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Purified arginine permease ofCandida albicans

is functionally active in a reconstituted system
Article in Yeast March 1998
DOI: 10.1002/(SICI)1097-0061(19980315)14:4<335::AID-YEA225>3.0.CO;2-J Source: PubMed

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. 14: 335345 (1998)

Purified Arginine Permease of Candida albicans is

Functionally Active in a Reconstituted System
PRANAB K. MUKHERJEE AND RAJENDRA PRASAD*
School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India
Received 11 June 1997; accepted 10 September 1997

We have for the first time purified arginine permease from a pathogenic yeast, Candida albicans, to homogeneity by
affinity chromatography using -arginine-linked agarose matrix as affinity column. The purified protein (PP) was of
66 kDa with no subunit structure. Two kinetically distinct binding affinities of PP were evident where high affinity
binding (S1) revealed a dependence on acidic pH while pH did not have dramatic effect on low affinity (S2) binding.
The specificity of -arginine binding to PP with regard to other amino acids, structural analogues and inhibitors, was
essentially similar to arginine transport observed in the intact cells of C. albicans (Rao et al., 1986). The purified
arginine permease was reconstituted into proteoliposomes and its functionality was tested by imposing a
valinomycin-induced membrane potential. All the characteristic features of -arginine transport displayed by the
reconstituted system were similar to those observed in intact cells. Thus homogeneous purified arginine permease
was also functionally active. ? 1998 John Wiley & Sons, Ltd.
Yeast 14: 335345, 1998.

Candida albicans; arginine permease; amino acid transport; affinity chromatography; functional
reconstitution

INTRODUCTION
Amino acids represent the chief nitrogen source
in yeasts, and are unidirectionally transported
in Saccharomyces cerevisiae via approximately 20
different amino acid permeases with narrow
specificities; one general system (capable of transporting nearly all amino acids) exists (Cooper,
1982; Horak, 1986). Most of these observations
have been based on kinetic and genetic studies but
not with purified proteins (PPs). Isolation and
purification of amino acid permeases of yeast has
always been difficult, mainly due to their relatively
low amounts in the membrane. Several yeast genes
coding for putative amino acid permeases have
been cloned but are yet to be characterized at the
*Correspondence to: R. Prasad, School of Life Sciences,
Jawaharlal Nehru University, New Delhi-110067, India.
Fax: (+91) 11 6165886; e-mail: rajendra@jnuniv.ernet.in.
Contract grant sponsor: University Grants Commission, New
Delhi.
Contract grant sponsor: Department of Science and
Technology, New Delhi.
Contract grant number: SP/SO/DO2/94.
CCC 0749503X/98/04033511 $17.50
? 1998 John Wiley & Sons, Ltd.

protein level. The deduced amino acid sequences of

these permeases show significant homology and
mostly have a putative 12 transmembrane helix
structure (Schmidt et al., 1994; Andre, 1995;
Grauslund et al., 1995; Isnard et al., 1996). The
gene sequences do provide some idea about their
coded proteins but pure and homogeneous protein
is essential to study the function of such proteins.
Studies on the dimorphic yeast, Candida
albicans, an opportunistic human pathogen, have
generated considerable interest in recent years
mainly due to an increase in the number of AIDS
patients (Odds, 1988; Prasad, 1991). The bud-tohyphal transition of C. albicans has been extensively studied since hyphae formation is believed to
have clinical significance (Odds, 1988; Prasad,
1991). The dimorphism of C. albicans is affected by
several environmental conditions, e.g., temperature, pH, and nitrogen source (Prasad, 1987).
Amino acids are the chief source of nitrogen,
which enter the C. albicans cells via different
permeases. We have earlier characterized several
amino acid permeases of C. albicans to show that

336
the uptake of some of the amino acids is mediated
by very specific permeases and at least for
-proline, the permease was found to be inducible
(Prasad, 1987). The electrochemical gradient of
protons generated by a plasma membrane (PM)bound ATPase drives the uptake of most of the
amino acids by yeast cells (Prasad, 1987; Serrano,
1988; Monk et al., 1991, 1994; Sychrova and
Chevallier, 1993; Sychrova et al., 1993). The transport of basic amino acids in C. albicans cells occurs
via two kinetically different systems, S1 and S2
(Rao et al., 1986). The genes responsible for basic
amino acid transport in C. albicans (CAN1) and
S. cerevisiae (CAN1) have been cloned (Hoffmann,
1985; Ahmad and Bussey, 1986; Sychrova and
Souciet, 1994). The expression of the C. albicans
basic amino acid permease gene in S. cerevisiae
showed that it actively transported arginine, lysine
and histidine (Sychrova and Chevallier, 1993). The
role of proline as a germ tube inducer and the
inducibility of its transporter in C. albicans, compared with the non-inducibility of arginine permease could help elucidate the role of amino acids
in governing the dimorphic transition (Dabrowa
et al., 1976; Dabrowa and Howard, 1981; Jauniaux
et al., 1987). A comparative study of the various
amino acid transporters also becomes important
since some drugs/toxic substances are known to be
transported through these transport systems
(Eddy, 1988; Capobianco et al., 1993; Jethwaney
et al., 1997). We have recently purified and functionally reconstituted the proline permease from
C. albicans (Jethwaney et al., 1997). We report
here the purification of the arginine permease from
the same organism to homogeneity. The purified
permease protein was also functionally reconstituted into proteoliposomes and the reconstituted
permease displayed kinetics and specificity similar
to those observed with intact cells (Rao et al., 1986).
MATERIALS AND METHODS
Strain and culture conditions
Candida albicans ATCC 10261 was grown and
maintained in YPD (1% yeast extract, 2% peptone,
2% dextrose) medium at 30)C in a rotary shaker.
Cells were harvested at mid-logarithmic phase by
low-speed centrifugation.
Isolation of crude membrane
Cells grown to mid-exponential phase in YPD
medium (1 g wet wt) were suspended in 2 ml
? 1998 John Wiley & Sons, Ltd.

. . .
grinding medium (250 m-sucrose, 10 m-Tris/
HCl, pH 75, 1 m-PMSF) and 2 g glass beads
(045050 mm diameter). The suspension was
mechanically disrupted in an MSK Braun cell
homogenizer by agitating it for a total of nine
cycles of 5 s each at 4000 vibrations min "1. The
homogenate was collected and centrifuged at
1000 g for 5 min at 4)C to remove unbroken cells
and glass beads. The supernatant was then centrifuged for 40 min at 15 000 g at 4)C and the
resulting crude membrane (CM) pellet was resuspended in 10 m-Tris/HCl (pH 75). The CM
fraction was stored at "70)C until further use.
Isolation of plasma membrane fraction
The CM fraction was diluted with Percoll (18%,
v/v) in 10 m-Tris/HCl (pH 75). The suspension
was centrifuged at 40 000 g for 40 min at 4)C in a
Sorvall TH-641 rotor (Hubbard et al., 1986). The
gradient so obtained had a translucent lipid layer
on the top and a PM band just below it. The PM
was aspirated and washed twice with 10 m-Tris/
HCl (pH 75) by centrifugation at 100 000 g for 30
min in the same rotor to remove traces of Percoll.
The purified PM pellet was resuspended in 10 mTris/HCl (pH 75) and the purity of PM was
routinely checked by assaying H + -ATPase at pH
66 and 90 (optimum pH for mitochondrial
ATPase). The ratio of the two activities (P=R66/
R90) was always high (>30) implying almost
no contamination by the mitochondrial fraction
(Gupta et al., 1991).
Solubilization and purification of -arginine
permease
The purified PM in a total volume of 3 ml
(34 mg protein/ml) was solubilized using Tween
40 (07%, v/v) as a detergent in the solubilization
buffer containing 10 m-Tris/HCl (pH 75), 1 mPMSF, 1 m-EDTA and 2 m-2-mercaptoethanol. The samples were incubated at 30)C for
5 min and then sonicated for 34 min in a bath
sonicator (Julabo, Seelbach) at 30)C. The solubilized proteins (710 ml) were separated from the
unsolubilized protein by centrifugation at 40 000 g
for 30 min in a Sorvall TH-641 rotor. The
soluble protein present in the supernatant was
dialysed for 12 h against 10 m-Tris/HCl (pH 75)
to remove additives like PMSF, EDTA, and
2-mercaptoethanol. The dialysis also resulted in
partial removal of the detergent. However, the

. 14: 335345 (1998)

 .
detergent concentration remained above its critical
micellar concentration value and the solubilized
protein stayed in the solution. The dialysed protein
fraction (78 mg) was loaded onto an -argininelinked agarose column (5 ml) equilibrated with
100 m-Tris/citrate (pH 50) containing 01%
Tween 40. The unbound protein was eluted with
10 bed volumes (50 ml) of this buffer. The bound
protein was then eluted with a step gradient of
0525 -NaCl (containing 01% Tween 40). Each
eluted fraction of 5 ml was dialysed for 12 h
against 10 m-Tris/HCl (pH 75) to remove NaCl
before the binding assay.
Binding assay
Binding was assayed by the radioactivity-based
equilibrium dialysis method, using the Dianorm
Equilibrium Dialysis Apparatus (Munchen,
Germany). One chamber was filled with the
reaction mixture containing either CM, PM
(200 g protein) or PP (12 g) and 10 -[14C]-arginine (37 MBq/mmol) in 100 m-Triscitrate,
pH 50 to a final volume of 1 ml. The other
chamber contained only buffer. The dialysis unit
was submerged in a water bath set at 30)C and the
chambers were rotated at 12 rpm for 1214 h after
which the liquid was taken out from both the
chambers, and the radioactivity in a 10 l aliquot
of each was counted in a Beckman liquid scintillation counter (model LS 1801) with Triton-based
scintillation cocktail (Cocktail-T, Spectrochem, India). Specific binding of the protein was calculated
from the difference in counts of the two chambers.
If the ligand -[14C]arginine was kept in the other
chamber with buffer, the binding results did not
change. For routine binding assay by S1 and S2
systems, 10 and 1 m of -[14C]arginine was
used at pH 50 and 70, respectively.
Evaluation of binding data
The concentration of bound ligand was calculated as follows. If L0 represents initial concentration of ligand, Lf the final concentration of free
ligand, and Lb the concentration of bound ligand,
then at equilibrium, at 30)C, L0 =Lf +Lf +Lb or,
Lb =L0 "2Lf.
SDS-polyacrylamide gel electrophoresis
The purity of PP was checked on a 620%
gradient of SDSPAGE or on native PAGE
? 1998 John Wiley & Sons, Ltd.

337
(Laemmli, 1970). The gels were developed by
either Coomassie Blue or silver stain (Blum et al.,
1987).
Protein measurements
Protein estimations were done by using the
modified micro-Bradford method (Pande and
Murthy, 1994), which gave minimum interference
by detergent.
Reconstitution of arginine permease
Proteoliposomes were reconstituted using a
detergentdialysis procedure as described earlier
(Jethwaney et al., 1997). Egg phosphatidylcholine
(20 mg/ml) and 40 mg n-octyl --glucopyranoside
(molar phospholipid to detergent ratio 05) were
dissolved in 1 ml ethanol, and dried under vacuum
with slow rotation. One millilitre of protein (1
2 g) in reconstitution buffer (50 m-Triscitrate
pH 50, 25 m-K2SO4, 5 m-MgSO4) was added
to the detergentphospholipid lipid film and the
flask was rotated without vacuum till the solution
became clear. The phospholipiddetergent solution was transferred to Dianorm Mini-Lipoprep=
apparatus and dialysed against 500 ml reconstitution buffer at 15)C for 2 h (with 1-hourly change)
across a 1000 kDa membrane separating the
protein-containing chamber from the buffer. To
remove extravesicular potassium ions, dialysis was
carried out for 30 min with fresh reconstitution
buffer, which had sodium sulphate in place of
potassium sulphate.
Measurement of -[14C]arginine uptake in
reconstituted proteoliposomes
Uptake of radioactive arginine into proteoliposomes was carried out by adding 100 l of
proteoliposome suspension to 1 ml reaction
mixture (50 m-Triscitrate pH 50, 5 m-MgSO4,
10100 --[14C]arginine) at 30)C. Valinomycin
was added (to a final concentration of 1 ) after
30 s to stimulate formation of membrane potential
due to potassium efflux out of the vesicles. Aliquots (100 l) were withdrawn at particular time
periods, filtered through nitrocellulose membrane
filters (pore size 022 m), washed with chilled
buffer (10 m-Triscitrate pH 50, 5 m-MgSO4,
same concentration of arginine as in reaction
mixture), dried and transferred to scintillation
cocktail. The radioactivity retained on the filter
was calculated based on the counts per minute
observed by liquid scintillation counting.

. 14: 335345 (1998)

. . .

338

Figure 1. SDSPAGE of fractions at various steps of purification, on linear density

gradient (620%). (A) Lane 1, crude membrane (CM); lane 2, plasma membrane (PM);
lane 3, purified protein (PP); Sigma molecular weight markers are indicated by short lines,
PP is indicated by arrows. The inset table shows specific activity of arginine binding (with
standard variations) to CM, PM and PP. The table in the lower panel represents a typical
purification table for the PP.

RESULTS AND DISCUSSION

Solubilization and purification of arginine
permease protein
We have exploited the high affinity transport
(25 Km for arginine uptake in intact cells)
property of arginine permease of C. albicans to
purify the protein in a single step to homogeneity
by using affinity chromatography. Pure PM fraction was used as the starting material for solubilization of membrane proteins in order to avoid any
interference due to soluble arginine metabolizing
enzyme(s). The non-ionic detergent Tween-40 was
routinely used where 80% solubilization of the
membrane proteins was achieved. Other detergents
like n-octyl---glucoside were equally successful.
Using equilibrium dialysis, binding activities of
arginine permease in CM, PM and solubilized
? 1998 John Wiley & Sons, Ltd.

protein fractions were monitored. There was a

15-fold increase in arginine binding activity from
CM to solubilized protein fraction.
The bound protein on affinity column was eluted
by a step gradient of NaCl (0525 ). The 25
eluted fraction gave the higher specific binding
activity, which was around 100-fold higher than
that observed for CM (see Table in Figure 1).
SDSPAGE of PP revealed a band of 66 kDa, with
an accompanying lower band of diffused appearance (Figure 1), while the electrophoretic mobility
of the PP under non-denaturing conditions showed
only one band of 66 kDa (data not shown).
Recently, a maltose transport protein was
identified in S. cerevisiae which gave two bands
on SDSPAGE. Although maltose proteins of
the two bands differed in size, their sequence
analysis revealed them to be homologous and

. 14: 335345 (1998)

339

Figure 2. (A) Specific binding of -[14C]arginine to arginine permease as

a function of pH (-, S1; /, S2). 100 m-Triscitrate was used for
buffers of pH range 4560, and 100 m-TrisCl for pH 6075. 10 and 1 m-arginine were used for S1 and S2 binding, respectively. (B)
Change in Km of S1 binding with increasing pH. At the pH-values
indicated, binding was measured as described in Materials and Methods.
The buffers used (100 m) were Triscitrate (pH 4560) and TrisCl
(pH 6075). Bars on each point show standard deviation.

highly hydrophobic (Van den Broeck et al., 1994).

The antibodies against glucose transporter (Hxt2)
also gave two bands on SDSPAGE even after the
blockage of glycosylation (Wendell and Bisson,
1993). A recently purified proline permease protein
of C. albicans also gave two diffused bands on
SDSPAGE (Jethwaney et al., 1997). It appears
from ours, as well as from others results that
? 1998 John Wiley & Sons, Ltd.

size heterogeneity could be common among

hydrophobic transport proteins of yeasts due
to their interaction with SDS (Isambert et al.,
1992; Bernardo et al., 1994; Wendell and Bisson,
1993). The molecular size determined from the
nucleotide sequence of CAN1 (basic amino acids
permease) of C. albicans predicts a putative protein
of 634 kDa (Sychrova and Souciet, 1994), but

. 14: 335345 (1998)

340

. . .

significant differences between predicted and apparent molecular weights of proteins have been
observed previously (Rodriguez et al., 1995). The
difference between the molecular weights of the
purified and the putative arginine permease could
be due to differences in glycosylation.
Substrate binding kinetics
Using intact cells, we have previously shown
that -arginine transport is mediated by a high
affinity S1 (Km =25 ) and a low affinity S2
(Km =200 ) system; both systems were insensitive to inhibition by NEM (Rao et al., 1986). Such
biphasic kinetics was also evident at the level of
PP. Based on the binding activity of PP at different
pH (Figure 2A), it was apparent that there was no
arginine binding by S1 beyond pH 70, the binding
affinities of the two systems S1 and S2 were
determined at their respective optimal pH.
Eadie-Hofstee plots of binding gave two
affinities, 25 (S1) and 287 (S2) (Figure 3).
This would imply that a single permease protein
has dual affinities. Similar to intact cells, the binding of -arginine at both affinities (S1 and S2)
was insensitive to NEM (Rao et al., 1986). In
this regard, it is pertinent to mention that we
have recently purified a proline permeases from
C. albicans which is highly sensitive to NEM
(Jethwaney et al., 1997).
Optimum pH
Amino acid transport in C. albicans cells as well
as in other yeasts is energized by an electrochemical gradient of protons generated and maintained
by plasma-membrane H + -ATPase (Opekarova
et al., 1987, 1993; Kotyk and Dvorakova, 1992;
Eddy and Hopkins, 1988; Sychrova and
Chevallier, 1993; Monk et al., 1991, 1994;
Sychrova et al., 1993; Serrano, 1988; Prasad,
1987). In intact C. albicans cells, the optimum pH
for arginine uptake was found to be 50 (data not
shown). Interestingly, the high affinity binding (S1)
has a sharp optimum pH while the low affinity
binding was not affected by change in external pH
(Figure 2A). The specific binding of S1 was drastically reduced at values higher than pH 50 and
the reduction in binding was associated with an
increase in Km (Figure 2B). The optimum pH of
50 for the high affinity binding system (S1)
matched well with that of functionally reconstituted PP into proteoliposomes (discussed below).
? 1998 John Wiley & Sons, Ltd.

Figure 3. Kinetic analysis of arginine binding by the PP for

(A) high affinity S1 and (B) low affinity S2 systems. Binding was
assayed as described in Materials and Methods, with various
external concentrations of -arginine (1 50 for S1 at pH
50 and 200 15 m for S2 at pH 70). All the values in the
graph are an average of three to four sets of experiments. Bars
on each point show standard deviation.

Incidentally, the optimum pH for arginine uptake

by intact cells of C. albicans was also at 50.
Arginine transport via the over-expressed C.
albicans CAN1 protein was studied at pH 45
(Sychrova and Chevallier, 1993; Sychrova and
Souciet, 1994); while C. albicans lysine permease
had a pH optimum of 50, which shifted to
62 when the protein was over-expressed in
S. cerevisiae (Sychrova et al., 1993). For the S.
cerevisiae LYP1p over-expressed system, increasing extracellular pH increased the accumulation
ratio of lysine without affecting the pH optimum
of LYP1p, while at the same time it did not
affect membrane potential (). It was shown
in that study that the pH effect was at the
permease protein level (Sychrova et al., 1993). Our
results also substantiate that the effect of pH is an

. 14: 335345 (1998)

341

Figure 4. Effect of different amino acids on (A) high affinity and (B)
low affinity systems of arginine permease. The arginine concentration
was 10 for high affinity system, and the amino acids or structural
analogues were 1 m, whereas for low affinity system, 1 m-arginine
and 100 m-analogues were used. For both low and high affinity
systems, competition experiments were done at respective optimum pH.
Bars indicate standard deviations for each point.

important consideration in ligand binding as well

as in translocation of -arginine by the arginine
permease.
Substrate specificity
The S. cerevisiae CAN1 and C. albicans CAN1
gene products both code for basic amino acid
permease but show differences in molecular size,
substrate specificity and kinetic parameters. The
? 1998 John Wiley & Sons, Ltd.

S. cerevisiae CAN1 product is predicted to have a

molecular weight of 657 kDa (Hoffmann, 1985)
and is specific for arginine and lysine, with a
preference for arginine; while the C. albicans
CAN1 product is a 634 kDa protein (Sychrova
and Souciet, 1994) specific for arginine, lysine and
histidine with very high affinity for arginine and
lysine (Sychrova and Chevallier, 1993).
In order to differentiate further between the
two systems of binding, substrate specificity

. 14: 335345 (1998)

342

. . .

was checked for both systems by using 100-fold

concentrations of other amino acids as well as of
-canavanine, an analogue of arginine. The high
affinity binding system (S1) appeared to be specific
since acidic and neutral amino acids could not
inhibit the binding; in contrast, the low affinity
binding system (S2) appeared to be rather nonspecific since unrelated amino acids, e.g. aspartic
acid and valine, could also inhibit the binding
(Figure 4). Interestingly, citrulline could only
inhibit the low affinity system. This assumes significance since it has been observed that citrulline
is transported only by the non-specific general
amino acid permease of S. cerevisiae (Grenson
et al., 1970). Both canavanine and ornithine competitively inhibit arginine binding to PP (Figure 5).
The binding specificity of S1 and S2 systems are
similar to those observed for arginine uptake in
intact C. albicans cells (Rao et al., 1986). Purified
arginine permease did not bind to -proline.
Similarly, purified proline permease protein did
not bind -arginine (data not shown). This further
emphasizes the specificity of respective PPs.
Reconstitution of arginine permease into
proteoliposomes
To demonstrate conclusively that the PP is
indeed an arginine permease, we reconstituted it
into proteoliposomes using the detergent dialysis
method (Jethwaney et al., 1997). In order to
optimize lipid requirement, we used phosphatidylcholine (PC), phosphatidylserine and phosphatidylethanolamine, as well as total lipids of C.
albicans in different molar ratios. A ratio of 1:3 of
PP and PC showed maximum accumulation of
-arginine and hence was used in subsequent
experiments. Valinomycin-induced membrane
potential has been used to induce amino acid
transport in proteoliposomes (Nakanishi et al.,
1994). In the present case energization was
attempted by artificially imposed K + gradient generated by the addition of valinomycin (Figure 6).
As shown in Figure 6, the addition of valinomycin
(10 ) to K + pre-loaded proteoliposomes enhanced [14C]-labelled -arginine uptake by almost
25-fold, while the absence of valinomycin failed
to energize arginine uptake. A similar effect was
observed when no K + was present inside or when
the outside concentration of K + was maintained
equal to that inside, showing that it is the K +
gradient-induced membrane potential () that
drives arginine transport into the vesicle (data not
? 1998 John Wiley & Sons, Ltd.

Figure 5. Effect of 05 m (/), 10 m (4) and 15 m (5)

concentrations of (A) -ornithine and (B) -canavanine on
arginine binding to purified arginine permease. (-) represents
the normal binding of arginine by the PP, in the absence of
ornithine or canavanine. Binding was checked at various external concentrations of arginine, in the presence of the indicated
concentration of canavanine or ornithine. The external concentration of arginine was between 05 and 10 in all the
experiments. The buffer used was 100 m-Triscitrate, pH 50.
Bars on each point show standard deviation.

shown). In the absence of the purified permease

protein, no uptake of -arginine into liposomes
was seen (Figure 6). There was also no uptake of
-proline into proteoliposomes reconstituted with
arginine permease protein. Both these controls
confirm the functionality of the reconstituted protein into proteoliposomes. We were unable to
significantly energize -arginine accumulation by
applied pH gradient (data not shown). This was
likely due to the unspecific membrane permeability
for protons leading to H + equilibration before
-arginine uptake could be started.

. 14: 335345 (1998)

Figure 6. [14C]--arginine uptake in proteoliposomes reconstituted with purified arginine permease. Uptake was initiated by
the addition of 10 -valinomycin after 30 s, as indicated by the
arrow (-). [14C]--arginine uptake in reconstituted proteoliposomes with no addition of valinomycin (,). -arginine uptake
in the liposomes without reconstituting it with the purified
protein ( 0 ). -arginine uptake in liposomes reconstituted with purified proline permease (4). Bars represent standard deviation of each value.

The kinetic parameters of arginine transport

into reconstituted proteoliposomes were assessed
by the initial velocities of uptake in the -arginine
concentration ranged between 1 and 200 .
The Km of -arginine transport in the reconstituted system was 66 , which was closer to the
high affinity system (S1) of arginine uptake
in intact cells (25 ) (Figure 7). While studies
with intact cells and PP binding assays showed the
possible existence of two transport systems S1 and
S2, the reconstituted system showed only the
low Km (high affinity) system which was active at
pH 5.
In order to ascertain the specificity of the reconstituted system, the uptake of -arginine was
measured in the presence of excess concentrations
of various amino acids. Figure 8 shows that the
reconstituted permease is very specific for arginine,
since only lysine and ornithine are able to competitively inhibit arginine uptake (5060%). -arginine
had no effect on -arginine uptake, indicating the
stereo-specificity of the permease. Interestingly, the
intact cells also showed similar specificity of
arginine uptake via the S1 system. The specificity
of arginine binding by the S1 system was equally
? 1998 John Wiley & Sons, Ltd.

343

Figure 7. Lineweaver-Burk plot of -arginine uptake in arginine permease reconstituted into proteoliposomes. The concentration of [14C]--arginine used was from 1 to 200 .
Uptake was studied as described in Materials and Methods.
Linear regression was plotted using the Sigmaplot= program.
Bars represent standard deviation of each value.

specific. The kinetic and specificity studies with

PP revealed a marked similarity with those
observed for arginine transport in intact cells.
Thus, based on our results it is apparent that the
PP is an arginine permease; however, the possibility of other arginine transporters with varied
specificity which may exist in C. albicans, cannot
be excluded.
There are very few permease proteins of
yeast which have been purified to homogeneity,
although genes encoding these transporters have
been analysed. The true functionality test, however, requires that the protein in question be
isolated in a homogeneous form. The work presented in this paper clearly demonstrates that by
using affinity chromatography with argininelinked agarose, and equilibrium dialysis as a
binding assay, arginine permease protein can be
purified to homogeneity. The isolated protein
exhibited similar specificities, kinetics and
sensitivity to inhibitors, as was shown earlier
for arginine uptake in intact cells. That the PP
was functionally active was evident when it was
reconstituted into proteoliposomes. In view of
the fact that very few membrane transporters
(particularly of amino acids) have been purified,
the results presented in this paper should provide a spring board for further purification and
characterization of amino acid transporters of
yeast.

. 14: 335345 (1998)

. . .

344

Figure 8. Effect of different amino acids on [14C]--arginine uptake by reconstituted

arginine permease. Percentage inhibition for each amino acid was calculated from the
uptake in the absence of any analogue. The arginine concentration was 10 and the
amino acids or structural analogues were 1 m. Uptake measurements were done as
described under Materials and Methods, at pH 50. Bars represent standard deviation of
each value.

ACKNOWLEDGEMENTS
P.K.M. is grateful to University Grants
Commission, New Delhi for providing financial
assistance in the form of Junior and Senior
Research Fellowships. The work was financed in
part by a Department of Science and Technology,
New Delhi (SP/SO/DO2/94) grant.
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