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INTRODUCTION |
The NifS protein is found in various organisms and is involved in
iron-sulfur protein biosynthesis. In the diazotroph Azotobacter vinelandii, the nifS gene was first identified as a
member of the nif operon that plays an essential role in the
biosynthesis of nitrogenase (1). Extensive biochemical analyses of
A. vinelandii NifS reveal that it is a
PLP1-containing enzyme
exhibiting desulfurase activity that produces elemental sulfur and
L-alanine from L-cysteine (2). These findings suggest that the physiological function of NifS is to supply inorganic sulfur for the assembly of the iron-sulfur cluster in nitrogenase (3,
4).
Many eukaryotic NifS homologues have been identified, of which the
Saccharomyces cerevisiae NifS homologue, Nfs1p, is the best
characterized (5-9). The nfs1 null mutant is lethal (5, 9),
and Nfs1p is localized mostly to the mitochondrial matrix (7-9).
Mitochondrial Nfs1p mediates the assembly of the iron-sulfur cluster of
both mitochondrial and cytosolic iron-sulfur proteins (6, 8) and also
regulates mitochondrial and cytosolic iron homeostasis (9). Kolman and
Söll (5) also show that a mutation in the NFS1 gene
suppressed a certain tRNA-splicing mutant (5), although the role of
Nfs1p in the process of tRNA splicing, which is thought to take place
in the nucleus, remains unclear.
Mouse and human counterparts to Nfs1p (mNfs1 and hNfs1, respectively)
have also been identified (7, 10). We showed that mNfs1 was found
mainly in the mitochondrial matrix (7). The full-length hNfs1
protein also possesses a mitochondrial-targeting presequence and has
been shown to be localized to mitochondria. Different sized hNfs1
proteins from a single transcript have also been detected in the
cytosolic and nuclear fractions (10), but the physiological
significance of these extramitochondrial hNfs1 proteins has not been elucidated.
The Escherichia coli NifS homologue, IscS, is a
multi-functional enzyme that possesses important physiological
functions not only in iron-sulfur cluster assembly (11, 12) but also in sulfur transfer to some intracellular compounds (13-18). These results
suggest that a fraction of Nfs1p may have some essential function other
than iron-sulfur cluster assembly, possibly in a different subcellular
location. In this study, we identified a potential nuclear
localization signal (NLS) sequence in the mature domain of yeast Nfs1p
and showed in vivo that this sequence was crucial for the
physiological function of extramitochondrial Nfs1p.
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EXPERIMENTAL PROCEDURES |
Yeast Strains and Growth Conditions--
The S. cerevisiae strains used in this study are listed in Table
I. A 2% galactose-containing medium
(SC-Gal) was used to express a gene under the galactose-inducible
(GAL1) promoter. A 2% glucose-containing medium (SC-D) and
a 2% lactate-containing medium (SC-lactate) were used to depress the
GAL1 promoter (19).
Mutagenesis of the NFS1 Gene and Plasmid Construction--
A
5'-terminal 500-base pair fragment of the NFS1 gene was
amplified by PCR from genomic DNA isolated from the wild-type strain W303-1B (20). It was cloned into the URA3-containing vector pYX013 (Ingenius) downstream of the GAL1 promoter. This
plasmid was linearized by cutting at a unique BlnI site in
the cloned NFS1 fragment and was integrated by homologous
recombination into the corresponding region in the chromosomal
NFS1 locus in W303-1B cells. Recombinants were selected on
uracil-depleted medium, and correct integration was confirmed by PCR.
One recombinant whose chromosomal NFS1 expression was
controlled by the inducible GAL1 promoter was named YN101
and used as a host strain to express the additional plasmid-borne Nfs1p
(either wild-type or mutant Nfs1p).
To construct a plasmid that constitutively expresses the wild-type
Nfs1p, the entire NFS1-coding region was amplified by PCR and then subcloned into a CEN4-based
TRP1-containing plasmid pTT-GAP (21) so the inserted
NFS1 could be expressed under the constitutive glycelaldehyde-3-phosphate dehydrogenase (GAP1) promoter.
This plasmid was named pTT-NFS1. To express Nfs1p with a
carboxyl-terminal hexahistidine peptide tag, the pTT-NFS1-h6 plasmid
was also constructed in a similar manner to pTT-NFS1, except for the
use of a (CATCAC)3-containing 3'-end primer for the initial
PCR amplification.
The NLS-like sequence RRRPR found in the Nfs1p clone was changed to
RRGSR by site-directed mutagenesis using a QuikChange site-directed
mutagenesis kit (Stratagene). The resulting plasmid was named
pTT-GSR-NFS1-h6, which expressed the mutant Nfs1p containing the RRGSR
sequence instead of the intact RRRPR sequence.
According to the sequence alignment of NifS-like proteins, a methionine
at residue 104 in yeast Nfs1p (Met104) was near the second
methionine of hNfs1, and another methionine at residue 118 (Met118) was conserved among the three eukaryotic NifS-like
proteins including hNfs1. Met104 and the Met118
in yeast Nfs1p were replaced with alanine by site-directed mutagenesis, and the resulting mutant NFS1-h6 genes were
integrated into pTT-GAP to make pTT-M2A-NFS1-h6 and
pTT-M3A-NFS1-h6, respectively. These plasmids were
introduced into YN101 to make strains that expressed the wild-type
Nfs1p under the inducible GAL1 promoter and that expressed
the plasmid-borne Nfs1p or mutant Nfs1p with or without a
(histidine)6-tag under the constitutive GAP1 promoter.
Plasmid pNS and pNS-NLS used for the nuclear transportation trap
analyses (22) were donated by K. Nagahari (Helix, Inc. Japan). The
mature Nfs1p (mNfs1p)-encoding region was inserted between the
XhoI and EcoRI sites in pNS to construct the
pNS-mNFS1 plasmid, which expresses an NES-LexAD-mNfs1p fusion protein.
Plasmid pNS-GSR-mNFS1, which expresses a NES-LexAD-GSR-mNfs1p fusion
protein, was constructed in a similar manner.
We constructed another series of the mutant NFS1 derivatives
by PCR, which were inserted into the ADE2-containing plasmid YIpDCE1, a kind gift from R. Stearman (National Institutes of Health,
Bethesda, MD) (19). The resulting plasmids were as follows: YIpNFS1 for
expression of the entire Nfs1p, YIpGSR-NFS1 for the entire Nfs1p with
the RRGSR sequence instead of the intact NLS sequence (GSR-Nfs1p),
YIpmNFS1 for mNfs1p, YIpGSR-mNFS1 for the mNfs1p with the RRGSR
sequence (GSR-mNfs1p), and YIpAla421-mNFS1 for the mNfs1p
with an Ala421 instead of the Cys421, which is
critical for its desulfurase activity (Ala421-mNfs1p). The
NFS1 derivatives in these plasmids were all expressed under
the constitutive yeast phosphoglycerol kinase promotor (19). Each
plasmid was linearized by cutting at a unique StuI site in the ADE2 gene in the vector backbone and then integrated
into the chromosomal ade2 locus of YN101,
YN101(pTT-NFS1-h6), or YN101(pTT-GSR-NFS1-h6) by homologous
recombination. ADE2+ colonies were selected on
the adenine-depleted SC-Gal, and cells with proper integration were
used for further analyses.
One XhoI restriction site in the pYK16 plasmid (kindly
provided by S. Nishikawa at Nagoya University) that was located
downstream of the EGFP gene was removed, and the resulting plasmid was
named pYK16EX. The following pYK16EX derivatives were used for the
expression of various Nfs1p-EGFP fusion proteins under the yeast
GAL1 promoter: pYK-NFS1 for the entire Nfs1p-EGFP,
pYK-GSR-NFS1 for Nfs1p-EGFP with the altered RRGSR-sequence in Nfs1p,
pYK-mNFS1 for mNfs1p-EGFP, and pYK16-GSR-mNFS1 for mNfs1p-EGFP with the
altered RRGSR sequence. These plasmids were transformed into S. cerevisiae W303 (diploid strain), and transformants were selected
on uracil-depleted medium.
Cell Staining of Various Nfs1p-EGFP Proteins--
Yeast cells
were grown in SC-Gal to A600 to 0.5 and
fixed with 5% formaldehyde in 0.1 M potassium phosphate
buffer (pH 6.5) for 2 h at room temperature. Cells were then
resuspended in phosphate-buffered saline and mounted on
poly-L-lysine-coated glass and observed by fluorescence
microscopy (Nikon).
Subcellular Fractionation and Immunological Detection of
Proteins--
For complete depletion of wild-type Nfs1p expressed
under the GAL1 promoter, cells were grown with SC-lactate
for 40 h at 30 °C before harvesting. Subcellular and
submitochondrial fractionations were performed as described previously
(21). Isolated mitochondria were finally resuspended in 20 mM Hepes-KOH (pH 7.5) and 0.6 M sorbitol.
Proteins in each fraction were separated by polyacrylamide gel
electrophoresis, transferred to a polyvinylidene difluoride membrane, and analyzed with an ECL Western blotting kit (Amersham Pharmacia Biotech). Various Nfs1p-related expression products were
detected with the purified anti-mouse Nfs1 antibody (7). Plasmid-borne
Nfs1p-h6 and GSR-Nfs1p-h6 were also detected with anti-(hexahistidine
peptide tag) monoclonal antibody (CLONTECH).
Assays of Mitochondrial Protein Enzymatic
Activity--
Mitochondrial fractions were assayed for the activities
of two iron-sulfur cluster-containing enzymes (aconitase and succinate dehydrogenase) and one non-iron-sulfur protein (malate dehydrogenase). Aconitase was assayed by measuring the formation of cis-aconitate at
240 nm (23). Succinate dehydrogenase activity coupling to cytochrome
c reduction was assayed by monitoring the increase in
absorbance at 550 nm (24). Malate dehydrogenase activity coupling to
-NAD+ reduction was assayed by monitoring the increase
in the product at 340 nm (25).
Miscellaneous Methods--
DNA manipulations were performed by
standard protocols for E. coli (26) and for S. cerevisiae (27). Protein estimation was performed with the
bicinchoninic acid assay kit (Bio-Rad) with bovine serum albumin as the standard.
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RESULTS |
Eukaryotic Nfs1 Proteins Have a NLS-like Sequence--
All of the
known eukaryotic Nfs1 proteins have a mitochondrial-targeting
presequence followed by a conserved sequence also found in bacterial
NifS proteins (7, 10). Analysis of the Nfs1p amino acid sequence with
the protein sorting signal searching program PSORT also identified an
NLS-like sequence, RRRPR, in the mature Nfs1p sequence (Fig.
1A). Sequence alignment of
three known eukaryotic (yeast, mouse, and human) Nfs1 proteins showed that this NLS-like sequence was completely conserved (Fig.
1B). In contrast, most bacterial NifS proteins, including
A. vinelandii NifS, had somewhat diverse sequences in the
corresponding region, including one neutral residue (glycine or
threonine) instead of the basic residue in the third or the fourth
position of the sequence (Fig. 1B). However, both the
E. coli and A. vinelandii IscS proteins possess
the RRKPR sequence, which is similar to the NLS-like sequence found in
eukaryotic Nfs1 proteins (Fig. 1B).

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Fig. 1.
The yeast Nfs1p amino acid sequence
(A) and comparison of the nuclear-targeting
signal-like sequence of Nfs1p with corresponding sequences of several
bacterial and eukaryotic counterparts (B).
A, the amino acid sequence of yeast Nfs1p is shown with its
presumptive mitochondrial targeting presequence (bold
letters with underline). An NLS-like sequence found by
the PSORT program is highlighted in a black box.
A lysine residue for pyridoxal phosphate binding is marked with an
asterisk, and residues for the class V-type PLP binding
sequence (48) are shown with bold letters in a
shadowed box. A cysteine residue essential for the cysteine
desulfurase activity (Cys421) is marked with a
plus. Numbers of amino acid residues are shown on the right
side of each column. Methionines at residue 104 and 118 of yeast Nfs1p,
both of which correspond to the second and the third methionine in
hNfs1, respectively, were shown with circles. B,
amino acid residues 291-348, which include the PLP binding and
NLS-like sequences, are aligned with corresponding regions of several
bacterial and eukaryotic NifS/IscS homologues. The top three sequences
are of the eukaryotic NifS homologues from yeast (yNfs1)
(5), mouse (mNfs1) (7), and human (hNfs1) (10),
respectively. The next two sequences are from E. coli (11)
and A. vinelandii (12) IscS proteins (described as
EcIscS and AvIscS, respectively). The bottom
three are from the bacterial NifS proteins of A. vinelandii
(AvNifS), Cyanothece PCC8801 (CyNifS),
Klebsiella pneumoniae (KpNifS), and
Rhodobacter sphaeroides (RsNifS) (7). The PLP
binding motifs are shown in brackets, and the putative
NLS-like sequence found in yeast Nfs1p and the corresponding regions in
other homologs are highlighted in a black box. Residues
conserved among at least seven of nine sequences were highlighted in
gray boxes.
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Ectopic mNfs1p-EGFP Lacking the Mitochondrial-targeting Sequence
Localized to Both Nucleus and Cytosol but Not
Mitochondria--
Several attempts to detect endogenous Nfs1p in the
nucleus by immunoblotting failed (7, 8), possibly because Nfs1p is only
present at extremely small levels in the nucleus. We tried to visualize
the intranuclear localization of Nfs1p by overexpressing Nfs1p-EGFP
fusion protein with or without the mitochondrial-targeting presequence
(Fig. 2). The fusion protein was
expressed in wild-type yeast diploid cells, and cellular localization
of the proteins was observed by fluorescent microscopy. When the Nfs1p
precursor protein, which contained the intact NLS-like sequence, was
fused to the amino terminus of EGFP, the fusion protein was clearly localized to mitochondria, consistent with previous Western blotting analyses (7, 8), whereas only faint fluorescence was observed in the
cytosol and nucleus (Fig. 2c). In contrast, when the
mNfs1p-EGFP was ectopically expressed, weak fluorescence could be
detected in the cytosol and nucleus but not in mitochondria (Fig.
2e). We also examined localization of the GSR-mNfs1p-EGFP
fusion protein, which contained an RRGSR sequence in place of the
intact NLS-like sequence in Nfs1p. This RRGSR sequence was designed to
mimic the corresponding region of bacterial NifS proteins (Fig.
1B). The resulting GSR-mNfs1p-EGFP also remained in the
cytosol (Fig. 2f). However, because of the very faint and
scattered fluorescence, it was unclear whether the fused protein was
excluded from the nucleus. Thus, we next took a more sensitive approach
to exploring the possible localization of Nfs1p to the nucleus.

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Fig. 2.
Fluorescent microscopic analyses of yeast
expressing various Nfs1p-EGFP proteins. Fixed yeast cells
expressing the Nfs1p-EGFP fusion proteins were observed with the
excited fluorescence at 488 nm for emission and 534 nm for detection.
Cells expressing the Nfs1p-EGFP proteins with (a) and
without (b) the mitochondrial targeting presequence for the
yeast CoxIV protein were used for controls. Nfs1p forms expressed in
the yeast cells were the precursor Nfs1p (c), GSR-Nfs1p
(d), mature Nfs1p (mNfs1p) (e), and GSR-mNfs1p
(f).
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Nuclear Transportation Trap Analysis Showed That a Fraction of
Nfs1p Fusion Protein Is Present in the Nucleus--
Using the nuclear
transportation trap method (22), we examined whether fusion of Nfs1p to
another protein could mediate translocation of that protein into the
nucleus. In this method, NES-LexAD, an engineered transcription factor
containing a nuclear export signal, is used to test whether a protein
of interest has the capacity to localize to the nucleus of yeast EGY48
cells (28), which harbor the LexAD-responsive LEU2 reporter
gene. If the fused protein contains an NLS, transformants are expected
to form Leu+ colonies. We constructed two mNfs1p fusion
proteins, one with an intact RRRPR sequence (NES-LexAD-mNfs1p) and the
other with a mutated RRGSR sequence (NES-LexAD-GSR-mNfs1p). When the
expression vector encoding each of these fusion proteins was introduced
into EGY48 cells, transformants were first selected for a
His+ phenotype. Expression of the fusion proteins was
confirmed immunologically with anti-LexAD monoclonal antibody (data not
shown). Then these transformants were further tested for the leucine
prototroph (Fig. 3). If the Nfs1p
contains a functional NLS, transformants were expected to form
His+/Leu+ colonies. As shown in Fig.
3C, transformants that expressed the NES-LexAD-mNfs1p showed
the Leu+ phenotype, whereas cells expressing the
NES-LexAD-GSR-mNfs1p had the His+/Leu
phenotype (Fig. 3D). These results suggested that the RRRPR
sequence potentially functions as an NLS in Nfs1p and that nuclear
localization is lost when the sequence is altered to RRGSR.

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Fig. 3.
Nuclear localization of Nfs1p analyzed by the
nuclear transportation trap assay. Yeast EGY48 cells (28)
transformed with a plasmid expressing either NES-LexAD (A),
NES-LexAD-NLS (B), NES-LexAD-mNfs1p (C), or
NES-LexAD-GSR-Nfs1p (D) fusion protein were spread onto the
Leu /His selection plates and grown at
30 °C for 3 days.
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Mutation of the NLS Sequence in Nfs1p Induced a Growth Defect in
Cells under Wild-type Nfs1p-depleted Conditions--
The
NFS1 gene has been shown to be essential for cell viability
(5, 8). Therefore, a yeast strain named YN101 in which the chromosomal
NFS1 gene was expressed under the control of the GAL1 promoter was constructed and used as a host strain for
in vivo complementation analyses. YN101 cells grow normally
like the wild-type W303-1B strain in SC-Gal but cannot grow in SC-D. When the plasmid-borne Nfs1p precursor with a hexahistidine tag (Nfs1p-h6) was constitutively expressed in YN101 cells, the cell growth
in SC-D was restored (section 1 in Fig.
4). In contrast, expression of the
hexahistidine-tagged mNfs1p (mNfs1p-h6) could not restore the cell
growth in SC-D (Ref. 8, data not shown). These results confirm that the
mitochondrial localization of Nfs1p is essential for cell growth. We
then examined whether the tagged mutant Nfs1p precursor (GSR-Nfs1p-h6)
could affect the complementation ability when expressed in YN101 cells.
As shown clearly in section 2 in Fig. 4, GSR-Nfs1p-h6 could
not restore cell growth under the wild-type Nfs1p-depleted conditions.
We also showed that two other Nfs1p mutants in which either the
alternative AUG, corresponding to the second or the third AUG used in
hNfs1 had been changed to GCG for alanine, could restore cell growth
under the wild-type Nfs1p-depleted conditions (sections 3 and 4 in Fig. 4).

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Fig. 4.
Complementation of YN101 with various
plasmid-borne NFS1 genes under chromosome-derived
NFS1-depleted conditions. Yeast strain YN101 was
transformed with various plasmids, each of which harbored the wild-type
or mutated NFS1 gene. Transformants selected on
SC-Gal-plated lacking both tryptophan and uracil were restreaked on
SC-Gal (A) and SC-D (B) plates lacking tryptophan
and uracil. Note that on the SC-D glucose-containing plate, the
GAL1 promoter-derived expression of chromosomal
NFS1 was depressed. 1, YN101(pTT-NFS1-h6);
2, YN101(pTT-GSR-NFS1-h6); 3,
YN101(pTT-M2A-NFS1-h6); 4,
YN101(pTT-M3A-NFS1-h6); 5, W303-1B;
6, YN101; 7, YN101(pTT-GAP); 8,
YN101(pTT-NFS1). W303-1B and YN101 cells were spread on separate
sections that had been supplemented with uracil and tryptophan (for
W303-1B) and with tryptophan (for YN101). Cells were grown at 30 °C
for 3 days. For details of the YN101 (pTT-M2A-NFS1-h6) and
YN101 (pTT-M3A-NFS1-h6), see "Discussion."
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After 40 h of growth in SC-lactate, the GAL1
promotor-driven expression of the wild-type Nfs1p in YN101 was
depressed to a level at which no cross-reactive proteins could be
detected by immunoblotting using anti-mouse-Nfs1 antibody (Fig.
5). Under the same growth conditions,
constitutively expressed plasmid-borne Nfs1p derivatives could be
detected both with the anti-mouse Nfs1 antibody and with the
anti-histidine-tag antibody. The GSR-Nfs1p-h6 fusion protein was found
to be localized to mitochondria and present in amounts comparable with
that of the control Nfs1p-h6 (Fig. 5).

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Fig. 5.
Western blot analysis of Nfs1p in various
strains grown under Nfs1p-depleted conditions. YN101-derived
transformants that contained various NFS1 genes
(a, YN101; b, YN101(pTT-NFS1); c,
YN101(pTT-NFS1-h6); d, YN101(pTT-GSR-NFS1-h6)) were grown in
SC-D. Cells were harvested, disrupted, and used for Western analysis.
The chromosome-derived wild-type Nfs1p and plasmid-borne Nfs1p were
detected with anti-mouse Nfs1 antibody (A), and Nfs1p-h6 and
its derivatives were detected with anti-His-tag antibody
(B).
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The Nfs1p Mutant with the RRGSR Sequence Exhibits Nearly Normal
Mitochondrial Iron-Sulfur Protein Activities--
We then assayed the
enzymatic activities of mitochondrial iron-sulfur proteins in the
strain YN101(pTT-GSR-NFS1-h6) to test whether the GSR-Nfs1p functions
in mitochondria equivalent to wild-type Nfs1p. The activities of two
mitochondrial iron-sulfur cluster-containing enzymes, aconitase and
succinate dehydrogenase, were assayed in mitochondrial fractions that
contained either Nfs1p-h6 or GSR-Nfs1p-h6 after growth of the cells in
the absence of galactose for 40 h. As a control, the activity of a
non-iron sulfur protein, malate dehydrogenase, was also assayed. As
shown in Fig. 6, the
GSR-Nfs1p-h6-containing mitochondria retained the activities of all
three enzymes examined, with slightly lower levels than
Nfs1p-h6-containing mitochondria. Mitochondria that contained no
plasmid-borne Nfs1p exhibited low levels of aconitase and succinate
dehydrogenase activity but a similar level of the malate dehydrogenase
activity to those containing the plasmid-borne Nfs1p-h6 (Fig. 6). These
results indicate that mitochondrial iron-sulfur proteins were assembled
in the active holo form in YN101(pTT-GSR-NFS1-h6) in which chromosomal
Nfs1p had been depleted but the plasmid-borne GSR-Nfs1p-h6 was
expressed. In other words, the mitochondrially localized GSR-Nfs1p-h6
was found to retain the ability to mediate cluster assembly of
mitochondrial iron-sulfur proteins, indicating that the mutation in the
NLS sequence did not cause any significant dysfunction in iron-sulfur
biogenesis in mitochondria.

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Fig. 6.
Enzymatic activities in mitochondrial
fractions containing Nfs1p-h6 or GSR-Nfs1p-h6. Malate
dehydrogenase, aconitase, and succinate dehydrogenase activities were
assayed in mitochondrial fractions prepared from YN101(pTT-NFS1-h6),
YN101(pTT-NFS1-h6), and YN101(pTT-GAP).
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Growth Defect of YN101(pTT-GSR-NFS1-h6) in SC-D Was Rescued by
Expression of Mature Nfs1p Containing an Intact NLS Sequence--
If
the nuclear localization of Nfs1p is essential for cell viability and
if the severe growth defect of YN101(pTT-GSR-NFS1-h6) cells in SC-D is
caused by the loss of nuclear localization of GSR-Nfs1p-h6, cell growth
should be recovered when another mutant Nfs1p with an intact NLS
sequence is simultaneously expressed outside the mitochondria. To
verify this hypothesis, we constructed a series of NFS1
derivatives (Fig. 7A) and
subcloned them into the shuttle vector YIpDCE1 (19). Then we integrated
each of these NFS1 derivatives into the ade2
locus of the genomic DNA of YN101(pTT-GSR-NFS1-h6) and YN101(pTT-GAP).
The Ade+ transformants, in which the proper integration had
been confirmed by the PCR method (19), were spotted in a dilution
series on either SC-Gal or SC-D (Fig. 7, B and
C).

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Fig. 7.
Complementation of the GSR-Nfs1p-h6 cell
viability defect under the wild-type Nfs1p-depleted growth
conditions. A, schematic representation of DNA
fragments that were integrated into the YN101(pTT-GAP) or
YN101(pTT-GSR-NFS1-h6) cells by homologous recombination described by
Stearman et al. (19). Thick boxes indicate the
various NFS1 derivatives. The mitochondrial localization
presequence is shown with a shadow (mt), and the
intact NLS (RRRPR) and the mutated (RRGSR) sequences are shown in
white and black boxes, respectively.
a, no Nfs1p; b, Nfs1p encoded by YIpNFS1;
c, mature Nfs1p by YIpmNFS1; d, mature Nfs1p with
the RRGSR sequence by YIpGSRmNFS1; e, mature Nfs1p in which
Cys421 was changed to Ala421 by
YIpAla421-mNFS1. Plasmid construction was described under
"Experimental Procedures." Plasmids were linearized by cutting at a
unique site in the ADE2 gene (indicated as gray thin
lines) and integrated into the ade2 locus of YN101(pTT-
GSR-NFS1-h6) (B) or YN101(pTT- GAP) (C) cells.
Complementation analysis of the transformants was performed by a serial
10-fold dilution of the cells plated onto the uracil-containing SC-Gal
(left) and the SC-D (right) plates. Cells were
incubated at 30 °C for 3 days.
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When the mature Nfs1p (mNfs1p) was simultaneously expressed in
YN101(pTT-GSR-NFS1-h6), cells could grow well in both SC-Gal and SC-D
(Fig. 7B, c). In the strain YN101(pTT-GAP), which contained no NFS1 gene, the growth of cells in the SC-D was not
restored when the mNfs1p was expressed (Fig. 7C,
c). Interestingly, when the GSR-mNfs1p was simultaneously
expressed in YN101(pTT-GSR-NFS1-h6), cell growth was not recovered
under the wild-type Nfs1p-depleted conditions (Fig. 7B,
d). Since mNfs1p, which lacks the presequence, was expressed
outside the mitochondria, these results indicate that the defective
function of GSR-Nfs1p-h6 can be complemented by extramitochondrial
mNfs1p, which contains an intact NLS sequence. This means that, in
addition to mitochondrial Nfs1p, a fraction of Nfs1p is required
outside the mitochondria, most likely in the nucleus, for cells to survive.
NifS proteins possess a conserved cysteine residue that is the critical
active site for desulfurase activity (2-4, 7). The corresponding
Cys421 in yeast Nfs1p was also shown to be essential for
its iron-sulfur cluster biogenesis activity in mitochondria (9). To
examine whether this Cys421 is also required for the
extramitochondrial function of Nfs1p, we constructed an additional
plasmid encoding mutated mNfs1p whose Cys421 was changed to
Ala421 (Ala421-mNfs1p). YN101(pTT-GSR-NFS1-h6)
cells transformed with this plasmid could grow on SC-Gal but could not
grow on SC-D (Fig. 7B, e), indicating that the
desulfurase activity was also important for the function of Nfs1p in
the nucleus.
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DISCUSSION |
Nfs1p has mostly been found in mitochondrial matrix in yeast cells
(7) and is essential for cell growth (5, 9). A mutant Nfs1p lacking the
mitochondrial-targeting presequence could not restore the growth of the
wild-type Nfs1p-depleted cells (Ref. 8 and this study). Since cellular
iron-sulfur proteins are involved in important metabolic processes such
as electron transfer reactions and transcriptional regulation (29-32)
and Nfs1p plays a significant role in iron-sulfur cluster assembly in
mitochondria (33), it is not surprising that the functional loss of the
mitochondrial Nfs1p should cause the loss of cell viability.
In this study, we demonstrated that the localization of Nfs1p to the
nucleus is also essential for cell viability. Although we failed to
detect any endogenous or ectopically expressed Nfs1p in the nucleus
directly by Western blotting or immunohistochemistry (Fig. 2), we were
able to show that Nfs1p is present in the nucleus using more sensitive
techniques (Figs. 4 to 7). Therefore, a small but significant fraction
of Nfs1p is localized to the nucleus in vivo and plays an
unknown essential role in cell viability. We also showed that the
desulfurase activity of Nfs1p was required for the function of Nfs1p in
the nucleus (Fig. 7) as well as in the mitochondria (9).
There are several possible functions for nuclear localized Nfs1p. Nfs1p
may be involved in iron-sulfur cluster assembly in the nucleus, as it
is in the mitochondrion. A [4Fe-4S] cluster-containing protein called
Ntg2p, a yeast homologue of endonuclease III, has been shown to be
present in the eukaryotic nucleus (34). However, it is difficult to
imagine that other mitochondrial components involved in the iron-sulfur
cluster assembly (Isu1p, Isu2p, Nfu1p, Isa1p, Isa2p, Yah1p) (32) also
have dual intracellular localization (in both mitochondrion and
nucleus) like the Nfs1p protein. In addition, it has been proposed that
the pre-assembled iron-sulfur cluster is transported from the
mitochondrial matrix to the cytosol by a certain transporter(s) located
in the mitochondrial membrane, which is necessary for the maturation of
the cytosolic iron-sulfur proteins (8, 32). Therefore, if the
extra-mitochondrial Nfs1p is related to the iron-sulfur cluster in the
nucleus rather than being involved in maturation, it may function in
the repair or maintenance of pre-assembled iron-sulfur clusters of
nuclear-localized iron-sulfur proteins such as Ntg2p.
Another possibility is that the nuclear-localized Nfs1p is involved in
nucleotide biosynthesis, because the allele of the NFS1 gene
was first identified as a suppresser gene named SPL1, mutation of which affected tRNA splicing (5). tRNA splicing occurs on
the inner surface of the nuclear membrane (35) and is greatly
influenced by specific modification of the ribonucleotides of tRNA.
Recent reports on the distinct functions of the E. coli IscS
protein (13-18) may shed some light on the function of
nuclear-localized Nfs1p. E. coli IscS was first shown to
possess a cysteine desulfurase activity that is involved in iron-sulfur
protein biosynthesis (11) and, subsequently, a sulfur transferase
activity by which a sulfur atom is transferred to a uridine to produce
a 4-thiouridine in tRNA (13-15). Following this analogy, it is quite
possible that nuclear Nfs1p is involved in nucleotide modification as a
sulfur donor. Therefore, a mutation in Nfs1p may affect its function in
the modification of specific ribonucleotides in the nucleus and on tRNA splicing.
Recently, IscS proteins found in E. coli and
Salmonella species have been reported to be involved in
thiamine and nicotinic acid biosyntheses (15, 36). The Bacillus
subtilis nifS gene product has also been reported to function in
NAD+ biosynthesis (37). Since the tRNA splicing process
includes an NAD+-dependent step in S. cerevisiae (38, 39), the nuclear Nfs1p is likely to be involved in
the process via its effect on NAD+ biosynthesis. Therefore,
nuclear localization of Nfs1p would be essential for cell survival,
because correct tRNA splicing is essential for yeast cell growth.
How is the localization of yeast Nfs1p to either the mitochondria or
the nucleus controlled? It has been reported that human NifS homologues
(hNfs1) of different lengths were synthesized from a single transcript
by differential translational initiation from alternative AUG codons
and that the smaller (presequence-truncated) forms were found in the
cytosol and nucleus, whereas the largest form was in mitochondria in a
proteolytically processed mature form (10). Yeast Nfs1p also possesses
alternative methionine residues at the positions corresponding to the
potential alternative translational initiation AUG codons of hNfs1
(Fig. 1A). However, our experimental data (section
3 and 4 in Fig. 4) suggest that the
amino-terminal alternative translation does not contribute to
localization of yeast Nfs1p to the nucleus. Internal initiation of
mRNA translation coupled to a physiological function is rarely observed in eukaryotes (40-42). Moreover, no internal ribosome entry
sites have been shown to function in growing yeast (43). Therefore, the
dual intracellular localization of the yeast NifS homologue may be
caused by a mechanism distinct from that of the human counterpart,
hNfs1. It has not yet been elucidated whether the hNfs1 in the nucleus
is essential for cell growth, but we assume that nuclear hNfs1 has some
significant function.
Recently, an increasing number of reports demonstrate localization of
one protein to different cellular locations in vivo (44-47). It has been proposed that, in some cases, re-localization of
a protein to another organelle occurs after the initial localization to
one organelle. For example, a single translation product of the yeast
fumarase gene, which is distributed between the cytosol and
mitochondrial matrix, is targeted to and processed in mitochondria before distribution (45), suggesting that the newly synthesized fumarase precursor can either be fully translocated cotranslationally into the mitochondrial matrix or it can be folded into an
import-incompetent state and released by retrograde movement through
the translocation pore into the cytosol. Dual distribution of Nfs1p may
follow a similar mechanism in which binding of the PLP to the mature
domain of the precursor might promote folding outside of the
mitochondria and prevent further translocation into the mitochondrial
matrix. Such Nfs1p molecules can be released into the cytosol and then be re-localized to the nucleus with the aid of its NLS. Alternatively, the dual localization of the Nfs1p might be caused by a simple balance
between the NLS and mitochondrial presequence functions after
translation in the cytosol.
Studies on the physiological function of the nuclear-localized Nfs1p in
yeast cells are now under way in our laboratory. Determination of
proteins that interact with Nfs1p in the nucleus may help to elucidate
the function of Nfs1p in this location.