From the Department of Environmental Health Sciences, Johns Hopkins University, School of Public Health, Baltimore, Maryland 21205
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ABSTRACT |
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The Saccharomyces cerevisiae SMF1
gene encodes a member of the well conserved family of Nramp metal
transport proteins. Previously, we determined that heavy metal uptake
by Smf1p was down-regulated by the product of the S. cerevisiae
BSD2 gene. We now demonstrate that this regulation occurs at the
level of protein stability. In wild type strains, the bulk of Smf1p is
normally directed to the vacuole and is rapidly degraded by vacuolar
proteases in a PEP4-dependent manner. In
bsd2 The Nramp family of polypeptides (for natural
resistance associated macrophage
protein) consists of a group of highly conserved integral membrane proteins thought to play an important role in heavy
metal transport. Homologues to Nramp have been identified in
animals, plants, and fungi, as well as in certain bacteria (1, 2).
Among the most studied are the Nramp1 and Nramp2 genes of rodents. Nramp1 is believed to control the
phagosomal accumulation of redox active iron or manganese ions, thereby
contributing to an oxygen radical defense against parasitic infection
(3-7). Nramp2 is expressed in all tissues and is needed for
proper iron absorption and utilization (7-9). In rats, the Nramp2
isoform (DCT1) is induced following iron starvation and exhibits a
broad substrate range including essential metals such as zinc, iron, manganese, and copper, as well as the nonessential metals cadmium and
lead (10). Transporters such as DCT1/Nramp that act on both essential
and toxic metals are expected to fall under tight cellular control.
The bakers' yeast Saccharomyces cerevisiae provides an
excellent model system in which to study the function and regulation of
eukaryotic metal transporters. The high affinity uptake of copper,
iron, and zinc in yeast is accomplished by the action of the
CTR1, FTR1, and ZTR1 gene products, respectively
(11-13). Each of these transport systems is induced under
metal-starvation conditions and correspondingly repressed at
physiological metal concentrations, and this regulation occurs at the
level of CTR1, FTR1, or ZTR1 gene transcription
(13-19). S. cerevisiae also contains two Nramp
homologues, SMF1 and SMF2 (1, 2, 20). Smf1p and
Smf2p share approximately 40% identity with mammalian Nramp proteins (1, 2, 20), and accordingly, murine Nramp2
complements the metal transport defect of smf mutant yeast
(21). Smf1p was identified by Supek et al. (6) as a
manganese transporter, and our subsequent studies demonstrated that
Smf1p and Smf2p additionally participate in the transport of
other heavy metals including copper, cobalt, and cadmium (22). Hence
like mammalian Nramp transporters, yeast Smf proteins exhibit a broad
specificity for both essential and nonessential toxic metals.
We have previously shown that metal transport by Smf1p is suppressed in
yeast by a process involving the product of the BSD2 gene
(22). When BSD2 is inactivated by mutation, the transport of
copper and cadmium by Smf1p greatly increases, and cells accumulate toxic levels of the metals (22). Bsd2p exhibits an endoplasmic reticulum (ER)1 localization
(22), yet the mechanism by which Bsd2p controls Smf1p was unclear. In
the present study, we demonstrate that Bsd2p and metal ions act
together to facilitate the rapid turnover of Smf1p. This
post-translation control of Smf1p effectively minimizes the
hyper-accumulation of toxic metals and also provides a rapid switch for
inducing metal uptake under conditions of metal starvation.
Yeast Strains and Growth Conditions--
Most of these studies
employed the isogenic wild type (AA255) and
bsd2
Stocks of strains were maintained on standard yeast
extract/peptone/dextrose media, and cultures for experimental analysis were obtained by growth in a synthetic minimal medium containing dextrose (SD) (23). All yeast transformations were carried out by
electroporation (24). A metal-depleted minimal defined medium (MDM) was
prepared through use of an ion exchange resin as described (25). MDM
was supplemented with 2.4 mM MgSO4, 30 mM KCl, 2.0 mM CaCl2 and 0.86 mM NaCl. As needed, MDM also contained 10 µM ZnCl2, 10 µM
Fe(NH4)2(SO4)2, 1.0 µM CuSO4, 10 µM
MnSO4, 1.0 µM CoSO4, or a
combination of these metals.
Molecular Biology--
Construction of the Smf1-HA expressing
plasmid pSF4 involved polymerase chain reaction (PCR) amplification of
SMF1 sequences
The ubc7
RNA blot analysis of SMF1 expression followed standard
procedures (28) and employed a probe spanning SMF1 sequences
Immunodetection Techniques and Biochemical Assays--
For
Western blot analysis, yeast cells expressing the Smf1-HA fusion
protein were grown to a mid-logarithmic phase
(A600 = 1.0) in SD medium or MDM as needed.
Extracts were prepared either by glass bead homogenization (29), or by
an alkaline lysis procedure (30). Samples were resolved by 12%
SDS-PAGE and analyzed by Western blot using a mouse anti-HA antibody
(BABCO) as described previously (22).
Immunofluorescence microscopy analysis was conducted essentially as
described (22). Briefly, strains transformed with a CEN
Smf1-HA plasmid were grown to a mid-logarithmic stage in SD medium,
fixed with formaldehyde, digested with zymolyase, and probed with a
mouse anti-HA antibody and a secondary antibody consisting of a goat
anti-mouse antibody coupled to FITC (Boehringer Mannheim) as described
(22). Nucleic acids were stained by DAPI (Sigma). FITC and DAPI
staining were monitored by fluorescence microscopy, whereas
visualization of yeast vacuoles used Nomarski optics.
Sucrose gradient fraction was conducted with cells grown to an
A600 of 0.5-1.0. For examination of Golgi, ER,
and vacuolar markers, cell lysates were prepared as described (22) and
were fractionated over linear or step gradients of 18-54% sucrose. Assays for GDPase and NADPH cytochrome c reductase were
carried out by standard procedures (29, 31). Assays for the vacuolar The Effects of bsd2 Mutations on Smf1p Protein Stability--
We
previously reported that the S. cerevisiae BSD2 gene
negatively regulates the accumulation of metal ions by the Smf1p metal transporter (22). In the present study, we monitored the accumulation and localization of this transporter using an epitope-tagged version of
the protein. Two copies of the hemagglutinin (HA) epitope were fused in
frame to Smf1p at the stop codon, creating a protein fusion that was
fully functional in complementing smf1
To investigate whether bsd2
We next addressed whether bsd2 mutations affect Smf1 protein
stability. Through the use of cycloheximide to inhibit new protein synthesis, we observed that Smf1-HA is normally a very unstable protein
and exhibits an apparent half-life between 10 and 20 min (Fig.
2). In comparison, Smf1-HA was greatly
stabilized in the bsd2 BSD2 Regulation of Smf1p Depends on Vacuolar but Not
Ubiquitin-mediated Proteolysis--
Two major protein degradation
pathways predominate in yeast, a ubiquitin-mediated pathway and a
pathway involving proteolytic breakdown in the vacuole. In the
ubiquitin-mediated pathway, proteins destined for degradation by the 26 S proteosome are tagged with ubiquitin via the action of a
ubiquitin-conjugating enzyme such as Ubc7p (33). The possible
involvement of this pathway in the degradation of Smf1p was examined
through use of an ubc7
To examine if Smf1p degradation involves the vacuole, we measured the
steady state level of Smf1-HA in strains containing mutations in
PEP4 necessary for vacuolar proteolysis (34). As shown in
Fig. 3A, Smf1-HA accumulated to a high level in the
pep4
The stabilization of Smf1p in a bsd2 mutant results in
hyper-accumulation of copper and cadmium (22). Because pep4
mutations also cause stabilization of Smf1p, we tested whether the
accumulated Smf1p in the pep4 mutant results in elevated
metal uptake. Unlike bsd2 Localization of Smf1p--
The subcellular localization of Smf1-HA
was examined by indirect immunofluorescence microscopy using a
secondary antibody coupled to FITC. As seen in Fig.
4A, Smf1-HA expressed in a
wild type strain exhibited rimming surrounding the nucleus (defined by
DAPI) and a light punctate staining pattern that was absent in control
cells not expressing Smf1-HA (not shown). This staining pattern
suggested localization to the ER and Golgi, and this was confirmed by
biochemical fractionation studies: Smf1-HA co-migrated with markers for
the Golgi and ER in sucrose gradient fractionation (Fig.
4B). It is noteworthy that our immunofluorescence studies failed to reveal the anticipated plasma membrane localization for
Smf1p. To ascertain if the intracellular localization of Smf1-HA represented rapid internalization of a cell surface protein, we tested
Smf1-HA localization in an end4 mutant defective for
endocytosis (35). We observed that Smf1-HA staining was not affected by the end4 mutation (not shown), demonstrating that the bulk
of Smf1p is not normally present on the cell surface. In a
bsd2 mutant, Smf1-HA exhibited a much brighter staining
pattern because of elevated levels of this polypeptide, but the
perinuclear and punctate staining pattern was still evident (Fig.
4A). A fraction of Smf1p appeared to be surface-localized in
some bsd2 cells, although this represented a minor component
(Fig. 4A).
Smf1-HA localization was additionally examined in a pep4 Metal Ions and the Rapid Turnover of Smf1p--
We addressed
whether metal ions can influence the stability of Smf1p. Treating the
growth medium with elevated concentrations of heavy metals such as
copper and manganese did not change the steady state levels of Smf1-HA
(not shown). However, depletion of the heavy metals zinc, cobalt,
copper, manganese and iron from the growth medium mimicked the effects
of a bsd2 mutation and resulted in high accumulation of
Smf1-HA (Fig. 7A). The
addition of manganese back to the growth medium was sufficient to
down-regulate Smf1-HA protein levels in the wild type strain (Fig. 7,
A and B), but not in the isogenic strain
containing a bsd2 mutation (Fig. 7B).
Down-regulation of Smf1-HA was also observed upon supplementation of
iron to the metal-depleted medium, although to a lesser extent than was
observed with manganese (Fig. 7A). The individual addition of zinc, copper or cobalt to the metal-depleted medium had no effect on
Smf1-HA levels (Fig. 7A).
We next tested whether the down-regulation of Smf1p by manganese ions
occurs at the level of gene transcription or protein stability. As seen
in Fig. 7C, manganese depletion had no effect on
SMF1 mRNA levels, and the same was seen with iron
depletion (not shown). To test for protein stability effects, the time
course of Smf1-HA degradation was monitored in strains grown under
manganese deplete or replete conditions. As seen in Fig. 7D,
Smf1-HA expressed in the wild type strain was stabilized in the
metal-depleted medium, and supplementation of manganese to the growth
medium resulted in instability of Smf1-HA in the wild type strain but
not in the bsd2 mutant (Fig. 7D). These findings
demonstrate that metal ions can induce degradation of Smf1p through a
mechanism dependent upon the BSD2 gene product.
To examine further the effect of metal ions on Smf1p, the localization
of Smf1-HA was examined in cells grown in metal-starved versus metal-replete conditions. These studies were
conducted with a pep4 mutant such that vacuolar Smf1-HA
could be readily discerned. Under metal-starvation conditions, intense
staining of Smf1-HA was observed at the cell surface (Fig.
8). Supplementation of metals back to
this metal-depleted medium resulted in a dramatic shift in Smf1-HA
localization where the bulk of this transporter was now found in the
vacuole and was absent from the cell surface (Fig. 8). Therefore, the
cellular localization of Smf1-HA is strongly influenced by the metal
ion status of the cell. When metal ions are ample, Smf1p is targeted to
the vacuole in a manner dependent on Bsd2p (Fig. 6, A and
B). Yet Bsd2p is not required for the recruitment of Smf1p
to the plasma membrane upon metal depletion as metal starvation still
triggered the cell surface localization of Smf1-HA in cells lacking
Bsd2p (Fig. 6C). Therefore, factors other than Bsd2p are
involved in the trafficking of Smf1-HA to the cell surface under
metal-starvation conditions.
These studies have addressed the regulation of metal transport by
the yeast Nramp protein Smf1p. This transporter was previously shown to
fall under negative control by the S. cerevisiae BSD2 gene
(22), and we now demonstrate that this repression occurs at the level
of Smf1p stability. The bulk of Smf1p is normally targeted to the
vacuole for degradation by vacuolar proteases, yet in bsd2
mutants, the transporter fails to arrive at the vacuole and the bulk of
the protein remains within the secretory pathway. Metal ions also play
an important role in Smf1p regulation. Exposure of cells to
physiological concentrations of metals such as manganese induces the
degradation of Smf1p in a Bsd2p-dependent manner. Under
conditions of metal starvation, the yeast Nramp protein fails to arrive
at the vacuole and accumulates at the cell surface. This switch in
localization to the cell surface occurs independent of Bsd2p.
Regulation of other metal transporters in yeast (e.g. the
Ctr1p (copper), Ftr1p (iron), and Zrt1p/Zrt2p (zinc) transporters) occurs predominantly at the level of gene transcription (13-19). Ctr1p
and Zrt1p additionally exhibit protein stability effects, but turnover
of these transporters is induced only at relatively high copper and
zinc concentrations, whereas the degradation of Smf1p occurs at
physiological concentrations of metals (30, 36). We have found no
evidence of transcriptional control of SMF1 by metals. The
strong regulation of Smf1p at the protein stability level may be
particularly important for a transporter that acts on both essential
(e.g. manganese) and nonessential toxic (e.g.
cadmium) metals. Targeting Smf1p to the vacuole ensures cessation of
harmful metal uptake, yet when cells are starved for essential metals,
Nramp activity can be rapidly induced by protein stabilization and
redistribution to the plasma membrane, bypassing the need for new Smf1p synthesis.
How is Smf1p directed to the vacuole? When metals are abundant, the
transporter appears to move directly through the secretory pathway to
the vacuole without plasma membrane routing since a block in
endocytosis fails to effect a plasma membrane localization for Smf1p
under these conditions. Furthermore, Smf1p targeting to the vacuole is
completely dependent on Bsd2p in the ER. Bsd2p does not appear to be a
general receptor for the transport of polypeptides to the vacuole
because bsd2 mutations do not impact on the delivery of
carboxypeptidase Y (CPY) to the vacuole (37). However, Chang and
co-workers have noted that a mutant allele of the plasma membrane
proton ATPase, Pma1p (Pma1-7p), is targeted to the vacuole in a
Bsd2-dependent manner (37). Presumably mutant Pma1-7p
adopts a specific conformation that is recognized by the Bsd2p-dependent machinery for vacuole targeting. We
therefore propose that Bsd2p (or an auxiliary factor dependent on
Bsd2p) recognizes a specific conformation of a subset of ion
transporters as they pass through the secretory pathway and triggers
the trafficking of these transporters to the vacuole for degradation.
Our studies are consistent with a model in which the metal bound or
active form of Smf1p adopts a conformation that is recognized by Bsd2p
for targeting to the vacuole. First, the trafficking of Smf1p to the
vacuole is absolutely dependent on the presence of metals. Second, our
recent studies indicate that vacuolar targeting requires a Smf1
polypeptide that is functional for metal
transport.2 It is conceivable
that the apo form of Smf1p adopts an alternative conformation that
fails to be recognized by Bsd2p and additionally favors trafficking to
the plasma membrane independent of Bsd2p. Although the mechanism
underlying the switch for Smf1p trafficking is unknown, it may involve
the unmasking of putative signal sequences on the Smf1 polypeptide for
plasma membrane localization.
mutants, Smf1p fails to enter the vacuole, and the
Nramp protein is stabilized. Metal ions themselves play an important
role in the post-translational regulation of Smf1p. The depletion of
heavy metals from the growth medium effects stabilization of Smf1p and
additionally results in accumulation of this transporter at the cell
surface. Supplementation of manganese alone is sufficient to trigger
rapid degradation of Smf1p in a Bsd2p-dependent manner.
Together the action of Bsd2p and metal ions provide a rapid and
effective means for controlling Nramp metal transport in response to
environmental changes.
INTRODUCTION
Top
Abstract
Introduction
References
MATERIALS AND METHODS
::HIS3 mutant (XL115) strains as
described previously (22). Strains XL112 (22) and XL120 are
smf1
::URA3 and
bsd2
::URA3 derivatives of AA255.
XL118 and XL119 are ubc7
::HIS3
mutants derived from AA255 and XL120, respectively. The isogenic wild type (L3852) and pep4
::URA3 (ACY17)
strains were kind gifts of A. Chang. XL121 and XL125 were obtained by
replacing the chromosomal BSD2 gene with HIS3 as
described (22) in strains L3852 and ACY17, respectively. XL126 and
XL124 are pep4::HIS3 mutants derived from AA255
and XL101, respectively.
230 to the stop codon using a primer that
changed the termination sequence to an NdeI site (CAT ATG in
frame with SMF1). The product was ligated to the pCRII
vector (Invitrogen), and following digestion with NdeI and
ApaI, the SMF1-containing fragment was used to
replace the BSD2 sequences of a CEN LEU2 plasmid
expressing Bsd2-HA (22). The Smf1-HA URA3 CEN plasmid pSF10
was obtained by inserting the Smf1-HA containing insert of pSF4 into
the XhoI and SacI sites of pRS416 (26).
mutation was introduced in yeast strains by the
PCR-mediated gene disruption method as described (27). A
ubc7
::HIS3 cassette was amplified by
PCR using the HIS3 plasmid pRS403 (26) as template, and
primers that spanned UBC7 sequences
140 to
100 and +533
to +494, with respect to the start codon, both fused at their 3' ends
to the sequence designated by Brachmann et al. (27) for
amplification of yeast auxotrophic markers. Proper deletion of
UBC7 was confirmed by colony PCR. The pep4
mutation was similarly introduced using primers that spanned
PEP4 sequences +24 to +64 and +1230 to +1270.
86 to +1833 amplified by PCR and radiolabeled with
32P.
-mannosidase involved monitoring fractions for absorbance at 400 nm
as described (32).
RESULTS
mutations when
expressed from its native promoter and present on a single copy
CEN vector (not shown). To monitor the effects of
bsd2 mutations on Smf1p levels, total extracts from cells
expressing the fusion protein were analyzed by immunoblot. As seen in
Fig. 1A, Smf1-HA expressed
from the CEN vector was increased by approximately
10-20-fold in a bsd2 null mutant compared with a
BSD2 wild type strain.
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Fig. 1.
Mutations in BSD2 cause an
increase in Smf1 protein levels. A, Western blot of
Smf1-HA. Extracts were prepared from the indicated yeast strains
transformed with either the Smf1-HA CEN plasmid pSF4 or with
pRS315 vector ( ) (26), as indicated. 5 µg of cell protein were
subjected to 12% PAGE, followed by Western blot analysis using an
anti-HA antibody. Migration of the 66-kDa Smf1-HA band was followed by
co-electrophoresis of molecular weight markers (left).
B, Northern blot analysis of SMF1 expression.
Total RNA from the indicated strains was subjected to Northern blot
analysis using SMF1 containing sequences as probe
(top), followed by rehybridization with a ACT1
probe encoding actin (38) as control (bottom). Strains
utilized were: wt = AA255; bsd2
= XL115;
and smf1
= XL112.
mutations result in a
transcriptional induction of the SMF1 gene, total RNA from
isogenic wild type and bsd2
mutants was subjected to
Northern blot analysis. We observed that mutations in BSD2
did not increase the expression of SMF1 at the mRNA
level (Fig. 1B).
mutant (Fig. 2). These results
demonstrated that the BSD2 gene product is involved in
controlling stability of the Smf1p polypeptide.
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Fig. 2.
bsd2 mutations
increase the stability of Smf1p. The indicated strains of yeast
transformed with the Smf1-HA plasmid pSF4 were treated with 100 µg/ml
cycloheximide for the designated time points prior to cell lysis and
analysis of Smf1-HA by Western blot as described in Fig. 1A.
Ten times more cell lysate was analyzed in wild type (wt)
samples compared with bsd2
samples. Strains utilized
were: wt = AA255; bsd2
= XL115.
mutant. As seen in Fig.
3A, the steady state levels of
Smf1-HA were increased in the ubc7
null strain,
suggesting that the proteosome participates to some degree in Smf1p
degradation. However, inactivation of BSD2 still caused a
rise in Smf1-HA protein levels in the ubc7 mutant (Fig.
3A). The strong additive effects of bsd2 and
ubc7 mutations indicated that BSD2 does not work
through the ubiquitin pathway to control Smf1p stability.
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Fig. 3.
The role of the ubiquitin-mediated and
vacuolar pathways of protein degradation in the control of Smf1p
stability. The indicated yeast strains transformed with pSF4 were
subjected to Western blot analysis of Smf1-HA as described in the
legends to Figs. 1A and 2. A, cells were lysed
without additional treatment. B, cells were treated with
cycloheximide for the given time points prior to preparation of cell
lysates. Ten times more cell lysate was analyzed in wild type
(wt) samples than the other strains. Strains utilized were:
panel A, left, wt = AA255;
bsd2 = XL120; ubc7 = XL118; and
bsd2 ubc7 = XL119; panel A, right, and
panel B, wt = L3852; bsd2 = XL121; pep4 = ACY17; and bsd2 pep4 = XL125.
mutant. Furthermore, an additional mutation in
BSD2 did not increase this high level of Smf1-HA. We next
compared the turnover rates of Smf1-HA in these strains. A
pep4 mutation was seen to increase Smf1-HA stability, and
there was no additive effect of double mutations in BSD2 and
PEP4 (Fig. 3B). Thus, BSD2 works
through vacuolar protein degradation to control Smf1p stability.
mutations, mutations in
PEP4 did not effect a rise in metal accumulation (not
shown). Thus the hyper-accumulated Smf1p in a pep4 mutant
appears nonfunctional for metal transport.
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Fig. 4.
Localization of Smf1-HA. A,
the wild type strain AA255 (wt) or bsd2 mutant
XL115 (bsd2
) were transformed with the Smf1-HA plasmid
pSF4 and were probed with either anti-HA and an FITC-conjugated
anti-rabbit antibody (FITC) or with DAPI for detection of
nucleic acids. Cells were analyzed by fluorescence microscopy at × 1000 magnification. B, extracts were prepared from strain
AA255 expressing Smf1-HA and were subjected to sucrose gradient
fractionation. Fractions were collected from the top to bottom and were
assayed for GDPase activity (
) in nanomoles of Pi
liberated per 20 min (a Golgi marker (G)); for NADPH
cytochrome C reductase (
) in nanomoles of cytochrome c
reduced per 10 min per µg of protein (an endoplasmic reticulum marker
(ER)); and for Smf1-HA (
) detected by Western blot,
quantitated by densitometric tracings, and shown as a percentage of the
maximal level of Smf1-HA.
mutant defective for vacuolar degradation. In this mutant, very intense
staining of Smf1-HA was found within the vacuole identified by Nomarski
optics (Fig. 5A), and in
sucrose gradient fractionation, the bulk of Smf1-HA co-migrated with
the vacuolar
-mannosidase marker (Fig.
6A). A quite distinct pattern
of Smf1-HA localization was achieved in an isogenic pep4
bsd2
double mutant. By immunofluorescence microscopy,
Smf1-HA was absent from the vacuole, and the protein exhibited a
punctate staining pattern (Fig. 5B). Smf1-HA expressed in
these cells failed to co-migrate with the vacuolar marker during sucrose gradient centrifugation and, instead, superimposed the markers
for Golgi and ER (Fig. 6B). Together, these studies
demonstrate that Smf1p is normally targeted to the vacuole for
degradation by PEP4-dependent proteases. Furthermore, this
delivery of Smf1p to the vacuole is dependent upon the product of the
BSD2 gene.
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Fig. 5.
Localization of Smf1-HA in a pep4
mutant. The pep4 mutant ACY17 (A)
or the bsd2 pep4 double mutant XL125
(B-C) expressing Smf1-HA were grown in a minimal
defined medium (MDM) supplemented with the heavy metal
mixture as described under "Materials and Methods"
(A-B) or in the same medium lacking these metals
(C) and were examined by fluorescence microscopy
(left) as indicted in legend to Fig. 4A.
Nomarski, Nomarski optics utilized for visualization of
vacuoles that appear as indentations.
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Fig. 6.
Subcellular fractionation of pep4
mutants expressing Smf1-HA. Cell lysates prepared from
the isogenic pep4 XL126 strain transformed with pSF4
(A) or the pep4
bsd2
strain
XL124 transformed with pSF10 (B) were subjected to sucrose
gradient fractionation as in Fig. 4B. Fractions were assayed
for
-mannosidase (
) in absorption units at 400 nm (a vacuolar
marker (V)); for GDPase activity (
), a Golgi marker
(G); for NADPH cytochrome c reductase (
), an
endoplasmic reticulum marker (ER); and for Smf1-HA (
) as
described in Fig. 4B.
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Fig. 7.
Effect of metal ions on the accumulation and
stability of Smf1p. Strain AA255 (A, C and
B, D, where indicated by wt) or the
bsd2 mutant bsd2 XL115 (B,
D, where indicated) were grown in MDM depleted of heavy
metals (
or Mn:
) or in the same medium supplemented individually
as indicated with iron, manganese, cobalt, copper, or zinc or
supplemented with a combination of these metals (all) as
described under "Materials and Methods."
A-B, Smf1-HA was detected by Western blot as in
Fig. 1A. C, SMF1 gene expression was
monitored by Northern blot as in Fig. 1B. D,
cells were treated with cycloheximide for the indicated time points
prior to analysis of Smf1-HA by Western blot as in Fig. 2. Five times
more cell lysate was analyzed in the case of "wt + Mn."
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Fig. 8.
Immunofluorescence microscopy of Smf1-HA
under metal-starvation conditions. The pep4 mutant
strain XL126 expressing Smf1-HA was grown in MDM specifically depleted
of heavy metals ( metals) or in the same medium
supplemented with the heavy metal mixture as described under
"Materials and Methods" (+metals). Cells were examined
by fluorescence microscopy (FITC) for detection of Smf1-HA,
by Nomarski optics for visualization of vacuoles and by DAPI staining
for nucleic acids, as in Figs. 4A and 5.
DISCUSSION
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ACKNOWLEDGEMENTS |
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We are indebted to Amy Chang, Scott Emr, Susan Michaelis, Cecile Pickard, and David Levin for invaluable discussions.
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FOOTNOTES |
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* This work was funded by the Johns Hopkins University NIEHS Center, National Institutes of Health, and by National Institutes of Health Grant ES 08996 (to V. C. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by National Institutes of Health training grant ES 07141.
§ To whom correspondence should be addressed: Johns Hopkins University, 615 N. Wolfe St., Rm. 7032, Baltimore, MD 21205. Tel.: 410-955-3029; Fax: 410-955-0116; E-mail: vculotta{at}jhsph.edu.
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ABBREVIATIONS |
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The abbreviations used are: ER, endoplasmic reticulum; MDM, metal-depleted minimal defined medium; PCR, polymerase chain reaction; SD, synthetic minimal medium containing dextrose; FITC, fluorescein isothiocyanate; DAPI, 4,6-diamidino-2-phenylindole; HA, hemagglutinin.
2 X. F. Liu and V. C. Culotta, manuscript submitted.
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REFERENCES |
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