©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Inositol Transport in Saccharomyces cerevisiae Involves Inositol-induced Changes in Permease Stability and Endocytic Degradation in the Vacuole (*)

(Received for publication, August 17, 1994; and in revised form, November 17, 1994)

Kent Lai Cynthia P. Bolognese Steve Swift Patricia McGraw (§)

From the Department of Biological Sciences, University of Maryland, Baltimore, Maryland 21228

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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 alpha2 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), (^1)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 alpha 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 alpha 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.


MATERIALS AND METHODS

Strains and Plasmids

The S. cerevisiae strains used in this study are listed in Table 1. Strain KL22 (itr1-T, ITR1-3F) was constructed by integration of the plasmid pITR1-3F406 at the ura3-52 locus of the itr1-deletion strain PMY218(itr1-T ) (Fig. 1B).




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.



Culture Medium and Growth of Yeast Strains

These have been described previously(4) . Unless stated otherwise, all chemicals were purchased from Sigma or J. T. Baker Chemicals. In order to block the endocytic pathway in the end mutants(6) , the culture was grown at 23 °C to mid exponential phase and then resuspended in medium prewarmed to 37 °C and the incubation was continued for a minimum of 10 min. At this point the culture was ready to subject to specific experimental manipulations. After the manipulation, the incubation continued at 37 °C and aliquots of culture were withdrawn at the indicated time intervals for either immunoblot analysis or assay of inositol uptake.

Construction of a Gene Coding for the Epitope-tagged Itr1 Permease

The construction of the plasmid pITR1-3F is shown in Fig. 1A. A DNA fragment containing the ITR1 gene (-796 bp to +1758 bp relative to the start site of the open reading frame (ORF)) was subcloned from plasmid pKL1(4) . The fragment was cloned into the BamHI and SalI sites of the vector pGEM3zf (Promega) to form plasmid pKLG3. Two primers, P.1 and P.2 (Table 2), were used to amplify the entire plasmid pKLG3 using reverse PCR (Fig. 1A). Primer P.1 contains a single FLAG (Kodak IBI) epitope sequence (5`-GAC TAC AAG GAC GAC GAT GAC AAG-3`) located immediately after the first ATG of the ITR1 ORF. Both primers P.1 and P.2 were designed to contain an SphI site at the 5` end so that the linear PCR product could be circularized to form plasmid pKL35. BamHI and SalI were used to isolate the epitope-tagged ITR1 gene fragment from pKL35. The fragment was subcloned into pRS415 (Stratagene) to form plasmid pITR1-F. Plasmid pITR1-F was transformed into the S. cerevisiae itr1-T deletion strain PMY218 (Table 1) to assay for complementation of the mutant inositol uptake phenotype. No complementation of uptake activity was detected. Examination of the plasmid sequence revealed that the SphI site used in the religation of the linear PCR product of plasmid pKL35 contained the sequence GCATGC. The presence of an additional ATG sequence before the authentic ATG of the ITR1 ORF may have caused inaccuracy in translational initiation. Plasmid pITR1-F was therefore digested with StuI, and the linearized plasmid was recircularized, deleting the SphI site between the two StuI sites. The resulting plasmid, pITR1-FS, conferred inositol uptake activity that was indistinguishable from wild type. V(max) of the transformant was 7.8 nmol/mg cells/min compared to 7.7 nmol/mg cells/min in wild type. Thus, the additional ATG sequence in the SphI site accounted for the unsuccessful complementation.



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.

Integration of the ITR1-3F Gene into an itr1-T Deletion Strain

The ITR1-3F gene from pITR1-3F was isolated with BamHI/SalI and subcloned into the BamHI/SalI sites of the integrating vector pRS406 (Stratagene). The resulting plasmid pITR1-3F406 was linearized at the URA3 gene in the plasmid with EcoRV (Fig. 1B). The linearized plasmid was used to promote integration at the ura3-52 locus of the itr1-T deletion strain (4) PMY218 to form strain KL22. KL22 was crossed to a wild type strain with a URA3 genetic background. Tetrad analysis of over 20 tetrads showed that the ratio of Ura:Ura spores was 4:0. This indicated the integration took place at the ura3 locus. KL22 was also crossed to an ura3 strain. Tetrad analysis of over 20 tetrads showed that the Ura phenotype segregated 2:2 indicating no additional integrations occurred at other sites in the genome. We also confirmed that one copy of the tagged gene was present in KL22 by PCR analysis. The 5` primer (P.3) hybridized to the region between +928 and +942. The 3` primer (PM2) annealed to region between +2827 and +2844. PCR results of the genomic DNA from KL22 using these two primers showed two bands of the expected sizes with the same intensity. One of them is the amplified product of the itr1-T gene, and the other one is the amplified product of the integrated ITR1-3F. Since the bands were of equal intensity, only one copy of ITR1-3F is present in the genome.

Immunoblot Analysis

Cells were grown as described previously (4) in 100 ml of inositol-deficient (I) medium to an OD of 1.0, then harvested, and washed with potassium phosphate buffer (pH 7). Half of the culture was resuspended in 50 ml of I medium, and the other half was resuspended in 50 ml of the same medium with the addition of 65 µM inositol (I). Cycloheximide (10 µg/ml) was added to both cultures at time = 0 relative to the start of the experiment. 15-ml aliquots of the cultures were removed at the indicated time intervals following the transfer. Whole cell lysates were prepared by resuspending the aliquots in 200 µl of lysis buffer (0.1% SDS, 1% Triton X-100 (Kodak), 0.5% sodium deoxycholate, 50 mM Tris-HCl, pH 7.5, 1% Nonidet P-40 (Calbiochem), 50 mM sodium fluoride, 5 mM EDTA, 5 mM EGTA, 0.02% sodium azide, 150 mM NaCl) in the presence of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml aprotonin). The cells were lysed by vortexing with an equal volume of glass beads (Glasperlen 0.45) until >90% lysis was observed microscopically. The cleared lysate was stored at -80 °C. Protein concentration of each lysate was determined with the Bio-Rad Protein Assay Kit. Bovine serum albumin was used to construct the standard curve for the assay.

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(2)HPO(4), 8 g/liter NaCl, 0.24 g/liter KH(2)PO(4), 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.

Purification of Vacuoles

The isolation of intact vacuoles is a well-established biochemical procedure and was accomplished as in Manolson et al.(7) .

Inositol Uptake Assay

The assay for inositol uptake was performed as described previously(4) . In order to increase the sensitivity of the inositol uptake assay at the basal level, the radiolabeled inositol stock solution (50 µM, 500 µl of total volume) was made by mixing 150 µl of myo-[1,2-^3H]inositol, 19 nmol/ml, 1 µCi/µl, DuPont NEN catalog no NET-906), 9.5 µl of 0.0025 M unlabeled myo-inositol (Sigma), and 340.5 µl of sterile distilled water. The protocol for the inositol uptake assay using this inositol stock solution was the same as previously described(4) .


RESULTS

Addition of Inositol Destabilizes the Itr1 Permease

In order to examine the effect of addition of inositol on the stability of the Itr1 permease directly, the published sequence of the FLAG epitope (8) was used to encode a triple FLAG tag at the N-terminal of the ITR1 gene. Epitope tagging offers specificity and speed in comparison to conventional polyclonal antibodies. In the case of the Itr1 permease, specificity was a particular concern because considerable sequence similarity exists at the amino acid level not only to the Itr2 permease, but also to other yeast sugar transporters(3) . Potential cross-reactions were therefore avoided by this approach. The construction of the ITR1-3F gene is detailed under ``Materials and Methods'' and Fig. 1.

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(max) 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.



Itr1 Permease Is Degraded in Mutants That Transcribe ITR1 Constitutively in the Presence of Inositol

The opi1 and opi3 mutants do not respond to inositol and transcribe ITR1 constitutively. The derepressed level of ITR1 mRNA is normal in the opi3 strain and about 20% higher than normal in the opi1 strain. The uptake activity in the mutants was subject to full repression. Repression occurred normally in the opi3 strain in 2 h and was delayed in the opi1 strain, reaching the basal level in 4 h. When protein synthesis was inhibited by cycloheximide, repression of uptake activity in the opi1 strain required only 2 h, indicating that increased amounts of the Itr1 permease are present in the opi1 strain and suggesting that synthesis of Itr1 permease is also constitutive. No change in repression kinetics in the wild type strain was observed when protein synthesis was inhibited(4) . The immunoblot data in Fig. 4show that the permease is stable in both the opi1 and opi3 strains in the absence of inositol (Fig. 4, A and B, lanes 2-4). Phosphorimage quantitative analysis showed that the derepressed level of Itr1 permease in the opi1 strain was about 2.8 times higher than in wild type, whereas in the opi3 strain permease levels were normal (data not shown). Fig. 4, A and B, lanes 5-7, show that the Itr1 permease is unstable once inositol has been added to the growth medium in the opi1 and opi3 strains. In the opi3 strain, as in wild type, permease is almost undetectable in the immunoblot assay after 1 h of growth in medium containing inositol. In the opi1 strain degradation occurs more slowly than in wild type, especially if protein synthesis is allowed to continue (Fig. 4, lanes 8-10).


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.



The Degradation of the Itr1 Permease Is Blocked in the Vacuolar Protease Mutant pep4

There are two known proteolytic pathways in yeast: one is thought to be cytosolic and the other is vacuolar. Cytosolic proteolysis usually involves the degradation of ubiquitinated proteins; however, ubiquitinated proteins have also been found in the vacuole(9, 10) , and recent data (11) suggest that the vacuole may be the last stop in the degradation pathway of some ubiquitinated proteins. Strains carrying mutations in genes required for ubiquitination were tested to see whether the stability of the Itr1 permease was increased in the presence of inositol. Hochstrasser and colleagues (12) identified two ubiquitination pathways; one pathway includes the genes UBC4 and UBC5, the other UBC6 and UBC7. A ubc6 ubc7 double mutant and a ubc5 strain both show the normal increase in degradation of the permease in response to inositol, and uptake of inositol was also unaffected by the ubc6, ubc7 and ubc5 mutations (data not shown).

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.



The Degradation of the Itr1 Permease Is Blocked by Mutation at END3 or END4

As seen in Fig. 7, inositol-induced degradation of the Itr1 permease was inhibited when 1 mM sodium azide or 20 µM CCCP was incorporated into the medium prior to the addition of inositol. Thus, proteolysis of Itr1 permease requires energy. In mammalian cells and other higher eukaryotes, lysosomal degradation is not energy-dependent(15) . Since degradation of the Itr1 permease is sensitive to energy inhibitors, either vacuolar degradation is energy dependent or the entire degradation process of the permease involves an additional step that is energy dependent or both. Proteins degraded in the vacuole reach the organelle by several pathways, which include autophagocytosis and endocytosis(16) . Autophagocytosis is a common route for the delivery of cytosolic proteins(16) . Extracellular ligands and plasma membrane proteins are often delivered to the lysosome by an energy-dependent process involving an endocytic pathway(17) .


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 pheromonealpha 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.



END3 and END4 Are Not Required for Inositol Uptake

In addition to a role in down-regulation of membrane receptors, receptor-mediated endocytosis is a mechanism for the uptake of nutrients such as iron in mammalian cells(21) . If down-regulation of inositol permease activity is mediated by an endocytic mechanism, does endocytosis also play a role in uptake? It is precisely during down-regulation, and at basal level, that inositol is being transported into the cell, although it is when inositol is absent that the uptake apparatus is fully derepressed. Is a permease endocytosed every time a molecule of inositol is transported in, or does the uptake mechanism function independently? In order to begin to address the relationship between uptake of inositol and endocytosis, derepression of uptake activity in the end3 and end4 strains was compared to both wild type and to strain KL22 carrying the epitope-tagged ITR1-3F allele. The data in Fig. 9shows that derepression of inositol uptake activity was normal in both the end3 and end4 strains, demonstrating that the uptake mechanism does not depend on the endocytic pathway defined by the end3/end4 mutations.


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.



Repression of Uptake Activity Does Not Require Degradation of the Itr1 Permease

Inositol-induced degradation of the Itr1 permease is blocked in the pep4, end3, and end4 strains. Is the inositol uptake activity of the permease still subject to repression in these strains, or does the block in degradation prevent down-regulation of activity? To address this question, we carried out a kinetic study of repression of inositol uptake in the pep4, end3, and end4 strains. The mutant strains were grown in medium without inositol, assayed to establish the derepressed level of activity, and then 65 µM inositol was added to the derepressed cultures to start the experiment. Uptake activity was assayed at different time points after the addition of inositol. The data in Fig. 10shows that full repression of uptake activity to basal level occurred in the end3, end4, and pep4 mutants although there was a slight delay in all three strains.


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.

Inactivation of Uptake Activity is Reversible When Endocytosis Is Blocked

The end3 and end4 mutations were identified by their failure to internalize alpha factor at 37 °C(6) . Strains with the mutations do not grow at 37 °C and are defective in down-regulation of the pheromone receptor as well as internalization of the alpha factor mating pheromone, although the precise defects that cause these phenotypes are not well understood at the present time. Other vesicle-mediated processes such as secretion and maturation of enzymes seem to be unaffected in end3 and end4 strains(6) , and results presented earlier in this paper indicate that targeting of the Itr1 permease to the plasma membrane also does not depend on the END3 or END4 gene products.

Since neither alpha 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. (^2)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.''



Rapid Turnover of the Itr1 Permease Continues during Basal Level Uptake: Stability Is Not Reestablished

We have established that uptake of inositol does not require endocytosis. However, during inositol-induced down-regulation, the rate of endocytic degradation of the Itr1 permease increases; the rate of degradation exceeds the rate of synthesis so that the concentration of permease is substantially reduced at basal level. At this point, questions arise about the regulation of uptake while inositol is actually being transported by the Itr1 permease into the interior of the cell. Once basal level is reached, does the permease regain stability, or does the permease remain short lived, requiring continuing protein synthesis to maintain basal level uptake of inositol.

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.''




DISCUSSION

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 (^3)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(2) 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.


FOOTNOTES

*
This work was supported by National Science Foundation Grant MCB-9118355 (to P. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 410-455-3484; Fax: 410-455-3875; mcgraw{at}umbc.edu.

(^1)
The abbreviations used are: PC, phosphatidylcholine; CCCP, carbonyl cyanide p-chlorophenylhydrazone; ORF, open reading frame; PCR, polymerase chain reaction.

(^2)
K. Lai and P. McGraw, unpublished results.

(^3)
S. Naronha, A. Moreira, and P. McGraw, unpublished results.


ACKNOWLEDGEMENTS

We thank Howard Riezman and Mark Hochstrasser for providing strains and Morrie Manolson for advice on vacuole purification and for the anti-Vph1 antibody.


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