditions, as ferrous iron is rapidly
oxidized to ferric iron, which is essentially insoluble at
physiological pH. The first step in iron transport is the reduction of
ferric iron mediated by a transmembrane electron transporter system
encoded by the FRE1 and FRE2 genes (6, 7). Although this step is essential for iron transport, Fre1p and Fre2p can
also reduce copper; thus, the term ferrireductase is a misnomer as they
are metalloreductases (8, 9). Although both FRE1 and
FRE2 are highly homologous and mediate the same reaction
they are differentially regulated. FRE1 is regulated by both
iron deprivation through the action of the iron transcription factor
Aft1p and copper deprivation through the action of the copper
transcription factor Mac1p. FRE2 is solely regulated by iron
deprivation through Aft1p (8, 10). In addition to these two cell
surface ferrireductases, a family of putative metalloreductases has
also been identified whose function is unknown (10).
High affinity iron transport is mediated by a bipartite system composed
of a ferroxidase and a transmembrane permease. The ferroxidase Fet3p is
a multicopper oxidase that catalyzes the oxidization of 4 mol of Fe(II)
with the concomitant reduction of 1 mol of molecular oxygen (11). The
S. cerevisiae ferroxidase has been biochemically
characterized both as the native protein (12) and as a secreted protein
lacking the transmembrane domain (13). The protein contains four copper
atoms and shows the spectroscopic properties of a multicopper oxidase
having copper in all three spectroscopic forms. Fet3p oxidizes iron,
with a Km of 0.15 µM, which is close
to the Km for cellular iron transport. Genetic
experiments suggest that oxidized Fe(III) is then transported by a
multitopic membrane protein encoded by the FTR1 gene (14). Ftr1p has six potential transmembrane domains as well as a potential iron-binding motif. This motif, composed of the amino acids REGLE, is
similar to a motif that has been implicated in the iron-binding region
of mammalian L chain ferritin (14). Mutation of these residues in
FTR1 abrogates its ability to transport iron.
Genes homologous to FET3 and FTR1 have been
identified in Schizosaccharomyces pombe (15). Co-expression
of either the S. cerevisiae genes FET3 and
FTR1 or the S. pombe homologues
fio1+ and fip1+ is
sufficient to confer high affinity iron transport in yeast strains with
defective chromosomal FET3 (15). These results suggest that
the permease and the oxidase are the only plasma membrane proteins
required for high affinity iron transport. Because Fet3p has
ferroxidase activity in vitro, a plausible hypothesis is
that it oxidizes Fe(II) to Fe(III), which is then transported across
the membrane by the permease Ftr1p. An alternative hypothesis that
Fet3p oxidizes Ftr1p has not been formally excluded. The motive force
behind transport is unclear, as is the mechanism by which iron is
released from the transporter. An attractive hypothesis is that
cytosolic reductants might provide a driving force for both iron
transport and release.
Once translated, Fet3p and Ftr1p must be synthesized concomitantly for
localization to the plasma membrane (14). If either protein is
synthesized by itself, it is restricted to an intracellular compartment. The requirement that both proteins must be simultaneously synthesized for proper processing suggests that these proteins might
function as a complex on the cell surface. The sorting process that
requires both proteins to be processed together, however, does not
require an active Fet3p. Mutated Fet3p, either due to a defect in
copper loading or to a mutation in a copper-binding domain, is capable
of being properly glycosylated and of accompanying Ftr1p to the cell
surface. Overexpression of soluble Fet3p lacking the transmembrane
sequence in a fet3 The high affinity iron transport system is primarily regulated
transcriptionally through the action of the DNA-binding protein Aft1p
(16). Deletion of AFT1 blocks transcription of both
FTR1 and FET3, whereas a mutant protein
Aft1pup results in constitutive expression of these high
affinity transport genes. Aft1p has been shown to bind DNA in an
iron-dependent manner, in which iron precludes binding
(17), whereas the mutant Aft1pup binds to DNA even in the
presence of iron. Thus, both biochemical and genetic studies
demonstrate that AFT1 encodes an iron-sensing transcriptional activator that regulates the expression of
FRE2, FET3, and FTR1. Aft1p also
regulates the expression of CCC2, a gene required for the
incorporation of copper into apo-Fet3p.
The Fet3p/Ftr1p transport system mediates iron transport only under
aerobic conditions because the transport system requires oxygen to
catalyze the oxidation of Fe(II) (18). An alternative iron transport
system, mediated by the FET4 gene product, effects iron
uptake in high iron conditions and is required for iron transport under
anaerobic conditions (19). Fet4p is a multitopic plasma membrane
protein capable of transporting a number of transition metals, and the
Fet4p transporter is sufficient to maintain growth in
fet3 A third potential yeast iron transporter has been defined through
studies on mammalian cells. Two independent studies identified homologous genes that function in intestinal and endosomal Fe(II) transport (21, 22). The genes (Nramp2, DCT1)
encode multitopic proteins that are H+ transition metal
transporters. When defective, these genes are responsible for the
phenotypes of anemia and defective transport of iron from the gut seen
in the mk mouse and Belgrade rat, respectively. When
expressed in oocytes, DCT1 results in increased uptake of iron and
other metals (22). Expression of Nramp2 in yeast also shows an effect
of iron accumulation (23). The mammalian genes have two yeast
homologues, SMF1 and SMF2, genes previously
identified as encoding manganese transporters (24).
SMF1/SMF2 may constitute a third cell surface iron transport
system, as deletion of SMF2 results in an exacerbation of
iron deprivation in yeast with deletions in FET3 and
FET4 (5).
Copper, like iron, is a redox-active metal, and although
essential, it is potentially toxic. Unlike iron, for which no storage mechanism has yet been defined in yeast, the cellular concentration of
copper is regulated both at the level of sequestration as well as the
level of uptake. Once taken up by cells, copper can be sequestered by
binding to metallothioneins, small proteins that bind copper and other
potentially toxic metals. Induction of metallothionein synthesis is
regulated transcriptionally by the product of the ACE1 gene,
which encodes a copper-dependent transcription factor (25).
Transport of copper is similar to iron in that it is highly regulated.
Copper is found in the environment as the oxidized, cupric
(Cu2+) form but is transported as the reduced, cuprous
(Cu+) form. Reduction of extracellular copper results from
the action of Fre1p and Fre2p (8, 9). The first yeast copper
transporter gene CTR1 was identified through its effects on
iron metabolism. Klausner and colleagues (26) had developed a clever
genetic scheme to identify yeast mutants defective in iron metabolism. The basis of this screen was to have the prototrophic gene
HIS3 placed under the control of the FRE1
promoter. Mutations that resulted in decreased cytosolic iron produced
increased transcription of the HIS3 gene, which overcame His
auxotrophy (26). Through this approach a mutant was discovered that had
reduced transport of both copper and iron. The primary defect in this
mutant was found to be in plasma membrane copper transport. Subsequent
analysis revealed that defective copper transport resulted in reduced
iron transport through a deficit in the copper loading of Fet3p.
The observation that in some yeast strains inactivation of
CTR1 does not lead to copper deprivation led to the
identification of a second copper transporter encoded by the
CTR3 gene (27). Although both Ctr1p and Ctr3p are membrane
proteins, they have low sequence homology. The proteins encoded by
these genes, however, appear to have redundant function. Each gene by
itself can maintain copper homeostasis and supply copper to all
intracellular targets. The origin of two high affinity copper
transporters with little homology is curious. A possibility is that the
gene products may transport other metals in addition to copper,
although a complete characterization of the metal specificity of Ctr1p
and Ctr3p has not yet been accomplished.
Deletion of CTR1 and CTR3 results in a dramatic
reduction in growth because of copper deprivation. The double deletion
mutant provides a phenotype that has been used in expression cloning to
identify other eucaryotic copper transporters. Using this approach, Zhou and Gitschier (28) identified a putative human copper transporter, hCTR1, which was homologous with CTR1 and could
functionally replace CTR1 in maintaining cellular copper
homeostasis in yeast. A similar approach was used to identify an
Arabidopsis copper transporter (29). The plant transporter
showed a high degree of homology to a then unknown yeast gene. That
gene, CTR2, appears to be a low affinity copper transporter
as overexpression of CTR2 can complement copper deficiency
of a ctr1 As transcription of FET3 and FTR1 is regulated by
iron, transcription of CTR1 and CTR3 is regulated
by copper through the action of the DNA-binding, metal-sensitive
transcription factor, Mac1p (25, 30). Similar to Aft1p, Mac1p
recognizes specific DNA sequences and binds to these sequences in the
absence of copper. Null mutations of both transcription factors prevent
transcription of target genes, and up-regulation mutants of both
transcription factors show constitutive metal independent activity.
Copper inhibition of Mac1p activity has been shown to be the direct
result of metal association with the protein (31). Low or intermediate
copper conditions stabilize Mac1p (31). High copper conditions,
however, produce rapid degradation of Mac1p (32). The degradation of Mac1p protects the cell from copper toxicity by reducing transcription of the copper transporter genes.
Ctr1p is regulated transcriptionally by Mac1p and post-translationally
by a copper-dependent proteolysis (33) (Fig.
2). Degradation appears to result from
activation of a surface-bound protease, as degradation occurs even in
the absence of Ctr1p endocytosis. This copper-activated protease is
unknown, but its action must be specific, as other surface membrane
proteins are not affected by high concentrations of copper.
The same themes observed in the physiology of copper and iron
transport have also been found in zinc transport, in which multiple zinc transporters are regulated by zinc at both transcriptional and
translational levels. The gene encoding the high affinity zinc
transporter, ZRT1, was identified on the basis of homology to a family of iron-regulated plant transporters (34). The plant transporters were initially identified by complementation of the low
iron growth deficit of a fet3 Examination of the concentration dependence of zinc transport in wild
type and zrt1 Northern analysis and reporter constructs suggested that
ZTR1 and ZRT2 are both regulated
transcriptionally. Using a genetic screen similar to that used to
identify AFT1, Zhao and Eide identified ZAP1 as a
gene that encodes a zinc-regulated transcription factor (37). When the
gene was deleted, severe zinc deficiency arose because of a lack of
transcription of both ZRT1 and ZRT2. Promoter analysis revealed that Zap1p binds to specific DNA sequences in the
5'-untranslated regions of ZRT1 and ZRT2.
Additionally, the transcription of ZAP1 is itself regulated
by zinc. There was increased transcription of ZAP1 in the
absence of zinc, as demonstrated by both Northern analysis and the use
of ZAP-reporter constructs. Unlike Mac1p, high levels of the zinc did
not lead to degradation of the transcription factor, Zap1p. Similar to
Ctr1p, however, the transporter Zrt1p shows post-translational
regulation. Under high zinc conditions the half-life of Zrt1p is
dramatically reduced. A difference between the degradation regulation
mechanism of Ctr1p and Zrt1p is that proteolysis of Zrt1p required
endocytosis (38). The half-life of Zrt1p is prolonged both under
conditions in which endocytosis is inhibited and in mutants defective
in vacuolar proteases. These results indicate that high zinc induces
the internalization of Zrt1p leading to its degradation.
As mentioned above, SMF1 and SMF2 have high
homology to the mammalian transition metal transporter
Nramp2. Although physiological studies show that Nramp2 can
transport a number of different transition metals with similar
affinity, analysis of the Belgrade rat revealed deficiencies in
manganese(39). Yeast strains deleted for SMF1 showed reduced
uptake of manganese whereas overexpression of SMF1 resulted
in increased levels of cellular manganese. Genetic studies revealed a
SMF1 homologue, SMF2, which also affected
manganese transport (40). Little is known regarding the normal
physiological function or metal preference of Smf1p or Smf2p.
SMF2 appears to have much greater preference for cobalt than
SMF1, and both are capable of transporting copper (41).
Because Nramp2 is an H+ transition metal symporter, it may
be expected that transport by Smf1p/Smf2p would also be
pH-dependent. The regulation of SMF1/SMF2 transcription is not known but does not appear to be affected by iron,
copper, or manganese.2 The
observation that Smf1p and Smf2p have broad metal specificity and are not transcriptionally regulated by manganese suggests that they
may not be the high affinity manganese transporters.
Smf1p-mediated transport activity, however, is regulated
post-translationally by the product of the BSD2 gene (42).
This gene encodes an endoplasmic reticulum membrane protein that was shown to be responsive to metal concentrations. In growth medium containing high metal concentrations, Bsd2p directs Smf1p to the vacuole for degradation. Cells deleted for Bsd2p have increased activity of Smf1p and Smf2p and accumulate high levels of
manganese, copper, cadmium, and cobalt.
Examination of the features of transition metal transport and
their regulation reveal consistent themes. The demand for metals is
sensed by a transcription factor, which then regulates the rate of
transporter transcription. This control of transport activity by
transcriptional regulation may extend to genes involved in the assembly
of the transporter, the notable example being the vesicular copper
transporter Ccc2p that is required for the assembly of holoFet3p. Based
on the observation that there are metal-sensing transcription factors
that regulate the accumulation of iron, copper, and zinc, we speculate
that there may well be a similar transcription molecule that may
regulate the activity of manganese transport. The only manganese
transporters discovered to date have broad metal specificity and do not
appear to be transcriptionally regulated by manganese. There may be
high affinity manganese transporters that are transcriptionally regulated.
Another consistent theme is that copper, manganese, and zinc
transporters show post-transcriptional regulation. When cells starved
for a specific metal are then exposed to high concentrations of metals
the activity of the transport system is modulated either by degradation
or alteration of vesicular traffic. To date no such regulation has been
seen for high affinity iron transport in S. cerevisiae. Once
induced, exposure to iron does not affect a rapid reduction in iron
transport activity. Identical experiments suggest that the high
affinity iron transport system in S. pombe can adapt and
reduce transport activity (15). These observations suggest that perhaps
there is a regulation mechanism in S. cerevisiae, which has
been overlooked. An alternative explanation is that S. cerevisiae can accommodate excess iron by rapid sequestration. The
mechanisms of iron storage in S. cerevisiae are unknown, but if S. cerevisiae does not rapidly repress iron transport
activity, then we predict that there must be a storage vehicle for iron deposition.
INTRODUCTION
Top
Introduction
References
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Fig. 1.
Model for regulation of metal transport in
yeast. High affinity metal transporters (TH)
are selective for the target metal and are transcribed according to
metal need by transcriptional activators (TA), which release
DNA upon metal binding. Low affinity transporters
(TL) are less responsive to metal need and are less
metal selective. Although Fet4p was identified as a low affinity iron
transporter, it can transport other metals. Smf1p/Smf2p also
transport a broad range of metals. ORF, open reading
frame.
Iron
strain does not produce surface
localization of Ftr1p, suggesting that the cytosolic or transmembrane
domain may be required.1 The
interaction domains of Fet3p and Ftr1p that are required for
interaction and transport to the surface are not known.
cells. Although there are increased amounts of
FET4 mRNA as a result of iron deprivation, the gene is
not regulated by Aft1p (20). The mechanism by which iron regulates the
expression of FET4 is yet to be elucidated.
Copper
ctr3
strain. CTR2 has higher homology with CTR3 than with CTR1. The
metal specificity of Ctr2p has also not been defined.
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Fig. 2.
Post-translational mechanisms for regulating
metal transport. a, metal-dependent degradation
of Ctr1p is independent of endocytosis. b,
metal-dependent degradation of Zrt1p is
endocytosis-dependent. c, Bsd2p mediates
degradation of Smf1p in metal-replete conditions. E.R.,
endoplasmic reticulum.
Zinc
/fet4
mutant (35). The
protein product of the homologous yeast gene, ZRT1, was
found to transport not iron, but zinc (34). Transcriptional analysis
showed that ZRT1 was highly regulated by zinc. Expression
studies revealed that Zrt1p effected high affinity zinc transport.
Zrt1p showed a marked preference for zinc, as only Cu+ and
Fe2+ showed competition with zinc and then only at
superphysiological concentrations (36).
cells revealed both an inducible high affinity transport system with a Km of 1.0 µM and a low affinity system with a Km
of 10 µM. The gene responsible for low affinity zinc
transport, ZRT2, was identified on the basis of sequence
homology with ZRT1. Sequence analysis showed both proteins
to be highly homologous (44% sequence identity) with eight potential
transmembrane domains. The zrt2
deletion strain was shown
to be viable in both high and low zinc medium, though a strain deleted
for both ZRT1 and ZRT2 showed a growth limitation for zinc. Analysis of both the zrt2
deletion strain and
strains in which ZRT2 was overexpressed demonstrated that
Zrt2p was responsible for low affinity zinc uptake (36). Zinc transport
by Zrt2p showed a similar inhibition spectrum by other transition
metals to Zrt1p. It is interesting that both Zrt1p and Zrt2p have the
same metal specificity, yet at a 10-fold difference in affinity. Both
transporters show a cluster of histidine residues in a variable loop
that is predicted to be cytosolic. Histidine residues are capable of
binding transition metals, and similar clusters are found in a wide
variety of transition metal transporters. That the clusters are found in what may be a cytosolic loop suggests that they are not involved in
the initial recognition of transport of metals from the extracellular surface but rather may be involved in a feedback regulation system.
Manganese
Speculation
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ACKNOWLEDGEMENTS |
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We thank members of the Kaplan laboratory for editing assistance and Valeria Culotta, David Eide, Dennis Winge, and Dennis Thiele for helpful discussions.
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FOOTNOTES |
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* This minireview will be reprinted in the 1999 Minireview Compendium, which will be available in December, 1999.
To whom correspondence should be addressed. Tel.: 801-581-7427;
Fax: 801-581-4517; E-mail: kaplan{at}bioscience.biology.utah.edu.
1 D. Radisky and J. Kaplan, unpublished results.
2 D. Radisky and J. Kaplan, unpublished data.
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REFERENCES |
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