(Received for publication, July 29, 1994; and in revised form, November 28, 1994)
From the
The Schizosaccharomyces pombe hmt1 gene encodes an ABC
(ATP-binding cassette)-type protein essential for Cd tolerance. Immunoblot analysis of subcellular fractions indicates
that the native HMT1 polypeptide is associated with the vacuolar
membrane. Vacuolar membrane vesicles were purified from strains that
hyperproduce, or are deficient in, the HMT1 protein. In vitro transport of radiolabeled substrates by these vesicles indicates
that HMT1 is an ATP-dependent transporter of phytochelatins, the
metal-chelating peptides involved in heavy metal tolerance of plants
and certain fungi. Vacuolar vesicles containing HMT1 are capable of
taking up both apo-phytochelatins and phytochelatin-Cd
complexes. HMT1 activity is sensitive to antibodies directed
against this protein and to vanadate, but not to inhibitors affecting
the vacuolar proton ATPase or ionophores that abolish the pH gradient
across the vacuolar membrane. Vacuolar uptake of Cd
and of a glutathione conjugate were also observed, but are not
attributable to HMT1. These studies highlight the importance of the
yeast vacuole in detoxification of xenobiotics.
ABC ()(ATP-binding cassette)-type proteins represent
one of the largest known families of membrane transporters(1) .
Members of this superfamily are characterized by the presence of a
highly conserved nucleotide-binding domain that is associated with a
more variable region capable of spanning the membrane multiple times.
DNA sequence analysis indicated that the Schizosaccharomyces pombe
hmt1 (heavy metal tolerance 1)
gene, essential for Cd
tolerance, encodes an ABC-type
protein(2) . Most eukaryotic ABC-type polypeptides exhibit a
duplicated structure, containing two transmembrane and two
nucleotide-binding domains. HMT1, on the other hand, has only one of
each. The few known eukaryotic ABC-type proteins that exhibit this
nonduplicated arrangement reside in internal membranes: rat PMP70 (3) and its human homologue (4) in the peroxisome and
TAP1 and TAP2 in endoplasmic reticulum and cis-Golgi(5) .
Previously we reported that an HMT1-
-galactosidase fusion protein
was localized to the fission yeast vacuole(2) .
ABC-type
proteins can mediate tolerance to a wide diversity of cytotoxic agents.
Multiple drug resistance proteins from mammals (reviewed in (6) ) and yeast (7, 8) decrease the toxicity
of a variety of anti-tumorigenic drugs. Leishmania IgptA
confers tolerance to methotrexate and arsenite(9) , and a
number of bacterial transporters are involved in resistance to
antibiotics and environmental toxins (reviewed in (10) ).
Resistance is achieved by export of the toxic substances from the cell.
The intracellular location of the HMT1--galactosidase chimera, on
the other hand, suggested that this protein might represent the first
example of an ABC-type transporter mediating resistance to a toxin by
sequestration in an intracellular membrane-bound compartment. The
nature of the substrate transported by HMT1, and its role in heavy
metal tolerance of fission yeast were not clear.
Plants and certain
fungi, including S. pombe, respond to heavy metals by inducing
synthesis of small peptides known as phytochelatins
(PCs)(11, 12, 13) . Unlike metallothioneins,
PCs are not produced by mRNA translation but are enzymatically
synthesized from glutathione (GSH, -Glu-Cys-Gly). PCs have the
general structure (
-Glu-Cys)
Gly, where n = 2-11, and like metallothioneins, chelate heavy
metals by formation of thiolate bonds. Yeast and plant cells exposed to
Cd
accumulate a low molecular weight (LMW) PC
Cd
complex, consisting mostly of PCs and Cd
, and a high
molecular weight (HMW) PC
Cd
S
complex,
containing acid labile sulfide (14, 15, 16) .
Genetic and biochemical analyses suggest that production of the sulfide
moiety in the HMW PC
Cd
S
complex involves
the purine biosynthetic pathway(17, 18) . The HMW
complex, a CdS crystallite coated by PC peptides(19) , has a
higher Cd
-binding capacity than LMW PC
Cd, and
the Cd
ions are less susceptible to acid
displacement(20) . Although detailed structures for HMW and LMW
PC
Cd complexes are not available, it is clear that both are
heterogeneous oligomeric aggregates containing multiple PC peptides of
variable length.
Since overexpression of hmt1 in yeast
cells confers enhanced Cd tolerance, as well as
accumulation of the metal(2) , it was postulated that HMT1 was
involved in vacuolar sequestration of heavy metals or metal-peptide
complexes. In this report we describe the localization of native HMT1
protein to vacuolar membranes and the discovery of an ATP-dependent PC
transport activity in vacuolar membrane vesicles. We present evidence
showing that HMT1 is responsible for this activity and describe the
substrate specificity and energy requirements of this transporter. In
addition, we observed Cd
and glutathione conjugate
transport activities in fission yeast vacuolar vesicles lacking
detectable amounts of HMT1 protein.
Plasmid pDH30(2) , containing an hmt1 cDNA in the pBluescript SK vector (Stratagene), was used for in vitro transcription/translation by the TnT T7 system
(Promega) in the presence of [S]Met (1141
Ci/mmol, DuPont). Canine pancreatic microsomal membranes (Promega) were
added according to the manufacturer's instructions. Translation
products were immunoprecipitated using Protein A-agarose or directly
separated by 10% SDS-polyacrylamide gel electrophoresis for
fluorography.
[H]GSSG was prepared from
[
H]GSH (300 mCi/mmol, DuPont)(28) . The
2,4-dinitrophenyl-S-glutathione conjugate (GSDNP) was
enzymatically synthesized from 1-chloro-2,4-dinitrobenzene (Sigma) and
[
H]GSH by glutathione S-transferase
(Sigma)(29) . [
H]GSDNP was separated from
its precursors by thin layer chromatography and quantified by
absorbance at 340 nm.
A 29-kDa peptide, encompassing most of the
HMT1 ATP-binding cassette domain, was synthesized in E. coli,
purified, and used to raise antibodies (antisera 622 and 623). Both
antisera recognized a 90-kDa protein in S. pombe (Fig. 1A) that was not detected by preimmune sera
and is consistent with the 90.5-kDa molecular mass predicted for HMT1.
The 90-kDa protein was associated with the P100 (100,000 g precipitable) particulate fraction of S. pombe extracts (lane g), but is not detected in the S100 supernatant (lane e), as expected of an integral membrane protein.
Figure 1:
A, immunoblot of S. pombe proteins using antiserum 622. Lanes contain 2 µg of protein
from vacuolar membrane vesicles prepared from: (a) LK100/pDH35 (hmt1 mutant complemented by an hmt1 cDNA in pART1); (b) Sp223/pART1 (hmt1
strain with empty vector); and (c) LK100/pART1. 40
µg of protein from: (d) a P100 (100,000
g precipitable) pellet derived from the supernatant overlying the
purified vacuole pellet and (e) the S100 (supernatant)
fraction of Sp223/pART1. 50 µg of protein from the P100 fractions
of extracts made from: (f) LK100/pDH35; (g)
Sp223/pART1; and (h) LK100/pART1. The blot was overdeveloped
to show the lack of HMT1 in LK100/pART1 lanes. The smaller bands (62
and 39 kDa) recognized by the antibody varied in intensity in different
experiments and are probably produced by proteolytic degradation of
HMT1. Similar effects have been reported with preparations of mammalian
P-glycoprotein(32) . B, Coomassie stained replica of
the portion of the gel (lanes a-c) shown in A that
contains vacuolar membranes.
Vacuoles were prepared from S. pombe spheroplasts as
described under ``Materials and Methods.'' Compared to the
particulate fraction of a total cellular extract, purified vacuolar
vesicles are highly enriched in vacuolar marker enzymes and
impoverished in markers for mitochondrial, Golgi, endoplasmic
reticulum, and plasma membranes (Table 1). In the immunoblot in Fig. 1A, lanes loaded with vacuolar membranes (Fig. 1A, lanes a and b) exhibited enrichment
of the hmt1 product when compared with total cellular
membranes (lanes f and g) despite containing 25-fold
less protein. The supernatant overlying the vacuolar pellet was
impoverished in HMT1 (lane d), suggesting that this protein is
sorted primarily to the vacuolar membrane. Cd treatment of wild type cultures did not alter levels of the
90-kDa protein (not shown).
The 90-kDa polypeptide was not detected
in total (lane h), or vacuolar (lane c), membranes
derived from the Cd-sensitive LK100 strain, which
bears an hmt1
allele with a nonsense
mutation in the 5` region of the gene(2) . Strains transformed
with the pDH35 plasmid accumulate high levels of the hmt1 mRNA (2) and 90-kDa peptide (Fig. 1A, lanes a and f and B, lane a).
Figure 2:
In vitro transcription/translation of hmt1 cDNA. A,
fluorograph of an SDS-polyacrylamide gel electrophoresis gel containing
[S]Met labeled in vitro translation
products derived from: (a) pBluescript SK+ canine
pancreatic microsomes; (b) pDH30 (pBluescript SK containing an hmt1 cDNA) without additives; (c) pDH30 + 1%
Triton X-100; (d) pDH30 + canine pancreatic microsomes. B, fluorograph of immunoprecipitated products from in
vitro transcription/translation of pDH30 in the presence of 1%
Triton X-100: (a) preimmune serum of rabbit 622; (b)
anti-HMT1 antiserum 622; (c) anti-HMT1 antiserum
623.
Figure 3:
Uptake
of S-labeled PC
Cd complexes by purified vacuolar
membrane vesicles. A, ATP-dependent uptake of LMW PC
Cd.
Vacuolar vesicles (20-40 µg of protein) prepared from the hmt1
mutant LK100 bearing the pART1 vector
containing an hmt1 cDNA (pDH35) (
,
) or no insert
(
,
) were incubated with 8 µM GSH
equivalents of LMW [
S]PC
Cd complex (50,000
cpm) in the presence (
,
) or absence (
,
) of 3
mM ATP and an ATP regeneration system. Aliquots were taken at
the times indicated and substrate uptake determined by the vesicle
filtration assay. Arrow indicates addition of Triton X-100 to
0.02%. B, comparison of uptake of LMW PC
Cd and HMW
PC
Cd
S
complex by vacuolar vesicles.
Vesicles from LK100/pDH35 were incubated with an equal number of counts
(50,000 cpm representing 6 µM GSH equivalents) of LMW
[
S]PC
Cd (
) or HMW
[
S] PC
Cd
S
(
) in the presence of ATP. Bars represent
S.E.
Figure 4:
Dependence of PCCd uptake on ATP
concentration. Double reciprocal plot of data used to calculate the K
of HMT1 for ATP. Linear regression
curve was calculated as described(35) . Inset: initial
velocity of LMW [
S]PC
Cd complex uptake
(calculated as in Table 2) by LK100/pDH35 vacuolar membrane
vesicles measured in the presence of an ATP regeneration system and
varying concentrations of ATP. Bars represent
S.E.
Figure 5:
Inhibition of PCCd uptake by
reduction of internal vesicle space. Uptake of LMW
[
S]PC
Cd complex was measured as described
in the legend to Fig. 3in the presence of 0.5, 0.75, 1.0, 1.25,
and 1.5 M sucrose. The initial uptake velocity (determined as
in Table 2) is plotted against the reciprocal of the sucrose
concentration. Bars represent S.E.
Unlike vesicles derived
from the HMT1 hyperproducing strain, LK100/pART1 vacuolar membrane
vesicles exhibited no ATP-dependent transport of the LMW
[S]PC
Cd complex (Fig. 3A).
Very little activity was observed with membrane vesicles derived from
Sp223/pART1 (not shown), probably because of the relatively low level
of HMT1 protein present in wild type vacuoles (compare lanes a and b in Fig. 1B) and low specific
activity of the in vivo labeled PC substrate. The differences
in PC uptake activity among the various strains was not due to
differential labilities of vacuolar vesicles. Transport competency of
the isolated vacuolar vesicles were similar with regards to
Cd
and GSDNP (see below). Additionally, vacuolar
vesicles from all three strains exhibited comparable levels of
ATP-dependent [
C]arginine uptake (not shown), an
activity that is also present in vacuoles of Saccharomyces
cerevisiae(36) and Neurospora
crassa(37) .
One of the polyclonal antibodies directed against HMT1 inhibited the PC transport activity in vitro. Incubation of vacuolar vesicles with 1 mg/ml IgG purified from antiserum 622 resulted in a 63% reduction in the initial velocity of PC uptake (determined as in Table 2) from 2.8 ± 0.6 to 1.3 ± 0.1 nmol/min/mg. Increasing antibody concentration did not further inhibit PC uptake. IgG purified from antiserum 623 had no effect on PC transport even at concentrations above 2 mg/ml IgG. The lack of PC uptake by vacuoles derived from the HMT1-deficient mutant, the high level of ATP-dependent PC transport observed in vacuolar vesicles prepared from the HMT1 hyperproducing strain, and the inhibition of PC uptake by antibodies directed against HMT1, all indicate that the hmt1 gene encodes the PC transporter.
Vacuolar vesicle uptake of labeled PCs was challenged with a series
of inhibitors (Table 2). Vanadate, which inhibits the activity of
a number of ABC-type proteins, significantly inhibited PC uptake with
an IC of 0.082 ± 0.004 mM. Bafilomycin A,
an inhibitor of vacuolar ATPase, nigericin, a proton-K
ionophore, and valinomycin, a K
ionophore, had
no significant effect, indicating that PC transport is not dependent on
the
pH across the vacuolar membrane.
Initial velocity
for ATP-dependent uptake of the LMW PCCd complex was 2.4
nmol/min/mg versus 0.7 nmol/min/mg for the HMW
PC
Cd
S
complex (Fig. 3B).
Estimating the precise difference in the rate with which these two
complexes were taken up is confounded by the variable number of PC
molecules per complex, over 25 in the HMW
PC
Cd
S
complex (19) compared with
3-8 in the LMW PC
Cd complex(38) . An additional
consideration is that the HMW PC
Cd
S
complex can spontaneously break down to form the LMW PC
Cd
form, thus it is unclear what proportion of counts taken up by the
vacuolar vesicles represents bona fide transport of the HMW complex.
Both of these factors lead to an overestimation of the rate of HMW
PC
Cd
S
transport, and an underestimation
of the difference in the relative affinity of HMT1 for these two
substrates.
Figure 6:
Vacuolar vesicle uptake of substrates
related to PC complexes. Vesicles derived from LK100/pDH35 (,
) and LK100/pART (
,
) were tested as in Fig. 2A in the presence (
,
) or absence
(
,
) of 3 mM ATP for uptake of the following
substrates: A, 8 µM
CdCl
(100 mCi/mmol); B, 50 µM [
H]GSDNP. Arrow indicates addition
of Triton X-100 to 0.02%. Bars represent
S.E.
Recent reports indicate that plant vacuoles
contain a transporter that recognizes various GSH
conjugates(40) , including GSDNP which is recognized with high
affinity. HMT1 function might be a part of a more generalized
xenobiotic detoxification pathway involving sequestration of GSH
conjugates produced by glutathione S-transferases. Vacuolar
membrane vesicles exhibited ATP-dependent
[H]GSDNP uptake (Fig. 6B) that
was abolished at 0 °C, or when ATP was substituted with AMP, ADP,
or AMP-PNP. Unlike PC transport, however, vacuolar vesicles derived
from LK100/pART1 or LK100/pDH35 exhibited no significant difference
regarding [
H]GSDNP uptake. Thus, fission yeast
vacuoles contain a GSH-conjugate transporter that is distinct from
HMT1. GSDNP transport was similar to HMT1-mediated PC uptake in that it
was not inhibited by proton ionophores or bafilomycin, but was
significantly reduced in the presence of 0.1 mM vanadate (not
shown).
It has been established that PCs are essential for heavy
metal tolerance in S. pombe(13) , and may also play an
important role in plants(12) . However, PC synthesis in and of
itself is not sufficient to confer heavy metal tolerance. Yeast strains
harboring a mutant hmt1 allele are Cd sensitive, notwithstanding their ability to synthesize PCs. In
this work we have shown that the native HMT1 protein is sorted to the
vacuolar membrane of S. pombe. We also report the discovery of
a novel PC transport activity in S. pombe vacuolar membrane
vesicles. PC uptake correlates with HMT1 abundance and is inhibited by
antibodies directed against this protein, indicating that HMT1 is most
likely responsible for PC transport into the vacuole. PC uptake by
vacuolar vesicles appears to be directly energized by ATP hydrolysis,
as expected of an ABC-type transporter. It is not dependent on activity
of the vacuolar proton ATPase, or sensitive to ionophores that
dissipate the
pH or membrane potential. Similar to other ABC-type
proteins, such as the cystic fibrosis transmembrane regulator (41) and P-glycoprotein(42) , HMT1-mediated PC uptake
is supported by ribonucleotides other than ATP and is inhibited by
vanadate.
HMT1 is capable of transporting both apo-PCs (measured as
uptake of [S]PC) and PC
Cd complexes
(measured as uptake of LMW [
Cd]PC
Cd). The
HMW PC
Cd
S
complex is also taken up by
vacuolar vesicles containing HMT1, albeit with lower efficiency. Some
ABC-type transporters exhibit wide substrate specificity. The multiple
drug resistance P-glycoprotein, for example, recognizes many
structurally-unrelated drugs, and the TAP1 and TAP2 transporters
mobilize peptides sharing very little, if any, amino acid sequence
similarity(43) . Thus, recognition of various types of PC
substrates by HMT1 would not be unprecedented. However, further study
is required to determine if the HMW PC
Cd
S
complex or apo-PCs represent bona fide substrates in vivo. The simplest model for the role of HMT1 in heavy metal tolerance
involves transport of PC-metal complexes into the vacuole as
illustrated in Fig. 7. The accumulation of PCs in the vacuoles
of Cd
-treated plant seedlings (45) might be
explained by the presence of an HMT1-like activity in the tonoplast.
Figure 7:
A model
of PC-mediated cadmium tolerance. Cd ions taken up by
the cell activate PC synthase (44) and induce synthesis of PCs,
which chelate cytoplasmic Cd
to form the LMW
PC
Cd complex. HMT1 actively transports the LMW PC
Cd complex
across the vacuolar membrane and sulfide is added within the vacuole to
generate the stable HMW PC
Cd
S
complex.
In this model, LMW PC
Cd functions as a cytoplasmic carrier,
whereas the HMW PC
Cd
S
complex is the
storage form of the metal. Since the ratio of Cd
to
PC is higher in the HMW complex than in the LMW complex, additional
Cd
could be supplied by the
Cd
/H
antiporter. Apo-PCs may also be
transported by HMT1, while Cd
enters through the
Cd
/H
antiporter.
HMT1 may not be dedicated exclusively to heavy metal tolerance.
Micronutrients such as copper or zinc, complexed with PCs, might be
stored in the vacuole via HMT1 transport and released to the cytoplasm
as required. PCCu and PC
Zn complexes, which can activate
some apo-metalloenzymes as efficiently as free metals(46) ,
have been shown to be a less toxic source of cofactors than free metal
ions(47) .
S. pombe vacuolar vesicles also exhibit
ATP-dependent uptake of Cd in the absence of PCs.
However, this activity is not attributable to HMT1 as it is present at
approximately the same level in a strain that hyperproduces this
protein and a mutant which does not contain detectable levels of the
HMT1 polypeptide. Cd
uptake appears to be mediated by
a Cd
/H
antiporter that depends on
the pH gradient generated by a vacuolar proton ATPase. A similar
activity residing in the tonoplast of oat roots has been
described(48) . The role of the Cd
transporter in heavy metal tolerance remains unclear.
Sequestration of free Cd
ions in the vacuole should
ameliorate their toxicity. However, LK100 cells, which contain an
active Cd
transporter, are still extremely sensitive
to Cd
. Plant cells exposed to Cd
contain very little, if any, of the free metal ion(47) .
Intracellular Cd
appears to be found almost
exclusively as PC
Cd complexes. This is in accordance with our
observations which indicate that S. pombe extracts contain
very little free Cd
(not shown). Furthermore, we find
that PC bound Cd
is not an efficient substrate for
the Cd
transporter. Thus, the major route for
sequestration of Cd
in the vacuole may be uptake of
PC
Cd complexes by HMT1. It is possible that the Cd
transporter is required when heavy metal first enters the cell
and PC levels are low, or when a metal challenge exhausts the
short-term capacity of the cell to synthesize stoichiometric amounts of
PCs. Alternatively, Cd
may represent an adventitious
substrate and the ``Cd
'' transporter may be
involved in some aspect of metal homeostasis unrelated to
Cd
tolerance. In S. cerevisiae and N.
crassa, a battery of antiporters maintain vacuolar pools of
Ca
, polyphosphate, and various amino acids, helping
to regulate the cytoplasmic levels of these substances (reviewed in (49) ). A metal antiporter may serve a similar function.
To our knowledge, HMT1 represents the first reported ABC-type transporter conferring tolerance to a toxic substance by sequestration in an intracellular membrane-bound compartment. The pfmdr1 gene product, implicated in chloroquine resistance, is localized to the digestive vacuole of Plasmodium falciparum(50) ; however, hyperproduction of this protein results in a 40-50-fold decrease in intracellular levels of the drug. S. pombe vacuoles also contain a glutathione conjugate transporter, which like HMT1 is insensitive to inhibitors of the vacuolar ATPase and sensitive to vanadate, suggesting by analogy that an ABC-type protein might be responsible. Recently, transport activities for glutathione conjugates (40) and bile acids (51) were found in the plant tonoplast. In both cases, it was hypothesized that ABC-type proteins were involved. Organisms are exposed to a large number of toxic compounds of both exogenous and endogenous origin. Conjugation of the toxins or their derivatives with ubiquitous intracellular metabolites such as glutathione, glucuronate, and sulfate is one way in which cells deal with this problem. In animals, a number of these conjugates are exported from the cell and then excreted from the organism. It appears that in plants and fungi, toxins ``tagged'' by conjugation (or chelation in the case of heavy metals) are sequestered in the vacuole, and that ABC-type transporters may play a major role in these detoxification pathways.