From the Department of Microbiology, Groningen Biomolecular
Sciences and Biotechnology Institute, University of Groningen, Kerklaan
30, 9751 NN, Haren, The Netherlands
 |
INTRODUCTION |
The first step in maltose metabolism in yeast is performed by the
maltose transport protein, which catalyzes the uptake of maltose in
symport with one proton. Subsequently, 1,4-
-glucosidase (maltase)
hydrolyzes internalized maltose into two molecules of glucose. Maltose
fermenting strains of Saccharomyces cerevisiae have one or
more MAL loci. Each locus comprises at least three genes,
MALX1 encodes the maltose transport protein,
MALX2 encodes the maltase, and MALX3 encodes an
activator of MALX1 and MALX2 (X
denotes one of five MAL loci, with X = 1, 2, 3, 4, or 6) (1). MAL gene expression in maltose-fermenting
"wild-type" strains is inducible and glucose-repressed. The latter
phenomenon is mediated by the main glucose repression/derepression
pathway, which constitutes the transcription factor Mig1p, at least one
protein kinase (Snf1p), and several other proteins whose functions are
unknown (2). While the main glucose repression/derepression pathway
inhibits the synthesis of the proteins involved in the first steps of
maltose metabolism, the presence of glucose activates a second
mechanism involved in the inactivation (also termed catabolite
inactivation) of the maltose transporter but not of maltase (3-7). The
inactivation seems to be due to targeting of the protein to the
vacuole, where it is proteolyzed (4, 5, 8).
If sugar-respiring or -derepressed (grown on nonfermentable carbon
sources) cells of S. cerevisiae are fed with glucose or other rapidly fermentable sugars such as fructose or sucrose, a number
of metabolic changes occur very rapidly, including inhibition of
gluconeogenesis (e.g. inactivation of
fructose-1,6-bisphosphatase and other gluconeogenic enzymes) and
stimulation of glycolysis (activation of phosphofructokinase) (for
review, see Ref. 2). The RAS-adenylate cyclase pathway has been
implicated in this metabolic switch, as the addition of the fermentable
sugar (first messenger) causes a rapid, transient increase in the level
of the second messenger cAMP, which in turn activates (specific) protein kinases. A role for cAMP-dependent protein kinase A
activity in catabolite inactivation of the high affinity glucose and
galactose transporters was suggested from studies in mutants with
varying kinase activities (9). Although these studies have been
disputed more recently (10), it has not been ruled out that
phosphorylation of the transport protein forms the trigger for the
controlled degradation of the protein. In this work, we have analyzed
catabolite inactivation of Mal61p by mutating putative protein kinase A
and C phosphorylation sites. We show that three putative
phosphorylation sites, Ser-295, Thr-363, and Ser-487, play a role in
catabolite inactivation of the maltose transporter. These comprise both
putative protein kinase A and C sites. We also show that inactivation
of the transport system and proteolytic degradation can be
uncoupled.
 |
MATERIALS AND METHODS |
Strains and Growth Conditions--
S. cerevisiae
6001B (MATa, SUC1, MAL11, MAL12, MAL13,
-ura3-52, leu2-3,112) and 6001B
11 (6001B,
mal11
::URA3) (11) were gifts from Dr. C. A. Michels (Queens College, New York). S. cerevisiae
RH144-3D (MATa, ura3, his4, leu2, bar1-1) and
RH266-1D (RH144-3D, end3) were gifts from H. Riezman
(University of Basel, Basel). Strains were grown in batch culture on YP
medium (1% (w/v) yeast extract, 2% (w/v) peptone (pH 6.6)),
supplemented with 2% (w/v) glucose or 2% (w/v) maltose, or on minimal
medium, containing per liter: 0.2 g of MgSO4, 3.0 g of NaH2PO4, 0.7 g of
K2HPO4, 2.5 g of
(NH4)2SO4 (pH 6.0), 1 ml of
Vishniac solution, 1 ml of vitamin solution, 0.5% (w/v) maltose, and
when appropriate 1 ml of uracil (40 mg/ml), L-leucine (125 mg/ml), L-histidine (20 mg/ml), or adenine (20 mg/ml). For
growth in chemostat culture, the cells were grown in the minimal medium
except that K2HPO4 was omitted and
NaH2PO4 was replaced by 1 g of
KH2PO4. Chemostat cultures were operated at a
dilution rate of 0.1 h
1, at 30 °C and pH 5.0 (kept
constant by titration with 1 M KOH).
DNA Manipulations--
DNA manipulations in Escherichia
coli were carried out in the strains JM101 (12) or DH5
(13) as
described previously (14). Yeast cells were transformed according to
the protocol of Gietz et al. (15). The plasmid YEpY18 (11)
is a yeast multicopy plasmid carrying the MAL61 gene under
control of the ADC1 promoter. A cassette gene of
MAL61 was constructed using mutagenic primers (see Table I)
and YEpY18 DNA as a template for polymerase chain reaction-based
synthesis of MAL61 fragments. Briefly, unique restriction sites were engineered by the polymerase chain reaction 400-500 base
pairs apart (see Fig. 1A), which allowed easy subcloning, mutagenesis, and sequencing of the appropriate fragments. The length of
the fragment containing MAL61 in YEpY18 was reduced by
digestion of a ScaI site 56 bases downstream the TGA stop
codon of the MAL61 gene and a SmaI site in the
plasmid, thereby reducing the size of the noncoding region by 900 bases. The resulting plasmid YEpY87 was used for mutagenesis of the
predicted phosphorylation sites in Mal61p. Site-directed mutagenesis of
the phosphorylation sites was carried out by the method of Kunkel
et al. (16) after subcloning of the appropriate fragments in
M13mp18, or, alternatively, by the polymerase chain reaction by using
mutagenic oligonucleotides in combination with primers that hybridize
at the unique restriction sites. Mutagenic primers are indicated in
Table I. All mutations were checked by restriction analysis and DNA
sequencing.
Maltase Assay--
Maltase activity was measured by following
the hydrolysis of p-nitrophenyl-
-D-glucoside
(pNPG)1 at 400 nm
and at 30 °C. The reaction mixture consisted of 1 mM pNPG in 100 mM potassium phosphate, pH 6.8. The
reaction was started by the addition of cell-free extract to a final
concentration of 60-70 µg of protein·ml
1; and
stopped by the addition of 2 M
Na2HCO3. Enzyme activity is expressed as
micromoles of pNPG converted·min
1·mg of
protein
1. Cell-free extract was prepared by vortexing 0.1 M potassium phosphate (pH 6.8)-washed cells for 4 min in
the presence of glass beads (diameter, 0.23-0.33 mm). Whole cells and
debris were removed by centrifugation (5 min; 13,000 × g). Protein concentrations were determined in the presence
of 0.5% (w/v) SDS using a modified Lowry assay (17).
Inactivation Studies--
Cells were harvested from a chemostat
culture and resuspended in an equal volume of minimal medium with or
without NH4+ plus carbohydrate (see
"Results") and 12.5 µg/ml cycloheximide to inhibit protein
synthesis (18). (NH4)2SO4 was
replaced by K2SO4 when appropriate. The
cultures were incubated at 30 °C, and two 1-ml samples were taken
every 10 min. One sample was used for immunodetection of Mal61p, and
the other sample was used for determination of the initial rate of
maltose uptake. The cells were washed once in buffer C (100 mM potassium citrate/PO4, pH 5.5) and
concentrated 20-fold. Transport of maltose in whole cells was measured
as described previously (11), at 30 °C using a final
[14C-U]maltose concentration of 45 µM.
Immunoblot Analysis--
After centrifugation, the cells were
resuspended in 200 µl of 50 mM Tris-HCl, pH 7.4, 1% SDS,
8 M urea. Cell-free extract was obtained by vortexing five
times, 1 min each, in the presence of glass beads (diameter 0.25-0.5
mm) with 1-min intervals on ice in between. Subsequently, the broken
cells were incubated for 10 min at 37 °C, and whole cells and debris
were removed by centrifugation (5 min; 6,000 × g).
Proteins were separated by electrophoresis in 10% SDS-polyacrylamide
and transferred to polyvinylidene difluoride (Millipore) sheets by
semidry electroblotting (19). Antibodies were raised against a purified
amino-terminal 6-histidine tag fusion protein
(N-(His)6-Mal61p). The antibodies were purified by
adsorption against cell-free extract of S. cerevisiae
6001B
11 (11), and used at serum dilutions of 1:15,000. Primary
antibodies were detected with a chemiluminescent detection system using
CSPDTM as substrate (Tropix Inc., Bedford, MA). Band intensities
were quantified using laser scanning densitometry.
 |
RESULTS |
MAL61 Cassette Gene and Mal61p Phosphorylation Mutants--
The
Mal61 protein of S. cerevisiae is phosphorylated in
vivo (4), but the kinases responsible for this phosphorylation as
well as the target sites are unknown. Potential phosphorylation sites
located in the cytoplasmic part of the protein were searched for with
the program "Prosite" from PC-GENE, and the corresponding residues
were replaced by alanines. These residues include putative protein
kinase C sites (Ser-295, Ser-317, and Ser-487) as well as a putative
cAMP-dependent protein kinase A site (Thr-363) (Fig. 1). As a control the serine residue at
position 311, with no predicted modification, was substituted for
alanine. To facilitate genetic manipulation of MAL61, a
cassette gene was constructed, containing unique restriction sites
approximately every 400 base pairs (Fig. 1A). The mutations
creating the new restriction sites were silent (Table
I), and the rate of maltose transport and
the expression level of the cassette protein were comparable to that of
wild-type Mal61p (results not shown). The cassette version of
MAL61 was used for further studies and the construction of
putative phosphorylation site mutants.

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 1.
A, maltose transport protein
mutants. The 1.85-kilobase pair DNA fragment of YEpY87 containing
the MAL61 gene of S. cerevisiae is shown.
Restriction sites introduced are indicated in italics.
The alcohol dehydrogenase promoter is shown as pADC1. The
MAL61 gene is depicted as a thick black bar and
the positions of the phosphorylation sites are indicated. Potential
phosphorylation sites are indicated as PKA (protein kinase
A) or PKC (protein kinase C). B, multiple
sequence alignment of the Mal61p homologs. Mal61p,
Mal31p, Mal11p, and Agt1p are the
maltose transporters of S. cerevisiae; Gal2p is
the S. cerevisiae galactose transporter; Hxt3p,
Hxt6p, Hxt7p, and Snf3p are glucose
transporters of S. cerevisiae; Lac12p is the
lactose transporter of Kluyveromyces lactis;
HepG2 is the human glucose transporter; and XylE
and AraE are the xylose and arabinose transporters of
E. coli.
|
|
Catabolite Inactivation of Mal61p--
The process of catabolite
inactivation is usually triggered by the addition of glucose (3-5). To
study catabolite inactivation of the maltose transport protein of
S. cerevisiae in a genetically well defined background, the
strain 6001B
11/YEpY87, expressing only Mal61p, was used in most
experiments. Cells were grown in a maltose-limited chemostat, which
ensures high levels of expression of Mal61p (11). Cells were harvested
from the chemostat and at time 0, the cells were shifted to a minimal
medium containing glucose and no ammonia. To ensure complete inhibition
of protein synthesis, cycloheximide was added to the inactivation
medium (18). The activity of Mal61p was estimated from the initial rate
of uptake of radioactive maltose, added at a low concentration (45 µM) in order to minimize effects of maltose metabolism.
The activity of the maltase was followed by the release of
4-nitrophenol from pNPG. Fig.
2 shows that under these conditions the
maltase activity decreased less than 10% over a period of 3 h,
whereas the maltose transport activity was reduced more than 85%
within 1 h. The t1/2 of inactivation of
Mal61p-mediated maltose transport activity was 19 min. Maltose
transport in the strain 6001B, expressing the MAL11 gene,
inactivates in the presence of glucose with a t1/2 of 40 min (Table
II). This indicates variations in
catabolite inactivation among naturally occurring homologs of Mal61p.
Extracts of 6001B
11(YEpY87) cells withdrawn at different time
intervals after the medium shift were also analyzed for the presence of the Mal61 protein by immunoblotting. It appeared that loss of maltose
uptake activity was paralleled by loss of Mal61p cross-reactivity on
immunoblots (Table II). Similar results were obtained with Mal61
protein, which carried an amino-terminal 6-histidine tag (N-(His)6-Mal61p). This construct will be
described elsewhere.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 2.
Catabolite inactivation of Mal61p and
Mal12p. Strain 6001B 11/YEpY87 (wild-type Mal61p), was grown in
a maltose-limited chemostat. At time 0, cells were harvested and
resuspended in a nitrogen-deficient medium containing 2% glucose plus
12.5 µg/ml cycloheximide. At the times indicated, the cells were
harvested and maltose transport ( ) and maltase enzyme activity ( )
were assayed.
|
|
View this table:
[in this window]
[in a new window]
|
Table II
Glucose-induced inactivation rate of wild-type and mutant maltose
transport proteins
Cells were grown in a maltose-limited chemostat. At time 0, cells were
harvested and resuspended in a nitrogen-deficient medium containing 2%
glucose plus 12.5 µg/ml cycloheximide, and transport of
[14C]maltose was assayed as described under "Materials and
Methods"; t1/2 values of inactivation were derived
from plots of log activity versus time (see Fig. 3), and
they represent the average ± S.D. for n independent
experiments.
|
|
Characterization of Phosphorylation Mutants--
All
MAL61 genes with mutations at Ser-295, Ser-311, Ser-317,
Thr-363, and Ser-487 as well as the triple mutant S295A,S311A,S317A were able to complement S. cerevisiae 6001B
11 for growth
on maltose, and could be grown in a maltose-limited chemostat. The
maltose transport rates catalyzed by the mutated maltose transporters were comparable to the rate catalyzed by wild-type Mal61p (5.3 ± 2.0 nmol maltose·min
1·mg
1 of protein).
In Fig. 3 catabolite inactivation of
Mal61p (wild type) is compared with the inactivation of Mal61p-S295A,
Mal61p-S311A, and the triple mutant, Mal61p-S295A,S311A,S317A. The
putative phosphorylation site at residue 295 is present in transporters from prokaryotic and eukaryotic species, whereas the sites at 311 and
317 are much less conserved (Fig. 1B). Wild-type Mal61p was
inactivated significantly faster than the single Mal61p-S295A and the
triple Mal61p-S295A,S311A,S317A mutant. The initial rate of maltose
uptake of Mal61p decreased to 50% within 20 min, whereas it took
30-35 min for the Ser-295 mutant to reach the same extent of
inactivation (Table II). The mutant
Mal61p-S311A showed no apparent protection against inactivation and had
a t1/2 similar to the wild type. The mutants of
Thr-363 and Ser-487 also displayed half-lifes in the presence of
glucose that were about twice that of wild-type Mal61p (Table II). The
quadruple Mal61p-S295A,S311A,S317A,T363A mutant was not able to
complement the 6001B
11 strain for growth in a maltose-limited
chemostat, although this mutant was able to complement for growth in
batch.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 3.
Catabolite inactivation of Mal61p
phosphorylation mutants. The inactivation experiment was performed
as described in the legend to Fig. 2. The uptake rate at time 0 (5.3 nmol min 1 mg 1 of protein) was taken as
100%. Strain 6001B 11/YEpY87 (wild type) ( ), YEpY105 (S295A)
( ), YEpY114 (S295A,S311A,S317A) ( ), YEpY120 (S311A) ( ).
|
|
View this table:
[in this window]
[in a new window]
|
Table III
Factors affecting catabolite inactivation of Mal61p
Strain 6001B 11/YEpY87 cells were grown in a maltose-limited
chemostat. At time 0 cells were harvested and resuspended in a medium
plus 12.5 µg/ml cycloheximide with or without nitrogen
(NH4+, as indicated) and containing sugar at the
concentration indicated; further details are the same as described in
the legend to Table II.
|
|
Immunoblots showed that in wild-type Mal61p the decrease in the level
of maltose transport protein occurred at a rate that was similar to the
decrease in maltose transport activity (Fig. 4, Table II). This was also true for the
T363A mutant. However, the Mal61p-S487A mutant protein disappeared more
slowly, the t1/2 of disappearance of the protein was
65 min compared to a t1/2 of 44 min for the decrease
in maltose transport activity. In the S295A and the triple mutant, no
significant decrease of the protein was observed. These results show
that in the T363A mutant both the decrease in maltose uptake activity
and the proteolytic degradation of Mal61p are retarded similarly,
whereas in the S295A and S487A mutants the loss of a putative
phosphorylation site has a more profound effect on the degradation than
on the inactivation.

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 4.
Proteolysis of Mal61p. Total cell
extracts were prepared from the same cells as used for the transport
assays. Proteins were separated by SDS-polyacrylamide gel
electrophoresis on a 10% gel and analyzed by immunoblotting with
antiserum against Mal61p. Each lane contained 25 µg of total protein.
Mal61p protein levels were estimated by laser scanning densitometry.
The density at t = 0 was taken as 100%.
|
|
Since phosphorylation of a serine introduces a negative charge at that
position, the effect of phosphorylation might be mimicked by changing
the serine to a negatively charged residue. In case of the Ser-295 to
glutamate substitution this could result in an increased inactivation
rate of Mal61p. Consistent with an enhanced inactivation (degradation)
rate of the protein, the Mal61p-S295E mutant was unable to complement
6001B
11 for growth on maltose (not shown). However, when this mutant
was expressed in a strain with a defect in the END3 gene,
which is necessary for endocytosis, low but significant maltose uptake
was observed (Fig. 5). This suggests that
in wild-type strains this mutant is subject to rapid "unregulated"
endocytosis, which reduces the concentration of Mal61p molecules in the
plasma membrane.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 5.
Maltose uptake in S. cerevisiae
endocytosis mutants. Mal61p-S295E was expressed in S. cerevisiae strain RH144-3D ( ) and its isogenic strains
RH266-1D (end3) ( ) using plasmid YEpY151. The cells were
grown on mineral medium plus 2% L-lactate and 0.1% yeast
extract to an OD at 660 nm of 1.5.
|
|
Factors Affecting Catabolite Inactivation of Mal61p--
To
estimate the half-life of the maltose transport protein in the absence
of glucose, cells were shifted to a medium containing maltose,
galactose, or ethanol instead of glucose. After a shift to maltose or
galactose, the transport activity did not decrease, but actually
increased. The maltose uptake activity remained virtually the same with
a t1/2 >5 h when cells were shifted to medium
containing ethanol (Table III, Fig. 6). A
shift to a medium containing 2% glucose plus ammonia yielded a
t1/2 of 25 min (Table III), which indicates that
omission of ammonia does not have a significant effect on the
inactivation rate. Overall, these results indicate that the intrinsic
degradation rate of Mal61p in the absence of glucose is very slow and
that inactivation of the protein is specifically triggered by glucose
and not by the presence of a fermentable substrate in general.
2-Deoxy-D-glucose (a nonmetabolizable analogue of glucose)
was also capable of triggering catabolite inactivation of the maltose
transporter (Table III). This suggests that glucose does not have to be
metabolized beyond glucose 6-phosphate to trigger catabolite
inactivation.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of various carbon sources on the
inactivation of Mal61p. The inactivation experiment was performed
as described in the legend to Fig. 2 except that different substrates
were used in the inactivation medium: 2% glucose ( ), 2% ethanol
( ), 500 µM maltose ( ), and 2% 2-DOG ( )
|
|
Finally, Medintz et al. (4) observed that inactivation of
Mal61p was dependent on growth conditions, i.e. the protein
inactivated more rapidly in nitrogen-starved medium than in rich
medium. Since their experiments were carried out with batch-grown
cells, whereas ours were done with cells harvested from maltose-limited
continuous cultures, we also analyzed the inactivation (decrease in
transport activity) of Mal61p and Mal61p-S295A in batch-grown cells.
The inactivation rates in batch were reduced to 16 min for wild-type Mal61p and 23 min for the Mal61p-S295A mutant. In the case of Mal11p
the effect was even larger with a t1/2 of 38 min for
cells from a continuous culture and 16 min for batch-grown cells. The expression levels of Mal61p in batch-grown cells were too low to
estimate protein degradation accurately (data not shown). Overall these
results indicate that the kinetics of inactivation of Mal61p strongly
depends on the growth conditions and that the differences in catabolite
inactivation between wild-type and mutant proteins are larger in slow
growing cells in the chemostat (D = 0.1 h
1) than in exponentially growing batch cells
(µmax
0.3 h
1).
 |
DISCUSSION |
The maltose transport protein belongs to a family of proteins from
prokaryotic as well as eukaryotic origin, of which the members
transport different sugars, i.e. MalX1p and Agt1p, maltose; HxtXp and HepG2, glucose; Gal2p, galactose; Lac12p, lactose; XylE, xylose; and AraE, arabinose (Fig. 1B) (20). Fig.
1B depicts the sequence comparison of the large cytoplasmic
loop between putative transmembrane helices VI and VII found in members
of this family. This region contains three of the four predicted phosphorylation sites studied here, i.e. Ser-295, Ser-317,
and Thr-363. We show that substitution of Ser-295, Thr-363, or Ser-487 for alanine has a dramatic effect on glucose-induced inactivation of
the maltose transport protein. The triple-mutant
Mal61p-S295A,S311A,S317A has the same inactivation time as the single
S295A mutant. The T363A mutation caused a similar increase in the
half-life of inactivation and Mal61 protein stability, increasing to
about twice that of wild-type Mal61p. The S295A and S487A mutations
also doubled the inactivation rate, but in these mutants the decrease
in protein levels was affected more strongly. The half-life of
Mal61p-S487A was about three times that of wild-type Mal61p, whereas no
significant decrease in Mal61p-S295A was observed. This finding
indicates that inactivation and degradation of Mal61p can be uncoupled
and that some form of modification (e.g. phosphorylation) of
the protein precedes the breakdown. Mal11p has an inactivation rate in
the presence of glucose of 38 min, which is comparable to the
inactivation rate of the Mal61p-S295A, Mal61p-T363A and Mal61p-S487A
mutants. Partial sequencing of the MAL11 gene revealed that
Ser-295, Thr-363, and Ser-487 are present in Mal11p, and the increased
inactivation time of Mal11p must therefore be caused by other
differences in primary sequence of Mal11p and Mal61p. The overall
identity between the two proteins is about
95%.2
Catabolite inactivation is not only induced by glucose but also by
2-deoxy-D-glucose (2-DOG). This compound is transported by
the glucose transport systems and is readily phosphorylated by yeast
hexokinases into 2-DOG-6-phosphate, but it is not metabolized further
(21). The catabolite inactivation triggered by 2-DOG is reminiscent to
the activation of the RAS-cAMP pathway for which metabolism of glucose
beyond glucose 6-phosphate is not needed for induction of the cAMP
signal (2). Alternatively, it is also possible that transport of
glucose is not required for catabolite inactivation and that external
glucose or 2-DOG is sensed by specific receptors in the plasma membrane
such as Snf3 or Rgt2 proteins as is the case for glucose repression
(22-24).
When cells are shifted to a medium containing galactose, maltose or
ethanol instead of glucose, maltose transport activity does not
decrease but, in the case of maltose and galactose, increases somewhat.
Since protein synthesis is completely inhibited in these cells,2 the increase in the presence of maltose and
galactose must reflect an increase in specific activity of the
transporter protein. The steady state maltose concentration in the
chemostat was approximately 100 µM,3 and a
switch to a medium containing a higher concentration of sugar
(galactose or maltose) may enable the cells to generate more metabolic
energy in the form of ATP as well as a higher proton motive force. This
higher proton motive force could be the reason for the increase in
transport activity. The concentration of maltose in the inactivation
medium was deliberately kept low (at 500 µM) in order to
avoid substrate induced lysis, as a result of unbridled uptake of
maltose (25), but it was higher than the steady state maltose
concentration in the chemostat.
In addition to Ser-295, Ser-317, and Ser-487, there are two other
protein kinase C phosphorylation sites predicted in Mal61p. One in the
N terminus at position 83 and one in the C terminus at position 572 (Fig. 1A). These sites have been removed by an internal
deletion at the N terminus and by deletion of the C terminus from amino
acid 572. Although plasmids carrying these mutations are able to
partially complement the S. cerevisiae strain 6001B
11 for
growth on maltose in batch, it was not possible to propagate these
strains in the chemostat using conditions described for the wild type
(results not shown). The S295E mutant was made in order to introduce an
acidic residue (negative charge) at a putative phosphorylation site
relevant for catabolite inactivation. We speculate that continuous
nonregulated inactivation of the S295E mutant forms the major cause for
the inability of the cells to grow on maltose. This is supported by the
observation that significant maltose uptake activity was measured when
the Mal61p-S295E mutant was expressed in a strain defective in
endocytosis (26).
We showed that mutation of both putative protein kinase C (Ser-295 and
Ser-487) and protein kinase A (Thr-363) phosphorylation sites have an
effect on catabolite inactivation of the maltose transporter. It should
be stressed, however, that protein kinase A and C recognition sites
cannot unequivocally be assigned (in fact they may overlap) (27), which
leaves quite some room for speculation about the kinase involved in
catabolite inactivation. Glucose addition to glucose-starved cells
mediates not only a transient increase in cAMP, thereby activating
protein kinase A (RAS-cAMP pathway), but it also leads to an increase
of the intracellular Ca2+ levels (28, 29). Since protein
kinase C is stimulated by Ca2+, the intracellular
concentration of this inorganic cation could mediate catabolite
inactivation as well.
It is possible that, in analogy with the Ste2p G protein-coupled plasma
membrane receptor, phosphorylation of the maltose transport protein at
one or more residues precedes subsequent ubiquitination of the protein
(30). Ubiquitination of the target protein is suggested to function as
the endocytosis signal for Ste2p (30). Involvement of ubiquitination in
the catabolite inactivation of maltose transport has recently also been
suggested (8). One could speculate that phosphorylation of Mal61p at specific sites alters the local structure through which those regions
become accessible for ubiquitination.
Catabolite inactivation of the maltose transporter in yeast was also
described in recent publications by Riballo et al. (5), Medintz et al. (4), and Lucero and Lagunas (8). The kinetics of catabolite inactivation reported by Riballo et al. (5) is quite different from that described here and also differs from the data
of Medintz et al. (4). Not only the half-life of
inactivation is different (60 min versus 20 min in our
study) but also the response to glucose is delayed up to 1 h in
some experiments (5). The cause of this lag phase is not understood,
but could be related to the use of less well defined S. cerevisiae strains and/or protein synthesis in the first hour of
inactivation. The Lagunas group (8) has also observed that the kinetics
of inactivation varies among strains, which makes it difficult to
compare the data on catabolite inactivation. Moreover, the strains used
by Riballo et al., carry the multicopy plasmid pRM1-1, which
specifies the MAL1 locus, and the cells are grown in batch
culture (5). We have observed that the half-life of Mal11p activity in
the presence of glucose is longer than that of Mal61p (Table II),
whereas the growth conditions (batch versus chemostat)
strongly affect the kinetics of catabolite inactivation of maltose
transport. Medintz et al. (4) reported catabolite
inactivation of maltose transport with two kinetic components,
i.e. one with a half-life of about 20 min and one with a
t1/2 of about 1 h (in nitrogen starvation medium); only the latter is claimed to be associated with degradation of the protein. These authors interpreted the rapid inactivation as the
result of a modification (phosphorylation) of the transporter. Our
studies confirm this notion as the loss of transport protein is slower
than the loss of transporter activity in both Mal61p-S487A and
Mal61p-S295A.