Download Zinc-induced Inactivation of the Yeast ZRT1 Zinc Transporter

THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 273, No. 44, Issue of October 30, pp. 28617–28624, 1998
Printed in U.S.A.
Zinc-induced Inactivation of the Yeast ZRT1 Zinc Transporter
Occurs through Endocytosis and Vacuolar Degradation*
(Received for publication, July 21, 1998)
Raad S. Gitan‡, Huan Luo‡, Jacquelyn Rodgers‡, Margaret Broderius§, and David Eide‡¶
From the ‡Nutritional Sciences Program, University of Missouri-Columbia, Columbia, Missouri 65211 and the
§Department of Biochemistry and Molecular Biology, University of Minnesota-Duluth, Duluth, Minnesota 55812
The plasma membrane is an essential barrier between the
extracellular milieu and the interior of the cell. The large
number of receptors and transport proteins embedded in this
membrane facilitate communication between the exterior and
interior of the cell, selective accumulation of nutrients, and
efflux of ions and other substrates. Controlling the activity of
these receptors and transporters is a critical part of how cells
respond to fluctuating extracellular signals and changing nutrient availability. While transcriptional regulation is one facet
of this control, there is a growing appreciation for the importance of protein trafficking in controlling the activity of plasma
membrane proteins. In mammalian cells, endocytosis of cytokine and growth factor receptors occurs following binding of the
ligand to the receptor (1). Ligand binding triggers the movement of these receptors into clathrin-coated pits, internaliza* This work was supported by the National Science Foundation and
the United States Department of Energy. The costs of publication of this
article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Nutritional Sciences
Program, 217 Gwynn Hall, University of Missouri-Columbia, Columbia,
MO 65211. Tel.: 573-882-9686; Fax: 573-882-0185; E-mail: deide
@showme.missouri.edu.
This paper is available on line at http://www.jbc.org
tion into endosomes, and subsequent delivery to lysosomes
where they are degraded.
In Saccharomyces cerevisiae, a similar mechanism of regulated endocytosis and degradation has been found for several
plasma membrane proteins. For example, binding of the a-factor mating pheromone to its receptor triggers endocytosis of the
ligand-receptor complex and subsequent degradation in the
vacuole, the lysosome-like compartment of the yeast cell (2).
This response attenuates the effects of mating pheromone during periods of prolonged exposure. Several nutrient transporters are endocytosed in response to the increased availability of
a preferred substrate. Glucose, the preferred carbon source for
yeast, signals the endocytosis of the GAL2 galactose permease
and the MAL61 maltose permease (3– 6). Likewise, NH1
4 signals endocytosis of the GAP1 amino acid permease because
ammonium is preferred over amino acids as a source of nitrogen (7, 8). Regulated endocytosis also controls the activity of
nutrient transporters in response to changing metabolic demands. The FUR4 uracil transporter is endocytosed in nutrient-starved cells or in cells in which protein synthesis is inhibited (9). This decrease in uracil uptake capacity may prevent
futile RNA synthesis. Finally, yeast transporters can be removed from the plasma membrane in response to high levels of
their corresponding substrate; this regulation presumably prevents overaccumulation of that substrate if it suddenly becomes abundant in the cell’s environment. For example, the
ITR1 inositol transporter is endocytosed in response to high
levels of inositol (10).
These examples illustrate the important role that regulated
endocytosis of plasma membrane proteins plays in controlling
nutrient uptake and responses to extracellular signals. Because of its facile genetics, yeast is an excellent model system
for study of this intriguing problem of protein trafficking. In
this report, we demonstrate the role of regulated endocytosis in
the homeostatic control of a metal ion, zinc. Zinc is an essential
nutrient for all organisms. This metal is a catalytic component
of over 300 enzymes (11) and also plays a structural role in
many proteins. For example, several motifs found in transcriptional regulatory proteins are stabilized by zinc including the
zinc finger, zinc cluster, and RING finger domains (12). Proteins containing these motifs are very common; for example,
almost 2% of the genes in the S. cerevisiae genome encode
proteins with zinc-dependent DNA-binding domains (13, 14).
Although zinc is an essential nutrient, it can be toxic if excess
amounts are accumulated. The precise cause of zinc toxicity is
unknown but the metal may bind to inappropriate intracellular
ligands or compete with other metals for enzyme active sites,
transporter proteins, etc. Therefore, in the face of fluctuating
extracellular zinc levels, cells must maintain an adequate intracellular zinc level to meet cellular requirements while preventing metal ion overaccumulation.
Mechanisms of regulating intracellular zinc levels or availabil-
28617
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The ZRT1 gene encodes the transporter responsible
for high affinity zinc uptake in yeast. ZRT1 is transcribed in zinc-limited cells and its transcription is repressed in zinc-replete cells. In this report, we describe
a second, post-translational mechanism that regulates
ZRT1 activity. In zinc-limited cells, ZRT1 is a stable,
N-glycosylated plasma membrane protein. Exposure to
high levels of extracellular zinc triggers a rapid loss of
ZRT1 uptake activity. Our results demonstrate that this
inactivation occurs through zinc-induced endocytosis of
the protein and its subsequent degradation in the vacuole. Mutations that inhibit the internalization step of
endocytosis also inhibited zinc-induced ZRT1 inactivation and the major vacuolar proteases were required to
degrade ZRT1 in response to zinc. Furthermore, immunofluorescence microscopy showed that ZRT1 is localized to the plasma membrane in zinc-limited cells and
that the protein is transferred to the vacuole via an
endosome-like compartment upon exposure to zinc.
ZRT1 inactivation is a relatively specific response to
zinc; cadmium and cobalt ions trigger the response but
less effectively than zinc. Moreover, zinc does not alter
the stability of several other plasma membrane proteins. Therefore, zinc-induced ZRT1 inactivation is a
specific regulatory system to shut off zinc uptake activity in cells exposed to high extracellular zinc levels
thereby preventing overaccumulation of this potentially toxic metal.
28618
Zinc-induced ZRT1 Inactivation
TABLE I
Yeast strains
Strain
Relevant genotype
Full genotype
Reference/source
DY1457
ZHY1
RH1965
DEY1531
RH144-3D
RH266-1D
AAY1048
AAY1046
GPY1100a
GPY418
RH3248
RG3248
Wild type
zrt1
end4
end4
END3
end3
SAC6
sac6
CHC1
chc1
pep4 prb1
pep4 prb1
MATa ade6 can1 his3 leu2 trp1 ura3
MATa ade6 can1 his3 leu2 trp1 ura3 zrt1::LEU2
MATa bar1 his4 leu2 lys2 trp1 ura3 end4::LEU2
MATa ade6 can1 his3 leu2 trp1 ura3 end4::LEU2
MATa bar1 leu2 his4 ura3
MATa bar1 leu2 his4 ura3 end3ts
MATa his3 leu2 lys2 ura3
MATa his3 leu2 lys2 ura3 sac6::LEU2
MATa can1 leu2 his4 trp1 ura3
MATa can1 leu2 his4 trp1 ura3 chc1ts
MATa ade2 can1 his3 leu2 trp1 ura3 pep4::HIS3 prb1::LEU2
MATa can1 his3 leu2 trp1 ura3 pep4::HIS3 prb1::LEU2
18
18
2
This study
23
23
24
24
25
25
H. Riezman
This study
MATERIALS AND METHODS
Yeast Strains and Growth Conditions—Strains used in this study are
described in Table I. DEY1531 and RG3248 are isogenic to DY1457 and
were generated by backcrossing the relevant mutant alleles
(end4::LEU2 and pep4::HIS3 prb1::LEU2, respectively) from their
nonisogenic parent strains into the DY1457 genetic background. Yeast
were grown in SD medium (0.67% yeast nitrogen base without amino
acids) supplemented with auxotrophic requirements and either 2%
glucose or 2% galactose. Where indicated, this medium was made zinc
limiting by adding 1 mM EDTA. A low zinc medium (LZM),1 prepared in
a similar manner as LIM (26), had the following composition: 0.17%
yeast nitrogen base without amino acids, (NH4)2SO4, or zinc (BIO101);
0.5% (NH4)2SO4; 10 mM trisodium citrate, pH 4.2; 2% glucose; 1 mM
Na2EDTA; and 1% adenine, histidine, leucine, and tryptophan. The
MnCl2 and FeCl3 concentrations in LZM were adjusted to final concentrations of 25 and 10 mM, respectively. Cell number in liquid cultures
was determined by measuring the optical density of the cell suspension
at 600 nm (OD600) and converting to cell number with a standard curve.
Plasmid and DNA Manipulations—Escherichia coli and yeast transformations were performed using standard methods (27, 28). Plasmids
1
The abbreviations used are: LZM, low zinc medium; HA, hemagglutinin antigen; PBS, phosphate-buffered saline; BSA, bovine serum
albumin.
used in this study are described in Table II. An epitope-tagged ZRT1
allele was constructed using overlap extension polymerase chain reaction (29). A 900-base pair SacI-PflMI fragment was generated in which
a sequence encoding a single hemagglutinin (HA) epitope (30) was fused
to the ZRT1 open reading frame immediately following the methionine
initiation codon. This fragment was inserted into SacI-PflMI digested
pMC5-HS (18) to generate pMC5-HSET. Plasmid pOE1-ET was constructed by isolating the epitope-tagged ZRT1 open reading frame from
pMC5-HSET by polymerase chain reaction using primers containing
either a BamHI or a SacI site on their 59 ends. This fragment was then
inserted into BamHI-SacI-digested pRS316-GAL1 (31). Plasmid pOE13ET was constructed by digesting pOE1-ET with AatII, which cuts
within the HA epitope sequence, and inserting a double-stranded oligonucleotide encoding two additional HA epitopes. A 2-kilobase XhoISacI fragment containing the GAL1 promoter and the ZRT1–3ET open
reading frame was transferred from pOE1–3ET to YIp306 (32) to generate YIp306 –3ET. This plasmid was digested with EcoRV prior to
transformation into yeast strains to direct integration of the plasmid at
the URA3 locus (33).
Zinc Uptake Assays and Atomic Absorption Spectrophotometry—Zinc
uptake assays were performed as described previously for iron uptake
(34) except that 65ZnCl2 (Amersham) and LZM-EDTA were substituted
for 59FeCl3 and LIM-EDTA, respectively. Cells were incubated for 5 min
in LZM-EDTA plus 1 mM 65Zn, collected on glass fiber filters (Schleicher
and Schuell), washed with 10 ml of ice-cold SSW (1 mM EDTA, 20 mM
trisodium citrate, 1 mM KH2PO4, 1 mM CaCl2, 5 mM MgSO4, 1 mM NaCl,
pH 4.2); cell associated radioactivity was measured on a Packard AutoGamma 5650 g-counter. Measurement of total cell-associated zinc levels was performed by atomic absorption spectrophotometry. Cells were
harvested, washed twice with an equal volume of distilled deionized
water, twice with an equal volume of 1 mM EDTA, resuspended in
one-fifth volume 6 M nitric acid, and incubated at 95 °C for 24 h. The
acid-digested samples were then assayed for zinc content on a Varian
Spectra AA-30 atomic absorption spectrophotometer.
Protein and Immunoblot Methods—Cells (100 ml, OD600 of 1–2) were
harvested by centrifuging 5 min at 1000 3 g and washed with an equal
volume of distilled water. These cells were resuspended in 20 ml 1 M
Tris-SO4, pH 9.3, 10 mM dithiothreitol, incubated at 30 °C for 10 min,
collected by centrifugation, washed once in spheroplasting buffer (1.2 M
sorbitol, 20 mM KHPO4 pH 7.4), resuspended in 10 ml spheroplasting
buffer plus zymolyase 20T (Seikagaku) (5 mg/g cells), and incubated at
30 °C for 30 – 45 min. Cells were then washed three times in an equal
volume of spheroplasting buffer, resuspended in 10 ml 0.6 M mannitol,
20 mM HEPES-KOH, pH 7.4, plus protease inhibitors (1 mM EDTA, 1
mM phenylmethylsulfonyl fluoride, 1 mM pepstatin A), and disrupted in
a Dounce homogenizer. The homogenate was centrifuged at 3000 3 g
for 5 min at 4 °C and the pellet of unbroken cells was discarded. Where
indicated, the supernatant was centrifuged 30 min at 123,000 3 g at
4 °C and the supernatant (soluble) and pellet (particulate/membrane)
fractions were collected. Immunoblots were performed as described (35)
using a monoclonal antibody specific to the HA epitope tag (12CA5,
BABCO). Horseradish peroxidase-conjugated goat anti-mouse or antirabbit secondary antibodies (Pierce) were used and detected by enhanced chemiluminescence (ECL, Amersham). Plasmid p352-HA (36)
and antibody 12CA5 were used for detection of the CTR1 protein and
pCB1 (37) and a rabbit anti-FET4 antiserum (38) were used for detection of the FET4 protein. Densitometric scanning was performed using
a CCD camera and IMAGE 1.44 software (National Institutes of
Health). Linearity of ECL signal intensity was confirmed with a control
immunoblot of serially diluted protein samples. EndoH and peptide
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ity include binding of the metal by metallothioneins (15), zinc
storage in intracellular compartments (16), and transport of the
metal out of the cell (17). The primary control point for zinc
homeostasis in S. cerevisiae is the regulation of zinc uptake
across the plasma membrane. Zinc uptake activity in yeast is
mediated by two systems. A high affinity system is active in
zinc-limited cells and the ZRT1 gene encodes the transporter for
this system (18). A low affinity transporter is active in zincreplete cells and is encoded by the ZRT2 gene (19). ZRT1 and
ZRT2 proteins share 67% sequence similarity and share a similarly high degree of relatedness to the IRT1 Fe21 transporter (20)
and the ZIP1, ZIP2, ZIP3, and ZIP4 zinc transporters of Arabidopsis thaliana (21). These proteins are members of a growing
family of metal ion transporters that have also been identified in
protozoans, nematodes, and humans.
Transcription of both ZRT1 and ZRT2 is induced in zinclimited cells, this induction is mediated by the ZAP1 transcriptional activator (22). In low zinc conditions, ZAP1 activates
expression of the ZRT1 and ZRT2 genes by as much as 30-fold.
Zinc inhibits ZAP1 function and limits expression in zinc-replete cells through an unknown mechanism. In this report, we
demonstrate that the ZRT1 zinc transporter is also regulated
by a separate, post-translational mechanism. We found that
under low zinc conditions, ZRT1 is a stable plasma membrane
protein. Exposure of cells to high zinc levels triggers internalization of the protein via endocytosis and its subsequent degradation in the vacuole. This post-translational regulatory system limits zinc uptake when cells are exposed to a sudden
increase in extracellular zinc levels, thereby preventing the
potentially harmful overaccumulation of the metal.
Zinc-induced ZRT1 Inactivation
28619
TABLE II
ZRT1 expression plasmids
Plasmid
Vector type
Promoter
No. of HA epitopes
Ref.
pMC5-HS
pMC5-HSET
pOE1
pOE1-ET
pOE1–3ET
Ylp306–3ET
ARS-CEN
ARS-CEN
ARS-CEN
ARS-CEN
ARS-CEN
Integrating
ZRT1
ZRT1
GAL1
GAL1
GAL1
GAL1
0
1
0
1
3
3
18
This study
18
This study
This study
This study
N-glycosidase F were purchased from New England Biolabs and used
following the manufacturers instructions.
Indirect Immunofluorescence Microscopy—Indirect immunofluorescence microscopy was performed essentially as described by Pringle et al.
(39) with the following modifications. Cells were fixed in 10 volumes of
cold methanol (220 °C) for 30 min. Fixed cells were treated with glusulase
to remove the cell wall and bound to polylysine-treated glass slides. The
cells were blocked in PBS 1 1 mg/ml BSA (PBS/BSA) by incubating at
room temperature for 2 h and stained with 12CA5 antibody (1:200 dilution in PBS/BSA) at room temperature for 16 h. Following washing of the
cells, goat anti-mouse IgG antibody coupled to ALEXA 488 (Molecular
Probes) was applied (1:200 dilution in PBS/BSA) and the cells were
incubated at 37 °C for 1 h. The slides were then washed 10 times in PBS
and viewed using epifluorescence and Nomarski optics.
indicate that the size heterogeneity observed for ZRT1 is due to
different degrees of N-linked glycosylation. It should be noted
ZRT1 size heterogeneity was not always observed (see below)
and the reason for this inconsistency is unknown.
Post-translational Control of ZRT1 Uptake Activity by Zinc—
Expression of the ZRT1 gene is regulated at the transcriptional
level by zinc; ZRT1 mRNA synthesis is induced by zinc limitation and repressed during culturing in zinc-replete media (18).
As we show here, ZRT1 activity is also regulated by zinc
through a separate post-translational mechanism. When zinclimited cells expressing a high level of ZRT1 were treated with
a high concentration of extracellular zinc, a rapid loss of zinc
uptake activity occurred that was not observed when cells were
maintained in a zinc-limiting medium (Fig. 2A). The loss of
activity upon zinc treatment was preceded by an initial lag
period of 10 –20 min. The post-translational nature of the effect
of zinc on ZRT1 activity was demonstrated in two ways. First,
treatment of cells with an inhibitor of protein synthesis, cycloheximide, did not prevent the loss of uptake activity in zinctreated cells (Fig. 2A, dashed lines). Second, zinc-induced loss
of uptake activity was also observed in zrt1 mutant cells expressing ZRT1 from the zinc-insensitive GAL1 promoter
(GAL1p-ZRT1) (Fig. 2B). In this experiment, expression from
the GAL1 promoter was repressed at time 0 by the addition of
glucose and protein synthesis was blocked with cycloheximide.
Similar data were obtained for the GAL1p-ZRT1 fusion in the
absence of cycloheximide (data not shown). From these results,
we conclude that (i) the ZRT1 zinc transporter is regulated by
zinc by a post-translational mechanism that is separate from
the previously characterized transcriptional control; (ii) new
protein synthesis is not required for the post-translational response to occur; and (iii) expression of ZRT1 from the GAL1
promoter can be used to study this response without the added
complexity of the zinc-responsive transcriptional control.
The half-lives (t1⁄2) of uptake activity loss were estimated
from the linear portions of the graphs in Fig. 2 (Table III). With
or without cycloheximide treatment, the t1⁄2 for loss of zinc
uptake activity decreased by greater than 7-fold following zinc
addition. Identical results were obtained with a zrt1 mutant
expressing ZRT1-ET from a high-copy plasmid (pMC5-HSET).
The t1⁄2 of uptake activity loss in zinc-treated cells expressing
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RESULTS
Immunological Detection of the ZRT1 Protein—To allow immunological detection of the ZRT1 protein, the ZRT1 gene was
epitope-tagged with either one or three tandem copies of the
hemagglutinin antigen (HA) epitope (YPYDVPDYA) at its
amino terminus immediately following the methionine initiation codon. These proteins (referred to as ZRT1-ET and ZRT1–
3ET, respectively) are functional as demonstrated by (i) their
ability to complement a zrt1 mutant for zinc-limited growth
and (ii) 65Zn uptake activity that is indistinguishable from wild
type ZRT1 (data not shown). On immunoblots, multiple protein
bands of approximately 40 – 45 kDa molecular mass were detected in cells expressing the ZRT1-ET allele (Fig. 1A). These
bands were not observed in cells expressing the untagged protein. These results demonstrate that the anti-HA antibody is
specific for the epitope-tagged ZRT1 protein and does not detect
other proteins in yeast. Similar results were obtained with the
ZRT1–3ET protein although the immunoblot signal intensity
was approximately 20-fold greater than that of ZRT1-ET (data
not shown). A slower migrating form of epitope-tagged ZRT1
was also frequently observed on immunoblots (Fig. 1A, arrow).
This may be a dimeric ZRT1 complex given that the molecular
mass of this band was 80 –90 kDa, i.e. twice the apparent
monomeric ZRT1 molecular mass.
Although ZRT1-ET was not detected in total cell homogenates, the protein was detectable in a subcellular fraction in
which cellular membranes were enriched (Fig. 1A). This result
suggests that ZRT1 is an integral membrane protein; further
experiments confirmed this hypothesis. Treatment with detergents (Triton X-100, n-octyl-b-D-glucopyranoside) released
ZRT1 into the soluble fraction whereas agents that can only
dissociate peripheral membrane proteins (i.e. 10 mM Na2CO3 at
pH 10, NaCl, NaBr, or urea) (40) did not (data not shown).
The multiple ZRT1 bands observed suggested that this protein may be subject to post-translational modification. To test if
ZRT1 was N-glycosylated, a particulate/membrane fraction isolated from cells expressing epitope-tagged ZRT1 was treated
with the glycosidases EndoH or peptide N-glycosidase F. EndoH cleaves the chitobiose core of high mannose oligosaccharides from N-linked glycoproteins, whereas peptide N-glycosidase F cleaves between the asparagine and the innermost
N-acetylglucosamine of the carbohydrate. Either treatment reduced the apparent molecular mass of ZRT1 from the 40 – 45kDa bands to a single band of 31 kDa (Fig. 1B). These data
FIG. 1. Characterization of the ZRT1 protein product. A, immunoblot detection of epitope-tagged ZRT1. Samples of total (T), soluble
(S), or particulate/membrane (P) proteins were prepared from wild type
cells (DY1457) expressing the untagged ZRT1 protein (pOE1, 2) or the
epitope-tagged ZRT1-ET protein (pOE1-ET, 1) from the GAL1 promoter and analyzed by immunoblotting (10 mg of protein/lane). The
positions of the molecular mass standards are shown and the arrow
indicates the slower migrating form of ZRT1. B, ZRT1 is N-glycosylated.
Particulate/membrane proteins were prepared from cells expressing
ZRT1-ET. Samples (10 mg of protein) were boiled in EndoH reaction
buffer (lanes 1 and 2) or in peptide N-glycosidase F reaction buffer
(lanes 3 and 4) and then incubated for 1 h at 37 °C in the absence (2) or
presence (1) of the corresponding enzyme (1000 units/reaction) prior to
immunoblot analysis.
28620
Zinc-induced ZRT1 Inactivation
TABLE III
Half-life of zinc uptake activity loss
The t1/2 values (in min) were calculated from the linear portions of the
graphs described in the legend to Fig. 2 and are representative of
several repetitions of these experiments. Plasmids pMC5-HSET and
pOE1-ET are described in Table II. CHX, cycloheximide.
Wild type
2Zn
1Zn
zrt1
2 CHX
1 CHX
pMC5-HSET
pOE1-ET
.150
23
.150
20
.150
21
.150
37
ZRT1 from the GAL1 promoter was reproducibly higher than
that observed in cells expressing the transporter from its own
promoter; the reason for this difference is not yet known. These
t1⁄2 values highlight the stability of ZRT1 uptake activity in low
zinc conditions and its remarkable rate of loss in the presence
of high zinc.
Degradation of the ZRT1 Protein in Response to Zinc—Immunoblots indicated that zinc-dependent inactivation of ZRT1
activity is accompanied by degradation of the ZRT1 protein.
Following cycloheximide treatment, the ZRT1 protein was stable in zinc-limited cells but rapidly degraded in zinc-treated
cells (Fig. 3A). As was the case for the loss of uptake activity,
FIG. 3. Inactivation of uptake activity is accompanied by degradation of the ZRT1 protein. A, a zrt1 mutant strain (ZHY1) expressing the epitope-tagged ZRT1 protein from the ZRT1 promoter
(pMC5-HSET) was grown to exponential phase in a zinc-limiting medium (LZM 1 10 mM ZnCl2), harvested, and reinoculated into SD glucose medium containing cycloheximide (100 mg/ml) and either 2 mM
ZnCl2 (1Zn) or 1 mM EDTA (2Zn). Cells were incubated at 30 °C,
harvested at the indicated times, and particulate/membrane fractions
were prepared for immunoblot analysis. B, signal intensities of the
bands in panel A were measured by densitometry and are plotted as the
percent of the t0 sample. C, effects of zinc on ZRT1 protein level when
expressed from the GAL1 promoter and on the levels of other plasma
membrane proteins. A zrt1 mutant (ZHY1) expressing ZRT1–3ET from
the GAL1 promoter (YIp306 –3ET) and wild type cells (DY1457) expressing either epitope-tagged CTR1 (p352-HA) or FET4 (pCB1) were
grown to exponential phase in SD galactose medium (CTR1 expression
was induced by the addition of 1 mM EDTA). The cells were harvested
and reinoculated into SD glucose medium supplemented with 100 mg/ml
cycloheximide and either 2 mM ZnCl2 (1Zn) or 1 mM EDTA (2Zn). Cells
were then incubated at 30 °C, harvested at the indicated times, and
total homogenates (ZRT1) or particulate/membrane fractions (CTR1
and FET4) were prepared for immunoblot analysis. The two FET4
bands detected here have been observed previously (38).
protein loss occurred after an initial lag period of approximately 30 min. Densitometric quantitation of the band intensities in Fig. 3A is shown in Fig. 3B, the t1⁄2 for protein loss was
determined from these data. In zinc-treated cells, the ZRT1
protein was degraded with a t1⁄2 39 min) that was 25% or less of
the t1⁄2 (.150 min) observed in zinc-limited cells. We noted that
the loss of protein in zinc-treated cells occurred more slowly
than the loss of uptake activity (i.e. compare a t1⁄2 for protein of
39 min and a t1⁄2 for uptake of 21 min). This result suggested
that inactivation of ZRT1 uptake activity is not the direct result
of proteolytic degradation but rather that degradation may
occur later in the inactivation process. Similar experiments
performed with expression of ZRT1 from the GAL1 promoter
yielded qualitatively similar results in which ZRT1 was less
stable in zinc-treated cells (Fig. 3C). As was observed for loss of
uptake activity, degradation of ZRT1 occurred more slowly (t1⁄2
of approximately 65 min) in the cells grown in galactose than in
glucose-grown cells.
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FIG. 2. Post-translational inactivation of ZRT1 zinc uptake activity in response to zinc. A, wild type cells (DY1457) were grown to
exponential phase in a zinc-limiting medium (LZM 1 10 mM ZnCl2),
harvested, and reinoculated into SD glucose medium supplemented
with either 2 mM ZnCl2 (filled symbols) or 1 mM EDTA (open symbols)
and in the presence (solid lines) or absence (dashed lines) of cycloheximide (100 mg/ml). Cells were harvested at the indicated times and
assayed for 65Zn uptake activity. B, a zrt1 mutant strain (ZHY1) expressing ZRT1-ET from the GAL1 promoter (pOE1-ET) was grown to
exponential phase in SD galactose medium, harvested, and reinoculated into SD glucose medium supplemented with 100 mg/ml cycloheximide and either 2 mM ZnCl2 (filled symbols) or 1 mM EDTA (open
symbols). Cells were incubated at 30 °C, harvested at the indicated
times, and assayed for 65Zn uptake activity. An experiment representative of several repetitions is shown. Each value is the mean of three
independent assays and the standard deviations were consistently
,10% of the corresponding mean.
Zinc-induced ZRT1 Inactivation
28621
FIG. 4. Relationship between cell-associated zinc levels, zinc
uptake activity, and ZRT1 protein level. A, a zrt1 mutant (ZHY1)
expressing ZRT1–3ET from the GAL1 promoter (YIp306 –3ET) was
grown to exponential phase in SD galactose medium. The cells were
harvested and resuspended in SD glucose medium supplemented with
100 mg/ml cycloheximide and either 2 mM ZnCl2 (filled symbols) or 1 mM
EDTA (open symbols). At the indicated times, cells were harvested and
assayed for 65Zn uptake activity (squares) or cell-associated zinc levels
(circles). B, a similar experiment to the one described in panel A was
performed except that aliquots of cells were treated with 1 mM EDTA or
with 1, 10, 100, or 1000 mM ZnCl2 for 4 h prior to harvest. Total cell
homogenates were also prepared for immunoblot analysis. An experiment representative of several repetitions is shown. Each value is the
mean of three independent assays and the error bars indicate 6 1 S.D.
To determine if the degradation of ZRT1 in response to zinc
was specific to ZRT1 or part of a more global change in plasma
membrane protein composition, we examined the stability of
other yeast plasma membrane proteins in zinc-treated cells.
The abundance of the CTR1 copper transporter, the FET4 iron
transporter, and the PMA1 H1-ATPase was not greatly altered
by zinc treatment (Fig. 3C, data not shown). Thus, zinc-induced
ZRT1 inactivation appears to be a specific response of the zinc
transporter.
Specificity of ZRT1 Inactivation for Zinc—The rapid decline
of ZRT1 zinc uptake activity is accompanied by an equally
dramatic increase in cell-associated zinc levels (Fig. 4A). The
maximum cell-associated zinc level (;1.3 nmol/106 cells), observed in this experiment after 90 min, was 6-fold higher than
the maximum steady-state level observed in our previous studies of zinc-responsive transcriptional control (;0.2 nmol/106
cells) (18). Given this extremely high level of metal ion accumulated by zinc-treated cells, it was important to determine if
ZRT1 inactivation and degradation was an indirect effect of
metal-induced stress or toxicity. Several experiments confirmed that ZRT1 inactivation is indeed a mechanism of zinc
homeostasis. First, there was no loss of cell viability caused by
any of the treatments described in Figs. 2– 4 (data not shown).
Second, loss of zinc uptake activity was not triggered by any of
several conditions that are known to cause inactivation of other
yeast plasma membrane transporters, i.e. deprivation of carbon
source, nitrogen, or phosphate, heat treatment, or cycloheximide treatment (5, 9) (Fig. 2, data not shown). Third, inactivation was triggered by much lower levels of zinc than the 2 mM
concentration used in our initial experiments. Treatment of
cells with as little as 1 mM zinc resulted in a significant loss of
uptake activity and ZRT1 protein (Fig. 4B). Higher levels of
zinc caused more dramatic effects on both ZRT1 activity and
protein level and was correlated with higher cell-associated
zinc levels; these results indicate that the degree of the response can be proportional to the zinc level over a certain range
of metal ion concentrations.
To determine the specificity of ZRT1 inactivation for zinc
over other metals, cells expressing ZRT1 from the GAL1 promoter were treated for 4 h with 0, 1, 10, or 100 mM concentrations of several other metal ions. MnCl2 and FeCl3 had no effect
on ZRT1 uptake activity, and NiCl2 caused only a slight decrease at the 10 or 100 mM concentrations (Fig. 5). CuCl2,
CdCl2, and CoCl2 treatments greatly decreased zinc uptake
activity albeit only at higher concentrations and/or to lesser
degrees than was observed for ZnCl2. The decrease in uptake
activity in response to copper correlated closely with loss of cell
viability (data not shown) but no loss of viability was observed
in the cadmium- or cobalt-treated cultures. Studies described
below demonstrated that ZRT1 inactivation in response to zinc
requires a functional endocytic pathway; ZRT1 inactivation is
defective in strains bearing mutations (e.g. end4) that inhibit
endocytosis. Inactivation of zinc uptake activity by Cd21 and
Co21 was inhibited in an end4 mutant strain (data not shown)
suggesting that ZRT1 inactivation is also triggered by Cd21
and Co21 ions.
Zinc-induced ZRT1 Inactivation Is Inhibited in Endocytosisdefective Mutants—The turnover of many yeast plasma membrane proteins occurs through endocytosis and subsequent vacuolar degradation. In response to a particular signal, these
proteins accumulate in clathrin-coated pits and are engulfed
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FIG. 5. Effects of other metals on inactivation of ZRT1 uptake
activity. A zrt1 mutant strain (ZHY1) expressing ZRT1–3ET from the
GAL1 promoter (YIp306 –3ET) was grown to exponential phase in SD
galactose medium. The cells were then harvested and resuspended in
SD glucose medium supplemented with 100 mg/ml cycloheximide and
either no metal (2) or the indicated concentration of ZnCl2, MnCl2,
FeCl3, NiCl2, CuCl2, CdCl2, or CoCl2. Cells were incubated at 30 °C for
4 h, harvested, and assayed for 65Zn uptake activity. An experiment
representative of several repetitions is shown. Each value is the mean
of three independent assays and the error bars indicate 1 S.D. M, 1 mM;
o, 10 mM; f, 100 mM.
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Zinc-induced ZRT1 Inactivation
into newly formed endosomes. The proteins are then transferred to the late endosome and delivered to the vacuole where
they are degraded by vacuolar proteases. To assess if this
pathway is responsible for zinc-induced ZRT1 inactivation, we
first examined the effects of zinc treatment on ZRT1 zinc uptake activity in yeast mutants defective for the initial internalization step of endocytosis. Several genes have been shown to
be important for this step to occur efficiently (for review, see
Ref. 41), including CHC1, END3, END4, and SAC6. CHC1
encodes the clathrin heavy chain protein; END3 and END4
encode proteins of unknown function; and SAC6 encodes fimbrin, an actin filament bundling protein. Initial zinc uptake
activity measured in each of these endocytosis mutants was
similar to its isogenic wild type parent, demonstrating that zinc
uptake by ZRT1 does not require endocytosis (Fig. 6). Upon zinc
treatment, inactivation of ZRT1 zinc uptake occurred more
slowly in each of the endocytosis-defective mutants relative to
its isogenic wild type parent. These data suggest that the loss
of uptake activity observed in zinc-treated cells is the direct
result of endocytosis of the ZRT1 protein.
To assess the role of the vacuolar proteases in the degradation of ZRT1, we examined the effects of mutations that eliminate the activity of the two major vacuolar endoproteinases,
proteinase A encoded by the PEP4 gene and proteinase B
encoded by the PRB1 gene. These two proteases are also required for activity of other hydrolases in the vacuole including
carboxypeptidase Y and aminopeptidase I (42). Inactivation of
ZRT1 uptake activity by zinc treatment was not altered in the
pep4 prb1 mutant but degradation was greatly inhibited (Fig.
7). These results indicate that vacuolar proteases are required
for degradation of ZRT1 to occur in response to zinc but are not
required for the loss of uptake activity.
Effects of Endocytosis- and Protease-defective Mutations on
ZRT1 Localization—The experiments described above suggest
that zinc-induced inactivation of ZRT1 activity involves endocytosis of the transporter and its subsequent delivery to the
vacuole for degradation. To test this hypothesis directly, immunofluorescence microscopy was used to determine the location of the ZRT1 protein in wild type, end4, and pep4 prb1
mutants with and without zinc treatment (Fig. 8). Little fluorescence was detected in cells expressing the wild type ZRT1
protein (data not shown). In wild type cells expressing the
epitope-tagged ZRT1–3ET protein, a bright rim of fluorescence
at the cell periphery was seen consistent with a plasma membrane localization of the protein. Plasma membrane ZRT1
staining was not affected by a 4-h incubation in zinc-limiting
conditions but completely disappeared following a 4-h incubation in 2 mM zinc. In the end4 mutant, plasma membrane
staining was also detected but was unaltered by zinc treatment. In the pep4 prb1 mutant, both plasma membrane and
the vacuolar staining was observed prior to zinc treatment; this
pattern was also observed following a 4-h incubation in zinclimiting medium. In zinc-treated cells, plasma membrane
staining was no longer apparent but vacuolar staining was still
observed.
Finally, vesicular intermediates of endocytosis can be
trapped in the cytoplasm by incubation at 15 °C (43). At this
temperature, plasma membrane internalization occurs normally but vesicle cargo accumulates in endosome-like compartments. These endosomal structures have been detected as intermediates in the endocytosis of other yeast plasma
membrane proteins (43, 44). To see if ZRT1 also passes through
this intermediate compartment en route to the vacuole, we
determined the localization of ZRT1 in cells incubated at 15 °C
with and without zinc treatment. Incubation at 15 °C for 4 h
without zinc treatment did not alter zinc uptake activity (data
not shown) nor did it alter the plasma membrane localization of
ZRT1 (Fig. 8). Inactivation of uptake activity occurred normally
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FIG. 6. Endocytosis-defective mutants are defective for zincinduced ZRT1 inactivation. Wild type (solid lines) or mutant
(dashed lines) cells of the indicated genotype were grown to exponential
phase under conditions that induced expression of ZRT1 (SD glucose 1
1 mM EDTA at 24 °C for panels A and B, SD galactose at 30 °C for
panels C and D). The cells were then harvested and resuspended in SD
glucose medium supplemented with 100 mg/ml cycloheximide and either 2 mM ZnCl2 (filled symbols) or 1 mM EDTA (open symbols) at 37 °C
for the temperature-sensitive chc1ts and end3 ts mutants (panels A and
B) or 30 °C (panels C and D). At the indicated times, cells were harvested and assayed for 65Zn uptake activity. Because these mutations
were generated in different strains than our standard wild type DY1457
strain, the results obtained with the corresponding isogenic parent are
also shown. A, CHC1 (GPY1100a) and chc1ts (GPY418)) transformed
with pMC5-HSET. B, END3 (RH144 –3D) and end3 ts (RH266 –1D)
transformed with pMC5-HSET. C, END4 (DY1457) and end4
(DEY1531) transformed with YIp306 –3ET. D, SAC6 (AAY1046) and
sac6 (AAY1048) transformed with YIp306 –3ET. An experiment representative of several repetitions is shown. Each value is the mean of
three independent assays and the error bars indicate 61 S.D.
FIG. 7. Mutants defective in vacuolar proteases are defective
for ZRT1 degradation. Wild type (DY1457, solid lines) and mutant
cells (RG3248, dashed lines) expressing ZRT1–3ET from the GAL1
promoter (YIp306 –3ET) were grown to exponential phase in SD galactose medium. The cells were then harvested and resuspended in SD
glucose medium supplemented with 100 mg/ml cycloheximide and either 2 mM ZnCl2 (filled symbols) or 1 mM EDTA (open symbols). At the
indicated times, cells were harvested, and assayed for 65Zn uptake
activity. Each value is the mean of three independent assays and the
error bars indicate 6 1 S.D. B, total cell homogenates were also prepared from zinc-treated cells for immunoblot analysis.
Zinc-induced ZRT1 Inactivation
in zinc-treated cells incubated at 15 °C (data not shown), consistent with endocytosis being unaffected at this temperature.
However, these zinc-treated cells accumulated ZRT1 protein in
cytoplasmic bodies similar to the endosome-like structures observed in previous studies. Taken together, these results demonstrate that ZRT1 inactivation results from removal of the
ZRT1 protein from the plasma membrane by endocytosis. The
subsequent degradation of the protein is a consequence of its
delivery to the vacuole. Vacuolar staining observed in the pep4
prb1 mutant in the absence of zinc treatment further suggests
that ZRT1 is endocytosed to the vacuole even in the absence of
zinc treatment, although at a slower rate than in zinc-treated
cells.
DISCUSSION
Previous studies demonstrated that zinc uptake in yeast is
regulated at the transcriptional level by intracellular zinc lev-
els (18, 22). A zinc-responsive transcriptional activator protein
called ZAP1 induces expression of the ZRT1 gene when cells
are grown under zinc-limiting conditions. Zinc represses the
activation function of ZAP1 through an unknown mechanism.
In this report, we describe a second mechanism that controls
zinc accumulation in yeast at a post-translational level; zinc
induces the endocytosis of the ZRT1 transporter and its subsequent vacuolar degradation.
The post-translational ZRT1 regulatory mechanism is
clearly separate from the transcriptional control system given
that inactivation of ZRT1 uptake activity occurs normally in a
zap1 deletion mutant.2 However, these two systems undoubtedly work together to maintain the homeostatic control of intracellular zinc levels. It is interesting to note that the transcriptional control system exerts its greatest effect on ZRT1
expression when cell-associated zinc levels vary between 0.01
and 0.07 nmol of Zn/106 cells (i.e. ;0.5– 4 3 107 atoms zinc/cell).
Approximately 90% repression of a ZRT1-lacZ fusion was observed when cell-associated zinc levels rose to 0.07 nmol/106
cells.3 In contrast, the post-translational response is triggered
only at cell-associated zinc levels of greater than 0.07 nmol of
Zn/106 cells (Fig. 4). Thus, we envision a two-tiered regulatory
system in which the transcriptional control can respond to
moderate changes in zinc availability and the post-translational control responds to more extreme variations. A likely
scenario in which the post-translational control would be important for maintaining zinc homeostasis is when zinc-limited
cells are suddenly exposed to high levels of zinc. The rapid
down-regulation of zinc uptake by ZRT1 endocytosis helps to
prevent overaccumulation of zinc and this would not be possible solely through the transcriptional control of a stable plasma
membrane protein. During inactivation of zinc uptake activity,
other systems may be induced to facilitate storage of the excess
zinc or mediate its efflux from the cell.
The model that we propose for inactivation of the ZRT1 zinc
transporter is as follows. In cells in which the intracellular zinc
levels are low-to-moderate (i.e. ,0.07 nmol/106 cells), the ZRT1
transporter is a stable plasma membrane protein and its level
is controlled by the transcriptional regulatory system. When
intracellular zinc levels rise above this level, the ZRT1 protein
is recruited into clathrin-coated pits as they form at the plasma
membrane and then internalized as the endosome pinches off
into the cytoplasm. The ZRT1-containing endosomes fuse with
the late endosome where the transporter is packaged into vesicles for delivery to the vacuole. Upon arriving in the vacuole,
proteases found in this organelle degrade the transporter. An
alternative model of the post-translational regulation of ZRT1
is that the transporter is a constitutively recycling protein. In
this scenario, ZRT1 is endocytosed at a constant rate and
transferred to the endosome. Under low zinc conditions, the
protein is returned to the plasma membrane with no net loss of
uptake activity. When cells are exposed to high zinc, however,
ZRT1 would be routed instead to the late endosome and then to
the vacuole for degradation. We think that this model is unlikely. If ZRT1 was a constitutively recycling protein and was
being rapidly endocytosed in the absence of zinc treatment,
incubating cells at 15 °C would cause the protein to accumulate
in the endosomes and ultimately deplete the transporter from
the plasma membrane. However, zinc uptake activity did not
decrease when cells were incubated at 15 °C in the absence of
zinc treatment. Thus, we found no evidence for constitutive
recycling of the ZRT1 protein between the plasma membrane
and intracellular endosomal compartments.
2
H. Luo and D. Eide, unpublished result.
Zhao, H., Butler, E., Rodgers, J., Spizzo, T., Duesterhoeft, S., and
Eide, D. (1998) J. Biol. Chem. 273, in press.
3
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FIG. 8. Effect of end4 and pep4 prb1 mutations on ZRT1 localization and abundance. Wild type (DY1457), end4 (DEY1531), and
pep4 prb1 (RG3248) strains expressing ZRT1–3ET from the GAL1 promoter (YIp306 –3ET) were grown to exponential phase in SD galactose
medium. The cells were then harvested and resuspended in SD glucose
medium supplemented with 100 mg/ml cycloheximide and either 2 mM
ZnCl2 (1Zn) or 1 mM EDTA (2Zn) and incubated at 30 °C (or at 15 °C
as indicated). At 0 (t0) and 4 h after reinoculation, cells were harvested,
fixed, stained for ZRT1 protein, and viewed by epifluorescence and
Nomarski optics. The intracellular indentations observed in the Nomarski images correspond to the vacuole.
28623
28624
Zinc-induced ZRT1 Inactivation
proteases nor did it require endocytosis; CTR1 degradation in
response to high copper appears to occur at the plasma membrane. These examples and our studies of zinc-induced ZRT1
inactivation highlight the importance of regulated protein trafficking in metal ion homeostasis.
Acknowledgments—We thank Alison Adams, Greg Payne, and
Howard Riezman for providing yeast strains used in this study. We also
thank John Cannon, Mary Lou Guerinot, Ann Thering, and Hui Zhao
for critical reading of the manuscript.
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The zinc-induced endocytosis model that we favor raises a
number of exciting new questions. First, while it is clear that
zinc induces endocytosis of ZRT1, it is unknown if this response
is induced by a mechanism that senses intracellular or extracellular metal ion levels. Second, it is unclear if the signal being
monitored is Zn21 ions per se, the activity of a zinc-dependent
or zinc-inhibited enzyme, or a more indirect consequence of
high metal accumulation. The observation that Co21 and Cd21
may also induce endocytosis of ZRT1 is potentially instructive.
Both Co21 and Cd21 have similar coordination chemistries to
Zn21 and will bind to protein ligands in a similar fashion but
generally cannot replace zinc as a functional enzymatic cofactor. Therefore, the simplest hypothesis is that Zn21 ions trigger
endocytosis directly and that Co21 and Cd21 mimic that signal.
The lower activity of Co21 and Cd21 in triggering the response
may be due to a greater specificity of the sensing mechanism
for Zn21, different uptake efficiencies for different metal ions,
or some other factor. A third unanswered question is how the
zinc signal is transmitted to ZRT1. This could occur through
the metal binding directly to the transporter or through an
indirect signal transduction pathway. We noted previously the
presence of a potential metal-binding site in a presumptive
cytoplasmic domain of ZRT1 (18); Zn21 binding to this site
could induce endocytosis of the protein.
How could zinc alter the rate of internalization of the transporter protein? Internalization of many plasma membrane proteins has been shown to be dependent on small, discrete sequences called “internalization signals” that are located in
cytoplasmic regions of the proteins (46). These signals include
the “tyrosine-based” signal (with the consensus NPXY or
YXXZ, where X is any amino acid and Z is a bulky hydrophobic
amino acid) and the “dileucine-based” single, i.e. a pair of
leucines or other bulky hydrophobic amino acids. Several such
elements can be identified in the ZRT1 amino acid sequence.
Studies have shown that functional internalization signals
bind directly to the AP-2 adaptin complex of clathrin-coated
pits and this binding event mediates recruitment of plasma
membrane proteins to these structures for internalization (47,
48). In the case of ZRT1, we propose that zinc increases the
activity of an internalization signal in a cytoplasmic domain of
ZRT1 and thereby increases its rate of endocytosis. The activity
of an internalization signal in ZRT1 could be modulated by
direct binding of zinc to the transporter, interaction of ZRT1
with other proteins, or covalent modification of the ZRT1 protein. Ubiquitination is required for efficient endocytosis of several yeast proteins (2, 6, 49) and may be involved in ZRT1
endocytosis as well.
Transcriptional regulation has long been known as an important component of metal ion homeostasis. Regulated protein
trafficking is now being recognized as an equally important
mechanism for controlling the activity and/or function of metal
ion transporters. Two examples of this regulation come from
recent studies of eukaryotic copper transporters. In cultured
mammalian cells, the Menkes copper-transporting P-type ATPases is localized to the trans-Golgi network where it supplies
the metal to secreted copper-dependent enzymes (45). Treating
these cells with copper causes the protein to relocalize to the
plasma membrane, presumably to facilitate direct copper efflux
from the cell. In yeast, the CTR1 copper transporter is endocytosed in response to copper in a manner analogous to what we
have observed for ZRT1 and zinc (44). Although copper uptake
was not measured in that study, CTR1 internalization probably
facilitates down-regulation of copper uptake activity and
thereby limits copper accumulation. Copper was also found to
induce the proteolytic degradation of CTR1. In contrast to
ZRT1, however, CTR1 degradation did not require vacuolar