375
Thiamin
S-12-2
Transport
in Yeast
and
Some
Aspects
of Its
Regulation A. IWASHIMA, Y. KAWASAKI, K.NOSAKA,
and H. NISHIMURA
Department of Biochemistry, Kyoto Prefectural University of Medicine, Kawaramachi Hirokoji, Kamigyo-ku, Kyoto 602 (Japan) I. Introduction Yeast Saccharomyces cerevisiae has been known to take up thiamin from the extracellular environment, although this organism can synthesize the vitamin de novo. Since Suzuoki[1] demonstrated for the first time that thiamin enters yeast cells by an active transport system in cell suspensions, several studies have been made on thiamin transport in various types of living cells[2]. Among eukaryotic cells, yeast has the most efficient transport system for thiamin, and moreover, it is available for genetic analysis. For these reasons we have been studying the thiamin transport system of S. cerevisiae from biochemical and genetic aspects. This communication describes the transport system for thiamin and its phosphate esters, and the regulation of the transport studied using mutant yeast cells. II.
General In
and
characteristics
resting
yeast
a Km
value
concentrated fold of
by
thiazole transport
of
and
thiamin for
the
moiety. system,
yeast
thiamin
analogs
Actually whereas
transport
This
by
conclusion
10,000-fold
inhibited
chemical
thiamin
is transported
0.18 ƒÊM.
approximately glucose
various
specific
of
cells
by on
the
structure
the
transport
transport
pyrimidine
system the
and
the
2, 4-dinitrophenol suggested
moiety
of alone
that the
a pH
the
taken
optimum
that uptake
[3].
thiamin is
with
observation
levels,
or
hydroxymethylpyrimidine hydroxyethylthiazole
by
extracellular cyanide
thiamin of
active
is supported
over
iodoacetate,
system an
The
the
system
molecule
than
by
the
yeast
4.5 is
is stimulated
14-
inhibitory
transport
up
of vitamin
effects is that
more of
the
thiamin
is not[4].
III. Photoaffinity labeling of yeast thiamin transport system with [3H]4-azido-2-nitrobenzoylthiamin Our previous studies demonstrated the occurrence of two thiamin-binding proteins in S. cerevisiae [5]: one is bound to plasma membrane (mTBP) which may be directly involved in the thiamine uptake, and the other is a soluble thiamin-binding protein (sTBP), a glycoprotein with a molecular weight of 140kDa in the periplasmic space. 4-Azido-2-nitrobenzoylthiamin (ANBT), a newly synthesized photoreactive thiamin derivative irreversibly inactivated both thiamin-binding activity of the plasma membranes and thiamin transport activity in yeast cells [6]. Furthermore, photoaffinity labeling of yeast plasma membranes with [3H]ANBT resulted in the specific covalent modification of a membrane component with an apparent molecular mass of 6-8 kDa [7]. On the other hand, no specific incorporation of the radioactivity was found in the plasma membranes in the cells of a thiamin transport mutant (PT-R2) isolated as a strain resistant to pyrithiamin, a strong thiamin antagonist [8]. This suggests that the small membrane component (6-8 kDa) takes part in the thiamin-binding of thiamin transport protein(s) in yeast plasma membranes, although the exact relationship of this component to the thiamin transport system will be needed to elucidate the precise mechanism of the transport process. IV. Transport of thiamin activity in yeast periplasm
phosphates
and a possible
role
for acid phosphatase
with thiamin-binding
Symposium (12)
376
We the
previously
uptake
protoplasts
[14C]
is
findings the
reported of
greatly
suggested
in
repressible and
on
isolated
was
to
in
S.
thiamin-binding
as
at
pH
5.0 and
thiamin
thus
was had
parent
yeast
cells
of
Thiamin
10482)
thiamin
of
the
thiamin
by
of of
genetic
activity
of
findings
thiamin
utilization
the
of
led
in
moiety
evidence
showing
the
uptake
of
by
pregrowth
of
that
[14C]
proteins
was
the
thiamin
is identical
to with
values
of
of
1.6
a magnitude that
the
periplasmic in
mutant
phosphates
lower acts
T-rAPase space
of
yeast
cells.
cells in
and
enzyme
that
phosphates
pho3
both (pho3)
phosphatase
Km
the
then were
mutant
conclusion
the
thiamin
periplasmic
sTBP
orders
the
encoded
exhibited
acid
indicates
phosphates thiamin
in
phosphatase
that this
2-3
to
These
and
defective
showed
which
[9].
yeast
protein
showed
were [12],
the
acid
a T-rAPase
It
but the
phosphatase
sTBP
these and
phosphates.
cells
phosphatase
in
of
findings
cells, by
acid
both
deglycosylated
which
These
acid
located
characteristic
respectively,
hydrolysis in
transport in
appeared
were
These striking
intact
thiamin.
Since
identity
and
as
S.
described
contrast
to
the
[13].
transport
repression
the
as
thiamin-binding
p-nitrophenylphosphate
phosphatase.
by
constitutive
Furthermore,
for
intact
thiamin.
purified
most
TPP,
for
reduced
of
activity.
affinity
and
mM)
supported
markedly
V. Regulation
activities
TMP
9.1
participating
further
above
the
for
high
catalyzes
cerevisiae,
sTBP
up
glycoproteins
electrophoresis.
The
is
phosphate
physiologically
This
without
by
to thiamin
membrane
possible
well
pyrophosphate(TPP)
taken
so-called
as
thiamin
hydrolyzed
cell
secretory
and
protoplasts
[14C]
exogenous
activities
purification,
cerevisiae.
activity
those(1.5 a
be
first
the
both
yeast
appreciably
that
thiamin,
gel
by
the
by
B1
and
are
reported
are
result,
the
found
T-rAPase
1.7 ƒÊM
[10] repressed
polyacrylamide
are
TMP across
is
by a
throughout
activities
they
probably
et al.
repressible
up
(TMP)
(T-rAPase)
As
is taken
translocation
phosphatase
[11].
copurified
than
by
cerevisiae
are
investigated
the
S.
acid
space
and
Schweingruber
PHO3
thiamin
whereas
TPP
followed
Recently,
[14C]
monophosphate
reduced, that
periplasm,
by
that
thiamin
Vitamin
thiamin
repressed
in
yeast to
be
exerted
transport almost
yeast
is repressed by
regulating
system
and
completely
of
mTBP T-rAPase
by
cells activity
in
exogenous
in
thiamin-containing [14].
As
a wild-type thiamin
in Table
of
S. cerevisiae(IFO
strain at
a
medium
shown
concentration
of
and 1 both
0.2 ƒÊM.
TABLE 1. Effect of thiamin added to the growth medium on activities of thiamin transport, thiamin repressible acid phosphatase and thiamin-synthesizing enzymes in the wild-type strain of S. cerevisiae and thi80 mutant
aSpecific
activity
is expressed
as pmoles
thiamin
bSpecific
activity
is expressed
as nmoles
Pi liberated
cSpecific
activity
is expressed
as nmoles
product
Each value is the mean for two experiments.
transported
per 106cells per minute . per 106cells per 5 min .
formed
per milligram
of protein
per 30 min .
A. IWASHIMA
Table
1 also
shows
that
the
activities
hydroxymethylpyrimidine These
and
findings
cerevisiae
indicate
are
that
controlled
by
of
0.2 ƒÊM
repressibility
of
these
is responsible was
found
phenotype
of
involved
the
may
thiamin.
be The
cells
formation
be
about
24%
mutant
intracellular
after
by
the
growth
in
the might
that
activities be
effector,
to
repress
content
of
TPP
minimal
further
level,
we
activities
of
with
markedly
compared revealed
TABLE
molecular reduced
with that
2.
those the
of
the
mutation
Activities
parent
activities
yeast
cells(IFO and
occurs
was with
of
10483)(Table in a single
enzymes,
in the footnote
the
about
half
that
enzymes
grown
but
remains
to
transport
Genetic
analysis
gene,
indicating
in the similar
for
2).
thi80
of
are
thiamin,
transport
the
constitutive four
cells
nuclear
thiamin
the
the
thiamin
a
altered
in
auxotrophic
enzymes,
for
that and
transport
a mutant
at
concentration
actually 0.2 ƒÊM
and
thiamin
pyrophosphokinase
suggests
after
thiamin
isolated
in the wild-type
as shown
cells
regulation
recently
of thiamin-synthesizing
are expressed
mutant
with
T-rAPase
even
and
T-rAPase
biosynthesis,
intracellular
activities
thiamin-synthesizing
is recessive
transport,
10482)
of
thiamin
S
a thiamin
possibilities
This
insufficient
in
hand, (IFO
grown
of
from
thiamin.
biosynthesis
strain
thiamin
strain.
supplemented
the
have
acid phosphatase
aSpecific
the
activity
TMP by
other
activities
several
of
thiamin
to an these
in
the product
the
parent
when are
wild-type of
due
medium
not shown). mechanism of
the
the
the
of
repressible
thiamin On
appreciable
there
a final
of
and
transport
thiamin,
TPP,
grown without thiamin(data Although the detailed at
thiamin
exogenous
of
had
formation
are
level.
from
[15],
Although
of
in
biosynthesis
as
1).
activities
derived
the
extract
T-rAPase thiamin
was
in
crude
system,
method
well
377
involved
in the
which
as
al.
enzymes
intracellular
chloride
(Table
a negative
the
thi80,
for
thi80
in thiamin
which
type
to
four
transport
by
enzymes
concentration
mutant
thiamin
triphenyltetrazolium
thiamin-synthesizing
which
the
mutant,
the
the
hydroxyethylthiazole
negatively
transport-constitutive selected
of
et
be
of
clarified thi3,
T-rAPase this
that
and thiamin
with wildwhen
thiamin, and
TPP,
mutant the
same
repressible
strain and thi3 mutant
of the Table
1.
Each value is the mean for two experiments.
protein encoded by THI3 is required for the synthesis of thiamin transport system, the four enzymes involved in thiamin biosynthesis as a positive regulatory factor.
VI. Conclusions We conclude that the coordinated
expression of structural genes for the thiamin
T-rAPase
and of
transport system
(probably containing mTBP), T-rAPase and enzymes involved in thiamin synthesis from hydroxymethylpyrimidine and hydroxyethylthiazole in S. cerevisiae is regulated positively by THI3, whereas the expression of these genes is controlled negatively by the intracellular TPP. On the other hand, T-rAPase(sTBP) is thought to play a role in the hydrolysis of thiamin phosphates in the
Symposium (12)
378
Vitamin
B1
periplasmic space prior to the uptake of their thiamin moieties by yeast cells. Since both thiamin transport system and T-rAPase activities are repressed by exogenous thiamin, the utilization of the thiamin moiety of these phosphates by yeast may be regulated by the intracellular TPP level both at the plasma
membrane
and the periplasmic
space of S. cerevisiae.
REFERENCES [1] Suzuoki, Z. (1955): Thiamine uptake by yeast cell. J. Biochem., 42, 27-39. [2] Rose, C. R. (1988): Transport of ascorbic acid and other water-soluble vitamins. Biochim. Biophys. Acta, 947, 335-366. [3] Iwashima, A., Nishino, H., and Nose, Y. (1973): Carrier-mediated transport of thiamine in baker's yeast. Biochim. Biophys. Acta, 330, 222-234. [4] Iwashima, A., Kawasaki, Y., and Kimura, Y. (1990): Transport of 2-methyl-4-amino-5hydroxymethylpyrimidine in Saccharomyces cerevisiae. Biochim. Biophys. Acta, 1022, 211-214. [5] Iwashima, A., Nishimura, H., and Nose, Y. (1979): Soluble and membrane-bound thiaminebinding proteins from Saccharomyces cerevisiae. Biochim. Biophys. Acta, 557, 460-468. [6] Sempuku, K., Nishimura, H., and Iwashima, A. (1981): Photoinactivation of the thiamine transport system in Saccharomyces cerevisiae with 4-azido-2-nitrobenzoylthiamine. Biochim. Biophys. Acta, 645, 226-228. [7] Nishimura, H., Sempuku, K., Kawasaki, Y., Nosaka, K., and Iwashima, A. (1989): Photoaffinity labeling of thiamin-binding component in yeast plasma membrane with [3H]4-azido2-nitrobenzoylthiamin. FEBS lett., 255, 154-158. [8] Iwashima, A., Wakabayashi, Y., and Nose, Y. (1975): Thiamine transport mutants of Saccharomyces cerevisiae. Biochim. Biophys. Acta, 413, 243-24. [9] Nishimura, H., Sempuku, K., and Iwashima, A. (1982): Thiamine transport in Saccharomyces cerevisiae protoplasts. J. Bacteriol., 150, 960-962. [10] Schweingruber, M. E., Fluri, R., Maundrell, K., Schweingruber, A-M., and Dumermuth, E. (1986): Identification and characterization of thiamine repressible acid phosphatase in yeast. J. Biol. Chem., 261, 15877-15882. [11] Nosaka, K., Nishimura, H., and Iwashima, A. (1988): Identity of soluble thiamin-binding protein with thiamin-repressible acid phosphatase in Saccharomyces cerevisiae. Biochim. Biophys. Acta, 967, 49-55. [12] Nosaka, K. (1990): High affinity of acid phosphatase encoded by PHO3 gene in Saccharomyces cerevisiae for thiamin phosphates. Biochim. Biophys. Acta, 1037, 147-154. [13] Nosaka, K., Kaneko, Y., Nishimura, H., and Iwashima, A.(1989): A possible role for acid phosphatase with thiamine-binding activity encoded by PHO3 in yeast. FEMS Microbiology Lett., 60, 55-60. [14] Nishimura, H., Nosaka, K., Sempuku, K., and Iwashima, A.(1986): Thiamine-binding activit of Saccharomyces cerevisiae plasma membrane. Experientia, 42, 607-608. [15] Iwashima, A., Nishino, H., Sempuku, K., and Nishimura, H. (1981): Interaction of basic dyes with thiamine transport system in Saccharomyces cerevisiae. Experientia, 37, 473-474.
Thiamin
S-12-2
Transport
in Yeast
and
Some
Aspects
of Its
Regulation A. IWASHIMA, Y. KAWASAKI, K.NOSAKA,
and H. NISHIMURA
Department of Biochemistry, Kyoto Prefectural University of Medicine, Kawaramachi Hirokoji, Kamigyo-ku, Kyoto 602 (Japan) I. Introduction Yeast Saccharomyces cerevisiae has been known to take up thiamin from the extracellular environment, although this organism can synthesize the vitamin de novo. Since Suzuoki[1] demonstrated for the first time that thiamin enters yeast cells by an active transport system in cell suspensions, several studies have been made on thiamin transport in various types of living cells[2]. Among eukaryotic cells, yeast has the most efficient transport system for thiamin, and moreover, it is available for genetic analysis. For these reasons we have been studying the thiamin transport system of S. cerevisiae from biochemical and genetic aspects. This communication describes the transport system for thiamin and its phosphate esters, and the regulation of the transport studied using mutant yeast cells. II.
General In
and
characteristics
resting
yeast
a Km
value
concentrated fold of
by
thiazole transport
of
and
thiamin for
the
moiety. system,
yeast
thiamin
analogs
Actually whereas
transport
This
by
conclusion
10,000-fold
inhibited
chemical
thiamin
is transported
0.18 ƒÊM.
approximately glucose
various
specific
of
cells
by on
the
structure
the
transport
transport
pyrimidine
system the
and
the
2, 4-dinitrophenol suggested
moiety
of alone
that the
a pH
the
taken
optimum
that uptake
[3].
thiamin is
with
observation
levels,
or
hydroxymethylpyrimidine hydroxyethylthiazole
by
extracellular cyanide
thiamin of
active
is supported
over
iodoacetate,
system an
The
the
system
molecule
than
by
the
yeast
4.5 is
is stimulated
14-
inhibitory
transport
up
of vitamin
effects is that
more of
the
thiamin
is not[4].
III. Photoaffinity labeling of yeast thiamin transport system with [3H]4-azido-2-nitrobenzoylthiamin Our previous studies demonstrated the occurrence of two thiamin-binding proteins in S. cerevisiae [5]: one is bound to plasma membrane (mTBP) which may be directly involved in the thiamine uptake, and the other is a soluble thiamin-binding protein (sTBP), a glycoprotein with a molecular weight of 140kDa in the periplasmic space. 4-Azido-2-nitrobenzoylthiamin (ANBT), a newly synthesized photoreactive thiamin derivative irreversibly inactivated both thiamin-binding activity of the plasma membranes and thiamin transport activity in yeast cells [6]. Furthermore, photoaffinity labeling of yeast plasma membranes with [3H]ANBT resulted in the specific covalent modification of a membrane component with an apparent molecular mass of 6-8 kDa [7]. On the other hand, no specific incorporation of the radioactivity was found in the plasma membranes in the cells of a thiamin transport mutant (PT-R2) isolated as a strain resistant to pyrithiamin, a strong thiamin antagonist [8]. This suggests that the small membrane component (6-8 kDa) takes part in the thiamin-binding of thiamin transport protein(s) in yeast plasma membranes, although the exact relationship of this component to the thiamin transport system will be needed to elucidate the precise mechanism of the transport process. IV. Transport of thiamin activity in yeast periplasm
phosphates
and a possible
role
for acid phosphatase
with thiamin-binding
Symposium (12)
376
We the
previously
uptake
protoplasts
[14C]
is
findings the
reported of
greatly
suggested
in
repressible and
on
isolated
was
to
in
S.
thiamin-binding
as
at
pH
5.0 and
thiamin
thus
was had
parent
yeast
cells
of
Thiamin
10482)
thiamin
of
the
thiamin
by
of of
genetic
activity
of
findings
thiamin
utilization
the
of
led
in
moiety
evidence
showing
the
uptake
of
by
pregrowth
of
that
[14C]
proteins
was
the
thiamin
is identical
to with
values
of
of
1.6
a magnitude that
the
periplasmic in
mutant
phosphates
lower acts
T-rAPase space
of
yeast
cells.
cells in
and
enzyme
that
phosphates
pho3
both (pho3)
phosphatase
Km
the
then were
mutant
conclusion
the
thiamin
periplasmic
sTBP
orders
the
encoded
exhibited
acid
indicates
phosphates thiamin
in
phosphatase
that this
2-3
to
These
and
defective
showed
which
[9].
yeast
protein
showed
were [12],
the
acid
a T-rAPase
It
but the
phosphatase
sTBP
these and
phosphates.
cells
phosphatase
in
of
findings
cells, by
acid
both
deglycosylated
which
These
acid
located
characteristic
respectively,
hydrolysis in
transport in
appeared
were
These striking
intact
thiamin.
Since
identity
and
as
S.
described
contrast
to
the
[13].
transport
repression
the
as
thiamin-binding
p-nitrophenylphosphate
phosphatase.
by
constitutive
Furthermore,
for
intact
thiamin.
purified
most
TPP,
for
reduced
of
activity.
affinity
and
mM)
supported
markedly
V. Regulation
activities
TMP
9.1
participating
further
above
the
for
high
catalyzes
cerevisiae,
sTBP
up
glycoproteins
electrophoresis.
The
is
phosphate
physiologically
This
without
by
to thiamin
membrane
possible
well
pyrophosphate(TPP)
taken
so-called
as
thiamin
hydrolyzed
cell
secretory
and
protoplasts
[14C]
exogenous
activities
purification,
cerevisiae.
activity
those(1.5 a
be
first
the
both
yeast
appreciably
that
thiamin,
gel
by
the
by
B1
and
are
reported
are
result,
the
found
T-rAPase
1.7 ƒÊM
[10] repressed
polyacrylamide
are
TMP across
is
by a
throughout
activities
they
probably
et al.
repressible
up
(TMP)
(T-rAPase)
As
is taken
translocation
phosphatase
[11].
copurified
than
by
cerevisiae
are
investigated
the
S.
acid
space
and
Schweingruber
PHO3
thiamin
whereas
TPP
followed
Recently,
[14C]
monophosphate
reduced, that
periplasm,
by
that
thiamin
Vitamin
thiamin
repressed
in
yeast to
be
exerted
transport almost
yeast
is repressed by
regulating
system
and
completely
of
mTBP T-rAPase
by
cells activity
in
exogenous
in
thiamin-containing [14].
As
a wild-type thiamin
in Table
of
S. cerevisiae(IFO
strain at
a
medium
shown
concentration
of
and 1 both
0.2 ƒÊM.
TABLE 1. Effect of thiamin added to the growth medium on activities of thiamin transport, thiamin repressible acid phosphatase and thiamin-synthesizing enzymes in the wild-type strain of S. cerevisiae and thi80 mutant
aSpecific
activity
is expressed
as pmoles
thiamin
bSpecific
activity
is expressed
as nmoles
Pi liberated
cSpecific
activity
is expressed
as nmoles
product
Each value is the mean for two experiments.
transported
per 106cells per minute . per 106cells per 5 min .
formed
per milligram
of protein
per 30 min .
A. IWASHIMA
Table
1 also
shows
that
the
activities
hydroxymethylpyrimidine These
and
findings
cerevisiae
indicate
are
that
controlled
by
of
0.2 ƒÊM
repressibility
of
these
is responsible was
found
phenotype
of
involved
the
may
thiamin.
be The
cells
formation
be
about
24%
mutant
intracellular
after
by
the
growth
in
the might
that
activities be
effector,
to
repress
content
of
TPP
minimal
further
level,
we
activities
of
with
markedly
compared revealed
TABLE
molecular reduced
with that
2.
those the
of
the
mutation
Activities
parent
activities
yeast
cells(IFO and
occurs
was with
of
10483)(Table in a single
enzymes,
in the footnote
the
about
half
that
enzymes
grown
but
remains
to
transport
Genetic
analysis
gene,
indicating
in the similar
for
2).
thi80
of
are
thiamin,
transport
the
constitutive four
cells
nuclear
thiamin
the
the
thiamin
a
altered
in
auxotrophic
enzymes,
for
that and
transport
a mutant
at
concentration
actually 0.2 ƒÊM
and
thiamin
pyrophosphokinase
suggests
after
thiamin
isolated
in the wild-type
as shown
cells
regulation
recently
of thiamin-synthesizing
are expressed
mutant
with
T-rAPase
even
and
T-rAPase
biosynthesis,
intracellular
activities
thiamin-synthesizing
is recessive
transport,
10482)
of
thiamin
S
a thiamin
possibilities
This
insufficient
in
hand, (IFO
grown
of
from
thiamin.
biosynthesis
strain
thiamin
strain.
supplemented
the
have
acid phosphatase
aSpecific
the
activity
TMP by
other
activities
several
of
thiamin
to an these
in
the product
the
parent
when are
wild-type of
due
medium
not shown). mechanism of
the
the
the
of
repressible
thiamin On
appreciable
there
a final
of
and
transport
thiamin,
TPP,
grown without thiamin(data Although the detailed at
thiamin
exogenous
of
had
formation
are
level.
from
[15],
Although
of
in
biosynthesis
as
1).
activities
derived
the
extract
T-rAPase thiamin
was
in
crude
system,
method
well
377
involved
in the
which
as
al.
enzymes
intracellular
chloride
(Table
a negative
the
thi80,
for
thi80
in thiamin
which
type
to
four
transport
by
enzymes
concentration
mutant
thiamin
triphenyltetrazolium
thiamin-synthesizing
which
the
mutant,
the
the
hydroxyethylthiazole
negatively
transport-constitutive selected
of
et
be
of
clarified thi3,
T-rAPase this
that
and thiamin
with wildwhen
thiamin, and
TPP,
mutant the
same
repressible
strain and thi3 mutant
of the Table
1.
Each value is the mean for two experiments.
protein encoded by THI3 is required for the synthesis of thiamin transport system, the four enzymes involved in thiamin biosynthesis as a positive regulatory factor.
VI. Conclusions We conclude that the coordinated
expression of structural genes for the thiamin
T-rAPase
and of
transport system
(probably containing mTBP), T-rAPase and enzymes involved in thiamin synthesis from hydroxymethylpyrimidine and hydroxyethylthiazole in S. cerevisiae is regulated positively by THI3, whereas the expression of these genes is controlled negatively by the intracellular TPP. On the other hand, T-rAPase(sTBP) is thought to play a role in the hydrolysis of thiamin phosphates in the
Symposium (12)
378
Vitamin
B1
periplasmic space prior to the uptake of their thiamin moieties by yeast cells. Since both thiamin transport system and T-rAPase activities are repressed by exogenous thiamin, the utilization of the thiamin moiety of these phosphates by yeast may be regulated by the intracellular TPP level both at the plasma
membrane
and the periplasmic
space of S. cerevisiae.
REFERENCES [1] Suzuoki, Z. (1955): Thiamine uptake by yeast cell. J. Biochem., 42, 27-39. [2] Rose, C. R. (1988): Transport of ascorbic acid and other water-soluble vitamins. Biochim. Biophys. Acta, 947, 335-366. [3] Iwashima, A., Nishino, H., and Nose, Y. (1973): Carrier-mediated transport of thiamine in baker's yeast. Biochim. Biophys. Acta, 330, 222-234. [4] Iwashima, A., Kawasaki, Y., and Kimura, Y. (1990): Transport of 2-methyl-4-amino-5hydroxymethylpyrimidine in Saccharomyces cerevisiae. Biochim. Biophys. Acta, 1022, 211-214. [5] Iwashima, A., Nishimura, H., and Nose, Y. (1979): Soluble and membrane-bound thiaminebinding proteins from Saccharomyces cerevisiae. Biochim. Biophys. Acta, 557, 460-468. [6] Sempuku, K., Nishimura, H., and Iwashima, A. (1981): Photoinactivation of the thiamine transport system in Saccharomyces cerevisiae with 4-azido-2-nitrobenzoylthiamine. Biochim. Biophys. Acta, 645, 226-228. [7] Nishimura, H., Sempuku, K., Kawasaki, Y., Nosaka, K., and Iwashima, A. (1989): Photoaffinity labeling of thiamin-binding component in yeast plasma membrane with [3H]4-azido2-nitrobenzoylthiamin. FEBS lett., 255, 154-158. [8] Iwashima, A., Wakabayashi, Y., and Nose, Y. (1975): Thiamine transport mutants of Saccharomyces cerevisiae. Biochim. Biophys. Acta, 413, 243-24. [9] Nishimura, H., Sempuku, K., and Iwashima, A. (1982): Thiamine transport in Saccharomyces cerevisiae protoplasts. J. Bacteriol., 150, 960-962. [10] Schweingruber, M. E., Fluri, R., Maundrell, K., Schweingruber, A-M., and Dumermuth, E. (1986): Identification and characterization of thiamine repressible acid phosphatase in yeast. J. Biol. Chem., 261, 15877-15882. [11] Nosaka, K., Nishimura, H., and Iwashima, A. (1988): Identity of soluble thiamin-binding protein with thiamin-repressible acid phosphatase in Saccharomyces cerevisiae. Biochim. Biophys. Acta, 967, 49-55. [12] Nosaka, K. (1990): High affinity of acid phosphatase encoded by PHO3 gene in Saccharomyces cerevisiae for thiamin phosphates. Biochim. Biophys. Acta, 1037, 147-154. [13] Nosaka, K., Kaneko, Y., Nishimura, H., and Iwashima, A.(1989): A possible role for acid phosphatase with thiamine-binding activity encoded by PHO3 in yeast. FEMS Microbiology Lett., 60, 55-60. [14] Nishimura, H., Nosaka, K., Sempuku, K., and Iwashima, A.(1986): Thiamine-binding activit of Saccharomyces cerevisiae plasma membrane. Experientia, 42, 607-608. [15] Iwashima, A., Nishino, H., Sempuku, K., and Nishimura, H. (1981): Interaction of basic dyes with thiamine transport system in Saccharomyces cerevisiae. Experientia, 37, 473-474.
