BIOCHEMICAL
Vcl. 83, No. 4, 1978
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Pages 1319-1324
August 29,1978
Site
of Action
in a-Mating
of Mating Type Cell
Takaharu
Tanaka
Central
Received
July
of Saccharomyces
and Hiroshi
Reseach
Mishima-Gun,
Factor
Kita
Institute,
Osaka,
cerevisiae
Suntory
Ltd.,
Shimamoto-Cho,
Japan
5,1978
Summary : The process of the entry of FITC-conjugated mating factor into a-mating type cells of Saccharom ces cerevisiae and its concentration into The nucleus were observe But, w end-mating type cells or diploid cells of S. cerevisiae were incubatea with the FITC-conjugated mating factor, it5- adsorption to the cell surface of the test organisms and its incorporation into the cell did not occur. The peptides formed by the cleavage of mating factor bycc-mating type cells of 2. cerevisiae were not adsorbed onto a-mating type cells. Introduction
:
by&-mating
type
indispensable cells
of
Mating cells
to the this
factor of
The factor
function
Bucking-Throm
of
(2)
The mating
the
and Hartwell
factor
is
only
thereby,
mating
of yeast (1)
between&-
works
and,
hormone
cerevisiae
of mating
DNA synthesis
As to the
a peptide
Saccharomyces
process
yeast.
by inhibiting
is
chemically
cell
a hypothesis
fluorescent
group,
the a-mating
type
adsorption
of
followed
by its
in the
nucleus
Abbreviation
FITC*,
of
cell
With the
the
entry of
used
the
into
following
the
mating
specificity
of
mating the
a-mating
: * FITC,
type
cell
division.
was proposed
amino
Fluorescein
factor
by
the
onto
the a-mating
are
finally,
with
factor
indicating
and,
cell,
mating
sequence, and was
coupled
Results factor
cytoplasm type
acid
(4,5,6)
was investigated.
FITC-conjugated
type
(3).
a peptide
(7).
is
on the a-mating
Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr synthesized
that
and a-mating
prevents
facor,
produced
for specific type
cell
concentration
described
in
this
paper.
Isothiocyanate
0006-291X/78/0834-1319$01.00/0 1319
Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
VoL83,No.
4,1978
BIOCHEMICAL
Materials
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
and Methods
Biochemical Materials ___ S. cerevisiae X-2180 1B (CL-mating type cell ) and X-2180 1A ( a-mating type cell ) and their diploid cell precursor from which two haploid cells were derived, were cultured in Burkholder's synthetic medium at 30°C with gentle stirring. Cell growth was followed by the increase in optical density at 550 nm. Coupling of FITC to Peptides ~ The mating factor and its two degradation products, Peptide A ( Trp-His-Trp-Leu-Gin-Leu ) and Peptide B ( Lys-Pro-Gly-Gin-Pro-Met-Tyr ), were treated with FXTC according to the method described by Coons (8). A peptide ( 0.5 umol ) was treated with 10 umol of FITC in 0.5 ml of 0.05 M sodium carbonate-bicarbonate buffer, pH 9.2. After incubation at 37°C for 60 min., the reaction mixture was chromatographed on a Sephadex LH 20 column ( 0.9 X 80 cm ) using 80 % methanol : acetic acid ( 10 : 1 ) as solvent. Fractions containing a FITC-conjugated peptide were lyophilized and the residue was dissolved in 1 ml of distilled water. Treatment of Yeast Cells with FITC-conjugated Peptide ~ Growing cells of a- and+mating type or of their,diploid were suspended in fresh Burkholder's medium to give 10' cells/ml. The suspensions were incubated at 30°C in the presence of the indicated amount of FITC-conjugated peptide. After 2 and 8 hours' incubation, 5 ul aliquots were withdrawn and were transferred onto glass slide. After drying under the stream of hot air, the specimens were covered with a mixture of glycerol : 5 M sodium bicarbonate ( 9 : 1 ) and were subjected to microscopic examination using a fluorescent microscope ( Olympus Ltd., Tokyo ). Chemicals ~ Sephadex LH 20 ( Pharmacia Fine Chemicals and FITC ( Division of Becton, Dickinson and Company ) were from Nakarai Chemicals Ltd., Kyoto. All the other reagents were analytical grade.
a
AB ) purchased used
Results Inhibition
of Growth
Factor type
cell
effect
factor.
to
have
When the
FITC-conjugated the
on&-mating
lapse
of
lasted
over
mating
factor
the
following
20 hours
in the than
cell
from
cells
factor,
3 hours
and more
inhibits
Fig.
of the
the
with
cell
of
20 wg/ml
has no
of
natural
treated
The growth
1 ug/ml
1320
were
it
mating
completely
addition.
40 hours
of a-mating but
as the
type
ceased
of
cell,
diploid
activity
presence
growth
Mating
1, FITC-conjugated
of -a-mating growth
the
and the
same biological
growing
mating
by FITC-conjugated
changes
type
obvious
Cell
factor
morphological
As is
proved
Type
mating
and induces
cerevisiae.
factor
a-Mating
Natural
biological 5.
of
only
with after
inhibition
FITC-conjugated of the
factor.
BIOCHEMICAL
Vol. 83, No. 4, 1978
AND BIOPHYSICAL
IO
20
30
RESEARCH COMMUNICATIONS
40
TiMEthour)
Fig.
1.
Effect
of FITC-conjugated
growth
of a-mating
At the
time
mating
factor
hours
cells.
C)--
- - - 0
cell
growth
of
the yeast,
a-mating cell
wh ich
-•
of
Th is
type
cell
:
1 ug/ml
:
20 vg/ml
was equivalent
observation
is
control
type
the
suggested
arrested
in its
the
growth
on the
cerevisiae.
arrow,
FITC-conjugated
to suspension
:
before
the a-mating
of 2.
by the
required
factor
at
of a-mating
complete
inhibition
of
to the
generation
time
possibility
that
the
Gl phase
the
of
the
cycle.
Binding
of
FITC-conjugated
of S. cerevisiae
-The
by a-mating
cells
the
were
cell
was added
0-o The three
type
indicated
type
l
mating
type
bud emergence
2 hours' and Peptide cell
at all.
and diploid ( Fig,
the
not
cell
of 2.
of the
at 3O'C.
and was bound
2A ).
adsorbed
In contrast
to the
to a-mating
to grow
mating
Type Cells
cell type
failed even
to the
factor.
1321
cell
mating surface
surface factor,
of a-mating
the
presence
factor
prevented
cell,%-mating
to adsorb in the
mating
The factor
The degraded
cerevisiae
2B ) and continued
concentrations
cell
to a-Mating
of FITC-conjugated
was followed
( Fig.
B, were
Factor
uptake
of
incubation
Mating
type mating of
high
within Peptide type cell factor
A
BIOCHEMICAL
Vol. 83, No. 4, 1978
Fig.
2.
Binding type a-
of cells
AND BIOPHYSICAL
FITC-conjugated of 5.
of
factor
on a- and+mating
cerevisiae.
( A ) and&mating
2 ug/ml
mating
RESEARCH COMMUNICATIONS
type
cells
FITC-conjugated
( B ) were
mating
factor
treated
at
with
30°C for
2 hours.
x 400.
Translocation Type
of
Cell
a-mating
to type
morphological factor into
the
to the
cytoplasm was found
( Fig.
3B ).
the mating
cell nuclei
to function surface of
the
in the
cell
cell
surface
in the
produced
Cell
duration
of
mating
After of
Surface
of
the
treatment
factor
out
8 hours'
and the from
the
a-mating
type
shown here
suggest
strongly
type
to function
type cells
cells when it
after
cells.
1322
its
of the mating
membrane
incubation,
the
bye-mating
a-Mating
increased,
distinctive
diffused
nuclei
in a-mating
and continues the
the
became
3A ).
The observations factor
from
FITC-conjugated
( Fig. only
Factor
-As
with
changes
factor
starts
Mating
Nuclei
cell
adsorbed the
the
the
mating
cells that
of LO cerevisiae is adsorbed translocation
to the to the
Vol. 83, No. 4, 1978
Fig.
3
8lOCHEMlCALAND
Translocation cell
of
surface
BIOPHYSICALRESEARCH
FITC-conjugated
mating
to the
nuclei
in a-mating
cells
were
incubated
5 hours
( A,)
COMMUNICATIONS
factor type
from cells
the of
S. cerevisiae. a-mating
type
mating
factor
under
the
for
conditions
with
FITC-conjugated
and 8 hours
described
for
Fig.
( B ),
respectively,
2.
Discussi'on The mating is
factor
cerevisiae
to inhibit
DNA synthesis
in a-mating
type
(2).
few reports
which
a-
reported
of 2.
and&-mating
presented bound diploid
(9)
peptides
could type
in this
only
to
cells
( tridecapeptide &-
cells
and aare
paper
from
type they
( unpublished no longer
able
and not
derived.
into data
factor. that
) mating
to be adsorbed
1323
the
changes there
between
mating
by&-mating
factor type
to
cells. the a-mating
is
cells
factor
and hexa-peptides type
are
The data
The mating
a hepta-
cells
morphological
in behavior
clearly
are
type
of much speculation,
the mating
cell,
degraded
induce
difference
demonstrate
which ) is
the
towards
the a-mating
by&-mating
and to
In spite
explain
cells
produced
by both
These type
two cells.
or
Vol. 83, No. 4, 1978
The mating cell
is
then
with
the
of have
the
factor
different
a-mating
type
To explain further
adsorbed
to
to
the
translocated It
mating
from
the
factor
factor.
of excess moved
BIOCHEMICAL
is
factor
the
cell
surface
to the
cell
biological
the
RESEARCH COMMUNICATIONS
cell
the a-mating
nucleus
interesting in
AND BIOPHYSICAL
surface during
that,
surrounding
to the surface function
of
prolonged
in spite
of
medium,
the
and no further
followed.
The mating
according
to
its
incubation the
presence
mating
nucleus
type
factor
adsorption factor
could
localization
cell. the
investigations
function are
of
the
mating
in
progress.
factor
more
precisely,
References Levi, J.D. (1956) Nature 177, 753. Bucking-Throm, E., Duntze,X, Hartwell, L.H. and Manney, T.R. (1973) Exp. Cell Res. 76, 99. Hartwell, L.H. (1974) Bact. Revs. 38, 164. Stbtzler, D., Kiltz, H. and Duntze, W, (1976) Eur. J. Biochem. 35, 357. Tanaka, T., Kita, H. and Narita, K. (1977) Proc. Japan Acad. 53, 67. Fnaka, T., Kita, H., Murakami, T. and Narita, K. (1977) J. Biochem ( Japan ) 82, 1681. Masui, Y., Chino, N.,Sakakibara, S., Tanaka, T., Murakami, T. and Kita, H. (1977) Biochem. Biophys. Res. Comm. 78, 534. Coons, A.H. and Kaplan, M.H. (1950) J. Exp. Med. i5T, 1. Tanaka, T. and Kita, H. (1977) J. Biochem. ( Japan) 82-, 1689.
1324
in
Vcl. 83, No. 4, 1978
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Pages 1319-1324
August 29,1978
Site
of Action
in a-Mating
of Mating Type Cell
Takaharu
Tanaka
Central
Received
July
of Saccharomyces
and Hiroshi
Reseach
Mishima-Gun,
Factor
Kita
Institute,
Osaka,
cerevisiae
Suntory
Ltd.,
Shimamoto-Cho,
Japan
5,1978
Summary : The process of the entry of FITC-conjugated mating factor into a-mating type cells of Saccharom ces cerevisiae and its concentration into The nucleus were observe But, w end-mating type cells or diploid cells of S. cerevisiae were incubatea with the FITC-conjugated mating factor, it5- adsorption to the cell surface of the test organisms and its incorporation into the cell did not occur. The peptides formed by the cleavage of mating factor bycc-mating type cells of 2. cerevisiae were not adsorbed onto a-mating type cells. Introduction
:
by&-mating
type
indispensable cells
of
Mating cells
to the this
factor of
The factor
function
Bucking-Throm
of
(2)
The mating
the
and Hartwell
factor
is
only
thereby,
mating
of yeast (1)
between&-
works
and,
hormone
cerevisiae
of mating
DNA synthesis
As to the
a peptide
Saccharomyces
process
yeast.
by inhibiting
is
chemically
cell
a hypothesis
fluorescent
group,
the a-mating
type
adsorption
of
followed
by its
in the
nucleus
Abbreviation
FITC*,
of
cell
With the
the
entry of
used
the
into
following
the
mating
specificity
of
mating the
a-mating
: * FITC,
type
cell
division.
was proposed
amino
Fluorescein
factor
by
the
onto
the a-mating
are
finally,
with
factor
indicating
and,
cell,
mating
sequence, and was
coupled
Results factor
cytoplasm type
acid
(4,5,6)
was investigated.
FITC-conjugated
type
(3).
a peptide
(7).
is
on the a-mating
Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr synthesized
that
and a-mating
prevents
facor,
produced
for specific type
cell
concentration
described
in
this
paper.
Isothiocyanate
0006-291X/78/0834-1319$01.00/0 1319
Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
VoL83,No.
4,1978
BIOCHEMICAL
Materials
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
and Methods
Biochemical Materials ___ S. cerevisiae X-2180 1B (CL-mating type cell ) and X-2180 1A ( a-mating type cell ) and their diploid cell precursor from which two haploid cells were derived, were cultured in Burkholder's synthetic medium at 30°C with gentle stirring. Cell growth was followed by the increase in optical density at 550 nm. Coupling of FITC to Peptides ~ The mating factor and its two degradation products, Peptide A ( Trp-His-Trp-Leu-Gin-Leu ) and Peptide B ( Lys-Pro-Gly-Gin-Pro-Met-Tyr ), were treated with FXTC according to the method described by Coons (8). A peptide ( 0.5 umol ) was treated with 10 umol of FITC in 0.5 ml of 0.05 M sodium carbonate-bicarbonate buffer, pH 9.2. After incubation at 37°C for 60 min., the reaction mixture was chromatographed on a Sephadex LH 20 column ( 0.9 X 80 cm ) using 80 % methanol : acetic acid ( 10 : 1 ) as solvent. Fractions containing a FITC-conjugated peptide were lyophilized and the residue was dissolved in 1 ml of distilled water. Treatment of Yeast Cells with FITC-conjugated Peptide ~ Growing cells of a- and+mating type or of their,diploid were suspended in fresh Burkholder's medium to give 10' cells/ml. The suspensions were incubated at 30°C in the presence of the indicated amount of FITC-conjugated peptide. After 2 and 8 hours' incubation, 5 ul aliquots were withdrawn and were transferred onto glass slide. After drying under the stream of hot air, the specimens were covered with a mixture of glycerol : 5 M sodium bicarbonate ( 9 : 1 ) and were subjected to microscopic examination using a fluorescent microscope ( Olympus Ltd., Tokyo ). Chemicals ~ Sephadex LH 20 ( Pharmacia Fine Chemicals and FITC ( Division of Becton, Dickinson and Company ) were from Nakarai Chemicals Ltd., Kyoto. All the other reagents were analytical grade.
a
AB ) purchased used
Results Inhibition
of Growth
Factor type
cell
effect
factor.
to
have
When the
FITC-conjugated the
on&-mating
lapse
of
lasted
over
mating
factor
the
following
20 hours
in the than
cell
from
cells
factor,
3 hours
and more
inhibits
Fig.
of the
the
with
cell
of
20 wg/ml
has no
of
natural
treated
The growth
1 ug/ml
1320
were
it
mating
completely
addition.
40 hours
of a-mating but
as the
type
ceased
of
cell,
diploid
activity
presence
growth
Mating
1, FITC-conjugated
of -a-mating growth
the
and the
same biological
growing
mating
by FITC-conjugated
changes
type
obvious
Cell
factor
morphological
As is
proved
Type
mating
and induces
cerevisiae.
factor
a-Mating
Natural
biological 5.
of
only
with after
inhibition
FITC-conjugated of the
factor.
BIOCHEMICAL
Vol. 83, No. 4, 1978
AND BIOPHYSICAL
IO
20
30
RESEARCH COMMUNICATIONS
40
TiMEthour)
Fig.
1.
Effect
of FITC-conjugated
growth
of a-mating
At the
time
mating
factor
hours
cells.
C)--
- - - 0
cell
growth
of
the yeast,
a-mating cell
wh ich
-•
of
Th is
type
cell
:
1 ug/ml
:
20 vg/ml
was equivalent
observation
is
control
type
the
suggested
arrested
in its
the
growth
on the
cerevisiae.
arrow,
FITC-conjugated
to suspension
:
before
the a-mating
of 2.
by the
required
factor
at
of a-mating
complete
inhibition
of
to the
generation
time
possibility
that
the
Gl phase
the
of
the
cycle.
Binding
of
FITC-conjugated
of S. cerevisiae
-The
by a-mating
cells
the
were
cell
was added
0-o The three
type
indicated
type
l
mating
type
bud emergence
2 hours' and Peptide cell
at all.
and diploid ( Fig,
the
not
cell
of 2.
of the
at 3O'C.
and was bound
2A ).
adsorbed
In contrast
to the
to a-mating
to grow
mating
Type Cells
cell type
failed even
to the
factor.
1321
cell
mating surface
surface factor,
of a-mating
the
presence
factor
prevented
cell,%-mating
to adsorb in the
mating
The factor
The degraded
cerevisiae
2B ) and continued
concentrations
cell
to a-Mating
of FITC-conjugated
was followed
( Fig.
B, were
Factor
uptake
of
incubation
Mating
type mating of
high
within Peptide type cell factor
A
BIOCHEMICAL
Vol. 83, No. 4, 1978
Fig.
2.
Binding type a-
of cells
AND BIOPHYSICAL
FITC-conjugated of 5.
of
factor
on a- and+mating
cerevisiae.
( A ) and&mating
2 ug/ml
mating
RESEARCH COMMUNICATIONS
type
cells
FITC-conjugated
( B ) were
mating
factor
treated
at
with
30°C for
2 hours.
x 400.
Translocation Type
of
Cell
a-mating
to type
morphological factor into
the
to the
cytoplasm was found
( Fig.
3B ).
the mating
cell nuclei
to function surface of
the
in the
cell
cell
surface
in the
produced
Cell
duration
of
mating
After of
Surface
of
the
treatment
factor
out
8 hours'
and the from
the
a-mating
type
shown here
suggest
strongly
type
to function
type cells
cells when it
after
cells.
1322
its
of the mating
membrane
incubation,
the
bye-mating
a-Mating
increased,
distinctive
diffused
nuclei
in a-mating
and continues the
the
became
3A ).
The observations factor
from
FITC-conjugated
( Fig. only
Factor
-As
with
changes
factor
starts
Mating
Nuclei
cell
adsorbed the
the
the
mating
cells that
of LO cerevisiae is adsorbed translocation
to the to the
Vol. 83, No. 4, 1978
Fig.
3
8lOCHEMlCALAND
Translocation cell
of
surface
BIOPHYSICALRESEARCH
FITC-conjugated
mating
to the
nuclei
in a-mating
cells
were
incubated
5 hours
( A,)
COMMUNICATIONS
factor type
from cells
the of
S. cerevisiae. a-mating
type
mating
factor
under
the
for
conditions
with
FITC-conjugated
and 8 hours
described
for
Fig.
( B ),
respectively,
2.
Discussi'on The mating is
factor
cerevisiae
to inhibit
DNA synthesis
in a-mating
type
(2).
few reports
which
a-
reported
of 2.
and&-mating
presented bound diploid
(9)
peptides
could type
in this
only
to
cells
( tridecapeptide &-
cells
and aare
paper
from
type they
( unpublished no longer
able
and not
derived.
into data
factor. that
) mating
to be adsorbed
1323
the
changes there
between
mating
by&-mating
factor type
to
cells. the a-mating
is
cells
factor
and hexa-peptides type
are
The data
The mating
a hepta-
cells
morphological
in behavior
clearly
are
type
of much speculation,
the mating
cell,
degraded
induce
difference
demonstrate
which ) is
the
towards
the a-mating
by&-mating
and to
In spite
explain
cells
produced
by both
These type
two cells.
or
Vol. 83, No. 4, 1978
The mating cell
is
then
with
the
of have
the
factor
different
a-mating
type
To explain further
adsorbed
to
to
the
translocated It
mating
from
the
factor
factor.
of excess moved
BIOCHEMICAL
is
factor
the
cell
surface
to the
cell
biological
the
RESEARCH COMMUNICATIONS
cell
the a-mating
nucleus
interesting in
AND BIOPHYSICAL
surface during
that,
surrounding
to the surface function
of
prolonged
in spite
of
medium,
the
and no further
followed.
The mating
according
to
its
incubation the
presence
mating
nucleus
type
factor
adsorption factor
could
localization
cell. the
investigations
function are
of
the
mating
in
progress.
factor
more
precisely,
References Levi, J.D. (1956) Nature 177, 753. Bucking-Throm, E., Duntze,X, Hartwell, L.H. and Manney, T.R. (1973) Exp. Cell Res. 76, 99. Hartwell, L.H. (1974) Bact. Revs. 38, 164. Stbtzler, D., Kiltz, H. and Duntze, W, (1976) Eur. J. Biochem. 35, 357. Tanaka, T., Kita, H. and Narita, K. (1977) Proc. Japan Acad. 53, 67. Fnaka, T., Kita, H., Murakami, T. and Narita, K. (1977) J. Biochem ( Japan ) 82, 1681. Masui, Y., Chino, N.,Sakakibara, S., Tanaka, T., Murakami, T. and Kita, H. (1977) Biochem. Biophys. Res. Comm. 78, 534. Coons, A.H. and Kaplan, M.H. (1950) J. Exp. Med. i5T, 1. Tanaka, T. and Kita, H. (1977) J. Biochem. ( Japan) 82-, 1689.
1324
in
