52
Membrane
P-9
Transport
F.M.HUENNEKENS,
Division Research
K.S.VITOLS,
of Biochemistry, Department Institute, 10666 North Torrey
INTRODUCTION: CHEMOTHERAPY
FOLATE
of Folate L.E.Po
Compourds PE, and
of Molecular and Experimental Pines Road, La Jolla, California
TRANSPORT
IN
CELL
REPLICATION
J.FAN
Medicine, The Scripps 92037 (USA)
AND
CANCER
One-carbon (C1) derivatives of tetrahydrofolate, the coenzyme form of the vitamin folic acid, play a key role in cell replication through their involvement in the biosynthesis of purine nucleotides, the pyrimidine nucleotide thymidylate, and the amino acids methionine and serine (reviewed in [1]). All eukaryotic cells and some prokaryotes (e.g., Lactobacillus cased), however, lack the ability to construct the parent folate structure (pteridine-aminobenzoate-glutamate), even though this task would appear to be trivial in view of the formidable synthetic capabilities of these cells. As a result, these cells depend upon an exogenous source of the vitamin (or its C1 derivatives) and a mechanism for internalization of these compounds. The latter is accomplished by an active transport process, which is augmented by the intracellular conversion of folates to polyglutamate forms that are unable to efflux from cells. Folate transport systems are also responsible for the uptake of antifolates, such as Methotrexate (MTA), that are used extensively in cancer chemotherapy (reviewed in [2]). Indeed, studies of Kessel et all [31 demonstrated that the efficacy of MTX against a series of malignant cell lines was directly proportional to their rate of uptake. For these reasons, folate transport has been the subject of numerous studies. Initial efforts were focused upon the kinetic characteristics of the transport systems in intact cells, but more recently attention has turned to components and mechanisms. The present report summarizes the current state of knowledge about the transport of folate and MTX in two well-studied models, L. cased and L1210 mouse leukemia cells.
Fig. 1.(Left) Structure of fluorescein methotrexate. In the structure shown, the fiunrescein (FITC) is linked via a diaminopentane spacer (DAP) to the ,r-cearboxyl of methotrexate (MTX). Fig. 2.(Right) Fluorescence F-MTX (from [6]).
microscopy of L. casei spheroplasts
labeled with the NHS, ester of
F.M.HUENNEKENS
et al.
53
Fig. 3. Electron microscopy of immunolabeled folate transport protein in L. casei spheroplasts.(Top) Labeling conducted prior to sectioning. CW, cell wall. (Bottom) Labeling conducted after sectioning. Original magnification, 32,000x. From [7].
FOLATE
TRANSPORT
L. for of
casei
folate a
IN
cells
have
compounds,
an
several for
examining
folate
been
the
studied
Folate
is
the
folate
compounds
in
10-2
in
V max
pmoles/min/mg
From
these
number
data,
transporter
is
s.
mechanism
is
thiamine,
of
the
appear an
transport
Expfession
obscure.
share
of
cations the
transport
is
the
by and
is
each
that
drives
the
the
biotin
and
transport
common
component, Electroneutrality
accomplished to
protein
x
energy-coupling
factor.
bound
3
molecule
separate some
ca.
turnover
folate
Folate,
utilizing
process of
each
the
x of
protein, 4•‹,
min-1
glycolysis,
energy-coupling
cotransport
10
6
amount
that
is
Kt is
transported
from
to
have
molecules/cell).
calculated
although still
104
for
although
proteins, possibly
x
min)
derived
the
at
substrate
per
process,
other
folate
transport
(outside->inside)
ATP,
transport
be
of
also
and
(2
can
molecule time
6
it
[4]).
but
with
[3H]folate
cells
(molecules
transit
MTX
folate of
pmoles/106
system in
nM),
value
protein,
membrane-associated
10-2
Kinetic
transport
5-methyltetrahydrofolate,
range.
binding
model
prokaryotes.
and
by
the of
convenient
(Kt=16
including
nM
of
(Cin:Cout)
(reviewed
substrate
basis these
uptake
folate
extensively
optimal
the
measured
a
casei
5-formyltetrahydrofolate values
for
provide
L.
the Since
ratios
transport
of
forms
vitamin.
system
they
characteristics have
the
concentration
hundred-fold,
requirement
that
for
efficient
achieving
CASEI
growth
property
assay
possess
vitamin,
absolute
a
microbiological
cells
LACTOBACILLUS
an
the is
by
the
transporter. regulated
by
a
repression/derepression mechanism, i.e., cells grown on levels of folate ranging from 10-9 to 10-6 M contain progressively lower amounts of the transporter. This may be due to a repressor protein containing bound folate that prevents transcription of the transporter gene.
Purification of the folate transport protein from L. casei involves extraction from membranes with Triton X-100, adsorption and elution from microgranular silica, and filtration through Sephadex G-25 [5] The isolated protein has a molecular weight of 18 kDa based upon SDS-PAGE analysis. It is characterized by an unusually high content of hydrophobic amino acids (and methionine) and the absence of carbohydrate. The amino acid sequence is not yet available. Visualizaion of the L. casei folate transport protein [6] can be accomplished by covalent labeling with a fiuorescein derivative of MTX (F-MTX), whose structure is shown in Fig. 1. Treatment of membrane fragments with the N-hydroxysuccinimide (NHS) ester of this probe, followed by SDS-PAGE of the detergent extract, yields a single fluorescent band (18 kDa). When intac cells are treated with the NHS-ester of F-MTX, however , no fluorescence is visible under the fluorescence microscope. This is attributed to the presence of cell walls that prevent photo-activation or-emission of the fiuorophore, since spheroplasts devoid of cell walls are readily labeled by this procedure (Fig. 2). In this instance, the probe is not attached to the 18 kDa transporter but rasher to a 33 kDa protein that appears to be located between the inner sod outer membranes. The nature of this latter protein or its role, if any, in folate transport is uhknown Visualization of the L. casei folate transport protein has also been accomplished via electron microscopy, using a double-antibody technique. Spheroplasts labeled prior to sectioning, roveal the presence of, transporters on the membrane (Fig. 3, top). However, when
Plenary
54
Lecture
9
spheroplasts are sectioned first and then labeled, antibody-reactive material is also detected in the cytoplasm (Fig. 3, bottom). This observation suggests that L. casei cells express a membrane and a cytoplasmic form of the folate transporter [7]. Both forms, after isolation, have molecular weights of 18 kDa. The membrane form is more hydrophobic, as indicated by amino acid composition and by the fact that it can be extracted readily into n-butanol. Structural differences between the two forms, and the possible relationship between the cytoplasmic and membrane forms, remain to be investigated.
FOLATE
TRANSPORT
L1210
cells,
express
a
primary
IN passaged
folate
but
pmoles/106
time
is
is is
s. or
to
as
Anion but
of
raised inactivation)
of
microscopy, below).
This
function
of
from
cell
cancer
of
L1210
(ca.
reagent
a
is
by
a
a
highly
biotin
All
these
a
to
are
understood.
omitted,
unchanged acid
of
including
the
The ƒÊM
the
cDNA
will
4,
is
transit
it
will
transport to
the
level
be
be
of
system,
this
process
transport
are
process.
intracellular
cAMP
phosphorylation
visualized,
the
via
NHS-ester
expression
the
the
is (and
fluorescence
of
of in
a
same higher [10].
Structure
the
of
F-MTX
(see
transporter
as
evaluation
of
a
cells
with Edman the
of
the
of a
36
its
of
glycosylphosphatidylinositol
sequence the
[PI-PLC]
and
make
to
(as the
by
occurrence
of and
shown
by
a
overlarger
transport
protein)
yet
(N-glycanase).
available
folate
anchor release
not
indicated
sequencing
L1210 ƒÊM
43
The is
the
a
observed.
F
character;
are
as
inhibitors;
as
Cloning,
(GPI) C
is
peptide:N-glycosidase
acid
beads gels
protease
protein
the
membrane
the
carbohydrate,
hydrophobic
amino
the
SDS-PAGE
kDa
are with
transformation
sequencing.
properties
of
multiple
The
components
washing, on
this [9].
treated
extract
proteolytic
with
prevents
are
extensive
culture
overcome
two
cells
detergent
10-liter
developed
the
migrates
to
a To
which
asparagine-linked
treatment
provide
a
presence
specific
level;
been
L1210
After
transporter
no
low protein. has
4). and
the
its the
in
which in
this
of
purification
probe,
out
phospholipase
folate the
by
(Fig.
the
consistent
All
its
transporter,
after is
of
of
contains
transporter.
absence
carbohydrate
Fig.
ca. with 5
"reduced
report,
this
for
applicability
beads.
any,
weight
membrane
much
the
the
this
mediates
can
50 ƒÊg
bond
carried
transporter
phosphatidylinositol-specific
a
termed In
the
hampered
(biotin-SS-MTX)
the
however,
the
is
MTX
of
if
composition
of
quantities
has
and
, the
of max protein
transport
anions
with
about for
degradation
N-terminus,
essentially
values
min-1,
drive
kinase
have
procedure
ester
are
molecular
amino
integral
the
been
mechanism
studying
also
disulfide
release
operations
The ƒÊM
expression
an
may
streptavidin-agarose to
significance,
blocked
the
when
for
only
of
containing
exposed
physiological
The
as
covalently
protein
contains
efficient
dithiothreitol
protein.
the
has
transporter
useful
It
(NHSS)
is with
when
Kt
V
5
system.
protein
be
transport
derivative
linker
preparation
kDa
of
intracellular
decreased
The
cycle.
cells)
N-hydroxysulfosuccinimide
treated
is
have
is
outside)
labeled
may cell
level
system
various
[4] is
cells
folate 1010
problem,
joined
or
the ƒÊM
cells
logistical key
growth
MTX
number
transport
the
proposed
protein.
technique
folate
patients.
Isolation of
of
system
L1210
labeling
this
cAMP-dependent
transport
individual
The
turnover
folate
of
system".
been
a
concentrations
5-methyltetrahydrofolate
(Kt>100 ƒÊM).
inside>concentration
that
the
in
which
and
properties,
transport
possible
in
micromolar
protein.
affinity"
has
folate
is
SYSTEM
on
substrate
the
contributions
exchange
It
[4])
pmoles/min/mg
transport
relative
the ƒÊM
[8].
vitro
poor
molecules/cell),
(concentration and
in
relatively
these
folate
Anion
Activity
a
capacity/low
the
identity
unclear.
0 .5
104
upon
"high
gradients
the
x
Based
folate/MTX" referred
(6
MICROMOLAR in
5-Formyltetrahydrofolate
curiously,
cells
THE
propagated
(reviewed
(Kt=1 ƒÊM).
folate,
12
or
system
-methyltetrahydrofolate 0.1
CELLS:
mice
transport
substrate
5 ƒÊM,
L1210 in
protein, the
indicate
failure that
of it
is
protein. transport kinetic
system
molecular A
in
human
characteristics weight
surprising,
of biotin-SS-MTX.
malignant as
(ca. and
the
80 potentially
cell
L1210
kDa),
lines
(e.g.,
counterpart. and important,
it
K562, The
contains observation
a
CCRF-CEM)
transporter,
considerable is
has however, amount
that
treatment
of of
F.M.HUENNEKENS
et al
55
K562 cells with DMSO, which induces transformation of these cells from the malignant to the normal state, is accompanied by down-regulation of this transporter [11] . This suggests that the p.M folate transport protein may be a "marker" for malignant transformation. Acquisition of this transport system would be advantageous to a cancer cell by allowing it to take up folate compounds more efficiently than a normal cell. As a compensation , however, the cancer cell would also become more sensitive to MTX.
FOLATE TRANSPORT IN LI210 CELLS: THE NANOMOLAR SYSTEM Evidence for the existence of a second folate transport system in eukaryotic cells was provided by the observation [12] that plasma membranes of KB cells (a human nasopharyngeal epidermoid carcinoma) were found to contain extremely large amounts (ca . 200 pmole/106 cells) of a folate binding protein (KD=1 nM). A soluble form of this protein , released into the medium, closely resembled folate binders that had been identified previously in serum , milk and other fluids. Because of its abundance, the KB protein was readily isolated [12] , and the amino acid sequence was deduced subsequently from its cDNA [13] . The protein contains a considerable amount of asparagine-linked carbohydrate [13] and it is anchored to the membrane by a GPI component [14]. Subsequent studies revealed that the KB membrane protein was able to transport folate compounds into the cells. Folate is the optimal substrate (K ,
Membrane
P-9
Transport
F.M.HUENNEKENS,
Division Research
K.S.VITOLS,
of Biochemistry, Department Institute, 10666 North Torrey
INTRODUCTION: CHEMOTHERAPY
FOLATE
of Folate L.E.Po
Compourds PE, and
of Molecular and Experimental Pines Road, La Jolla, California
TRANSPORT
IN
CELL
REPLICATION
J.FAN
Medicine, The Scripps 92037 (USA)
AND
CANCER
One-carbon (C1) derivatives of tetrahydrofolate, the coenzyme form of the vitamin folic acid, play a key role in cell replication through their involvement in the biosynthesis of purine nucleotides, the pyrimidine nucleotide thymidylate, and the amino acids methionine and serine (reviewed in [1]). All eukaryotic cells and some prokaryotes (e.g., Lactobacillus cased), however, lack the ability to construct the parent folate structure (pteridine-aminobenzoate-glutamate), even though this task would appear to be trivial in view of the formidable synthetic capabilities of these cells. As a result, these cells depend upon an exogenous source of the vitamin (or its C1 derivatives) and a mechanism for internalization of these compounds. The latter is accomplished by an active transport process, which is augmented by the intracellular conversion of folates to polyglutamate forms that are unable to efflux from cells. Folate transport systems are also responsible for the uptake of antifolates, such as Methotrexate (MTA), that are used extensively in cancer chemotherapy (reviewed in [2]). Indeed, studies of Kessel et all [31 demonstrated that the efficacy of MTX against a series of malignant cell lines was directly proportional to their rate of uptake. For these reasons, folate transport has been the subject of numerous studies. Initial efforts were focused upon the kinetic characteristics of the transport systems in intact cells, but more recently attention has turned to components and mechanisms. The present report summarizes the current state of knowledge about the transport of folate and MTX in two well-studied models, L. cased and L1210 mouse leukemia cells.
Fig. 1.(Left) Structure of fluorescein methotrexate. In the structure shown, the fiunrescein (FITC) is linked via a diaminopentane spacer (DAP) to the ,r-cearboxyl of methotrexate (MTX). Fig. 2.(Right) Fluorescence F-MTX (from [6]).
microscopy of L. casei spheroplasts
labeled with the NHS, ester of
F.M.HUENNEKENS
et al.
53
Fig. 3. Electron microscopy of immunolabeled folate transport protein in L. casei spheroplasts.(Top) Labeling conducted prior to sectioning. CW, cell wall. (Bottom) Labeling conducted after sectioning. Original magnification, 32,000x. From [7].
FOLATE
TRANSPORT
L. for of
casei
folate a
IN
cells
have
compounds,
an
several for
examining
folate
been
the
studied
Folate
is
the
folate
compounds
in
10-2
in
V max
pmoles/min/mg
From
these
number
data,
transporter
is
s.
mechanism
is
thiamine,
of
the
appear an
transport
Expfession
obscure.
share
of
cations the
transport
is
the
by and
is
each
that
drives
the
the
biotin
and
transport
common
component, Electroneutrality
accomplished to
protein
x
energy-coupling
factor.
bound
3
molecule
separate some
ca.
turnover
folate
Folate,
utilizing
process of
each
the
x of
protein, 4•‹,
min-1
glycolysis,
energy-coupling
cotransport
10
6
amount
that
is
Kt is
transported
from
to
have
molecules/cell).
calculated
although still
104
for
although
proteins, possibly
x
min)
derived
the
at
substrate
per
process,
other
folate
transport
(outside->inside)
ATP,
transport
be
of
also
and
(2
can
molecule time
6
it
[4]).
but
with
[3H]folate
cells
(molecules
transit
MTX
folate of
pmoles/106
system in
nM),
value
protein,
membrane-associated
10-2
Kinetic
transport
5-methyltetrahydrofolate,
range.
binding
model
prokaryotes.
and
by
the of
convenient
(Kt=16
including
nM
of
(Cin:Cout)
(reviewed
substrate
basis these
uptake
folate
extensively
optimal
the
measured
a
casei
5-formyltetrahydrofolate values
for
provide
L.
the Since
ratios
transport
of
forms
vitamin.
system
they
characteristics have
the
concentration
hundred-fold,
requirement
that
for
efficient
achieving
CASEI
growth
property
assay
possess
vitamin,
absolute
a
microbiological
cells
LACTOBACILLUS
an
the is
by
the
transporter. regulated
by
a
repression/derepression mechanism, i.e., cells grown on levels of folate ranging from 10-9 to 10-6 M contain progressively lower amounts of the transporter. This may be due to a repressor protein containing bound folate that prevents transcription of the transporter gene.
Purification of the folate transport protein from L. casei involves extraction from membranes with Triton X-100, adsorption and elution from microgranular silica, and filtration through Sephadex G-25 [5] The isolated protein has a molecular weight of 18 kDa based upon SDS-PAGE analysis. It is characterized by an unusually high content of hydrophobic amino acids (and methionine) and the absence of carbohydrate. The amino acid sequence is not yet available. Visualizaion of the L. casei folate transport protein [6] can be accomplished by covalent labeling with a fiuorescein derivative of MTX (F-MTX), whose structure is shown in Fig. 1. Treatment of membrane fragments with the N-hydroxysuccinimide (NHS) ester of this probe, followed by SDS-PAGE of the detergent extract, yields a single fluorescent band (18 kDa). When intac cells are treated with the NHS-ester of F-MTX, however , no fluorescence is visible under the fluorescence microscope. This is attributed to the presence of cell walls that prevent photo-activation or-emission of the fiuorophore, since spheroplasts devoid of cell walls are readily labeled by this procedure (Fig. 2). In this instance, the probe is not attached to the 18 kDa transporter but rasher to a 33 kDa protein that appears to be located between the inner sod outer membranes. The nature of this latter protein or its role, if any, in folate transport is uhknown Visualization of the L. casei folate transport protein has also been accomplished via electron microscopy, using a double-antibody technique. Spheroplasts labeled prior to sectioning, roveal the presence of, transporters on the membrane (Fig. 3, top). However, when
Plenary
54
Lecture
9
spheroplasts are sectioned first and then labeled, antibody-reactive material is also detected in the cytoplasm (Fig. 3, bottom). This observation suggests that L. casei cells express a membrane and a cytoplasmic form of the folate transporter [7]. Both forms, after isolation, have molecular weights of 18 kDa. The membrane form is more hydrophobic, as indicated by amino acid composition and by the fact that it can be extracted readily into n-butanol. Structural differences between the two forms, and the possible relationship between the cytoplasmic and membrane forms, remain to be investigated.
FOLATE
TRANSPORT
L1210
cells,
express
a
primary
IN passaged
folate
but
pmoles/106
time
is
is is
s. or
to
as
Anion but
of
raised inactivation)
of
microscopy, below).
This
function
of
from
cell
cancer
of
L1210
(ca.
reagent
a
is
by
a
a
highly
biotin
All
these
a
to
are
understood.
omitted,
unchanged acid
of
including
the
The ƒÊM
the
cDNA
will
4,
is
transit
it
will
transport to
the
level
be
be
of
system,
this
process
transport
are
process.
intracellular
cAMP
phosphorylation
visualized,
the
via
NHS-ester
expression
the
the
is (and
fluorescence
of
of in
a
same higher [10].
Structure
the
of
F-MTX
(see
transporter
as
evaluation
of
a
cells
with Edman the
of
the
of a
36
its
of
glycosylphosphatidylinositol
sequence the
[PI-PLC]
and
make
to
(as the
by
occurrence
of and
shown
by
a
overlarger
transport
protein)
yet
(N-glycanase).
available
folate
anchor release
not
indicated
sequencing
L1210 ƒÊM
43
The is
the
a
observed.
F
character;
are
as
inhibitors;
as
Cloning,
(GPI) C
is
peptide:N-glycosidase
acid
beads gels
protease
protein
the
membrane
the
carbohydrate,
hydrophobic
amino
the
SDS-PAGE
kDa
are with
transformation
sequencing.
properties
of
multiple
The
components
washing, on
this [9].
treated
extract
proteolytic
with
prevents
are
extensive
culture
overcome
two
cells
detergent
10-liter
developed
the
migrates
to
a To
which
asparagine-linked
treatment
provide
a
presence
specific
level;
been
L1210
After
transporter
no
low protein. has
4). and
the
its the
in
which in
this
of
purification
probe,
out
phospholipase
folate the
by
(Fig.
the
consistent
All
its
transporter,
after is
of
of
contains
transporter.
absence
carbohydrate
Fig.
ca. with 5
"reduced
report,
this
for
applicability
beads.
any,
weight
membrane
much
the
the
this
mediates
can
50 ƒÊg
bond
carried
transporter
phosphatidylinositol-specific
a
termed In
the
hampered
(biotin-SS-MTX)
the
however,
the
is
MTX
of
if
composition
of
quantities
has
and
, the
of max protein
transport
anions
with
about for
degradation
N-terminus,
essentially
values
min-1,
drive
kinase
have
procedure
ester
are
molecular
amino
integral
the
been
mechanism
studying
also
disulfide
release
operations
The ƒÊM
expression
an
may
streptavidin-agarose to
significance,
blocked
the
when
for
only
of
containing
exposed
physiological
The
as
covalently
protein
contains
efficient
dithiothreitol
protein.
the
has
transporter
useful
It
(NHSS)
is with
when
Kt
V
5
system.
protein
be
transport
derivative
linker
preparation
kDa
of
intracellular
decreased
The
cycle.
cells)
N-hydroxysulfosuccinimide
treated
is
have
is
outside)
labeled
may cell
level
system
various
[4] is
cells
folate 1010
problem,
joined
or
the ƒÊM
cells
logistical key
growth
MTX
number
transport
the
proposed
protein.
technique
folate
patients.
Isolation of
of
system
L1210
labeling
this
cAMP-dependent
transport
individual
The
turnover
folate
of
system".
been
a
concentrations
5-methyltetrahydrofolate
(Kt>100 ƒÊM).
inside>concentration
that
the
in
which
and
properties,
transport
possible
in
micromolar
protein.
affinity"
has
folate
is
SYSTEM
on
substrate
the
contributions
exchange
It
[4])
pmoles/min/mg
transport
relative
the ƒÊM
[8].
vitro
poor
molecules/cell),
(concentration and
in
relatively
these
folate
Anion
Activity
a
capacity/low
the
identity
unclear.
0 .5
104
upon
"high
gradients
the
x
Based
folate/MTX" referred
(6
MICROMOLAR in
5-Formyltetrahydrofolate
curiously,
cells
THE
propagated
(reviewed
(Kt=1 ƒÊM).
folate,
12
or
system
-methyltetrahydrofolate 0.1
CELLS:
mice
transport
substrate
5 ƒÊM,
L1210 in
protein, the
indicate
failure that
of it
is
protein. transport kinetic
system
molecular A
in
human
characteristics weight
surprising,
of biotin-SS-MTX.
malignant as
(ca. and
the
80 potentially
cell
L1210
kDa),
lines
(e.g.,
counterpart. and important,
it
K562, The
contains observation
a
CCRF-CEM)
transporter,
considerable is
has however, amount
that
treatment
of of
F.M.HUENNEKENS
et al
55
K562 cells with DMSO, which induces transformation of these cells from the malignant to the normal state, is accompanied by down-regulation of this transporter [11] . This suggests that the p.M folate transport protein may be a "marker" for malignant transformation. Acquisition of this transport system would be advantageous to a cancer cell by allowing it to take up folate compounds more efficiently than a normal cell. As a compensation , however, the cancer cell would also become more sensitive to MTX.
FOLATE TRANSPORT IN LI210 CELLS: THE NANOMOLAR SYSTEM Evidence for the existence of a second folate transport system in eukaryotic cells was provided by the observation [12] that plasma membranes of KB cells (a human nasopharyngeal epidermoid carcinoma) were found to contain extremely large amounts (ca . 200 pmole/106 cells) of a folate binding protein (KD=1 nM). A soluble form of this protein , released into the medium, closely resembled folate binders that had been identified previously in serum , milk and other fluids. Because of its abundance, the KB protein was readily isolated [12] , and the amino acid sequence was deduced subsequently from its cDNA [13] . The protein contains a considerable amount of asparagine-linked carbohydrate [13] and it is anchored to the membrane by a GPI component [14]. Subsequent studies revealed that the KB membrane protein was able to transport folate compounds into the cells. Folate is the optimal substrate (K ,
