Vol. 71, No. 2,1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
STEROL REPLACEMENT IN SACCRAROMYCES CEREVISIAE. EFFECT ON CELLULAR PERMEABILITY Francis Laboratoire
Received
AND SENSITIVITY
EARST and Richard
TO NYSTATIN.
JUND
de G6n6tique Physiologique, I.B.M.C. du C.N.R.S. 15, rue R. Descartes, 67000 Strasbourg, France
March
26,1976 SUMMARY
Hore than 80% of the cellular ergosterol can be replaced by cholesterol in a sterol requiring mutant strain of Saccharo~ces cerevisiae. The effect of this replacement, as well as the effect of sterol starvation on the uptake and exit of cytosine and o-aminoisobutyric acid (wAIBA) was studied in an attempt to elucidate the role of sterols in cellular permeability. Neither the exit of cytosine nor the exit of a-AIBA was affected by changes in the sterol content of the cell. Cells grown on cholesterol or on ergosterol had very similar rates of cytosine uptake, but a lower rate was found for sterol-starved cells. This difference may be a consequence of the cellular growth rate. However, nystatin induces a much slower exit of cr-AIBA in cells grown on cholesterol than in cells grown on ergosterol. This strongly suggests that a change in membrane structure has taken place.
Ergosterol cerevisiae rol
is
cells,
is present
sented study
in
to show that of the
effect
the predominant
comprising
generally
the yeast
cytoplasmic
this
is
also
membranes effect
well
cations
(8)
is also
thought
plasma
cells
of membrane ty of this
Copyright All rights
is
established
lipids
(9).
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
the membrane state
order
(6).
present
cholesterol considerably
prevents
pre-
(2).
membrane
The
systems
and rigidity
(3, 4).
phospholipid
(5),
: this
anions
in plasma
Experiments
(6,
7),
membranes
performed
on myco-
the crystallisation
the non electrolyte
Cobon and Haslam have 535
has been
of cholesterol of water
that
Ergoste-
synthesis
of artificial
incorporation
permeability.
pool.
of model
increase
Cholesterol
sterol
Saccharomyces
and evidence
the permeability
their
and diminishes
sterols
after
for
demonstrated
microorganism
for
growing
total
(1)
the permeability
and non electrolytes
have
membrane
required
considerably
to influence
of the
on the physical
that
demonstrated
in aerobically
90%
has shown that
diminishes
has been
sterol
of sterols
made of phospholipids It
sterol
demonstrated
permeabilithat
changes
BIOCHEMICAL
Vol. 71, No. 2, 1976
in ergosterol tion the
levels
temperature sterol
in yeast
produce
of mitochondrial
concentration
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
a concomitant
ATPase
(IO).
in the mitochondria
change
An inverse and the
in the phase relationship
transibetween
transition
temperature
the sterol
requiring
was established. The present yeast
communication
mutant
erg
ly the natural sterol
starvation
acid
were
test
structural
rent
sterols.
reports
1. In this yeast
sterol
on cellular
investigated. differences
data
strain,
obtained
cholesterol
ergosterol.
antibiotic
of the cellular
MATERIAL
was shown
The effects
permeability
The polyene
from
of this
to cytosine nystatin membrane
to replace
efficient-
replacement
and of
and a-aminoisobutyric was used as a probe in cells
grown
to
on diffe-
AND METHODS
Yeast strains and genetic analysis. The mutant strain FK erg 1 (ATCC 28382) and the parent strain FL 100 (ATCC 28383) as well as the strain RJ 16 sp2 carrying the fcg 2-3 (permease less) and j%g 1-l (cytosine desaminase less) mutations are isogenic and have been previously described (II, 12). The genetic analyses were performed according to the methods of Mortimer and Hawthorne (13) 0 Media and culture methods. Yeast nitrogen base (Difco) 6.7 g/l, supplemented with 20 g/l glucose was the basic minimal medium. The sterols, 1.5 mg/l, were dispersed by 0.25 g/l Tergitol np40 (Sigma). Sterol analysis. The cells growing in exponential phase were harvested and washed with distilled water containing 0.25 g/l Tergitol np40. The non saponifiable lipids were extracted and separated on silica gel G plates as previgusly described (14). The sterol fraction was acetylated by using radioactive ) Cl acetic anhydride (15). Ergosterol and cholesterol acetates were separated on silica gel G plates impregnated with silver nitrate, developed overnight in a cyclohexane-benzene (3-2 V/V) solvent system. The respective amounts of radioactivity of different spots were counted in presence of 10 ml of a mixture of toluene diphenyloxazole 5 g/l which allowed the measurement of the absolute amount of each compound. Uptake and efflux measurement. All experiments were carried out at 30°C on exponentially growing cells in minimal medium. Uptake and efflux of cytosine were measured as previously described (12). For the measurement of the efflux of ci-aminoisobut ric acid (a-AIRA), the cells were incubated for 45 min. in the presence of v I4 C I labelled substrate, then filtered through a membrane filter (1.2 u pore size), washed once with fresh medium and resuspended in fresh medium. At different times during the experiment, I ml samples were removed, filtered and washed with 2 x 10 ml ice cold water. The filters were then dried and their radioactivity counted in toluene plus 5 g/l diphenyloxasole (Intertechnique liquid scintillation spectrometer SL 30).
536
Vol. 71, No. 2, 1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
RESULTS Sterol
analysis. Whereas
weight),
the sterol
0.15X,
whether
cholesterol rol
the sterol content
culture
(vs the amount
data strain,
hypothesis strain
in minimal
a leaky
(see Material
with
cholesterol
acetyl
ergosterol).
Active
transport
of cytosine
A recombinant cytosine
(inactivation
velocities
of cytosine
cholesterol ponding ble
reciprocal
were
lowered
values 0.53
rates
plots
for
and 0.15
were
l/1000
strain
1 fey l-l
or after time).
not
l
strain
the mutant
of cytosine
uptake
were
in the recombinant
strain
erg
strain
l-l
537
grown
latter
with
4.9 mg/l
of
after
were
found
in
the metabolism
I-lmutation).
strain
after
of
Initial growth
starvation
on
(corres-
as calculated
from
the dou-
different,
but
the Vmax
type
2 mM for
sterol
at different
This
fey
on cholesterol after
1 mutant
was associated
of sterol
to the wild
cultured
measured 1 fou
in this
are
This
and separated
to bypass
by the
30 set-‘)
8
l-l.
significatively
in comparison
the mutant
extracted
18 hours
after
The mutant
supplemented
The Km values
1) were
concentration
experiment.
was used
measured
were
strain
to ergosterol.
1 fcu
ergoste-
in the erg
of the counts
erg
uptake
(fig.
for
than
or
cholesterol.
114C( radioactivity
desaminase
in the mutant
and 0.33
The sterols
of cytosine
the doubling
(mM internal
pectively,
erg
or on ergosterol, to twice
medium
in the
strain
with
of following
than
14% residual
occured
in minimal
(less
of
greater (1 mg/l)
in the mutant
converted
and Methods). only
was never ergosterol
of ergosterol
on the basis
(40 mCi/mM).
with
supplemented
was partially
8 generations
acetylation acetyl
strain
a mean value
medium
FL 100 is 0.9% (w/dry
strain
was present
synthesis
was eliminated
cholesterol
type
1 mutant
hand,
sterols)
cholesterol
was grown
114Cl
of total
that
or that
of the erg
On the other
of growth
suggests
of the wild
medium was supplemented
(1 mg/l).
generations
content
strain.
The observed
the wild
type
and ergosterol starvation.
temperatures
on cholesterol
strain, res-
Initial from
3” to 36”
or ergosterol.
The
Vol. 71, No. 2,1976
BlOCHEMlCkL
AND BIOPHYSICAL RESEARCH COhWJNlCATlONS
Figure I. Double reciprocal plots of strain erg 1 fey 1-l as a function of tration in cells is expressed in mM of - - -culture medium supplemented with time 240 min.) ;-A- -medium supplemented doubling time 195 min.) ;- -+I8 hours time 400 min.) ;--wild type strain
initial velocities of cytosine uptake in external concentration. Cytosine concencell water per 30 sec. incubation. 2 ug/ml ergosterol (Vmax 0.328, doubling with 2 ug/ml cholesterol (Wax 0.535, sterol starvation (Vmax 0.148, doubling (Vmax 1.57, doubling time 140 min.).
0.50 t
plots of initial velocity Figure 2. Arrhenius of temperature. I ml samples of the erg 1 fq or ergosterol (M) are preincubated m-B), peratures before uptake measurement.
538
of cytosine uptake as a function 1-I cells grown on cholesterol for 5 min. at the different tem-
Vol. 71, No. 2, 1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
corresponding
plots
2) show biphasic
transition Efflux
Arrhenius temperature
of 114Cl
enters
the yeast
concentrated sine
cell
for sterol were
(fey
(12).
testing
by active
2-3)
cellular
out
is
transport
(fey strain,
for
on two recombinant
l-l). cytosine wAIBA in erg
sterols. strains
acid
the same
in minukr
(cPAIBA). compound,
Extracellular
(16).
cerevisiae
enters
and leaves were
grown
The assays
having
the cytocytosi-
the cell
chosen
on cholesterol,
ergo-
cytosine
permeability
the mutations
.
by free
therefore
for
carrying
is also
desaminase-less
and cytosine 1 cells
which
cytosine cells
In a cytosine
I time
with
a non metabolized
in Saccharomycea
permeability starved
a-aminoisobutyric
(wAIBA)
The two compounds,
and in cells carried
acid
mutation
ne permease-less diffusion
and (14Cl
and accumulated
desaminase-less
kinetics
at 23".
cytosine
a-aminoisobutyric
(fig.
. 10
.
erg
* 20 time
. in
1
* 50
*
* 40
min.
Figure 3. The cells are incubated for 45 min. in minimal medium plus 0.50 mM of [14C] cytosine, then washed and transfered in fresh medium without cytosine, at time zero. The exit is measured as described in Methods. Cytosine efflux from strain J@C~ l-l fey 2-3 grown without sterol (M), 3: and from strain erg 1 fey 1-l fcu 2-3 grown on cholesterol (Q-U), ergosterol (*-++), and after a 15 hours period of sterol starvation (a+-). 3B : Cytosine efflux from strain erg 1 fey 1-l fCu 2-3 in the presence of nysztin. Nystatin is added at time zero at a final concentration of 10 rig/ml. - Cells grown on cholesterol ; efflux measured in presence w) and absence (W) of nystatin. - Cells grown on ergosterol ; efflux measured in presence (W) and absence (U-0) of nystatin.
539
BIOCHEMICAL
Vol. 71, No. 2,1976
fey
1-l
fey
strain
erg
constant
fey
1
l-l
(k min.
lesterol, tial
or fey 1-l
2-3,
fey
than
1 fey l-l
strain
fey
l-l
Effect
fey
2-3
strain
of nystatin
and other
The study
cells
are
nystatin
cursors
(18).
nystatin
if
The effect a-AIBA
(fig.
of addition
effect 3B).
In contrast, type
mented
ergosterol
it
with
strain
in the presence
was greatly
decreased
effective have
indicated
resistant
to nystatin
ergosterol
its
exit
of with
by cholesterol
on erg
accumulate
and on the wild
growing cells.
in the cytosine
fast
of a-AIBA
in minimal
or cholesterol of nystatin
in erg
the cells
540
were
4B).
to (14). and
in the presence
or not
cultured
exit
of
had no signi-
permease-less
A rapid
cultured
pre-
of cytosine
was induced
1 cells
these
medium
Nystatin
medium whether (fig.
sterols
ergosterol
culture
efflux
diffusion exit
step
are more resistant
on the
type
of
crucial
to membrane
in the
1 cells
the
has shown that
mutants
(10 mg/l)
the growth
binding
and thus
requiring
replaced
when
in
supplemented
that
mutants
grown
observed
in
FL 100 and the
in inhibiting
is
a very
exit
no significant
substance
free
to that strain
of this
investigated
on cytosine
ini-
and a-AIBA.
studies
of nystatin
or ergosterol,
in the wild
occured
is
the
in cholesterol
medium had been
of cytosine
ergosterol
ergosterol
cholesterol ficant
Moreover,
similar
of cho-
Moreover
of cytosine
a-AIBA,
growth
same rate
higher
type
1’4Cl
with
the
in
4).
to synthesize
was therefore
the
Several
of yeast
very
exit
3A).
The kinetics
cells.
cytosine
in the presence
(fig.
was always
is highly
action
was grown
When the wild
whether
that
kinetics
medium
with
(fig.
fungi.
unable
3A).
on the exit
in the antifungal (17).
strain
therefore
preloaded
or cholesterol
order
depleted
are
(fig.
were
The polyene yeasts
sterol
fey 2-3
was detected,
ergosterol
the
grown
erg
was observed
first
in ergosterol
strain
label
follows
concentration
cells
114Cl
It
cytosine
grown
1 mutant
2-3.
whether
or in
intracellular
erg
2-3
= 0.040)
ergosterol
fey
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cells
by the polyene it
was supple-
of a-AIBA on ergosterol,
on cholesterol
(fig,
also but 4A).
5
A
10
20
in
4.
ini”.
40
60
a-aminoisobutyric
50
(0 tin*
20 in
min.
30
40
;!, c 10
bdium ozida , lo-)M
tint*
20
fered in fresh medium without described in Methods. 4k: Exit measured in erg 1 cells cultured on ergosterol (U+), and cholesterol (-M). Nystatin (10 rig/ml) is added at time 22 min. 4B : Exit measured in wild type strain after growth in minimal medium supplemented with ergosterol (U) and cholesterol CO--@. Nystatin is added at time 12 min. 4c : Exit measured from wild type strain. 511s grown in standard medium @-@ ; cells grown on proline 4 mg/ml as sole ammonium source &+I ; cells grown in standard medium and pretreated with IO-3M sodium azide during 19 min. before addition of nystatin (0-O).
in
i:, min.
nystitim
(a-AIBA) exit induced by nystatin, from strain incubated for 45 min. in minimal medium plus acid, The cells are then washed and transa-AIBA (time zero), and the exit is measured as
acid
FL 100 and er 1. The cells are 0.32 mM of [l B C] CX-aminoisobutyric
Figure
Time
30
10i
3
30
0
40
BIOCHEMICAL
Vol. 71, No. 2,1976
AND BIOPHYSICAL RESEARCH COMMUNlCATlONS
DISCUSSION It mately
can be concluded 80%
of the cellular
on the cellular acid
(a-AIBA).
take
(fig.
vation
permeability The differences
are more likely
linked
differences
starvation.
It
like
a-AIBA
which
is
from
of these cannot
grown
in
always grown
fact
that
rules
out
cells
than
is no release
a-AIBA
exit is
nystatin
growing
grown
ced by nystatin
(17).
membrane
or in absence
cellular
energetics
This
proline
other
Experiments
as the in vitro
pretreated
as sole
in contrast with
was demonstrated
cells
to its
data
with
mycoplasma
measurement
of diffusion
542
whether
(20)
the cells
Furthermore than
the
ergosterol target
of fast
of growing
10m3M sodium
azide
loss
by
type
that
of effect
on the selective
with
the wild
by the fact
ammonium source. lack
nystatin
the primary
inhibitor
a si-
of sensitivity
of erg 1 mutant is
for
on choleste-
against since
anf
integration
of sterols.
is a more efficient
cells.
with
(19),
a com-
induces
difference
to the polyene,
growth
for
to the structural This
of
sterol
20 min.)
nystatin
of cholesterol
a better
in yeast
by nystatin,
in agreement
as well
that
of a-AIBA
or in cells
cellular
found
have been grown
4A).
effect
cholesterol
since
of slow
(fig.
same sensitivity
promotes
which
points
life
that
exit
as after
were
(half
The fact
cells
membrane
of the
the possibility action,
rapidly
on ergosterol,
the
cholesterol
nystatin
from
by a protective
on ergosterol,
cell
kinetics
star-
as reflec-
of the down-hill as well
up-
sterol
of the cells
sterols
exit
by the cell.
the cell
presents
both
similar the
of a-AIBA
on the outside
are
(10)
exit
be explained
strain
sine
released
those
sterols
fixation
4C),
not
leaves
The kinetics for
that
which
lower
than
similar
significant
cytosine
gnificantly to1
is
rates.
of cytosine
and after
physiology
effect
or a-aminoisobutyric
velocities
cholesterol
to the general
of approxi-
has no stricking
in initial
on ergosterol,
are very
the replacement
such as cytosine
observed
in growth
and a-AIBA
that
by cholesterol
of compounds
grown
cytosine
the above data
ergosterol
1) in cells
ted by the
pound
from
there
Acceleration on the
exit
of of cyto-
of metabolites
and yeast of liposomes
(fig.
indu-
mitochondria (6)
point
to
Vol. 71, No. 2, 1976
BIOCHEMICAL
an important
role
experiments,
as well
different admit
of sterols
that
a very
properties content
in permeation
as the
conclusion.
of completely
data
small
amount
reported
on pig
be responsible blocked
for
mutants
is
would
two sets
sufficient
experiments, the basic
However
erythrocytes
these
of sterol
In our
RESEARCH COMMUNICATIONS
and diffusion.
One way to conciliate
on the membrane. would
AND BIOPHYSICAL
membrane
permit
probing
(19)
point
to a
of data
would
be to
to endow
then,
our present
its
the residual properties.
of this
specific ergosterol
The isolation
hypothesis.
ACKNOWLEDGEMENTS We are ssions and to was supported Scientifique
grateful to Prof. F. Lacroute for helpful and stimulating discuB. Winsor for help in editing this manuscript. This investigation by grant no 75.7.1337 from the DClCgation G&-&ale 1 la Recherche et Technique and by the Fondation 1 la Recherche Mbdicale. REFERENCES
1. 2. 3. 4.
5. 6.
7. 8.
Longley R.P., A.H. Rose and B.A. Knights (1968) Biochem. J. 108, 401-412. Mopurgo G., G. Serlupi-Crescenzi, G. Tecce, F. Valente and D. Venettacci (1964) Nature (London) g, 897-899. Butler K.W., I.C.R. Smith and H. Schneider (1970) Biochim. Biophys. Acta, 219, 514-517. Boggs J.M. and J.C. Hsia (1972) Biochim. Biophys. Acta, 290, 32-42. Finkelstein A. and A. Cass (1967) Nature, 216, 717-718. and L.L.M. Van Deenen (1972) Biochim. Biophys. Demel R.A., K.R. Bruckdorfer Acta, 255, 321-330. Papahadjopoulos D. and J.C. Watkins (1967) Biochim. Biophys. Acta, x, 639-652. Vanderkooi J.M. and A. Martonosi (1971) Arch. Biochem. Biophys. 147, 632645.
9. IO.
De Kruyff B., R.A. Demel and L.L.M. Acta, 255, 331-347. Cobon G.S. and J.M. Haslam (1973)
Van Deenen Biochem.
(1972)
Biophys.
Biochim. Res.
Biophys.
Commun. 2,
320-
325.
11.
Karst
F. and F. Lacroute
(1974)
Biochem.
Biophys.
Res.
Conuaun. 2,
370-
376.
12. 13. 14.
Chevallier M.R., R. Jund and F. Lacroute (1975) J. Bacterial. 122, 641. Mortimer R.K. and D.G. Hawthorne (1966) Genetics, 2, 165-178. Karst F. and F. Lacroute (1973) Biochem. Biophys. Res. Commun. 52,
629741-
747.
15.
Benveniste
P.,
M.J.E.
Hewlins
et B. Fritig
(1969)
Eur.
J. Biochem.
2, 526-
536.
16. 17. 18.
19. 20.
Kotyk A. and L. Rihova (1972) Biochim. Biophys. Acta, 288, 380-389. Kinsky S.C. (1971) Antibiotics, 1, 122-141, Springer, New-York. Bard M. (1972) J. Bacterial. 111, 649-657. Biochim. Biophys. Acta, 226, 726Deuticke J. and C.H.R. ZEllner7972) 731. Rottem S., V.P. Cirillo, B. De Kruyff, M. Shinitzky and S. Razin (1973) Biochim. Biophys. Acta, 323, 509-519.
543
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
STEROL REPLACEMENT IN SACCRAROMYCES CEREVISIAE. EFFECT ON CELLULAR PERMEABILITY Francis Laboratoire
Received
AND SENSITIVITY
EARST and Richard
TO NYSTATIN.
JUND
de G6n6tique Physiologique, I.B.M.C. du C.N.R.S. 15, rue R. Descartes, 67000 Strasbourg, France
March
26,1976 SUMMARY
Hore than 80% of the cellular ergosterol can be replaced by cholesterol in a sterol requiring mutant strain of Saccharo~ces cerevisiae. The effect of this replacement, as well as the effect of sterol starvation on the uptake and exit of cytosine and o-aminoisobutyric acid (wAIBA) was studied in an attempt to elucidate the role of sterols in cellular permeability. Neither the exit of cytosine nor the exit of a-AIBA was affected by changes in the sterol content of the cell. Cells grown on cholesterol or on ergosterol had very similar rates of cytosine uptake, but a lower rate was found for sterol-starved cells. This difference may be a consequence of the cellular growth rate. However, nystatin induces a much slower exit of cr-AIBA in cells grown on cholesterol than in cells grown on ergosterol. This strongly suggests that a change in membrane structure has taken place.
Ergosterol cerevisiae rol
is
cells,
is present
sented study
in
to show that of the
effect
the predominant
comprising
generally
the yeast
cytoplasmic
this
is
also
membranes effect
well
cations
(8)
is also
thought
plasma
cells
of membrane ty of this
Copyright All rights
is
established
lipids
(9).
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
the membrane state
order
(6).
present
cholesterol considerably
prevents
pre-
(2).
membrane
The
systems
and rigidity
(3, 4).
phospholipid
(5),
: this
anions
in plasma
Experiments
(6,
7),
membranes
performed
on myco-
the crystallisation
the non electrolyte
Cobon and Haslam have 535
has been
of cholesterol of water
that
Ergoste-
synthesis
of artificial
incorporation
permeability.
pool.
of model
increase
Cholesterol
sterol
Saccharomyces
and evidence
the permeability
their
and diminishes
sterols
after
for
demonstrated
microorganism
for
growing
total
(1)
the permeability
and non electrolytes
have
membrane
required
considerably
to influence
of the
on the physical
that
demonstrated
in aerobically
90%
has shown that
diminishes
has been
sterol
of sterols
made of phospholipids It
sterol
demonstrated
permeabilithat
changes
BIOCHEMICAL
Vol. 71, No. 2, 1976
in ergosterol tion the
levels
temperature sterol
in yeast
produce
of mitochondrial
concentration
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
a concomitant
ATPase
(IO).
in the mitochondria
change
An inverse and the
in the phase relationship
transibetween
transition
temperature
the sterol
requiring
was established. The present yeast
communication
mutant
erg
ly the natural sterol
starvation
acid
were
test
structural
rent
sterols.
reports
1. In this yeast
sterol
on cellular
investigated. differences
data
strain,
obtained
cholesterol
ergosterol.
antibiotic
of the cellular
MATERIAL
was shown
The effects
permeability
The polyene
from
of this
to cytosine nystatin membrane
to replace
efficient-
replacement
and of
and a-aminoisobutyric was used as a probe in cells
grown
to
on diffe-
AND METHODS
Yeast strains and genetic analysis. The mutant strain FK erg 1 (ATCC 28382) and the parent strain FL 100 (ATCC 28383) as well as the strain RJ 16 sp2 carrying the fcg 2-3 (permease less) and j%g 1-l (cytosine desaminase less) mutations are isogenic and have been previously described (II, 12). The genetic analyses were performed according to the methods of Mortimer and Hawthorne (13) 0 Media and culture methods. Yeast nitrogen base (Difco) 6.7 g/l, supplemented with 20 g/l glucose was the basic minimal medium. The sterols, 1.5 mg/l, were dispersed by 0.25 g/l Tergitol np40 (Sigma). Sterol analysis. The cells growing in exponential phase were harvested and washed with distilled water containing 0.25 g/l Tergitol np40. The non saponifiable lipids were extracted and separated on silica gel G plates as previgusly described (14). The sterol fraction was acetylated by using radioactive ) Cl acetic anhydride (15). Ergosterol and cholesterol acetates were separated on silica gel G plates impregnated with silver nitrate, developed overnight in a cyclohexane-benzene (3-2 V/V) solvent system. The respective amounts of radioactivity of different spots were counted in presence of 10 ml of a mixture of toluene diphenyloxazole 5 g/l which allowed the measurement of the absolute amount of each compound. Uptake and efflux measurement. All experiments were carried out at 30°C on exponentially growing cells in minimal medium. Uptake and efflux of cytosine were measured as previously described (12). For the measurement of the efflux of ci-aminoisobut ric acid (a-AIRA), the cells were incubated for 45 min. in the presence of v I4 C I labelled substrate, then filtered through a membrane filter (1.2 u pore size), washed once with fresh medium and resuspended in fresh medium. At different times during the experiment, I ml samples were removed, filtered and washed with 2 x 10 ml ice cold water. The filters were then dried and their radioactivity counted in toluene plus 5 g/l diphenyloxasole (Intertechnique liquid scintillation spectrometer SL 30).
536
Vol. 71, No. 2, 1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
RESULTS Sterol
analysis. Whereas
weight),
the sterol
0.15X,
whether
cholesterol rol
the sterol content
culture
(vs the amount
data strain,
hypothesis strain
in minimal
a leaky
(see Material
with
cholesterol
acetyl
ergosterol).
Active
transport
of cytosine
A recombinant cytosine
(inactivation
velocities
of cytosine
cholesterol ponding ble
reciprocal
were
lowered
values 0.53
rates
plots
for
and 0.15
were
l/1000
strain
1 fey l-l
or after time).
not
l
strain
the mutant
of cytosine
uptake
were
in the recombinant
strain
erg
strain
l-l
537
grown
latter
with
4.9 mg/l
of
after
were
found
in
the metabolism
I-lmutation).
strain
after
of
Initial growth
starvation
on
(corres-
as calculated
from
the dou-
different,
but
the Vmax
type
2 mM for
sterol
at different
This
fey
on cholesterol after
1 mutant
was associated
of sterol
to the wild
cultured
measured 1 fou
in this
are
This
and separated
to bypass
by the
30 set-‘)
8
l-l.
significatively
in comparison
the mutant
extracted
18 hours
after
The mutant
supplemented
The Km values
1) were
concentration
experiment.
was used
measured
were
strain
to ergosterol.
1 fcu
ergoste-
in the erg
of the counts
erg
uptake
(fig.
for
than
or
cholesterol.
114C( radioactivity
desaminase
in the mutant
and 0.33
The sterols
of cytosine
the doubling
(mM internal
pectively,
erg
or on ergosterol, to twice
medium
in the
strain
with
of following
than
14% residual
occured
in minimal
(less
of
greater (1 mg/l)
in the mutant
converted
and Methods). only
was never ergosterol
of ergosterol
on the basis
(40 mCi/mM).
with
supplemented
was partially
8 generations
acetylation acetyl
strain
a mean value
medium
FL 100 is 0.9% (w/dry
strain
was present
synthesis
was eliminated
cholesterol
type
1 mutant
hand,
sterols)
cholesterol
was grown
114Cl
of total
that
or that
of the erg
On the other
of growth
suggests
of the wild
medium was supplemented
(1 mg/l).
generations
content
strain.
The observed
the wild
type
and ergosterol starvation.
temperatures
on cholesterol
strain, res-
Initial from
3” to 36”
or ergosterol.
The
Vol. 71, No. 2,1976
BlOCHEMlCkL
AND BIOPHYSICAL RESEARCH COhWJNlCATlONS
Figure I. Double reciprocal plots of strain erg 1 fey 1-l as a function of tration in cells is expressed in mM of - - -culture medium supplemented with time 240 min.) ;-A- -medium supplemented doubling time 195 min.) ;- -+I8 hours time 400 min.) ;--wild type strain
initial velocities of cytosine uptake in external concentration. Cytosine concencell water per 30 sec. incubation. 2 ug/ml ergosterol (Vmax 0.328, doubling with 2 ug/ml cholesterol (Wax 0.535, sterol starvation (Vmax 0.148, doubling (Vmax 1.57, doubling time 140 min.).
0.50 t
plots of initial velocity Figure 2. Arrhenius of temperature. I ml samples of the erg 1 fq or ergosterol (M) are preincubated m-B), peratures before uptake measurement.
538
of cytosine uptake as a function 1-I cells grown on cholesterol for 5 min. at the different tem-
Vol. 71, No. 2, 1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
corresponding
plots
2) show biphasic
transition Efflux
Arrhenius temperature
of 114Cl
enters
the yeast
concentrated sine
cell
for sterol were
(fey
(12).
testing
by active
2-3)
cellular
out
is
transport
(fey strain,
for
on two recombinant
l-l). cytosine wAIBA in erg
sterols. strains
acid
the same
in minukr
(cPAIBA). compound,
Extracellular
(16).
cerevisiae
enters
and leaves were
grown
The assays
having
the cytocytosi-
the cell
chosen
on cholesterol,
ergo-
cytosine
permeability
the mutations
.
by free
therefore
for
carrying
is also
desaminase-less
and cytosine 1 cells
which
cytosine cells
In a cytosine
I time
with
a non metabolized
in Saccharomycea
permeability starved
a-aminoisobutyric
(wAIBA)
The two compounds,
and in cells carried
acid
mutation
ne permease-less diffusion
and (14Cl
and accumulated
desaminase-less
kinetics
at 23".
cytosine
a-aminoisobutyric
(fig.
. 10
.
erg
* 20 time
. in
1
* 50
*
* 40
min.
Figure 3. The cells are incubated for 45 min. in minimal medium plus 0.50 mM of [14C] cytosine, then washed and transfered in fresh medium without cytosine, at time zero. The exit is measured as described in Methods. Cytosine efflux from strain J@C~ l-l fey 2-3 grown without sterol (M), 3: and from strain erg 1 fey 1-l fcu 2-3 grown on cholesterol (Q-U), ergosterol (*-++), and after a 15 hours period of sterol starvation (a+-). 3B : Cytosine efflux from strain erg 1 fey 1-l fCu 2-3 in the presence of nysztin. Nystatin is added at time zero at a final concentration of 10 rig/ml. - Cells grown on cholesterol ; efflux measured in presence w) and absence (W) of nystatin. - Cells grown on ergosterol ; efflux measured in presence (W) and absence (U-0) of nystatin.
539
BIOCHEMICAL
Vol. 71, No. 2,1976
fey
1-l
fey
strain
erg
constant
fey
1
l-l
(k min.
lesterol, tial
or fey 1-l
2-3,
fey
than
1 fey l-l
strain
fey
l-l
Effect
fey
2-3
strain
of nystatin
and other
The study
cells
are
nystatin
cursors
(18).
nystatin
if
The effect a-AIBA
(fig.
of addition
effect 3B).
In contrast, type
mented
ergosterol
it
with
strain
in the presence
was greatly
decreased
effective have
indicated
resistant
to nystatin
ergosterol
its
exit
of with
by cholesterol
on erg
accumulate
and on the wild
growing cells.
in the cytosine
fast
of a-AIBA
in minimal
or cholesterol of nystatin
in erg
the cells
540
were
4B).
to (14). and
in the presence
or not
cultured
exit
of
had no signi-
permease-less
A rapid
cultured
pre-
of cytosine
was induced
1 cells
these
medium
Nystatin
medium whether (fig.
sterols
ergosterol
culture
efflux
diffusion exit
step
are more resistant
on the
type
of
crucial
to membrane
in the
1 cells
the
has shown that
mutants
(10 mg/l)
the growth
binding
and thus
requiring
replaced
when
in
supplemented
that
mutants
grown
observed
in
FL 100 and the
in inhibiting
is
a very
exit
no significant
substance
free
to that strain
of this
investigated
on cytosine
ini-
and a-AIBA.
studies
of nystatin
or ergosterol,
in the wild
occured
is
the
in cholesterol
medium had been
of cytosine
ergosterol
ergosterol
cholesterol ficant
Moreover,
similar
of cho-
Moreover
of cytosine
a-AIBA,
growth
same rate
higher
type
1’4Cl
with
the
in
4).
to synthesize
was therefore
the
Several
of yeast
very
exit
3A).
The kinetics
cells.
cytosine
in the presence
(fig.
was always
is highly
action
was grown
When the wild
whether
that
kinetics
medium
with
(fig.
fungi.
unable
3A).
on the exit
in the antifungal (17).
strain
therefore
preloaded
or cholesterol
order
depleted
are
(fig.
were
The polyene yeasts
sterol
fey 2-3
was detected,
ergosterol
the
grown
erg
was observed
first
in ergosterol
strain
label
follows
concentration
cells
114Cl
It
cytosine
grown
1 mutant
2-3.
whether
or in
intracellular
erg
2-3
= 0.040)
ergosterol
fey
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cells
by the polyene it
was supple-
of a-AIBA on ergosterol,
on cholesterol
(fig,
also but 4A).
5
A
10
20
in
4.
ini”.
40
60
a-aminoisobutyric
50
(0 tin*
20 in
min.
30
40
;!, c 10
bdium ozida , lo-)M
tint*
20
fered in fresh medium without described in Methods. 4k: Exit measured in erg 1 cells cultured on ergosterol (U+), and cholesterol (-M). Nystatin (10 rig/ml) is added at time 22 min. 4B : Exit measured in wild type strain after growth in minimal medium supplemented with ergosterol (U) and cholesterol CO--@. Nystatin is added at time 12 min. 4c : Exit measured from wild type strain. 511s grown in standard medium @-@ ; cells grown on proline 4 mg/ml as sole ammonium source &+I ; cells grown in standard medium and pretreated with IO-3M sodium azide during 19 min. before addition of nystatin (0-O).
in
i:, min.
nystitim
(a-AIBA) exit induced by nystatin, from strain incubated for 45 min. in minimal medium plus acid, The cells are then washed and transa-AIBA (time zero), and the exit is measured as
acid
FL 100 and er 1. The cells are 0.32 mM of [l B C] CX-aminoisobutyric
Figure
Time
30
10i
3
30
0
40
BIOCHEMICAL
Vol. 71, No. 2,1976
AND BIOPHYSICAL RESEARCH COMMUNlCATlONS
DISCUSSION It mately
can be concluded 80%
of the cellular
on the cellular acid
(a-AIBA).
take
(fig.
vation
permeability The differences
are more likely
linked
differences
starvation.
It
like
a-AIBA
which
is
from
of these cannot
grown
in
always grown
fact
that
rules
out
cells
than
is no release
a-AIBA
exit is
nystatin
growing
grown
ced by nystatin
(17).
membrane
or in absence
cellular
energetics
This
proline
other
Experiments
as the in vitro
pretreated
as sole
in contrast with
was demonstrated
cells
to its
data
with
mycoplasma
measurement
of diffusion
542
whether
(20)
the cells
Furthermore than
the
ergosterol target
of fast
of growing
10m3M sodium
azide
loss
by
type
that
of effect
on the selective
with
the wild
by the fact
ammonium source. lack
nystatin
the primary
inhibitor
a si-
of sensitivity
of erg 1 mutant is
for
on choleste-
against since
anf
integration
of sterols.
is a more efficient
cells.
with
(19),
a com-
induces
difference
to the polyene,
growth
for
to the structural This
of
sterol
20 min.)
nystatin
of cholesterol
a better
in yeast
by nystatin,
in agreement
as well
that
of a-AIBA
or in cells
cellular
found
have been grown
4A).
effect
cholesterol
since
of slow
(fig.
same sensitivity
promotes
which
points
life
that
exit
as after
were
(half
The fact
cells
membrane
of the
the possibility action,
rapidly
on ergosterol,
the
cholesterol
nystatin
from
by a protective
on ergosterol,
cell
kinetics
star-
as reflec-
of the down-hill as well
up-
sterol
of the cells
sterols
exit
by the cell.
the cell
presents
both
similar the
of a-AIBA
on the outside
are
(10)
exit
be explained
strain
sine
released
those
sterols
fixation
4C),
not
leaves
The kinetics for
that
which
lower
than
similar
significant
cytosine
gnificantly to1
is
rates.
of cytosine
and after
physiology
effect
or a-aminoisobutyric
velocities
cholesterol
to the general
of approxi-
has no stricking
in initial
on ergosterol,
are very
the replacement
such as cytosine
observed
in growth
and a-AIBA
that
by cholesterol
of compounds
grown
cytosine
the above data
ergosterol
1) in cells
ted by the
pound
from
there
Acceleration on the
exit
of of cyto-
of metabolites
and yeast of liposomes
(fig.
indu-
mitochondria (6)
point
to
Vol. 71, No. 2, 1976
BIOCHEMICAL
an important
role
experiments,
as well
different admit
of sterols
that
a very
properties content
in permeation
as the
conclusion.
of completely
data
small
amount
reported
on pig
be responsible blocked
for
mutants
is
would
two sets
sufficient
experiments, the basic
However
erythrocytes
these
of sterol
In our
RESEARCH COMMUNICATIONS
and diffusion.
One way to conciliate
on the membrane. would
AND BIOPHYSICAL
membrane
permit
probing
(19)
point
to a
of data
would
be to
to endow
then,
our present
its
the residual properties.
of this
specific ergosterol
The isolation
hypothesis.
ACKNOWLEDGEMENTS We are ssions and to was supported Scientifique
grateful to Prof. F. Lacroute for helpful and stimulating discuB. Winsor for help in editing this manuscript. This investigation by grant no 75.7.1337 from the DClCgation G&-&ale 1 la Recherche et Technique and by the Fondation 1 la Recherche Mbdicale. REFERENCES
1. 2. 3. 4.
5. 6.
7. 8.
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543
