Null mutations in the SNF3gene of Saccharomyces cerevisiaecause a diVerent phenotype than previously isolated missense mutations

Abstract

Missense mutations in the SNF3 gene of Saccharomyces cerevisiae were previously found to cause defects in both glucose repression
and derepression of the SUC2 (invertase) gene. In addition, the growth properties of snf3 mutants suggested that they were
defective in uptake of glucose and fructose. We have cloned the SNF3 gene by complementation and demonstrated linkage of the
cloned DNA to the chromosomal SNF3 locus. The gene encodes a 3-kilobase poly(A)-containing RNA, which was fivefold more abundant
in cells deprived of glucose. The SNF3 gene was disrupted at its chromosomal locus by several methods to create null mutations.
Disruption resulted in growth phenotypes consistent with a defect in glucose uptake. Surprisingly, gene disruption did not
cause aberrant regulation of SUC2 expression. We discuss possible mechanisms by which abnormal SNF3 gene products encoded
by missense alleles could perturb regulatory functions.

Full text

Vol.
6,
No.
11
MOLECULAR
AND
CELLULAR
BIOLOGY,
Nov.
1986,
p.
3569-3574
0270-7306/86/113569-06$02.00/0
Copyright
C
1986,
American
Society
for
Microbiology
Null
Mutations
in
the
SNF3
Gene
of
Saccharomyces
cerevisiae
Cause
a
Different
Phenotype
than
Do
Previously
Isolated
Missense
Mutations
LENORE
NEIGEBORN,
PAMELA
SCHWARTZBERG,
ROBERT
REID,
AND
MARIAN
CARLSON*
Department
of
Genetics
and
Development
and
Institute
for
Cancer
Research,
Columbia
University
College
of
Physicians
and
Surgeons,
New
York,
New
York
10032
Received
8
April
1986/Accepted
29
July
1986
Missense
mutations
in
the
SNF3
gene
of
Sacharomyces
cerevisiae
were
previously
found
to
cause
defects
in
both
glucose
repression
and
derepression
of
the
SUC2
(invertase)
gene.
In
addition,
the
growth
properties
of
snf3
mutants
suggested
that
they
were
defective
in
uptake
of
glucose
and
fructose.
We
have
cloned
the
SNF3
gene
by
complementation
and
demonstrated
linkage
of
the
cloned
DNA
to
the
chromosomal
SNF3
locus.
The
gene
encodes
a
3-kilobase
poly(A)-containing
RNA,
which
was
fivefold
more
abundant
in
cells
deprived
of
glucose.
The
SNF3
gene
was
disrupted
at
its
chromosomal
locus
by
several
methods
to
create
null
mutations.
Disruption
resulted
in
growth
phenotypes
consistent
with
a
defect
in
glucose
uptake.
Surprisingly,
gene
disruption
did
not
cause
aberrant
regulation
of
SUC2
expression.
We
discuss
possible
mechanisms
by
which
abnormal
SNF3
gene
products
encoded
by
missense
alleles
could
perturb
regulatory
functions.
Glucose
repression,
or
carbon
catabolite
repression,
is
a
global
regulatory
system
affecting
the
expression
of
many
genes.
Our
studies
of
glucose
repression
in
Saccharomyces
cerevisiae
have
focused
on
the
SUC2
gene.
Expression
of
SUC2
is
regulated
only
by
glucose
repression
and
is
modu-
lated
over
a
greater
than
200-fold
range.
The
SUC2
gene
encodes
both
secreted
and
intracellular
forms
of
invertase
via
two
mRNAs
with
different
5'
ends
(4,
7,
18).
Secreted
invertase
is
encoded
by
a
glucose-repressible
1.9-kilobase
(kb)
mRNA.
This
secreted
enzyme
is
responsible
for
the
extracellular
hydrolysis
of
sucrose
and
raffinose.
The
intra-
cellular
invertase
is
encoded
by
a
constitutive
1.8-kb
mRNA
and
has
no
obvious
physiological
function
(22).
We
previously
isolated
mutations
in
six
genes,
SNFI
through
SNF6
(sucrose
nonfermenting),
essential
for
regu-
lated
SUC2
expression
(6,
17).
The
snJf3
mutants
were
unable
to
grow
on
raffinose
and
were
defective
in
growth
on
sucrose,
but
none
showed
pleiotropic
defects
in
utilization
of
galactose
or
nonfermentable
carbon
sources.
Our
11
snf3
mutants
displayed
a
range
of
phenotypes
with
respect
to
regulation
of
SUC2
expression:
derepressed
secreted
invertase
activity
ranged
from
10
to
35%
of
the
wild-type
level.
Moreover,
all
showed
constitutive
(glucose-
insensitive)
synthesis
of
secreted
invertase,
ranging
up
to
20%
of
the
derepressed
wild-type
level
(17;
P.S.,
unpub-
lished
results).
Further
studies
suggested
that
regulation
of
SUC2
transcription
was
aberrant;
some
snJ3
mutations
af-
fected
expression
of
a
gene
fusion
in
which
the
LEU2
promoter
is
controlled
by
the
SUC2
upstream
regulatory
region
(23).
A
puzzling
phenotype
of
the
snJ3
mutants
was
that
all
were
more
defective
in
growth
on
sucrose
and
raffinose
than
would
have
been
predicted
from
their
invertase
activity.
We
suggested
previously
(17)
that
the
snf3
mutants
might
be
defective
in
uptake
of
glucose
and
fructose,
which
are
released
by
the
extracellular
hydrolysis
of
sucrose
and
raffinose.
Although
one
can
imagine
that
such
a
defect
in
glucose
uptake
could
account
for
the
invertase
constitutivity
*
Corresponding
author.
of
snf3
mutants,
it
is
not
obvious
how
such
a
defect
per
se
could
also
impair
derepression
of
secreted
invertase.
We
report
here
the
cloning
of
the
SNF3
gene.
A
3-kb
mRNA
encoded
by
SNF3
was
identified,
and
its
level
was
shown
to
be
regulated
by
glucose
repression.
The
gene
was
disrupted
at
its
chromosomal
locus
by
several
methods.
Disruption
resulted
in
phenotypes
consistent
with
a
defect
in
glucose
uptake,
but
surprisingly
did
not
cause
the
aberrant
regulation
of
invertase
expression
that
was
observed
with
the
missense
mutations.
MATERIALS
AND
METHODS
Strains
and
genetic
methods.
Table
1
lists
the
S.
cerevisiae
strains
used
in
this
study.
Standard
methods
were
used
for
genetic
analysis
(25)
and
transformation
(13).
Scoring
for
carbon
source
utilization
was
done
as
described
previously
(6),
except
that
1
p.g
of
antimycin
A
per
ml
was
added
to
rich
medium
instead
of
incubating
the
plates
anaerobically.
Com-
plementation
of
snJ3
mutations
by
plasmids
was
determined
by
testing
for
anaerobic
growth
on
synthetic
complete
me-
dium
(25)
lacking
uracil
and
containing
the
specified
carbon
source.
Isolation
of
plasmids
carrying
SNF3.
The
library
(gift
of
P.
Novick
and
M.
Rose)
contained
genomic
DNA
from
an
isogenic
derivative
of
strain
S288C
cloned
in
YCp5O.
Plas-
mids
able
to
complement
snf3-72
were
isolated
by
methods
described
previously
(8).
Subclones.
pRRC5
and
pLN19
(see
Fig.
1)
are
subclones
in
vectors
YCp5O
and
pCGS40
(11),
respectively.
pLNC2
was
constructed
by
deleting
the
BamHI
fragment
from
pPSC5.
pLN10
is
a
subclone
of
the
BamHI
fragment
in
pCGS40.
All
other
subclones
are
derivatives
of
YIp5
(3).
pLN204-30
contains
the
3.4-kb
SalI-EcoRI
fragment
cloned
in
YIp5.
pRR1,
pRR2,
and
pRR4
were
derived
from
pLN204-30
by
insertion
of
the
HIS3
BamHI
fragment.
Analysis
of
diploid
transformants
with
SNF3
gene
disrup-
tions.
Genomic
DNAs
from
transformants
were
digested
with
EcoRI
and
analyzed
by
Southern
blot
hybridization
(26)
with
radiolabeled
pLN204-9
DNA.
Strains
carrying
a
disrup-
tion
gave
rise
to
a
4.3-kb
fragment
from
the
wild-type
SNF3
3569

3570
NEIGEBORN
ET
AL.
TABLE
1.
List
of
S.
cerevisiae
strainsa
Strain
Genotype
MCY501
MATa
ade2-101
gal2
SUC2
MCY638
MATa
ura3-52
his4-539
lys2-801
SUC2
MCY657
MATxL
snJ3-72
ura3-52
lys2-801
SUC2
MCY714
MATat
snJ3-217
ura3-52
SUC2
MCY1093
MATa
ura3-52
his4-539
lys2-801
SUC2
MCY1407
MATa
snj3-A4::HIS3
Ahis3
ura3-52
Iys2-801
SUC2
MCY1443
MATa
snJ3-2::HIS3
Ahis3
ura3-52
lys2-801
SUC2
MCY1257
MA
TalMA
Ta
Ahis3lAhis3
ura3-521
+
lys2-8011lys2-801
+
Iade2-101
SUC21SUC2
a
All
strains
are
isogenic
or
congenic
to
S288C.
locus
and
a
larger
fragment
from
the
disrupted
locus
(5
kb
for
the
insertions
and
4.6
kb
for
the
substitution).
Invertase
assays.
Glucose-repressed
cells
were
prepared
by
growing
cells
to
exponential
phase
in
medium
containing
2%
glucose,
and
derepressed
cells
were
obtained
by
shifting
repressed
cells
to
medium
containing
0.05%
glucose,
as
described
previously
(8).
Secreted
invertase
activity
was
assayed
in
whole
cells
(8).
Assays
for
I8-galactosidase
and
maltase.
3-Galactosidase
was
assayed
in
permeabilized
cells
(12).
For
maltase
assays,
cells
were
broken
by
vortexing
with
glass
beads
and
assayed
as
described
previously
(14)
in
the
presence
of
40
,ug
of
phenylmethylsulfonyl
fluoride
per
ml
and
1
mM
EDTA.
Protein
concentrations
were
determined
by
the
Bio-Rad
protein
assay.
RESULTS
Cloning
the
SNF3
gene.
Plasmids
carrying
the
SNF3
gene
were
isolated
from
a
library
by
complementation
of
the
defect
in
raffinose
utilization
caused
by
snf3.
A
library
of
genomic
DNA
cloned
in
the
centromere-containing
vector
YCp5O
was
used
to
transform
strain
MCY657
(snjf3-72
ura3)
to
uracil
independence.
Transformants
were
then
screened
for
anaerobic
growth
on
rich
medium
containing
raffinose.
Seven
different
plasmids
carrying
overlapping
cloned
seg-
ments
were
isolated
from
the
Raf+
transformants
by
passage
through
Escherichia
coli
cells
(8).
Figure
1
shows
the
restric-
tion
maps
of
two
of
these
plasmids,
pPSC4
and
pPSC5.
To
verify
that
the
cloned
DNA
complemented
the
snjf3
muta-
tions,
we
showed
that
transformation
of
strains
MCY657
(snJ3-72)
and
MCY714
(snJ3-217)
with
pPSC4
restored
raf-
finose
utilization
and
proper
invertase
derepression
(Table
2).
The
cloned
DNA
was
shown
to
direct
integration
of
pLN185
(Fig.
1)
to
the
SNF3
chromosomal
locus.
pLN185
was
used
to
transform
MCY638
(SNF3
ura3),
and
a
Ura+
transformant
was
crossed
to
MCY657
(snf3
ura3).
Tetrad
analysis
of
the
resulting
diploid
showed
that
the
Ura+
and
Snf1
phenotypes
cosegregated
2:2
in
19
tetrads,
demonstrat-
ing
that
pLN185
had
integrated
into
the
genome
at
a
site
tightly
linked
to
the
SNF3
locus.
Localization
of
the
SNF3
gene
on
the
cloned
DNA.
Subclones
were
constructed
in
episomal
vectors
and
tested
for
SNF3
function
by
complementation
(Fig.
1).
Each
plas-
mid
was
used
to
transform
MCY657
(snf3-72),
and
four
Ura+
COMPLEME
NTATION
H
R
RRR
S
XB
Bg
R
I.M.
a
I
I
a
I
2.
. '
-
1E
R
B
I
ie.-I
i,*
pRRC5,
pLN19
pLN10,
pLN185
pLN204
-9
pLN204-1
7
pLN204-48
pLN
204-6
pRRI
|HIS3
pRR2
pR
R4
FIG.
1.
Restriction
maps
of
SNF3
clones.
Plasmids
are
described
in
Materials
and
Methods.
Only
the
S.
cerevisiae
DNA
segment
(solid
line)
is
shown,
except
that
the
YEp24
sequence
(shaded
bar)
is
indicated
in
pPSC5
and
pPSC4.
Restriction
sites
within
the
HIS3
fragment
(open
box)
are
not
shown.
The
ability
of
each
episomal
plasmid
to
complement
the
raffinose-nonfermenting
phenotype
of
a
snf3
mutant
is
indicated.
The
wavy
arrow
indicates
the
approximate
position
and
direction
of
transcription
of
the
SNF3
RNA.
Restriction
sites:
B,
BamHI;
Bg,
BglII;
H,
HindIII;
R,
EcoRI;
S,
SaII;
X,
XhoI
PLASMID
pPSC5
pPSC4
pLNC2
+
........
1J.-,
MOL.
CELL.
BIOL.
HIS3
-1111.

SNF3
GENE
OF
S.
CEREVISIAE
3571
transformants
were
tested,
for
raffinose
utilization.
Se-
quences
within
the
4.3-kb
EcoRI
fragment
of
pLN19
were
sufficient
to
complement
the
snf3
defect.
The
inability
of
pLNC2
and
pLN10
to
complement
indicated
that
the
SNF3
gene
spanned
the
BamHI
site.
To
confirm
that
sequences
within
the
4.3-kb
EcoRI
fragment
complemented
the
defect
in
single
copy,
the
centromere-containing
plasmid
pRRC5
was
used
to
transform
strains
carrying
snJ3-72,
snJ3-217,
or
snJ3-39.
pRRC5
complemented
the
defects
in
sucrose
and
raffinose
utilization
and
regulation
of
secreted
invertase
expression
(Table
2).
Structure
and
regulated
expression
of
the
SNF3
RNA.
To
identify
the
RNA
encoded
by
SNF3,
poly(A)-containing
RNAs
from
both
glucose-repressed
and
derepressed
cultures
of
a
wild-type
(SNF3)
strain
were
examined
by
Northern
blot
hybridization
analysis.
A
3-kb
RNA
was
detected
with
three
contiguous
probes,
pLN204-17,
pLN204-48,
and
pLN204-6
(Fig.
2).
Because
the
complementation
analysis
indicated
that
the
SNF3
gene
spanned
the
BamHI
site,
this
3-kb
RNA
must
be
encoded
by
SNF3.
The
approximate
position
of
the
SNF3
RNA
is
indicated
in
Fig.
1;
the
direction
of
transcription
was
inferred
from
studies
of
gene
fusions
expressed
in
bacteria
(J.
Celenza
and
M.
Carlson,
unpublished).
A
second
RNA
was
also
detected:
probes
pLN204-9
and
pLN204-17
hybridized
to
a
4.2-kb
RNA,
which
could
not
be
encoded
in
its
entirety
by
the
SNF3
complementing
region.
Comparison
of
the
amounts
of
SNF3
RNA
in
glucose-
repressed
and
-derepressed
cells
indicated
that
the
level
of
SNF3
RNA
was
regulated
by
glucose
repression
(Fig.
2).
In
three
experiments,
the
SNF3
RNA
was
about
fivefold
more
abundant
in
derepressed
cells
than
in
glucose-repressed
cells.
In
derepressed
cells
the
SNF3
RNA
was
5-
to
10-fold
less
abundant
than
the
URA3
RNA.
Disruption
of
the
chromosomal
SNF3
locus.
The
phenotype
of
a
null
mutation
at
the
SNF3
locus
was
not
apparent
from
the
previous
isolation
of
snJ3
alleles
(17).
Therefore,
the
cloned
SNF3
gene
was
used
to
disrupt
the
chromosomal
SNF3
locus.
Insertion
and
substitution
mutations
were
con-
structed
in
pRR1,
pRR2,
and
pRR4
(Fig.
1)
and
were
then
introduced
into
the
genome
at
the
SNF3
locus
by
the
method
of
Rothstein
(21).
pRR1,
pRR2,
and
pRR4
DNAs
were
cleaved
with
EcoRI
and
SaiI,
and
the
fragments
were
used
to
transform
the
diploid
strain
MCY1257
(Ahis3lAhis3
SNF31SNF3)
to
histidine
prototrophy;
a
diploid
was
used
in
TABLE
2.
Secreted
invertase
activity
in
strains
carrying
SNF3
plasmids
Relevant
Secreted
invertase
activitya
genotype
Plasmid
Repressed
Derepressed
snj3-72
YCp50
1
40
snj3-72
pPSC4
<1
200
snJ3-72
pRRC5
<1
160
snj3-217
YCp50
<1
25
snf3-217
pPSC4
<1
310
snj3-217
pRRC5
<1
220
snj3-39
YCp5O
50
80
snf3-39
pRRC5
6
110
SNF3
pCGS40
<1
220
SNF3
pLN19
<1
190
a
Micromoles
of
glucose
released
per
minute
per
100
mg
(dry
weight)
of
cells.
Values
are
the
average
from
assays
of
two
transformants,
and
standard
errors
were
<15%.
Cultures
were
grown
in
synthetic
complete
medium
(25)
lacking
uracil;
assay
values
are
therefore
not
directly
comparable
to
those
shown
in
Table
3,
for
which
cultures
were
grown
in
rich
medium.
M
R
D
so
-
-
SNF3
_~~-URA3
FIG.
2.
Northern
blot
analysis
of
the
SNF3
RNA.
Poly(A)-
containing
RNAs
were
prepared
(24)
from
cells
of
strain
MCY501
grown
under
repressing
(lane
R)
or
derepressing
(lane
D)
conditions,
separated
by
electrophoresis
in
a
O.9o
agarose
gel
containing
formaldehyde
(15),
and
transferred
to
nitrocellulose
(27).
RNAs
homologous
to
32P-labeled
(20)
pLN204-48
DNA
were
detected
by
hybridization
(5)
and
autoradiography.
The
relative
abundance
of
RNAs
was
estimated
with
a
Joyce-Loebl
Chromoscan
3
densitometer.
Lane
M,
Restriction
fragment
markers.
case
SNF3
proved
to
be
an
essential
gene.
Recombination
events
occurring
on
either
side
of
the
HIS3
gene
would
result
in
the
replacement
of
the
wild-type
sequence
with
the
mutation.
To
verify
the
presence
of
the
appropriate
mutation
at
the
SNF3
locus
of
one
homolog,
genomic
DNA
from
His'
transformants
was
analyzed
by
Southern
blot
hybridization
(see
Materials
and
Methods).
This
analysis
also
confirmed
that
SNF3
is
a
unique
gene.
Diploids
carrying
each
disruption
were
subjected
to
tetrad
analysis.
In
each
case
all
four
spores
of
seven
tetrads
were
viable;
thus,
disruption
of
SNF3
was
not
lethal
in
haploids.
Histidine
prototrophy
cosegregated
2:2
with
inability
to
utilize
raffinose,
indicating
that
disruption
conferred
a
raf-
finose-nonfermenting
phenotype.
The
mutants
also
grew
more
slowly
on
sucrose
than
did
the
wild
type.
These
new
alleles
have
been
designated
as
follows:
the
insertion
derived
from
pRR1,
snJ3-J::HIS3;
the
insertion
from
pRR2,
snf3-
2::HIS3;
and
the
substitution,
snJ3-A4::HIS3.
Each
mutation
was
recessive to
the
wild-type
SNF3
allele
and
failed
to
complement
snj3-72
and
snJ3-217.
pRRC5
complemented
snJ3-A4::HIS3.
These
results
prove
that
we
have
cloned
the
authentic
SNF3
gene
and
disrupted
the
SNF3
locus.
Disruption
of
SNF3
does
not
cause
aberrant
regulation
of
SUC2
expression.
The
three
disruptants
were
assayed
for
secreted
invertase
activity
under
glucose-repressing
and
derepressing
conditions
(Table
3).
Surprisingly,
no
signifi-
cant
defect
in
regulation
of
SUC2
expression
could
be
detected:
secreted
invertase
was
glucose
repressed
and
was
derepressed
to
wild-type
levels
in
response
to
glucose
dep-
rivation.
For
comparison,
Table
3
also
shows
the
aberrant
regulation
of
secreted
invertase
expression
(low-level
constitutivity
and
reduced
derepression)
observed
in
snf3
missense
mutants.
Heteroallelic
diploids
of
genotype
snJ3-A4::HIS31snf3-39
and
snf3-A4:
:HIS31snf3-217
exhibited
defective
invertase
VOL.
6,
1986

3572
NEIGEBORN
ET
AL.
TABLE
3.
Secreted
invertase
activity
in
snj3
mutants
Secreted
invertase
activitya
Relevant
genotype
Repressed
Derepressed
SNF3
<1
230
snJ3-l::HIS3
<1
170
snj3-2::HIS3
<1
220
snJ3-A4::HIS3
<1
210
snf3-39b
40
70
snI3_72b
14
50
snJ3_142b
7
40
snI3-217b
5
25
SNF31SNF3
<1
200
sn]3-A4::HIS31SNF3
<1
210
snJ3-391SNF3
2
160
snJ3-2171SNF3
<1
180
snf3-A4::
HIS31snJ3-39
40
73
snj3-A4::HIS31snJ3-217
5
24
snf3-A4::
HIS31snJ3-A4::
HIS3
3
160
snf3-2171snj3-217
6
29
snf3-391snf3-39
38
68
a
Micromoles
of
glucose
released
per
minute
per
100
mg
(dry
weight)
of
cells.
Values
are
the
average
of
at
least
two
determinations.
Cultures
were
grown
in
rich
medium
(YEP).
b
Values
are
from
Neigeborn
and
Carlson
(17).
regulation
(Table
3)
and
growth
properties
similar
to
those
of
mutants
homozygous
for
snJ3-39
and
snJ3-217,
respectively.
The
finding
that
sn]3-A4::HIS3
is
recessive
to
snf3-39
and
snJ3-217
suggests
that
the
missense
alleles
encode
SNF3
gene
products
with
abnormal
function.
In
addition,
the
phenotypic
resemblance
of
these
diploids
to
the
parent
missense
mutant
argues
that
the
snj3
mutation
is
solely
responsible
for
the
observed
phenotype.
Both
snJ3-39
and
snJ3-217
are
recessive
to
the
wild-type
SNF3
allele
with
respect
to
growth
phenotypes
and
regulation
of
secreted
invertase
synthesis
(Table
3).
snJ3
mutants
are
defective
in
utilization
of
low
concentra-
tions
of
glucose.
The
snf3
null
mutants
were
defective
in
sucrose
and
raffinose
utilization
despite
normal
derepression
2%o
Glucose
a
c
of
secreted
invertase.
These
findings
suggested
that
the
snJ3
mutations
caused
defects
in
the
utilization
of
the
low
con-
centrations
of
glucose
and
fructose
that
result
from
the
extracellular
hydrolysis
of
sucrose
and
raffinose.
We
there-
fore
examined
the
growth
of
single
colonies
of
snJ3
null
mutants
on
rich
medium
containing
2
or
0.1%
glucose.
Colonies
of
snf3-2::HIS3
and
snJ3-A4::HIS3
mutants
grew
more
slowly
than
wild-type
colonies
on
medium
with
0.1%
glucose
(Fig.
3).
No
difference
in
colony
size
was
discernible
on
plates
containing
2%
glucose.
This
reduced
ability
to
grow
on
low
concentrations
of
glucose
would
be
consistent
with
a
defect
in
glucose
uptake.
Effects
of
snf3
mutations
on
expression
of
a
GALIO-lacZ
gene
fusion
and
maltase.
Evaluation
of
the
growth
properties
of
snf3
missense
(17)
and
null
mutants
provided
no
evidence
for
effects
of
snf3
mutations
on
the
expression
of
other
glucose-repressible
genes
besides
SUC2;
these
mutants
were
able
to
grow
on
galactose,
glycerol,
and
ethanol.
For
a
more
sensitive
assay,
we
examined
the
effects
of
snJ3
mutations
on
expression
of
the
GAL1O-lacZ
gene
fusion
on
the
episomal
plasmid
pRY123
(28).
snJ3
mutants
transformed
with
pRY123
were
grown
under
inducing
and
repressing
conditions
and
assayed
for
3-galactosidase
activity
(Table
4).
GAL1O-lacZ
expression
was
inducible
to
within
two-
or
threefold
the
wild-type
level
in
all
snf3
mutants.
Small
defects
in
glucose
repression
were
observed
in
the
snf3-72,
snf3-217,
and
snf3-A4::HIS3
mutants.
The
snJ3-39
mutant,
however,
showed
dramatic
insensitivity
to
glucose,
produc-
ing
nearly
1,000-fold
more
,B-galactosidase
than
the
wild
type
in
the
presence
of
2%
glucose
and
80-fold
more
in
the
presence
of
5%
glucose.
We
also
examined
the
effect
of
snf3-39
on
expression
of
the
glucose-repressible
enzyme
maltase.
Our
strains
are
derived
from
the
maltose-nonfermenting
strain
S288C
and
were
therefore
transformed
with
aplasmid
that
comple-
mented
the
defect,
pM1.2F
(gift
of
M.
Charron
and
C.
Michels).
Assays
of
maltase
activity
showed
that
the
snf3-39
mutant
was
inducible
to
normal
levels,
but
exhibited
16-fold
more
maltase
activity
under
glucose-repressing
conditions
0.13/
Glucose
b
c
FIG.
3.
Growth
of
snJ3
mutants
on
0.1%
glucose.
Strains
were
streaked
for
single
colonies
on
solid
rich
medium
containing
either
2
or
0.1%
glucose
(without
antimycin),
and
the
plates
were
incubated
at
30°C
for
2
days.
(a)
MCY1407
(snJ3-A4::HIS3);
(b)
MCY1443
(snf3-2::HIS3);
(c)
wild-type
strain
MCY1093
(SNF3).
MOL.
CELL.
BIOL.
a

SNF3
GENE
OF
S.
CEREVISIAE
3573
than
did
the
wild
type
(Table
4).
Thus,
snJ3-39,
which
was
the
allele
that
caused
the
most
severe
defect
in
glucose
repression
of
SUC2,
also
caused
defects
in
glucose
repres-
sion
of
two
other
genes.
Effects
of
multiple
copies
of
SNF3
on
invertase
expression.
To
assess
the
effect
of
multiple
copies
of
the
SNF3
gene
on
SUC2
expression,
wild-type
strain
MCY1093
was
trans-
formed
with
the
multicopy
plasmid
pLN19
(Fig.
1).
Trans-
formants
were
grown
under
conditions
of
glucose
repression
and
derepression
with
selection
for
maintenance
of
the
plasmid.
No
significant
alteration
of
SUC2
expression
was
observed
(Table
2).
DISCUSSION
We
have
cloned
the
SNF3
gene
and
identified
the
3-kb
RNA
that
it
encodes.
The
level
of
this
RNA
was
regulated
by
glucose
repression.
The
unexpected
finding
of
this
study
was
that
disruption
of
the
SNF3
gene
resulted
in
a
phenotype
different
from
those
of
previously
isolated
missense
mutants.
The
11
missense
mutants
showed
aberrant
regulation
of
SUC2
expression
in
response
to
glucose
availability,
includ-
ing
defects
in
both
repression
and
derepression.
In
contrast,
disruption
of
the
gene
caused
no
perturbation
in
the
regula-
tion
of
SUC2
by
glucose
repression.
Thus,
regulation
was
normal
in
mutants
carrying
a
disrupted
SNF3
locus,
which
presumably
encoded
no
functional
product,
and
was
aber-
rant
in
missense
mutants.
Regulation
of
two
other
glucose-
repressible
genes,
a
GAL1O-lacZ
fusion
gene
and
the
maltase
gene,
was
strikingly
defective
in
cells
carrying
the
snf3-39
mutation.
To
explain
these
results,
we
suggest
that
these
missense
mutants
each
produce
an
abnormal
SNF3
gene
product
that
is
reasonably
stable
within
the
cell,
and
we
further
propose
that
regulation
is
normal
in
the
absence
of
the
SNF3
gene
product
but
that
abnormal
SNF3
products
can
cause
aber-
rant
regulation.
The
variations
in
phenotypes
among
the
missense
mutants
would
then
result
from
differences
in
the
mutant
gene
products.
The
absence
of
any
detectable
regu-
latory
defect
in
snJ3
null
mutants
suggests
that
the
SNF3
gene
is
not
essential
for
normal
regulation
of
SUC2
expres-
sion.
A
possible
explanation
for
the
regulatory
anomalies
in
the
missense
mutants
is
that
the
SNF3
product
interacts
physically
with
one
or
more
proteins
that
play
regulatory
roles
in
glucose
repression
and
that
mutant
SNF3
proteins
TABLE
4.
Expression
of
a
GAL1O-lacZ
fusion
and
maltase
in
snJ3
mutantsa
1-Galactosidase
activityb
Maltase
activityc
Relevant
Gal-Gly-Glu
Gal-Gly-Glu
Mal-Glu
genotype
(5%),
(2%),
Ga[-Gly,
(5%)'
Mal,
repressed
repressed
repressed
SNF3
0.17
0.37
990
70
8,100
snj3-39
14
350
390
1,100
7,500
snf3-72
0.14
3.9
860
NDd
ND
snjf3-217
0.18
1.2
640
ND
ND
sn13-A4::
HIS3
0.09
2.6
580
ND
ND
aCultures
were
grown
to
mid-log
phase
in
synthetic
complete
medium
lacking
uracil
and
containing
the
indicated
carbon
sources:
Gal,
2%
galactose;
Gly,
3%
glycerol;
Glu,
2
or
5%
glucose,
as
indicated;
Mal,
2%
maltose.
b
Units
of
activity,
normalized
to
the
OD6wo
of
the
culture,
were
calculated
as
described
by
Miller
(16).
Values
are
the
average
of
assays
of
two
transformants,
and
standard
errors
were
<25%.
c
Nanomoles
of
p-nitrophenyl-a-D-glucopyranoside
cleaved
per
minute
per
milligram
of
protein.
Values
are
the
average
of
assays
of
two
transformants,
and
standard
errors
were
<15%.
d
ND,
Not
determined.
perturb
the
function
of
such
neighboring
proteins.
If
that
is
indeed
the
case,
it
may
be
possible
to
obtain
mutations
in
the
genes
encoding
such
proteins
by
pseudoreversion
of
snf3
missense
mutants.
The
snf3
missense
mutations,
which
clearly
cause
a
change
of
function
rather
than
a
simple
loss
of
function,
are
nonetheless
recessive
to
the
wild-type
allele.
This
finding
suggests
that
the
wild-type
SNF3
gene
product
either
func-
tionally
displaces
the
mutant
product
or
perhaps
interacts
with
the
mutant
product
to
restore
normal
function.
There
is
a
simple
explanation
for
the
fact
that
none
of
our
snJ3
missense
mutations
conferred
the
null
phenotype.
In
our
original
mutant
searches,
raffinose-nonfermenting
mu-
tants
were
screened
for
defects
in
derepression
of
secreted
invertase,
and
mutants
showing
normal
derepression
of
invertase
were
not
retained
for
further
study.
The
defect
of
snJ3
null
mutants
in
sucrose
and
raffinose
utilization,
despite
normal
levels
of
secreted
invertase,
and
their
defect
in
utilization
of
0.1%
glucose
suggest
that
SNF3
is
essential
for
glucose
and
fructose
uptake.
Subsequent
analysis
of
the
kinetics
of
glucose
uptake
has
shown
that
snf3
mutants
are
defective
in
the
high-affinity
glucose
uptake
system
(L.
Bisson,
L.
Neigeborn,
M.
Carlson,
and
D.
Fraenkel,
manuscript
in
preparation),
which
is
normally
derepressed
in
response
to
glucose
limitation
(2).
One
pos-
sibility
is
that
SNF3
encodes
a
structural
component
of
the
uptake
system.
We
speculate
that
the
aberrant
glucose
repression
in
snf3
missense
mutants
results
from
a
defect
in
sensing
or
signal-
ling
the
availability
of
glucose
in
the
environment.
A
role
for
SNF3
in
glucose
uptake
is
consistent
with
the
notion
that
abnormal
SNF3
products
could
affect
membrane
or
mem-
brane-associated
proteins
with
sensory
or
signalling
func-
tions.
In
bacteria,
a
protein
of
the
phosphoenolpyruvate-
sugar
phosphotransferase
system,
enzyme
IIIGIc,
is
involved
in
glucose
uptake
and
also
activates
adenylate
cyclase,
and
therefore
plays
a
role
in
the
regulation
of
expression
of
catabolite-repressible
genes
(see
reference
19
for
a
review).
In
S.
cerevisiae
there
is
evidence
for
interaction
between
the
high-affinity
glucose
uptake
system
and
hexokinase
PII(B),
which
is
putatively
a
regulatory
protein
for
glucose
repres-
sion
(9,
10):
the
functioning
of
the
high-affinity
glucose
uptake
system
is
dependent
on
the
presence
of
hexose
kinases
(1).
Previous
genetic
evidence
that
mutations
in
other
trans-acting
genes
required
for
regulated
SUC2
ex-
pression,
SNFI,
SNF2,
SNF4,
and
SSN6,
are
epistatic
to
snJ3
missense
mutations
(17)
also
suggests
that
the
regula-
tory
defect
in
snf3
mutants
occurs
early
in
the
regulatory
circuitry.
ACKNOWLEDGMENTS
We
thank
P.
Novick
and
M.
Rose
for
providing
the
library,
R.
West
for
pRY123,
and
M.
Charron
and
C.
Michels
for
pM1.2F.
This
work
was
supported
by
Public
Health
Service
grant
GM-34095
from
the
National
Institutes
of
Health
and
an
Irma
T.
Hirschl
Research
Career
Award
to
M.C.
LITERATURE
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L.
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D.
G.
Fraenkel.
1983.
Involvement
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glucose
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by
Saccharomyces
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Natl.
Acad.
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USA
80:1730-1734.
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Bisson,
L.
F.,
and
D.
G.
Fraenkel.
1984.
Expression
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dependent
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in
Saccharomyces
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biological
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