Institut für Mikrobiologie,
Heinrich-Heine-Universität, D-40225 Düsseldorf, Germany
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INTRODUCTION |
In prokaryotic and eukaryotic cells,
aminoglycoside antibiotics promote mistranslation by specific
interactions with small rRNA (2, 8, 14, 25, 31). At low
concentrations of aminoglycosides, mistranslation leads to phenotypic
suppression of mutant phenotypes in Saccharomyces cerevisiae
(37, 44). Additional inhibitory activities of
aminoglycosides are caused by binding to specific mRNA sequences and
include the block of group I intron splicing (46) and the
inhibition of binding of the human immunodeficiency virus Rev protein
to its cognate RNA sequences (47).
Mutations lowering or increasing the susceptibility to
aminoglycosides in yeast are known. Mutations in
PMA1, encoding a plasma-membrane ATPase (38), as
well as mutations in the mitochondrial and cytoplasmic rRNAs (8,
14, 25) confer aminoglycoside resistance. Resistance has also
been generated experimentally by overexpression of the yeast
NEO1 gene, encoding a homolog of a cytoplasmic ATPase that is involved in ribosome assembly (39), or by expression of
bacterial genes encoding aminoglycoside-modifying enzymes (e.g.,
aph or hph genes encoding bacterial
phosphotransferases [12]), which can serve as dominant
selectable markers in expression plasmids (18, 19).
Aminoglycoside supersensitivity can arise because of mutations causing
defects in N glycosylation (3, 13) and O glycosylation
(45a). crl mutants are supersensitive to
hygromycin B and other aminoglycosides but are resistant to
cycloheximide (28). Similarly, pdr1 mutants are
sensitive to some aminoglycosides but resistant to other compounds and
show some respiratory defects (40). An increase in
aminoglycoside sensitivity can also arise because of the presence of
certain omnipotent translational suppressors (26);
mutations in MOF4, whose gene product is involved in reading frame maintenance, increase the sensitivity to paromomycin
(10). Furthermore, specific mutations in 18S rRNA
significantly enhance the susceptibility of yeast to the aminoglycoside
streptomycin (8).
We previously described ags mutant strains of S. cerevisiae, which show enhanced sensitivity to aminoglycosides,
including G418, hygromycin B, destomycin A, gentamicin X2,
apramycin, kanamycin B, lividomycin A, neamine, neomyin, paromomycin,
and tobramycin (15). The ags phenotype was not
associated with any of the sensitivities and additional phenotypes
caused by previously described mutations. Genetic analyses suggested
that the aminoglycoside supersensitivity of ags strains was
caused by mutations in three recessive genes, one of which
(AGS1) was found to map close to the centromere of chromosome III. The second gene involved in supersensitivity
(AGS2) appeared to be linked to AGS1 on
chromosome III, while the third gene, designated AGS3, was
unlinked to AGS1 and situated on an undefined chromosome.
Here we describe the cloning and characterization of two genes
complementing the supersensitivity of the ags strains. We
show that a small unknown protein (Ags1p) and known proteins (Pho80p,
Pho85p, and Pho4p) contribute to a basal level of resistance to
aminoglycosides in wild-type yeast cells.
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MATERIALS AND METHODS |
Strains and growth conditions.
The strains used are listed
in Table 1. Cells were grown in complex
YPD or synthetic SD medium (43). Sensitivities to
antibiotics were determined on YPD plates containing a linear gradient
of 0 to 25 µg of hygromycin B per ml or 0 to 300 µg of G418
(Geneticin; Sigma) per ml. The ags phenotype was routinely
tested on YPD plates containing 1, 5, and 10 µg of hygromycin B per
ml; ags mutants were inhibited by hygromycin B at 5 and 10 µg/ml, while wild-type strains were able to grow at these hygromycin
B concentrations. ags1 pho80 double mutants were obtained as
segregants of a cross of W10-2B and VG51.
Genes complementing the ags phenotype.
The
ags strain RC1707 (15) was transformed with an
S. cerevisiae genomic library constructed either in the
replicating vector YRp7 (33) or in the centromeric vector
YCp50 (41), using the lithium acetate transformation
protocol (22). Transformants grown selectively for 2 days at
30°C on supplemented SD medium were replica plated to YPD medium
containing 20 µg of G418 per ml. Resistant transformants were
retested to confirm enhanced resistance to hygromycin B and G418.
Furthermore, chromosomal DNA of the resistant transformants was
prepared, and plasmids were recovered by transformation into
Escherichia coli (43). Retransformation of the
obtained plasmids into RC1707 confirmed that antibiotic resistance was
plasmid borne. Of 73,400 transformants obtained with the YCp50 library,
7 transformants carried a plasmid conferring resistance; of 33,200 transformants obtained with the YRp7 library, 13 were identified as
conferring resistance.
Plasmids.
Twelve of 13 G418- or hygromycin B-resistant
transformants obtained with the YRp7 genomic library carried a plasmid
with an insert of 8.5 kb, which was designated pA8. In the case of the YCp50 library, all seven resistant transformants carried a plasmid with
a genomic insert of about 12 kb; this plasmid was designated pB12.
Subclones of the genomic inserts were constructed in the centromeric
vector YCplac22 (16). The YCplac22 subclone carrying the
4.7-kb EcoRI-SalI fragment derived from pA8,
which conferred identical antibiotic resistance, was designated
YCplac22-4.7. Similarly, the YCplac22 subclone carrying the 8-kb
EcoRI fragment derived from pB12 conferred the same
resistance as pB12 and was designated pB6A. (All constructed subclones
are summarized in Fig. 2.) pMD1.9 was constructed from pB6A by
inserting the 1.6-kb SacI-SpeI AGS1
fragment into YCplac22 between the SacI and XbaI sites of YCplac22. pSW17 was constructed by insertion of the 4.7-kb EcoRI-SalI fragment (YCplac22-4.7) carrying
PHO80 into YCplac33 (16). pSW18 was constructed
by insertion of the 6.5-kb EcoRI fragment (YCplac22-6A)
carrying AGS1 into YCplac33 (16).
Gene disruptions.
Gene disruptions were performed with
strain CEN-PK141 by a PCR method (19); strain CEN-PK141 is
isogenic to CEN-PK2 (7) except that it is a
MATa/MAT
diploid and is heterozygous for
the auxotrophic markers. To delete the entire coding region of
AGS1, the primers AGS-L (5'-ATG GAC ATG GAC AAC ACG
GAT ATC TCC CCA ACC AAC CAT CCA GCT GAA GCT TCG TAC GC-3')
and AGS-R (5'-CTA TTT GCG GTT CAG GAC GTC TAT CTG TGC GTT TAG CGA GGC ATA GGC CAC TAG TGG ATC TG-3') were used first to
amplify the loxP-kanMX-loxP (Kanr) module
(italic indicates homology to AGS1; boldface indicates homology to loxP) on plasmid pUG6 (19). The PCR
product was transformed into CEN-PK141, the resulting diploid
transformant was sporulated, and the deletion was confirmed by Southern
blotting with the 3.1-kb NcoI-PstI fragment
downstream of AGS1 as a probe. The disrupted diploid was
sporulated, and haploid ags1
segregants were identified
by their G418 resistance.
Other procedures.
Total RNA was prepared as described
previously (42), separated by denaturing gel
electrophoresis, transferred to nylon filters, and probed with a
32P-labelled DNA fragment carrying AGS1 (1-kb
BamHI-SalI fragment of YCplac22-1) according to
standard procedures. Invertase (encoded by SUC2) was
analyzed by activity staining following native acrylamide gel
electrophoresis (32); alternatively, proteins in cell
extracts were denatured and separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (5% acrylamide),
and invertase was detected by Western blotting with a polyclonal rabbit
anti-invertase antibody (1:1,000) followed by goat horseradish
peroxidase-coupled anti-rabbit immunoglobulin G (1:5,000). Likewise,
carboxypeptidase Y (CPY) in cellular proteins was detected after
SDS-PAGE (7.5% acrylamide) with a monoclonal anti-CPY antibody (1:500)
(Molecular Probes) followed by goat horseradish peroxidase-coupled
anti-mouse immunoglobulin G antibody (1:10,000) (Dianova)
(48).
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RESULTS |
Genomic clones complementing the Ags
phenotype.
We previously characterized strain RC1707, which harbors at least three
mutations leading to increased sensitivities to several aminoglycoside
antibiotics (15). To identify genes involved in basal
aminoglycoside resistance of wild-type yeast strains, we transformed
RC1707 with genomic libraries (33, 41) and selected
transformants able to grow at 10 to 20 µg of G418 per ml,
concentrations at which wild-type strains, but not ags
strains, are able to grow. Resistant transformants were analyzed
further by allowing plasmid loss during nonselective growth in YPD
medium; in all cases the plasmid-free derivative strains were as
antibiotic sensitive as the parental strain RC1707. Furthermore,
plasmids carried by the resistant transformants were recovered by
transformation of chromosomal DNA into E. coli; RC1707
transformants carrying the reisolated plasmid were as resistant as the
original transformants. The latter experiments indicated that in all
cases, aminoglycoside resistance of the isolated transformants was
plasmid borne.
By these criteria, 13 plasmids conferring resistance were identified in
a genomic library constructed in the multicopy vector YRp7
(33); in addition, 7 plasmids conferring resistance were isolated from a genomic library constructed in the centromeric vector
YCp50 (41). Restriction analyses revealed that 12 of the 13 isolated YRp7 derivatives were identical and carried a genomic insert
of 8.5 kb; a representative plasmid was designated pA8. Similarly,
restriction analyses of the YCp50 clones showed that they contained an
identical genomic insert of about 12 kb; a representative plasmid was
designated pB12. RC1707 transformants carrying plasmid pA8 or pB12
showed increased G418 resistance compared to the untransformed RC1707
strain; these transformants, however, were less resistant than the
wild-type strain (Fig. 1). An identical
pattern of sensitivity and resistance of strains was obtained by using
hygromycin B (data not shown).
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FIG. 1.
Sensitivity of yeast strains to G418. The orientation of
the G418 gradient is indicated at the top. Growth of wild-type strain
ER110-6B and growth of ags mutant RC1707 untransformed or
transformed with genomic clone pB12 or pA8 are compared.
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Subcloning experiments.
To delimit the genomic region in pA8
and pB12 which was responsible for complementation of the
Ags phenotype, we constructed various plasmid subclones
and tested their abilities to confer G418 and hygromycin B resistance
in transformants of strain RC1707 (Fig.
2).
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FIG. 2.
Complementation of aminoglycoside supersensitivity of
strain RC1707 by genomic clones. The extent of genomic inserts in the
indicated plasmid is shown by the black bars. The ability of the clones
to confer resistance to G418 is indicated by +,  , or +/; the
complementing activity of the original clone is designated +. A
restriction map of the genomic region along with the coding region of
AGS1 (top) or PHO80 (bottom) (open arrows) is
shown. B, BamHI; K, KpnI; X, XbaI; R,
EcoRI; S, SalI; Sc, SacI; Se,
SpeI; Sh, SphI. The EcoRI sites
flanking the genomic insert in pB6A do not occur in the published
sequence of chromosome III; therefore, these sites are in
parentheses.
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A YRp7 derivative carrying a 4.7-kb EcoRI-SalI
genomic fragment derived from pA8 (pA84) was found to convey levels of
resistance similar to those with pA8. To explore if resistance was due
to the high copy number of pA84, we transferred the 4.7-kb fragment to
the centromeric vector YCplac22, resulting in plasmid YCplac-4.7 (Fig.
2). RC1707 transformants carrying YCplac-4.7 were as resistant as
transformants carrying pA8 and pA84, indicating that complementation by
the genomic 4.7-kb fragment can occur at low plasmid copy numbers. From
the activities of additional subclones, we concluded that a 2.7-kb
KpnI-SalI genomic fragment contained in
YCplac22-2.7, which is derived from A8, is sufficient to confer G418
and hygromycin B resistance (Fig. 2).
A 6-kb genomic EcoRI fragment, which is contained in pB12,
was subcloned into YCplac22 (resulting in plasmid pB6A); resistances of
transformants carrying pB12 or pB6A were identical. The genomic insert
of pB6A was subcloned further, and a subclone containing a 1.9-kb
SacI-SpeI fragment (pMD-1.9) was found to confer
the same resistance levels as pB6A and pB12 (Fig. 2). A subclone
containing an even smaller, 1-kb BamHI-SalI
fragment (YCplac22-1) was able to complement the Ags
phenotype partially.
Sequences of genomic clones.
The sequence of the 1-kb
BamHI-SalI fragment of YCplac22-1 and the
terminal sequences of the 2.7-kb KpnI-SalI
fragment of YCplac22-2.7 were determined.
Computer analyses revealed that the sequence of the 1-kb
BamHI-SalI fragment of YCplac22-1 was identical
to that of a segment on chromosome III between the centromere and the
LEU2 gene. A single complete open reading frame was detected
on this fragment, extending from bp 105841 to 105578 of chromosome III;
this reading frame has the potential to encode a protein of 88 amino
acids (Fig. 3). We had previously mapped
a gene, designated AGS1, involved in aminoglycoside
resistance close to the centromere of chromosome III (15).
Because of the complementation results, as well as the altered
transcript size in RC1707 and the phenotype of the disruptant strain
(see below), we designate the identified open reading frame
AGS1. Ags1p did not show significant homologies to any other
protein in the databases. The Ags1 protein is rich in hydrophobic and
acidic residues but does not contain known sorting signals or
transmembrane regions (Fig. 3A). Computer analyses (http://expasy.hcuge.ch/sprot/prosite.html) predict that the N-terminal
20 amino acids are in a 
-turn or coil configuration, while residues
20 to 88 adopt a -sheet conformation.
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FIG. 3.
Translational product and transcript of AGS1.
(A) Theoretical protein encoded by AGS1. Hydrophobic
residues are shaded, acid residues are marked by closed circles, and
basic residues are marked by open circles. (B) AGS1 and
ags1 transcripts. Total RNAs of strain RC1707 (lane 1),
strain RC1707(pB6A) (lane 2), and the wild-type strain BJ1991 (lane 3)
were probed with a 1-kb BamHI-SalI probe of
YCplac22-1 carrying AGS1. The migrations of 16S and 25S
rRNAs are indicated.
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Computer analyses of the terminal sequences of the genomic fragment
contained in YCplac22-2.7 revealed that it corresponds to a fragment in
close vicinity to the centromere of chromosome XV. Only a single open
reading frame (YOL001W) was detected in this region, extending from bp
325249 to 326130 of chromosome XV. YOL001W corresponds to the
PHO80 gene, which encodes a cyclin activating the Pho85p
kinase involved in phosphate regulation (36). We initially
referred to this gene as AGS3, because it is located on a
different chromosome than AGS1 (15), but because of its discovered identity to PHO80, we refer to it here by
this established name.
AGS1 transcript.
To characterize the
AGS1 transcript, we isolated total RNA from a wild-type
strain (BJ1991), the ags strain RC1707, and a transformant
of RC1707 carrying the genomic clone pB6A. Northern blots were prepared
and probed with the SalI-BamHI fragment carrying AGS1 (Fig. 3B).
In the AGS1 wild-type strain BJ1991, a single transcript of
about 1 kb was obtained, indicating that AGS1 is expressed
(Fig. 3B, lane 3). From the size of its coding region, we deduce that the 5' and 3' untranslated regions of the AGS1 transcript
amount to about 0.7 kb. In contrast, the ags strain RC1707
contained a shortened transcript of about 0.75 kb (Fig. 3B, lane 1),
suggesting that an extensive deletion has occurred in the
AGS1 gene of this strain. As expected, the RC1707
transformant carrying the genomic clone pB6A expresses the deleted
ags1 transcript, as well as the wild-type AGS1
mRNA (Fig. 3B, lane 2).
The transcript of the PHO80 (AGS3) gene has been
characterized previously (27).
Construction and phenotype of ags1 strains.
To
verify the function of AGS1, its coding region was replaced
by a loxP-kanMX-loxP module in the diploid strain CEN-PK141 (see Materials and Methods). Replacement of the AGS1 coding
region by the loxP-kanMX-loxP module introduces a new
PstI site at the location of the deleted AGS1
(Fig. 4, top). Thus, on Southern blots
with the 3.1-kb PstI-NcoI fragment downstream of
AGS1 as a probe, DNA of wild-type cells is expected to
contain a 7.7-kb PstI fragment, while the size of this
fragment is reduced to 3.8 kb in
ags1::loxP-kanMX-loxP strains. Two
G418-resistant diploids containing both the 7.7- and 3.8-kb
PstI fragments were obtained (Fig. 4, bottom, lanes 1 and
2). The diploids were sporulated, tetrads were dissected, and haploid
segregants were analyzed.
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FIG. 4.
AGS1 disruption in the diploid CEN-PK141. A
schematic of the disruption of AGS1 (open arrow) by the
loxP-kanMX-loxP module is shown at the top. N,
NcoI; P, PstI. A Southern blot demonstrating the
disruption of one of the two AGS1 alleles is shown at the
bottom. Genomic DNAs of two disruption strains (lanes 1 and 2) and the
original strain CEN-PK141 (lane 3) were cut with PstI and
analyzed by Southern blotting with the 1-kb
BamHI-SalI AGS1 fragment as a probe.
The migrations of the wild-type AGS1 fragment (7.7 kb), the
ags1 fragment (3.8 kb), and standard fragments (4.3 and
5.1 kb) are as indicated.
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As expected, G418 resistance due to the
ags1::loxP-kanMX-loxP
disruption segregated 2:2 with G418 sensitivity (AGS1).
Interestingly, the ags1 haploids were found to be
supersensitive to hygromycin B at a concentration of 10 µg/ml. This
phenotype was verified on hygromycin B gradient plates, as shown for
the two haploid ags1 segregants W10-2A and W10-2B (Fig.
5A). Thus, deletion of AGS1
reproduces the aminoglycoside supersensitivity phenotype observed
originally in strain RC1707. Reintroduction of the intact AGS1 gene into W10-2B by transformation with plasmid pSW18
reconstituted the wild-type resistance level, while a control plasmid
(YCplac33) did not complement the ags1 defect (Fig. 5A).
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FIG. 5.
Hygromycin B resistance of yeast strains. The indicated
strains were streaked on a linear gradient of hygromycin B (0 to 120 µg/ml) in YPD medium and grown for 2 days at 30°C. The effects of
the ags1 mutation (A), the pho80,
pho85, and pho4 mutations (B) and a combination
of ags1 and pho80 (C) were tested.
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Besides aminoglycoside supersensitivity, ags1 mutants did
not show any other phenotype compared to AGS1 strains;
growth rates on media containing glucose or sucrose as carbon sources,
as well as the microscopic appearances of cells, were identical in
ags1 and AGS1 wild-type strains.
Phenotypes of pho80 (ags3) and
pho85 strains.
As shown above, we had identified the
PHO80 gene on one type of genomic clone complementing the
supersensitivity phenotype of strain RC1707. To test if
PHO80 is involved in aminoglycoside resistance of yeast, we
compared the hygromycin B sensitivities of a pho80 mutant
(SH8249) and an isogenic wild-type strain (SH8245). Furthermore, since
PHO80 encodes a cyclin activating the Pho85p protein kinase,
we also examined whether aminoglycoside resistance was affected in an
isogenic pho85 mutant (SH8405). As shown in Fig. 5B, both
pho80 and pho85 mutants were significantly more sensitive than the wild type to hygromycin B; an increased sensitivity of both mutants to G418 was also observed (data not shown). An identical result was obtained with a different isogenic set of strains
(BY391 [pho85] and BY490 [pho80]) derived
from strain BY263 (30) and with strain VG51
(pho80). These results suggested that the activity of the
Pho80p-Pho85p complex is required to maintain wild-type levels of
resistance against aminoglycosides.
One of the functions of the Pho80p-Pho85p complex is to phosphorylate
the Pho4 transcription factor and thereby to inactivate it under
high-phosphate conditions (36). To examine whether an
unrefrained activity of Pho4p was responsible for the increased sensitivity of the pho80 and pho85 mutants, we
tested isogenic pho80 pho4 and pho85 pho4 double
mutants for their sensitivity to hygromycin B and G418. In such double
mutants, wild-type resistance levels were restored (Fig. 5B). These
results suggested that an elevated activity of Pho4p is responsible for
an increase in sensitivity to aminoglycoside antibiotics.
To test if ags1 and pho mutations have an
additive effect in increasing aminoglycoside sensitivity, we crossed
strain W10-2B (ags1) with strain VG51 (pho80)
and identified segregants. Among 12 tetrads, 2 tetrads (WK4a-7 and
WK4b-5) which contained two wild-type spores and two ags1
pho80 double mutant spores were identified. Among such double
mutants, in each tetrad, one segregant (WK4a-7A or WK4b-5A) indeed was
more sensitive than the single mutants, while a second segregant
(WK4a-7C or WK4b-5C) showed no enhanced sensitivity compared to the
pho80 mutant (Fig. 5C). Thus, unknown genes present in the
genetic background of the parental strains appear to influence the
aminoglycoside sensitivity of the double mutants.
ags1 mutants have a defect in N glycosylation.
It
has been reported that defects in N glycosylation lead to an increased
sensitivity to hygromycin B and an increased resistance to
orthovanadate (5, 13). To test if AGS1 is
involved in N glycosylation, we examined the N glycosylation status of
two secreted proteins, CPY and invertase (Suc2p), in ags1
mutants. N-glycosyl chains in CPY are short, consisting essentially of the core moiety of the dolichol-linked precursor structure; mutations in the core region lead to inefficient transfer of glycosyl chains from
the dolichol precursor to CPY (4, 48). On the other hand,
invertase N-glycosyl chains are extended to contain heterogeneous lengths of outer chains (3, 32).
The electrophoretic migration of CPY in ags1 mutants was not
different from that of CPY produced by wild-type strains (Fig. 6A), indicating that AGS1 is
not involved in the biosynthesis and transfer to the CPY protein of the
dolichol-linked N-glycosyl precursor structure. As expected,
underglycosylated CPY appeared in an alg5 mutant, which
synthesizes a defective core structure (21).
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FIG. 6.
CPY and invertase (SUC) in yeast strains. (A) CPY in
cellular proteins separated by SDS-PAGE was detected by immunoblotting
with an anti-CPY antibody. The positions of mature CPY (mCPY) and
underglycosylated CPY forms (1 and 2) are indicated. Lane 1, W10-2A
(ags1); lane 2, W10-2B (ags1); lane 3, W10-2C
(AGS1); lane 4, W10-2D (AGS1); lane 5, G2-10
(GDA2); lane 6, G2-11 (gda2); lane 7, PRY98
(alg5). (B) Invertase in cellular proteins was separated by
nondenaturing gel electrophoresis followed by staining of its activity
in the gel. Lanes are as in panel A. (C) Invertase in cellular proteins
separated by SDS-PAGE was detected by immunoblotting with an
anti-invertase antibody. The migration of invertase in the wild-type
strain W10-5A (lane 2) is indicated (mSUC). Lane 1, W10-5C
(ags1); lane 3, G2-10 (GDA2); lane 4, G2-11
(gda2). The migration of molecular mass markers (84 and 116 kDa) is indicated, and the expected migration of core-glycosylated
invertase (about 90 kDa) is marked by an asterisk.
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A defect in the addition of outer chains to the N-glycosyl core
structure can be easily monitored by activity staining of invertase
which has been separated on native polyacrylamide gels (32).
Using this system, we found that invertase of ags1 mutants migrated significantly faster (Fig. 6B, lanes 1 and 2) than invertase of wild-type strains (Fig. 6B, lanes 3 and 4). Abnormal migration of
invertase is more pronounced than in gda2 mutants
(1), which have a partial defect in outer chain addition.
These results could be confirmed by immunoblotting, by which denatured
cellular proteins separated by SDS-PAGE were analyzed with an
anti-invertase antibody. As expected, the bulk of invertase appeared as
a heterogeneous smear around 150 kDa in wild-type cells (Fig. 6C, lane
2). In contrast, most invertase in the ags1 mutant produced
distinct smaller bands (Fig. 6C, lane 1), with a major invertase
species at 90 kDa, which is the size of core-glycosylated invertase
(35). Again, the gda2 defect, although detectable
in this system (Fig. 6C, lane 4), did not result in a phenotype as
significant as observed for the ags1 mutant. Thus, it
appears that the extension of N-glycosyl chains beyond the core
structure is defective in ags1 mutants. No defects in N
glycosylation of invertase and CPY were observed in a pho80
strain (VG51) (data not shown), suggesting that aminoglycoside supersensitivities in ags1 and pho80 strains are
caused by different mechanisms.
Defective N glycosylation leads not only to supersensitivity to
hygromycin B but also to resistance to orthovanadate (5, 32). To determine if ags1 mutants show this phenotype,
we grew AGS1 wild-type strains (W10-2C and W10-2D) and
ags1 mutant strains (W10-2A and W10-2B) on YPD agar
containing 10 mM orthovanadate. While both wild-type strains were
unable to grow, the ags1 mutants developed visible colonies
after 2 days of growth (data not shown). Thus, ags1 mutants
are vanadate resistant, as expected for a defect in N glycosylation.
Chitinase secreted by S. cerevisiae is highly O glycosylated
(35). Neither ags1 nor pho80 mutants
showed different electrophoretic mobilities of chitinase, indicating
that defects in both genes do not perturb O glycosylation (data not
shown).
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DISCUSSION |
An analysis of mutant strain RC1707 (15) revealed two
genes, AGS1 and AGS3 (PHO80), whose
expression is required to maintain basal levels of resistance to
aminoglycoside antibiotics in yeast. The susceptibility to
aminoglycoside antibiotics varies greatly among subtypes and species of
yeasts, ranging from extreme sensitivity, as in S. cerevisiae RC1707, to high resistance, as in the human fungal
pathogen Candida albicans. The reasons for these differences in aminoglycoside sensitivities are not known. Elucidation of resistance mechanisms may guide the development of antifungal agents;
furthermore, sensitive strains may serve as improved host strains,
since plasmids carrying genes encoding aminoglycoside-modifying enzymes, such as the phosphotransferases encoded by aph and
hph, can be selected at low concentrations of antibiotics.
AGS1 was found to encode a small protein of 88 amino acids
which had not been assigned a function previously (and which because of
its small size is not a subject of a systematic functional analysis
project [34]). Our evidence indicates that
AGS1 is mutated in strain RC1707, since (i) the
AGS1 transcript in RC1707 is shortened, indicating an
extensive deletion; (ii) introduction of the wild-type AGS1
gene into RC1707 leads to the reappearance of the wild-type
AGS1 transcript and restores hygromycin B and G418
resistance; and (iii) deletion of AGS1 in a wild-type strain leads to hygromycin B supersensitivity. Further analyses of the ags1 strain revealed that besides increased
susceptibility to hygromycin B, it showed a higher resistance to
vanadate and underglycosylated invertase, indicating a defect in N
glycosylation. It has been reported previously that defects in N
glycosylation lead to increased hygromycin B sensitivity and vanadate
resistance; this phenotype is observed with mutants defective in the
biosynthesis of the core region or outer N-glycosyl chains (5,
13). ags1 mutants did not show defects in growth or
microscopic appearance, indicating that general cellular functions are
not perturbed by the lack of Ags1p function. Particularly, growth on
sucrose as the sole carbon source was not affected in ags1
strains, suggesting that underglycosylation of invertase is not due to
a specific defect in invertase secretion. Thus, AGS1 appears
to encode a hitherto undescribed component of the N glycosylation
machinery. To explore whether core or outer chain biosynthesis is
affected in ags1 mutants, we examined the glycosylation
status of CPY, which only contains unextended, core-sized N-glycosyl
chains (4). The ags1 mutant did not show any
defect in CPY glycosylation, indicating that neither the activity of
oligosaccharyltransferase nor the biosynthesis of the core region is
affected by the Ags1 protein. In contrast, glycosylation of invertase,
whose N-glycosyl chains are composed of core and outer chain regions
(3, 35), was severely defective in ags1 mutants,
leading to the accumulation of an invertase species with migration
identical to that of the core-glycosylated form. Thus, it appears that
a biosynthetic step in the elaboration of outer chains requires the
Ags1 protein. Possibly, the ags1 mutation may lead to
defects similar to those in mnn9 and och1
mutants, which are also defective in outer chain biosynthesis (3,
32). Because Ags1p, although hydrophobic, does not contain a
signal sequence or a transmembrane region, it may influence N
glycosylation in Golgi vesicles from their cytoplasmic sides, e.g., by
interaction with the Och1p mannosyltransferase (32).
Remarkably, a small hydrophobic protein is also required for the
activity of the oligosaccharyltransferase complex in the endoplasmic
reticulum (9). The molecular mechanism by which defects in N
glycosylation lead to aminoglycoside sensitivity is not known. It
appears that fully extended N-glycosyl chains of one or more proteins
are required for basal levels of aminoglycoside resistance, possibly by
permitting a high level of export or by preventing easy import of
aminoglycosides. ags1 mutants may be useful host strains for
the production of heterologous pharmaceutical glycoproteins, because
the lack of extended N-glycosyl chains may increase specific activities
and reduce antigenic properties of proteins secreted by yeast
(11).
Surprisingly, AGS3 was found to be identical to the
PHO80 gene, which is known to encode a protein acting as a
cyclin to activate the Pho85p protein kinase (29, 30). The
Pho80p-Pho85p complex is known to phosphorylate and thereby inactivate
the Pho4p transcriptional activator, which is required to activate
expression of PHO5, which encodes acid phosphatase (reviewed
in references 24 and 36). Since
the Pho80p-Pho85p complex is active only under high-phosphate conditions, PHO5 is expressed only in media containing low
concentrations of phosphate. We found that both pho80 and
pho85 mutants were supersensitive to aminoglycosides but
that simultaneous mutation of PHO4 increased resistance
levels. Thus, it appears that the supersensitivity phenotype requires
an elevated activity (lack of phosphorylation) of the Pho4p protein;
conversely, under high-phosphate conditions, Pho80p and Pho85p prevent
activation of Pho4p and prevent aminoglycoside supersensitivity. The
molecular mechanism by which activation of Pho4p leads to enhanced
sensitivity is open to speculation. It is difficult to envisage a
function of the PHO5-encoded acid phosphatase in this
process, because Pho5p is secreted into the periplasm. A complex
scenario in which aminoglycosides are phosphorylated in the cytoplasm,
transported in the periplasm, and then dephosphorylated (activated) by
acid phosphatase cannot be excluded at present and needs to be verified
experimentally. Other explanations may be related to recent findings
which have demonstrated that Pho proteins are involved in processes
other than the biosynthesis of phosphatase. Pho85p is able to associate with cyclins other than Pho80p to function as a cyclin-dependent kinase, e.g., during the cell cycle (30). Since we observed that pho85 mutants are more sensitive to hygromycin B than
pho80 mutants, it is possible that an unknown cyclin is
partially redundant with Pho80p. The Pho80p-Pho85p kinase is known to
phosphorylate targets other than Pho4p, e.g., enzymes involved in
glycogen metabolism (20, 45). Also, it has been known for a
long time that, in contrast to wild-type cells, pho80
(tup7) mutants are able to utilize 5'-mononucleotides
(6), but the molecular mechanisms of this phenotype are
unknown. Our finding that the pho4 mutation only partially
restores resistance to hygromycin B in a pho85 strain could
be due to an additional function of Pho85p with regard to
aminoglycoside susceptibility that is independent of Pho4p.
We are grateful to S. Harashima, B. Andrews, C. Abeijon, and F. Hilger for providing strains.
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Abstract
Introduction
