(Received for publication, August 17, 1994; and in revised form, November 17, 1994)
From the
Uptake of inositol by Saccharomyces cerevisiae is mediated by a specific inositol permease encoded by the ITR1 gene. Removal of inositol from the growth medium results in an increase in ITR1 mRNA abundance. The increase in ITR1 mRNA is accompanied by an increase in de novo synthesis of the Itr1 permease leading to an increased capacity for uptake. When inositol is added to the growth medium inactivation of uptake activity occurs, and both transcription of ITR1 and uptake activity are repressed to a basal level of function. The transcriptional regulation of ITR1 depends on the INO2, INO4, and OPI1 genes. In addition, repression is also achieved by regulation of ITR1 expression at the post-translational level. In this study, we show that there is a change in the stability of the Itr1 permease after the addition of inositol to the growth medium. Immunoblot analysis using a monoclonal antibody against an epitope attached to the Itr1 permease showed that the addition of inositol causes a dramatic increase in the rate of degradation of the permease. After the repressed (basal) level is achieved, turnover continues to be rapid. The increased rate of degradation was also observed in strains with mutations that block conjugation to ubiquitin. Degradation was not observed in strains defective in the END3/END4 endocytic pathway or in the production of vacuolar proteases (PEP4). Thus, inactivation of the Itr1 permease is accompanied by endocytic internalization followed by degradation in the vacuole. Inactivation may be a separate process that precedes and signals endocytic degradation. Since the end3/end4 mutations did not affect uptake activity under derepressed conditions, endocytosis is not required for normal inositol uptake.
How does the removal of proteins by proteolysis function in the
regulation of cellular metabolism? Repression of transcription is a
mechanism that effectively acts to regulate the level of synthesis of a
protein. The intracellular concentration of a short lived protein is
rapidly decreased if new synthesis is repressed. In the yeast Saccharomyces cerevisiae the 2 repressor, a
constitutively short lived cell type-specific transcriptional
regulator, is an excellent example of this type of
regulation(1, 2) . However, if a protein involved in a
metabolic pathway is long lived, repression at the transcriptional
level alone will not efficiently remove the activity. Simultaneous
inactivation or destabilization of the protein would allow rapid
elimination of the activity. In this paper we investigate the role of
proteolysis in the regulation of inositol transport in S.
cerevisiae.
Uptake of inositol by S. cerevisiae is mediated by the major inositol permease (Itr1p or Itr1 permease) encoded by the ITR1 gene(3) . A second permease encoded by the ITR2 gene plays a minor role(3, 4) . Both transcription of the ITR1 gene and inositol uptake activity are regulated in response to the presence or absence of inositol in the growth medium (3, 4) . Abundance of the ITR1 mRNA increases when inositol is removed from the medium. This increase in ITR1 mRNA abundance is accompanied by an increase in the de novo synthesis of the Itr1 permease as well as an increase in inositol uptake activity. When inositol is added to the growth medium, the level of ITR1 mRNA drops off quickly and uptake activity is fully repressed within two hours(4) .
Previously we demonstrated that uptake of the phospholipid precursor inositol is coordinated to membrane phospholipid biosynthetic activity through the action of the INO2, INO4, and OPI1 gene products(4) . The INO2, INO4, and OPI1 gene products regulate the expression of genes encoding enzymes required for the biosynthesis of the major membrane phospholipids. Expression of the INO1 gene, encoding an enzyme required for endogenous biosynthesis of inositol, is also regulated by INO2, INO4, and OPI1(5) . Derepression of ITR1 transcription and inositol uptake activity require the two pleiotropic positive regulators encoded by INO2 and INO4. The negative regulator encoded by OPI1 is required for repression of ITR1 transcription when inositol is added in the growth medium(4) .
In addition
to this genetic regulation, repression of ITR1 transcription
also requires on-going synthesis of phosphatidylcholine (PC), ()thus coordinating uptake activity to activity in the PC
biosynthetic pathway(4) . Expression of the INO1 gene
also responds to the level of uptake activity; INO1 transcript
levels increase in the presence of external inositol if the capacity
for uptake is decreased. A double itr1itr2 mutation causes
nearly full derepression of INO1; an itr1 mutation
leads to derepression at about 50% of the normal derepressed level of
expression(4) . This finding indicates that the cell precisely
controls the internal inositol concentration through a balance between
inositol uptake and inositol biosynthesis.
As we uncovered the significant role played by inositol uptake in the regulation of membrane phospholipid biosynthesis and inositol biosynthesis, we were intrigued by the mechanisms involved in the negative regulation of the uptake activity. Our data showed that in the absence of inositol the normal derepressed level of inositol uptake activity could be sustained for a minimum of 2 h without de novo protein synthesis. Thus, as it waits for inositol, the Itr1 permease is a stable long-lived protein. Since full repression of uptake activity occurred within 2 h after the addition of inositol to the medium, we suspected that the permease was inactivated in the presence external inositol. Additional evidence for an inactivation mechanism came from analysis of opi1 and opi3 mutations. Transcription of ITR1 is constitutive in strains harboring these mutations(4) . However, inositol uptake activity was subject to full repression. This clearly indicated that the decreased level of ITR1 transcript was not the only mechanism involved in the repression of uptake activity. Since inhibition of translation for 2 h by cycloheximide did not result in repression of uptake activity, and in fact had no effect on uptake activity, we could rule out negative regulation at the translational level as the mechanism of repression(4) .
In this paper we
track the permease itself and show that negative regulation of inositol
uptake activity results in endocytosis of the Itr1 permease followed by
degradation in the vacuole. Mutations that block receptor-mediated
endocytosis (end3 and end4) of factor, the
yeast mating pheromone, also block degradation of the Itr1 permease.
Since inositol uptake was normal in the presence of the end3 and end4 mutations, we conclude that uptake of inositol
does not depend on an endocytic mechanism. Additional investigation of end3/end4 suggests that these mutations that are known to
block internalization of
factor also block internalization of the
Itr1 permease. Since uptake activity is nonetheless inactivated in
their presence, a separate inactivation mechanism that precedes and
signals the increase in the rate of degradation may exist. We also
found that the permease is not restabilized once inositol uptake
activity reaches basal level, and turnover continues to be rapid. At
this point however, since no further decrease in activity is observed,
the rate of synthesis must equal the rate of degradation.
Figure 1:
Plasmid structures. A, plasmid
pKLG3 containing the ITR1 gene was used as a template for PCR
to introduce a single FLAG epitope sequence to the N
terminus of the ITR1 ORF. Plasmid pITR1-F contains the single
epitope-tagged ITR1 gene in the yeast shuttle vector pRS415.
Plasmid pITR1-FS is a derivative of pITR1-F with the SphI site
removed. Plasmid pITR1-FS was used as a template for PCR to replace the
single FLAG
epitope sequence with three copies of the
sequence. The resulting construct pITR1-3F contains three
FLAG
epitope sequences at the N terminus of the ITR1 ORF. B, the plasmid pITR1-3F406 contains the
subcloned ITR1 gene from pITR1-3F in the yeast
integrative vector pRS406. pITR1-3F406 was linearized within the URA3 gene with EcoRV. The linearized fragment was
then integrated at the ura3 locus of the itr1-delete
strain PMY218 to form strain KL22. B, BamHI; S, SalI; G, BglII; Bc, BclI; E, EcoRV; St, StuI; Sp, SphI; Sm, SmaI.
Primers P.3 and P.4 (Table 2) were used to amplify a 350-bp
fragment from plasmid pITR1-FS. This fragment contained the N-terminal
sequence of the ORF of the epitope-tagged ITR1 gene. Primer
P.3 includes three FLAG epitopes (3F) immediately after
the ATG initiation codon of the ITR1 ORF. At the same time, StuI and BglII were used to delete the 350-bp
N-terminal fragment of the ITR1 gene in the original plasmid
pITR1-FS. The deleted fragment was replaced by the amplified 3F
fragment to form plasmid pITR1-3F, carrying an ITR1 ORF
that contains three FLAG
epitopes at the N terminus. The
detection signal improved considerably in the immunoblot procedure
described below.
100
µg of total protein was loaded into each well of a 12.5%
SDS-polyacrylamide gel electrophoresis gel. Gels were run in duplicate
(Mini-Protean II Cell, Bio-Rad, 45 min at 200 V), and one gel was
stained to confirm the quality and quantity of the protein samples. The
other gel was blotted (Mini Trans-Blot Electrophoretic Transfer Cell,
Bio-Rad, 1 h at 100 V) to Nytran membrane (Schleicher & Schuell) in
Transfer Buffer (192 mM glycine, 25 mM Tris-HCl, pH
8, 20% methanol). Blocking of the nytran blot was done for 1 h at room
temperature in Rinse Buffer (0.1% Triton X-100, 150 mM NaCl,
10 mM Tris-HCl, pH 8, 1 mM EDTA) containing 5% Nonfat
Dry Milk powder (Carnation) and 1 mM sodium azide. Primary
antibody (M2 anti-FLAG monoclonal antibody, Kodak IBI
13026), was added at 10 µg/ml to the blocking solution, and the
incubation was continued overnight at 4 °C. After incubation, the
blot was washed three times (10`) with 20 ml of phosphate-buffered
saline, pH 7.4, (1.44 g/liter Na
HPO
, 8 g/liter
NaCl, 0.24 g/liter KH
PO
, 0.2 g/liter KCl) and
blocked in 20 ml of phosphate-buffered saline containing 5% NonFat Dry
Milk powder and 1 mM sodium azide. Secondary antibody was
added as 10 µCi of NEN
I-antimouse IgG (2-10
mCi/mg or 128 mCi/ml, NEX-161) and incubated for 4 h at room
temperature. The blot was washed three times (10`) with 20 ml of
phosphate-buffered saline containing 0.3% Tween-20, dried, and
autoradiographed. Quantitation of radioactivity was by Phosphor Image
Analysis
. The amount of the primary and secondary antibody
used was in the range where signal intensity was observed to be
linearly proportional to the protein concentration. The abundance of
Itr1 permease relative to the total protein was calculated by dividing
the bound radioactivity to the amount of the protein loaded and
expressed at counts/min/microgram protein. Fully derepressed Itr1p
abundance relative to total protein was arbitrarily assigned a value of
1.0.
Since the
addition of an epitope tag to a protein can lead to a change in the
activity/specificity of the protein, the inositol uptake activity of
epitope-tagged Itr1 permeases was determined. An initial attempt that
involved insertion of a single FLAG sequence at the
C-terminal of the Itr1 permease eliminated inositol uptake activity
(data not shown). When a single FLAG
sequence was inserted
at the N-terminal (ITR1-F), uptake activity was
indistinguishable from wild type (V
7.7 nmol/mg
cells/min), confirming that the additional sequence did not affect the
function of the permease. The signal-to-noise ratio in analyses with
the Itr1-F permease and the anti-FLAG
M2 monoclonal
antibody was poor, however, so two additional epitopes were inserted in
order to maximize the signal. The initial construction of a functional
Itr1-3F permease failed because the construct contained an
extraneous initiation codon 10 bp in front of the authentic ATG in the ITR1-F gene (see ``Materials and Methods''). Removal
of the extra ATG allowed translation of a fully active Itr1-F
permease.
Kinetic analysis of derepression and repression of Itr1-3F permease activity was done to determine if the regulation of inositol uptake activity in the strain carrying the ITR1-3F gene was the same as in wild type (Fig. 2). The plasmid pITR1-3F carries the ITR1-3F gene on the CEN/ARS plasmid pRS415. Strain PMY218, with a deletion allele at the ITR1 locus (itr1-T), was transformed with pITR1-3F. In strain KL22, a single copy of the ITR1-3F gene is integrated into the yeast genome of the itr1 deletion strain (PMY218) at the ura3 locus (see ``Materials and Methods'' for more details of the constructions). As can be seen in Fig. 2B, establishment of the basal repressed level of uptake activity was delayed in the strain carrying pITR1-3F in comparison to wild type. Although, in theory, this CEN/ARS construct should behave like a single copy allele, it took 3 h after the addition of inositol to achieve full basal level repression instead of 2 h. We had previously observed delayed repression in a strain (opi1) that overexpresses the ITR1 gene(4) . When the ITR1-3F gene was integrated into the yeast genome as a single copy in strain KL22, repression was normal; thus regulation of the Itr1-3F permease in strain KL22 was the same as the wild type Itr1 permease (Fig. 2B).
Figure 2:
Derepression and repression of inositol
uptake activity. A, derepression. Wild type (PMY169) and KL22
(carrying the epitope-tagged ITR1 gene) cells were grown in
I medium to mid-log stage and then washed and
transferred to I
medium. Inositol uptake activity was
assayed as described under ``Materials and Methods ''at the
indicated time intervals after the transfer. B, Repression.
Wild type, KL22, and the itr1 strain PMY218 transformed with
the plasmid pITR1-3F were grown in I
media and
then transferred to I
media. Inositol permease
activity was assayed at the indicated times. The growth conditions and
assay procedure are described under ``Materials and
Methods.''
Itr1 permease activity continues at the normal derepressed rate for at least 2 h in the absence of protein synthesis(4) . Fig. 3shows an immunoblot analysis of the Itr1 permease before and after the addition of inositol. Derepression of inositol uptake activity requires protein synthesis(3, 4) , and Fig. 3A, lanes 2-6, shows the accumulation of newly synthesized permease, reaching the fully derepressed level in 1.5 h. Fig. 3B, lanes 2-5, shows that the derepressed level of protein was maintained without protein synthesis for 2 h. Therefore, when inositol is not present, the Itr1 permease is a stable long-lived protein. When 65 µM inositol was added to the growth medium, an immediate change in the stability of the permease was observed. Fig. 3B, lanes 6-9, shows that after 15 min a decrease in permease level of approximately 50% was observed, and permease was undetectable in the immunoblot assay 1 h after the addition of inositol. Fig. 3B, lanes 10-13, shows that when protein synthesis was allowed to proceed, degradation of the permease was observed and permease was undetectable 2 h after the addition of inositol. From these data, we concluded that inositol triggers an increase in the rate of degradation of the Itr1 permease.
Figure 3:
Derepression and repression of the Itr1
permease. A, derepression. KL22 cells were grown in
I medium to mid-log stage. At time = 0, the
cells were harvested, washed, and transferred to I
medium. Total cell extracts were prepared at the indicated time
intervals and analyzed by immunoblot as described under
``Materials and Methods.'' B, Repression. KL22 cells
were grown in I
medium to mid-log stage. At time
= 0, the cells were harvested, washed, and transferred to medium
with or without 65 µM inositol (I
or
I
). Cycloheximide (cyc) was added to the
cultures shown in lanes 2-9 at a final concentration of
10 µg/ml to inhibit de novo protein synthesis. Total cell
extracts were prepared at the indicated time
intervals.
Figure 4:
Inositol-induced degradation of the Itr1
permease occurs in mutants that transcribe ITR1 constitutively. A, S. cerevisiae strain PMY255 (opi1) was grown in I medium to mid-log
stage. At time = 0, the cells were harvested, washed, and
transferred to medium with or without 65 µM inositol
(I
or I
). Inositol-induced
degradation of the Itr1 permease was assayed by immunoblot as described
under ``Materials and Methods.'' B, S.
cerevisiae strain PMY256 (opi3) was grown and assayed as
described in A.
Inside the acidic vacuoles of yeast, there are
hydrolytic enzymes(13) . One major vacuolar endopeptidase,
proteinase A, is encoded by the gene PEP4. This protease is
essential for the proteolyic processing and maturation of several other
major vacuolar proteases such as proteinase B and carboxypeptidase
Y(14) . We examined the change in the abundance of Itr1p in a pep4 mutant after the addition of inositol. As shown in Fig. 5A, no decrease in the abundance of Itr1p was
observed when inositol was added to the medium. Therefore, degradation
of the permease requires vacuolar proteases and most likely takes place
in the vacuole. In order to obtain direct evidence, vacuoles were
purified from wild type and pep4 strains, and the Itr1
permease was observed to accumulate only in the vacuoles of the pep4 strain (Fig. 6). In addition, no proteolysis of
the N-terminal epitope-tagged region of the permease takes place prior
to delivery to the vacuole. Therefore, the anti-FLAG antibody should accurately reflect the location of the permease
in the pathway it takes from the plasma membrane to the vacuole. The
kinetic analysis in Fig. 5B shows that inositol uptake
activity was not affected by the pep4 mutation.
Figure 5:
Degradation of the Itr1 permease is
blocked by pep4 mutation. A, strain PMY259 (pep4) was grown in I medium to mid-log
stage. At time = 0 the cells were harvested, washed, transferred
to either I
or I
medium as
indicated. Cycloheximide was added to a final concentration of 10
µg/ml. Total cell extracts were prepared at the indicated time
intervals and analyzed by immunoblot as described under
``Materials and Methods.'' B, wild type (PMY168) and
PMY259 cells were grown in I
medium to mid-log stage
and then washed and transferred to I
medium. Inositol
uptake activity was assayed as described under ``Materials and
Methods'' at the indicated time intervals after the
transfer.
Figure 6:
Purified pep4 vacuoles contain
Itr1 permease. Cells were grown in either I medium or YEPD
(rich medium) plus inositol (I
), followed by vacuolar
isolation as described under ``Materials and Methods.''
Immunoblots were done in duplicate, and the presence of vacuolar
protein in each lane was confirmed with antibody against the vacuolar
protein Vph1 (bottom panel)(7) . The blot in the top panel was probed with anti-Flag antibody to detect the
Itr1p.
Figure 7:
Inositol-induced degradation of Itr1p in
KL22 is sensitive to energy inhibitors. KL22 cells were grown up in
I medium to mid-log stage. At time = 0 the
cells were harvested, washed, and transferred to medium containing 65
µM inositol (I
) with or without energy
inhibitors as indicated. [Sodium azide] (az) was 1
mM and [CCCP] was 20 µM. After 2 h of
incubation in I
medium in the presence of the
inhibitors, degradation of the Itr1 permease was assayed by immunoblot
as described under ``Materials and
Methods.''
If the delivery of the Itr1 permease to the vacuole is by an
endocytic pathway, we would expect no inositol-induced degradation of
the permease in mutants with lesions that block endocytosis.
Receptor-mediated endocytosis has been shown to act in yeast in the
down-regulation of the mating pheromone
receptors(18, 19, 20) . Recently, two mutants (end3 and end4) were identified by Raths et al.(6) based on their failure to internalize the yeast mating
pheromone factor and its cognate receptor at 37 °C. The data
shown in Fig. 8demonstrates that both END3 and END4 are required for inositol-induced degradation of the
permease. Fig. 8A shows that the Itr1 permease is
stable in the absence of inositol at 37 °C (lane 2) and
that inositol-induced degradation occurs normally. Fig. 8, B and C show that the stability of the permease in the
absence of external inositol is not affected by the end3 (Fig. 8B, lanes 1-3)) or end4 (Fig. 8C, lanes 1-3) mutations,
either at 23 or at 37 °C. However, both mutations blocked
inositol-induced degradation of the permease (Fig. 8, B and C, lanes 4-6). The abundance of the
Itr1 permease was virtually unchanged 4 h after exposure to inositol in
both the end3 and the end4 strains, and the block in
degradation was observed at both the permisssive and non-permissive
temperatures.
Figure 8:
Degradation of the Itr1 permease requires END3 and END4. The strains were grown in
I medium to mid-log stage. At time = 0 the
cells were harvested, washed, and transferred to I
medium or to medium with 65 µM inositol
(I
) at room temperature or at 37 °C. Cycloheximide
was added at a final concentration of 10 µg/ml when indicated, and
incubation was continued for 2 h. Total cell extracts were prepared at
the designated time intervals and Itr1 permease abundance was assayed
by immunoblot as described under ``Materials and Methods.'' A, wild type strain PMY168. B, end3 strain
PMY257. C, end4 strain
PMY258.
Figure 9:
END3
and END4 are not required for inositol uptake. Wild type
(PMY168), KL22, RH266-1D (end3), and RH268-1C (end4) cells were grown in I medium to
mid-log stage and then washed and transferred to I
medium. Inositol uptake activity was assayed as described under
``Materials and Methods'' at the indicated time intervals
after the transfer.
Figure 10:
Repression of uptake activity does not
require degradation of the Itr1 permease. Wild type (PMY168),
RH266-1D (end3), RH268-1C (end4), and
PMY259 (pep4) cells were grown in I media
and then transferred to I
media. Inositol permease
activity was assayed at the indicated times. The growth conditions and
assay procedure are described under ``Materials and
Methods.''
Repression of uptake activity would be expected to be independent of the process of permease degradation in two circumstances: (i) if the permease is removed from its functional cellular compartment, presumably the plasma membrane system, and does not have access to its ligand inositol, or (ii) if the permease is actually inactivated prior to degradation. The first circumstance is the likely explanation for down-regulation in the pep4 strain. In the pep4 mutant, there is nothing obvious that would prevent the permease from being endocytosed and delivered to the vacuole. No degradation of the permease occurs because of the defect in production of proteinase A, but the permease is effectively removed from its functional compartment. The fact that down-regulation also was observed in the end mutants provides an opportunity to investigate the existence of a separate inactivation step that precedes and may signal the endocytic degradative process.
Since neither factor nor its receptor appear to be internalized in end3 and end4 strains, the mutations may also prevent
internalization of the Itr1 permease. Alternatively, the permease may
be internalized by a mechanism that does not involve the END3 or END4 gene products; the mutations might block some
subsequent early step in the transport of the permease to the vacuole.
If the mutations prevent internalization of the permease, uptake
activity would not be down-regulated in the mutant strains unless the
permease is inactivated by a mechanism that does not require
endocytosis or degradation. In a previous study(4) , we found
that once cells are exposed to inositol, inactivation is irreversible
without new protein synthesis. Full repression to basal level activity
normally takes 2 h after the addition of inositol to the growth medium.
When we challenged derepressed wild type cells with inositol for only
30 min then transferred the cells back to inositol-free medium,
inositol uptake activity continued to drop until basal level was
reached at the end of 2 h. (
)In view of our present results,
these data suggest that as soon as the presence of external inositol is
detected, and once internalization and endocytosis are initiated, the
pathway is irreversible; the permease cannot be recycled. One would
predict that if end3/end4 block internalization of the Itr1
permease and pep4 does not, the process might be reversible in
the end3/end4 strains but not in the pep4 strain.
The data in Fig. 11show that down-regulation of inositol uptake is not reversible in the pep4 strain. Therefore, we concluded that the exposure of the cells to inositol for 30 min is sufficient to irreversibly initiate a complete endocytosis and vacuolar proteolysis of the existing Itr1 permease. When the end3 and end4 mutants were subjected to the same analysis, an immediate recovery of the uptake activity was observed when inositol was removed at the end of 30 min (Fig. 11). The recovery was followed by a moderate drop in activity that may be explained by the prolonged exposure of the strains to cycloheximide. Although not yet conclusive, the evidence presented suggests that the entire repression process can be functionally dissected into a minimum of two different stages. The first stage involves inactivation of the Itr1 permease by a mechanism that can be reversed when inositol is removed from the medium if the permease has not been internalized and delivered to the vacuole. The second stage and beyond involves the internalization of the permease and its degradation in the vacuole.
Figure 11:
Inactivation of uptake activity is
reversible if endocytosis is blocked. Wild type (PMY168),
RH266-1D (end3), RH268-1C (end4), and
PMY259 (pep4) cells were grown to mid-log stage in
I medium so uptake activity was derepressed. At time
= 0 the cells were harvested, washed, and transferred to medium
with 65 µM inositol (I
). After 30 min,
inositol was removed by washing and resuspending the cells in
inositol-free medium prewarmed to 37 °C. Cycloheximide was added at
a concentration of 10 µg/ml. The incubation was then continued at
37 °C. Uptake activity was assayed at the indicated time points as
described under ``Materials and
Methods.''
In order to address this
question, it was necessary to increase the sensitivity of the inositol
uptake assay in order to be able to detect any increase or decrease in
uptake activity at basal level. This was accomplished by increasing the
amount of labeled inositol relative to unlabeled inositol in the
inositol assay solution (see ``Materials and Methods''). The
data in Fig. 12show basal level activity of about 0.01 nmol/mg
cells/min when the cells are growing in medium containing inositol
(I to I
). However, when protein
synthesis was blocked by cycloheximide (I
to
I
+ cyc), an immediate drop in uptake activity
was observed. When cells are transferred from medium with inositol to
medium without inositol, derepression of uptake activity ordinarily
takes place. Derepression requires protein synthesis, so if
cycloheximide is added to the culture, derepression is not observed. Fig. 12shows that when cells were transferred from medium with
inositol to medium without inositol but with cycloheximide
(I
to I
+ cyc), basal level
uptake activity was maintained. Therefore, the permease regains
stability when inositol is removed because, even though derepression of
activity does not occur, uptake activity continues at the same level
without new protein synthesis. We therefore conclude that stability of
the Itr1 permease is not reestablished once basal level is attained,
and turnover continues to be rapid, requiring new protein synthesis in
order to maintain constant basal level uptake.
Figure 12:
De novo synthesis of permease is required
to maintain basal level uptake activity. Wild type (PMY168) cells were
grown in I medium to mid-log stage. At time = 0
the cells were harvested, washed, and transferred to fresh media as
indicated. Cycloheximide was added at a concentration of 10 µg/ml.
Uptake activity was assayed at the indicated time points using an assay
solution with an increased ratio of radiolabeled to unlabeled inositol
in order to assay uptake at basal level as described under
``Materials and Methods.''
The regulatory system we have described combines mechanistic features of down-regulation of hormone receptors by receptor-mediated endocytosis with aspects of catabolite inactivation. In catabolite inactivation, as observed in the yeast maltose uptake system, when the catabolite is itself available, the alternative supply system is inactivated. Maltose, a glucose dimer, is catabolized to glucose by maltase, and the maltose catabolite glucose is subsequently metabolized by the cell. When glucose becomes available directly, the maltose catabolite glucose triggers the shut-down of the maltose uptake system. We demonstrated that in the absence of its ligand inositol, the yeast inositol uptake system was derepressed to its maximum capacity, and high levels of the Itr1 permease were observed. This regulation is different from what occurs in systems that are regulated by catabolite inactivation. For example, the maltose uptake system is derepressed when its ligand maltose is present, and glucose is absent. In this case, the uptake system is inactivated not by its ligand maltose, but by the catabolite glucose. The inositol uptake system is subject to ligand inactivation rather than catabolite inactivation. It is inositol itself that triggers inactivation of the inositol uptake system.
There are probably mechanistic similarities between catabolite inactivation and ligand inactivation. For example, Lagunas and colleagues (22) have shown that catabolite inactivation of the maltose uptake system is accompanied by proteolytic degradation of the maltose permease, although no information on the proteolytic pathway is available at the present time. Another similarity between the two regulatory mechanisms may be in how they are triggered. A likely model for the interaction of glucose with the maltose permease would be that glucose binds to the permease at a site that is different from the region of the permease that binds maltose, triggering inactivation. The results presented in this paper show that uptake of inositol did not involve endocytosis and that turnover of the Itr1 permease continued to be rapid at basal level. It is unlikely that each time a molecule of inositol is transported in, an Itr1 permease is endocytosed and degraded. Rather, a more likely model would allow ongoing uptake, with each permease in the population possessing a specific probability that a second regulatory binding site for inositol will be occupied. In the model, occupation of this second regulatory site by inositol would render the permease susceptible to inactivation and endocytosis. Inactivation may involve modification of the permease and be mediated by one or more proteins. Although the endocytic pathway is not well understood at the molecular level, it is expected that numerous proteins that are required for yeast endocytosis will be identified in the future.
The purpose of the down-regulation of the inositol uptake system is different from that of the maltose uptake system and other uptake systems that are regulated by catabolite inactivation. In catabolite inactivation, the uptake system (or pathway) is not needed and the tighter the shut-down, the greater the conservation of cellular resources. In the case of down-regulation of inositol uptake, the purpose is certainly not to achieve complete inactivation. It is when inositol is present, and the system is down-regulated, that the uptake system actually functions to provide the cell with inositol. The purpose of the down-regulation is to change the way the uptake system functions, rather than to eliminate it. How is the function of the uptake system changed? One change is to reduce uptake capacity. We have demonstrated that uptake capacity is reduced by endocytosis and degradation of a large segment of the permease population. The second change is in the stability of the permease. We showed that the Itr1 permease is not restabilized at basal level, but continues to be rapidly turned over, so that ongoing protein synthesis is required to maintain uptake activity at the basal level. Are these reasonable changes? The reduction in the permease level may simply reflect the actual number of permease molecules needed to satisfy the inositol requirements of the cell when external inositol is available for uptake. Some other benefit must explain the much higher level of Itr1 permease that waits for the appearance of the ligand inositol.
A second question to be addressed is to ask what could justify the continued rapid turnover rate of the permease at basal level? Why is it not restabilized? The rapid turnover may reflect a mode of regulation necessary when external inositol is present and uptake is functioning and may provide a mechanism that allows uptake activity to be rapidly responsive to changes in the rate of synthesis of new permease. There is evidence that the intracellular inositol concentration is rigorously regulated. First, internal inositol pools have been measured by Carman and colleagues (23) and found to be about 100-fold lower than intracellular amino acid pools. Second, the enzyme PI synthase that catalyzes the synthesis of phosphatidylinositol is regulated primarily by the available concentration of its substrates cytidine diphosphate diacylglycerol and inositol(24) . Therefore, the concentration of inositol that is available to PI synthase regulates the rate of synthesis of PI and ultimately the level of PI in the cell. A third indication that the internal level of inositol is important is that excretion of inositol is observed in strains that harbor mutations that may elevate the internal inositol pools, suggesting elimination of excess internal inositol is critical(25) .
Inositol uptake activity is also regulated at the level of synthesis by transcriptional regulation of the ITR1 gene. Transcription of ITR1 is regulated by the INO2, INO4, and OPI1 genes and coordinated to phospholipid biosynthetic activity(4) . One model that would require rapid turnover would be a system where uptake activity is fine tuned at the transcriptional level. If the Itr1 permease is turned over rapidly, rapid reduction of uptake activity would occur soon after a reduction in the rate of synthesis through negative regulation of transcription. In this model, ITR1 mRNA would also turn over rapidly, allowing permease levels and uptake of inositol to quickly reflect changes in synthesis. Translational control of synthesis may also play a role, but neither evidence for translational control nor genes involved in translational control have been identified as yet, whereas the characterization of the transcriptional control system is well established, and thus constitutes the more convincing model at present.
Data presented in this paper show that mutations that block receptor-mediated endocytosis of the yeast pheromone receptors also block the degradative pathway of the Itr1 permease. The endocytic mechanism may be quite similar to receptor-mediated endocytosis. Certain differences in the two processes must exist, however, because of the difference in the functions of the two proteins, one being a permease the others being pheromone receptors. There is no evidence that pheromones are transported into the interior of the cell for any reason but to achieve down-regulation of the receptor population with the cognate ligand internalized and degraded in the process(26) . Activation of the G-protein signaling pathway does not require internalization of mating pheromone, and no other internal function for pheromone has been identified(26) . The difference between the function of mating pheromones and inositol is that inositol is transported into the cell where it then has a function as an essential cellular metabolite. The Itr1 permease is a transporter that provides a nutrient to the cell, whereas the pheromone receptor is not. We found that uptake of inositol is independent of endocytosis, implying an uptake pore exists in the Itr1 permease tertiary structure that facilitates the uptake process. On the other hand, mating pheromone is not found inside the cell when the internalization step of endocytosis is blocked, implying that there is no uptake pore in the mating pheromone receptor.
One aspect of
the inositol uptake regulatory system is that the Itr1 permease, with a
long half-life, is most abundant in the cell when there is nothing for
it to do. This implies some great advantage to the cell in having the
capacity to rapidly take advantage of any opportunity to take up
external inositol. Is there anything in the physiology of the organism
that could rationalize this kind of regulation? It may be that when the
organism is used to produce ethanol, the yield is compromised if it is
also necessary to produce inositol. Inositol is of course a component
of PI, a major membrane phospholipid accounting for between 20 and 40%
of the membrane phospholipid composition(27) . In addition,
yeast sphingolipids all contain inositol(28) . Synthesis of
inositol may reduce the yield of ethanol in an organism that is used to
produce ethanol. A number of yeasts that are used commercially strictly
to produce ethanol are inositol auxotrophs. They cannot make inositol
under any circumstance. These yeasts include the champagne yeast
ATCC834 ()and a number of other yeasts used in the brewing
industry listed with ATCC. Schizosaccharomyces pombe is also a
natural inositol auxotroph and was purported to come from an African
beer. A significant amount of ethanol that is commercially produced is
made from corn mash, a source that is also very rich in
inositol(29) . On the other hand, S. cerevisiae has
been used both to make alcohol and to make bread. When leavening is the
function of the yeast, the yield of CO
from carbohydrate
may be sufficient to support a more flexible use of glucose, and thus
the compromise retaining the capacity to synthesize inositol, but also
maintaining an uptake system that is immediately ready to relieve the
metabolic demand.