277
FEMS MicrobiologyLetters28 (1985) 277-279
Published by Elsevier
FEM 02142
Inactivation of active glucose transport in Candida wickerhamii is
triggered by exocellular glucose
(Yeast; glucose transport systems; interconversion; regulation)
I. Spencer-Martins and N. van Uden
Laboratory of Microbiology, Gulbenkian Institute of Science, 2781 OeirasCodex, Portugal
Received15 March1985
Accepted19 April 1985
1. SUMMARY
Under conditions of derepression the yeast
Candida wickerhamii formed a high-affinity glu-
cose proton symport. Glucose and glucose analogues induced inactivation of the glucose proton
symport and its interconversion into a low-affinity
facilitated diffusion system. The specific inactivation rate increased with the concentration of the
inactivating sugar and did not obey saturation
kinetics. This dependence was still pronounced at
sugar concentrations far above saturation of the
glucose transport systems. This suggested that the
inactivation and interconversion mechanism was
triggered by interaction of the inactivating sugar
with receptor sites located on the cell surface.
suits which suggest that the process of inactivation
and interconversion is triggered by interaction of
glucose and glucose analogues with a receptor site
located on the cell surface.
3. MATERIALS AND METHODS
3.1. Yeast strain and growth conditions
C. wickerhami( IGC3244 was maintained on
glucose (2%, w/v), peptone (1%, w/v), yeast extract (0.5%) agar. The glucose proton symport was
induced by growing the yeast at 25°C with mechanical shaking in a liquid medium [2] containing
0.5% ,w/v cellobiose [1]. The presence of the proton symport was verified by the use of a pH
electrode as described [1].
2. INTRODUCTION
3.2. Inactivation and interconversion
The yeast C. wickerhamii growing in glucose
medium transported glucose by facilitated diffusion while derepression of the cells induced the
formation of a glucose proton symport and the
disappearance of the facilitated diffusion system.
In buffer with glucose the symport suffered inactivation while the facilitated diffusion system
re-emerged [1]. Here we present experimental re-
Inactivation of the glucose proton symport and
its interconversion into the facilitated diffusion
system was induced by suspending cellobiosegrown cells in 100 mM Tris-citrate buffer, pH 5 at
25°C-in: the presence of glucose or a glucose
analogueat the desired concentration. Samples
were taken at time intervals and initial rates of
glucose uptake measured by the use of D-[13H]glucose as described [1].
0378-1097/85/$03.30 © 1985 Federationof EuropeanMicrobiologicalSocieties
278
During the interconversion process the high-affinity proton symport and the low-affinity facilitated diffusion system co-exist in proportions that
change with time. Let V1 and K] be the maximum
velocity of the initial uptake rate of glucose and
the Michaelis cons!ant of the proton symport and
V2 and K 2 the kinetic parameters of the facilitated
diffusion system. During the interconversion process the measured initial uptake rate of glucose at
any concentration S is the sum of two rates:
S
S
13= VI KI..[_ S F V2--g2..[_ S
(1)
To estimate V] and V2 at different times during the
interconversion process the initial glucose uptake
rate was measured at a low glucose concentration
(SL = 0.04 mM) at which the contribution of the
symport predominates, and at a high glucose concentration (S n = 10 mM) at which facilitated diffusion contributes significantly to the total rate. A
first estimate of V 1 w a s obtained by assuming that
at SL the contribution to the total rate made by
the facilitated diffusion system was zero. This value
for V1 was substituted in Eqn. (1) to obtain a first
estimate of V2 at S H, which was then used to
obtain a second estimate of V1 at SL and so forth.
For each interconversion experiment the values for
K] and K 2 used in the calculations were obtained
from Lineweaver-Burk plots at time zero (only the
symport present) and after completion of the interconversion (only facilitated diffusion present).
The calculated values of V1 and V2 were used as
measures of the capacities of the two transport
systems during interconversion.
4. RESULTS AND DISCUSSION
When derepressed cells were suspended in buffer
containing glucose or glucose analogues (2-deoxyo-glucose, 6-deoxy-D-glucose, 3-O-methyl-D-glucose or D-xylose) at suitable concentrations, the
glucose proton symport activity decreased with
time while the facilitated diffusion system emerged.
Fig. 1 shows results obtained with 0.25% ( w / v )
2-deoxyglucose as the inactivating sugar and glucose as the substrate for transport.
The rate of inactivation of the symport was a
_~100'
I
O
E
0
{3_
o3
r"
>~50
/
a,-,
0
(9
\-
n,,-
o
2'o
6b
Time (rain)
Fig. 1. Inactivation and interconversion of glucose transport
systems in C. wickerhamii 3244. Derepressed cells were incubated in buffer with 0.25% w / v 2-deoxy-D-glucose. The
maximum velocities of the glucose proton symport (O) and of
the facilitated diffusion system (O) were estimated as described
in ~a~TEmM~S AND METHODS.
function of the concentration of the inactivating
sugars (Fig. 2). The inactivation curves were neither
clearly exponential nor linear and seemed to be
composed, at high concentrations of the inactivat-
A
loo'
"\
8
E
9c 50
0
/
\.
E
> 20
\.
10
sb
ld0
T i m e (rnin)
Fig. 2. Inactivation of the glucose proton symport of C
wickerhamii 3244 in the prescace of 2-deoxy-D-glucose. Derepressed cells were incubated in buffer with 2-deoxy-D-glucose: l , 0.1% w/v; O, 0.5% w/v, *, 2% w/v; A, 10% w/v. The
maximum transport velocities were estimated as described in
MATERIALS
AND
METHODS.
279
ing sugar, of a rapid and a slow phase. From the
initial parts of the curves we calculated specific
(exponential) rates of inactivation and plotted these
values against the concentration of the inactivating
sugar. The specific inactivation rates were a linear
function of the concentration of the inactivating
sugar (Fig. 3). We feel it would be premature to
try to present a kinetic model of the proces s. The
important result that can be extracted at this time
from the experimental data is the fact that the
dependence of the inactivation rate on the concentration of the inactivating sugar extended into
concentration ranges (> 550 mM) far above those
needed to saturate the glucose transport systems of
C. wickerhamii, the glucose half-saturation constants of which are around 0.18 mM (proton symport) and 1.7 mM (facilitated diffusion) [1].
We conclude that the inactivation of the proton
8
•
to
'o>
:6
.w
symport and its interconversion into a facilitated
diffusion system does not require transport of the
inactivating sugar and is probably triggered by
interactions of the latter with receptor sites on the
cell surface, rather than by the action of intracellular glucose or a glucose catabolite. The name
'catabolite' interconversion we proposed earlier [1]
for this process, in analogy with the catabolite
inactivation of enzymes of, yeasts [3,4] and other
microorganisms [5], is therefore not quite appropriate.
The rapid inactivation of the high-affinity proton symport and its substitution by the low-affinity facilitated diffusion system at high extraceUular
glucose concentrations makes physiological sense.
So does the location on the cell surface of the
receptor sites that register the extracellular glucose
concentrations and initiate the inactivation and
interconversion process. Were they located inside
the cell registering the intracellular concentration
of glucose or of glucose catabolites, the access of
the inactivation and interconversion mechanisms
to information regarding the extracellular glucose
concentrations would be severely limited by the
capacity of the glucose carriers.
There is some evidence suggesting that similar
mechanisms may regulate high and low affinity
sugar transport in Saccharomyces cereoisiae [6,7]
and in Neurospora crassa [8].
REFERENCES
t...
oo
o
oil
o'.6
Inactivating sugar (M)
Fig. 3. Dependence of the specific rate of inactivation of the
glucose proton symport of C. wickerhamii IGC3244 on the
concentration of the inactivating sugar: •, 2-deoxy-D-glucose;
O, glucose.
[1] Spencer-Martins, I. and van Uden, N. (1985) Biochim.
Biophys. Acta 812, 168-172.
[2] Van Uden, N. (1967) Arch. Mikrobiol. 58, 155-168.
[3] Gancedo, C. (1971) J. Bacteriol. 107, 401-405.
[4] Holzer, H. (1976) Trends Biochem. Sei. 1, 178-181.
[5] Switzer, R.L. (1977) Annu. Rev. Microbiol. 31, 135-157.
[6] Matern, H. and Holzer, H. (1977) J. Biol. Chem. 252,
6399-6402.
[7] Bisson, L.F. and Fraenkel, D.G. (1984) J. Bacteriol. 159,
1013-1017.
[8] Schneider, R.P. and Wiley, W.R. (1971) J. Bacteriol. 106,
487-492.