A Mammalian Lysosomal Membrane Protein Confers Multidrug Resistance upon Expression in Saccharomyces cerevisiae*

Douglas L. HogueDagger , Lilli Kerby, and Victor Ling

From the British Columbia Cancer Research Centre, Vancouver, British Columbia V5Z 1L3 Canada

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mouse transporter protein (MTP) is a highly conserved polytopic membrane protein present in mammalian lysosomes and endosomes. The role of MTP in regulating the in vivo subcellular distribution of numerous structurally distinct small molecules has been examined in this study by its expression in a drug-sensitive strain of the yeast Saccharomyces cerevisiae. Surprisingly, the expression of MTP in membranes of an intracellular compartment resulted in a cellular resistance or hypersensitivity to a range of drugs that included nucleoside and nucleobase analogs, antibiotics, anthracyclines, ionophores, and steroid hormones. The intracellular bioavailability of steroid hormones was altered by MTP, as determined using an in vivo glucocorticoid receptor-driven reporter assay in yeast, suggesting that the MTP-regulated drug sensitivity arose due to a change in the subcellular compartmentalization of steroid hormones and other drugs. MTP-regulated drug sensitivity in yeast was blocked to varying degrees by compounds that inhibit lysosomal function, interfere with intracellular cholesterol transport, or modulate the multidrug resistance phenotype of mammalian cells. These results indicate that MTP is involved in the subcellular compartmentalization of diverse hydrophobic small molecules and contributes to the inherent drug sensitivity or resistance of the mammalian cell.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The mammalian cell is exquisitely compartmentalized into organelles, distinct vesicle populations, cytoplasm, and the extracellular space. Metabolic and catabolic processes present within each of these compartments require the presence of one or more different types of small molecules. Therefore, the transport and/or retention of these small molecules between these subcellular compartments must be tightly regulated to maintain the integrity of these cellular processes. The lipid membrane architecture of cellular organelles, vesicles, and the plasma membrane dictates that the compartmentalization of small hydrophilic molecules occur by protein-mediated transport processes. Many transporters of hydrophilic molecules, such as amino acids (1), hexoses (2), and nucleosides (3), have been identified in the plasma membrane. Membrane proteins that mediate the flux of nucleotide, nucleotide sugars, and nucleotide sulfate into the Golgi and endoplasmic reticulum (4), amino acid neurotransmitters and biogenic amines into storage vesicles (1, 5), and substrates across the inner mitochondrial membrane (6) have also been identified and characterized.

In contrast, mechanisms or transport processes responsible for regulating a subcellular compartmentalization of small hydrophobic molecules, which can readily permeate lipid membranes, are not as well understood. Plasma membrane transporters of this type include the multidrug efflux pump P-glycoprotein (7), the multidrug resistance-associated protein (8), the phosphocholine translocase MDR2 (9), bile acid transporters (10, 11), and fatty acid transporters (12). P-glycoprotein and multidrug resistance-associated protein found to be present within intracellular compartments presumably provide a similar function to their plasma membrane counterparts (8, 13). However, little else is known of proteins that mediate the intracellular transport of small hydrophobic molecules.

Mouse transporter protein (MTP)1 is a murine 4-transmembrane spanning protein present in lysosomes and late endosomes (14),2 and its transcript (or that of its mammalian homologs) is expressed in most, if not all, tissues and cell types (15).3 MTP exhibits 98% identity with its human homolog and thus represents a highly conserved mammalian protein. It is 233 amino acids in length and possesses a hydrophilic C terminus containing several tyrosine-based sorting motifs (YXX*, where * represents a bulky hydrophobic residue) that have been demonstrated, in other membrane proteins, to be responsible for localization to intracellular compartments (16). A phenotypic selection assay using Saccharomyces cerevisiae originally identified a truncated MTP that could mediate a cellular uptake of thymidine by its inappropriate expression at the plasma membrane (15). Based upon this observation, MTP was predicted to be involved in the subcellular compartmentalization of nucleosides.

We are interested in defining further the functional role of MTP in the subcellular compartmentalization of various types of small molecules within the mammalian cell. However, it is exceedingly difficult to examine the in vivo distribution of small molecules in mammalian cells, the exception being those molecules that are intrinsically fluorescent, such as anthracyclines (17). The yeast S. cerevisiae has previously been used as a heterologous expression system to study the function of MTP (15) and other mammalian membrane proteins, including the anion exchanger AE1 protein (18), Golgi nucleotide-galactose transporters (19), the cystic fibrosis transmembrane conductance regulator (20), and a vesicular copper transporter (21). On this basis, we have utilized a sensitive in vivo reporter system that employs a drug-sensitive strain of S. cerevisiae to study the physiological role of MTP in regulating the subcellular distribution of small molecules (22). The S. cerevisiae protein data base contains no proteins that exhibit any significant identity with MTP; therefore, interference by a yeast ortholog of MTP would not be expected.3

The drug-sensitive yeast strain lacks the plasma membrane proteins Pdr5 and Snq2, which are ATP-binding cassette transporters that mediate a cellular resistance to a wide variety of structurally diverse drugs by acting as drug efflux pumps (23-28). In essence, these transporters act to provide an extracellular compartmentalization of drugs. The absence of these two transporters allows for the accumulation of toxic concentrations of drug within the cell that ultimately leads to cell death. Should the expression of MTP in this yeast regulate the distribution of these toxic drugs between subcellular compartments, a subcellular sequestration and altered cytotoxicity of the drug may arise. Evidence to support a functional role for MTP in regulation of the subcellular distribution of several structurally distinct small molecules (or their metabolites) is presented.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

MTP Expression Vectors-- The open reading frame of MTP was amplified from pMTP (15) by the polymerase chain reaction method using oligonucleotides that corresponded to the 5' (5'-CGCATCCATGGTGTCCATGACTTTC-3', NcoI site underlined) and 3' (5'-GGCATGGTACCTCAGGCAGGCAGGTAAGGA-3', KpnI site underlined) termini. The polymerase chain reaction product was subjected to Taq DyeDeoxy terminator cycle sequencing using an Applied Biosystems model 310 DNA sequencer. The open reading frame of MTP was subcloned into the yeast expression vector pYPGE15 to generate pYP-MTP. The pYPGE15 vector, a gift from Joe Brunelli (Washington State University), contains a URA3 auxotrophic marker, 2 µm origin, a constitutive phosphoglycerate kinase promotor, and a CYC1 transcriptional terminator.

Cell Culture-- The yeast strains YYM4 (MATa ura3-52 leu2-Delta 1 his3-Delta 200 ade2-101oc lys2-801amb Delta pdr5::TRP1 Delta snq2::hisG) and YPH499 (MATa ura3-52 leu2-Delta 1 his3-Delta 200 ade2-101oc lys2-801amb trp1-Delta 63) were kindly provided by Karl Kuchler (University and Biocenter of Vienna). Yeast strain YNK410 (MATa ura3-52 leu2-Delta 1 his3-Delta 200 ade2-101oc lys2-801amb trp1-Delta 63 Delta pdr5::GT3Z) was obtained from Anastasia Kralli (Biozentrum, University of Basel). Yeast strains were grown at 30 °C on YPD or selective media that contained auxotrophic supplements for plasmid maintenance as described by Kaiser et al. (29).

Cellular Fractionation of Yeast-- Yeast was grown in selective medium to an A600 of 1.0 and diluted 5-fold into 500 ml of YPD. The culture was grown to an A600 of 1.0, whereupon sodium azide was added (final concentration, 10 mM) and culture was chilled in ice water. Cells were collected by centrifugation (4000 × g for 10 min) and suspended in 10 ml of SB-Zymolyase (1.4 M sorbitol, 50 mM potassium phosphate, pH 7.5, 10 mM sodium azide, 40 mM beta -mercaptoethanol, 0.3 mg/ml Zymolyase 100T) and incubated at 30 °C for 40 min. Spheroplasts were pellet by centrifugation (700 × g for 10 min) and suspended in 5 ml of lysis buffer (0.8 M sorbitol, 10 mM MOPS, pH 7.2, 1 mM EGTA) that contained Complete protease inhibitor mixture (Roche Molecular Biochemicals). Spheroplasts were lysed by Dounce homogenization, and homogenate was subjected to centrifugation (2500 × g, 10 min, 4 °C) to pellet unbroken cells. The supernatant was collected, and 1-ml portions of the homogenate were layered onto a discontinuous gradient composed of 1 ml each of 12, 18, 24, 30, 36, 42, 48, 54, and 60% (w/v) sucrose in lysis buffer. Gradients were centrifuged (100,000 × g for 16 h at 4 °C), and 1-ml fractions were collected from the bottom of the gradient. Total cellular membranes were collected by centrifugation (100,000 × g for 60 min at 4 °C) of the Dounce homogenate.

Drug Sensitivity Assays-- The sensitivity of yeast to different chemical compounds was initially screened using a paper disc assay. Yeast was grown in appropriate selective medium and plated (0.15 A600 units) in top agar (0.7% agarose in selective medium) onto selective medium-agar plates. A 5-µl aliquot of drug dissolved in either ethanol or Me2SO was spotted onto a paper disc (Whatman no. 5, 7 mm diameter), which was then laid on the top agar. Plates were incubated at 30 °C for 3 days and examined for cleared zones of growth inhibition. Ethanol or Me2SO alone spotted onto paper discs did not inhibit growth.

A quantitative drug sensitivity assay was performed using a method similar to that of Egner et al. (22). Various concentrations of drug prepared in 55 °C selective medium-agar were plated in triplicate into 24-well plates. Actively growing cultures of cells were diluted to A600 values of 0.12, 0.012, and 0.0012, and then 10-µl aliquots of each dilution were spotted in triplicate onto drug-containing medium. Plates were incubated at 30 °C for 3 days. A quantitative measure of the drugs effect was determined based upon the concentration of drug that inhibited colony formation by 50% (IC50 value). Modulator studies were performed as above except using a single concentration of drug in the absence or presence of modulating drug.

Steroid Hormone Response Assays-- Assays were performed using the yeast strain YNK410, which harbors the genomically integrated reporter gene GT3Z (22), which had been transformed with the glucocorticoid receptor expression plasmid pRS314-GN795 (30) and either pYPGE15 or pYP-MTP. Cell cultures were grown overnight at 30 °C in selective media, diluted 1:10 into fresh selective media that contained dexamethasone, progesterone, or ethanol (carrier), and then grown for 14 h. Hormone-induced expression of beta -galactosidase (beta -gal) in cell lysates was measured using a glass bead lysis/o-nitrophenyl-beta -D-galactosidase assay method (29). Reactions were terminated after identical periods of incubation, and A420 was measured. Protein content of cell lysates was determined, and specific activity of beta -gal in cell lysate was expressed as nmol/min/mg of protein.

Immunological Methods-- Yeast protein samples were denatured in 4 M urea prior to being subjected to electrophoretic separation by SDS-polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene fluoride membranes. Membranes were sequentially incubated with primary antibodies and peroxidase-conjugated anti-rabbit IgG (Jackson Immunologicals) as described previously (15) and visualized using the enhanced chemiluminescence method (Amersham Pharmacia Biotech). Rabbit antiserum used was specific for DPAP A (Tom Stevens, University of Oregon), Pep12p (Scott Emr, University of California, San Diego, CA), Vma4p (Daniel Klionsky, University of California, Davis, CA), Pma1(Andre Goffeau, Universite Catholique de Louvain), Sec61p (Randy Schekman, University of California, Berkeley, CA), and MTP (15).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression of MTP in YYM4 Yeast-- The open reading frame of the MTP cDNA was cloned into a high copy yeast expression vector, and its expression in YYM4 cells was examined by immunoblotting analysis. MTP was present as a low abundance protein in the membranes of cells transformed with the MTP-containing vector, but was not detected in cells transformed with the parental pYPGE15 vector (Fig. 1A). The cellular location of MTP in yeast was assessed by immunoblot analysis of subcellular fractions isolated by means of discontinous sucrose gradient centrifugation (Fig. 1B). MTP appeared to colocalize to fractions most tightly associated with antibody markers for Golgi (DPAP A) and prevacuolar vesicles (Pep12p), but not with those for plasma membrane (Pma1), endoplasmic reticulum (Sec61p), and vacuole (Vma4p). These results indicated that the localization of MTP was restricted to intracellular membranes, most probably those of Golgi or Golgi-derived vesicles and/or the endosome-like prevacuolar vesicles (31).


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Fig. 1.   Expression and subcellular localization of MTP in S. cerevisiae. YYM4 yeast transformed with pYPGE15 or pYP-MTP were examined for the presence of MTP. A, total membrane fractions were prepared, and equal quantities were subjected to SDS-polyacrylamide gel electrophoresis and immunoblot analysis with anti-MTP antibody. Arrow indicates immunoreactive species. B, YYM4 yeast transformed with pYP-MTP were lysed and subjected to subcellular fractionation by centrifugation on a discontinous 12-60% (w/v) sucrose gradient. Equal portions of gradient fractions were subjected to SDS-polyacrylamide gel electrophoresis and immunoblot analysis using anti-MTP antibody or antibodies specific for markers of the plasma membrane (Pma1), Golgi (DPAP A), endoplasmic reticulum (Sec61p), vacuole (Vma4p), and prevacuolar (Pep12p) compartments.

Drug Sensitivity of MTP-expressing Yeast-- MTP-expressing yeast were initially screened for sensitivity to a variety of compounds using a paper disc assay. Drug sensitivity, relative to control YYM4 yeast, was assigned based on the diameter of the zone of growth inhibition (Fig. 2). The expression of MTP in YYM4 yeast conferred increased resistance or a hypersensitivity to a range of compounds that included antibiotics, anthracyclines, neutral and carboxylic ionophores, nucleoside and nucleobase analogs, steriod hormones, dihydropyridines, phenothiazines, and cationic lipophilic molecules (Table I). MTP-expressing yeast did not exhibit altered sensitivity toward cyclohexamide, vancomycin, tamoxifen, or cisplatin, as compared with control yeast. The parental strain, YPH499, exhibited none or little sensitivity to any of the compounds examined (not shown).


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Fig. 2.   Assessment of drug sensitivity of MTP-expressing yeast using the disc assay. YYM4 yeast containing pYPGE15 vector (control) or pYP-MTP (MTP) were plated in top agar and subjected to paper disc assays to determine MTP-mediated drug resistance. In this example, 5-µl aliquots of the following drug solutions (in Me2SO) were deposited onto individual paper discs: nicardipine (194 mM), trifluoperazine (312 mM), progesterone (636 mM), or ethidium bromide (220 mM). Zones of growth inhibition were examined after 3 days of incubation at 30 °C.

                              
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Table I
Effect of expression of MTP on the drug sensitivity of YYM4 yeast
YYM4 yeast transformed with pYPGE15 or pYP-MTP was subjected to paper disc drug assays. Zones of growth inhibition for each drug were compared to pYPGE15-transformed yeast. Hypersensitivity led to increased diameters of zones, resistance led to decreased diameters, and no change resulted in identical diameters.

The degree of cellular drug resistance or sensitivity conferred by the expression of MTP toward a number of compounds was quantitated using a colony formation assay, an example of which is presented in Fig. 3. The concentrations required to inhibit colony formation by 50% (IC50) determined for control and MTP-expressing yeast are shown in Table II. MTP conferred an increased resistance of approximately 1.5-15-fold to drugs that included carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), nicardipine, erythromycin, progesterone, and rhodamine 123. Conversely, MTP-expressing yeast was 2-50-fold more sensitive than control cells to trifluoperazine, TPP bromide, 5-fluorouracil, and 5-fluorouridine. The expression of MTP within the membranes of the Golgi and/or prevacuolar compartments could significantly modify the cytotoxicity of many structurally distinct drugs, suggesting that this effect occurred via a change in the subcellular distribution and subsequent intracellular bioavailability of these drugs.


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Fig. 3.   Quantitation of drug sensitivity of MTP-expressing yeast by plating assay. A quantitative measure of the drug resistance or sensitivity was determined by measuring the colony formation of serially diluted cultures of YYM4 yeast containing pYPGE15 vector (control) or pYP-MTP (MTP) that had been aliquoted onto selective media that contained either drug carrier (Me2SO) or increasing concentrations of drug solution. Shown are examples using drug carrier (Me2SO), nicardipine (670 µM), trifluoperazine (440 µM), progesterone (500 µM), erythromycin (1500 µM), and 5-fluorouridine (40 µM).

                              
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Table II
Quantitation of the MTP-mediated resistance/hypersensitivity in YYM4 yeast
YYM4 yeast transformed with pYPGE15 or pYP-MTP were subjected to the quantitative plating assay using graded concentrations of each drug. The IC50 is indicated.

Modulation of MTP-mediated Drug Sensitivity-- The profile of drug resistance mediated by MTP exhibited a striking similarity to that exhibited by multidrug-resistant (MDR) mammalian cells that overexpress the P-glycoprotein (also termed MDR1). P-glycoprotein is a ATP-binding cassette transporter that mediates resistance to rhodamines, anthracyclines, steroid hormones, and carboxylic ionophores (7, 32). Cells that overexpress P-glycoprotein also exhibit a collateral sensitivity or hypersensitivity toward various hydrophobic molecules (33); however, P-glycoprotein does not mediate resistance or hypersensitivity toward 5-fluorouracil or 5-fluorouridine (34). The MDR phenotype in mammalian cells can be blocked by the presence of one of many different modulators, which are hydrophobic molecules that generally possess a tertiary nitrogen (35).

We assessed the ability of several MDR modulators to block the drug hypersensitivity or resistance mediated by the expression of MTP in YYM4 yeast through the use of the quantitative plating assay, an example of which is shown in Fig. 4. The modulators, which included amiodarone, dipyridamole, reserpine, and verapamil, did not exhibit toxicity to YYM4 yeast at the concentrations utilized (not shown). All modulators completely failed to reverse the hypersensitivity to 5-fluorouridine or the resistance to nicardipine (Table III). Conversely, all modulators completely blocked resistance to progesterone. Resistance to CCCP, erythromycin, and rhodamine 123 was blocked to various degrees by each modulator. These results indicated that these MDR modulators were, to varying extents, able to block MTP-mediated resistance/hypersensitivity. This modulation effect was dependent upon drug-modulator combinations present, a characteristic that is shared with the interaction of these modulators with P-glycoprotein (32, 35).


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Fig. 4.   Modulation of drug sensitivity mediated by MTP. The ability of MDR modulators to block MTP-mediated drug resistance or sensitivity was assessed by the inclusion of modulator in quantitative plating assays that employed a single drug concentration. In this example, YYM4 yeast containing pYPGE15 vector (control) or pYP-MTP (MTP) was aliquoted onto selective media that contained progesterone (500 µM) alone or in addition to verapamil (1570 µM), dipyridamole (400 µM), reserpine (470 µM), or amiodarone (650 µM).

                              
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Table III
Effect of modulators on MTP-mediated drug resistance/hypersensitivity
YYM4 yeast transformed with pYP-MTP was plated onto medium that contained no drug, 5-fluorouridine (40 µM), CCCP (39 µM), erythromycin (2990 µM), rhodamine 123 (160 µM), nicardipine (420 µM), or progesterone (500 µM) in the presence or absence of modulators as described in Fig. 4. The ability to form colonies, as compared to in the absence of modulator, was assessed.

Regulation of Intracellular Steroid Bioavailability by MTP-- The mechanism by which steroid hormones exert their cytotoxicity in yeast is not known but is thought to be related to their interference in the pathways of biosynthesis or cellular transport of sterols. The yeast ATP-binding cassette transporter Pdr5 can reduce the cellular accumulation of steroid hormones by transporting these molecules into the extracellular space, thereby circumventing their toxic effects (22, 25, 27). Decreased cellular accumulation of steroid hormones mediated by Pdr5 has been directly demonstrated by an in vivo reporter assay in the YNK410 yeast strain (22, 25). YNK410 yeast harbors (i) a genomically integrated cassette composed of glucocorticoid response elements upstream of a beta -gal reporter gene, and (ii) a disrupted PDR5 gene (the absence of the Pdr5 transporter allows for an intracellular accumulation of steroid hormones). Expression of a glucocorticoid receptor in YNK410 yeast drives transcription of the beta -gal reporter gene in a steroid hormone-dependent fashion and therefore provides a direct measurement of the intracellular bioavailability of steroid hormone.

MTP was able to mediate a cellular resistance to steroid hormone toxicity; therefore, we used the YNK410 beta -gal reporter system to measure whether this resistance was associated with an alteration in the intracellular bioavailability of steroid hormones. Exposure of MTP-expressing cells to 10 µM dexamethasone or progesterone induced approximately 15- and 25-fold greater levels of beta -gal activity, respectively, than compared with control cells under identical conditions (Fig. 5A). This effect of dexamethasone and progesterone was dose-dependent, because greater levels of beta -gal expression were observed in the presence of MTP than in its absence over a 100-fold concentration range (Fig. 5B). The apparent saturation and decline of beta -gal expression that was observed at higher concentrations of dexamethasone has previously been documented in this assay and the ability of dexamethasone to induce greater overall levels of beta -gal expression than progesterone in both MTP-expressing and -nonexpressing cells correlated with the different affinities of these steroid hormones for the glucocorticoid receptor (36). These experiments revealed that the expression of MTP in YNK410 cells resulted in an increased intracellular bioavailability of progesterone and dexamethasone that subsequently allowed for greater steroid hormone interaction with the glucocorticoid receptor.


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Fig. 5.   Effect of MTP expression on steroid hormone response. YNK410 cells harboring pRS314-GN795 and either not expressing (solid) or expressing (hatched) MTP were grown in steroid hormone-containing medium, and cell lysates were prepared and assayed for beta -gal activity. A, activity in cells grown in 10 µM dexamethasone or progesterone. Presented are mean measurements for three independent assays. S.E. values are indicated (bars) or were too small to be observed. B, activity in cells grown in increasing concentrations of dexamethasone or progesterone. Shown is a single representative assay.

The correlation of steroid resistance with an increased intracellular bioavailability in MTP-expressing cells suggests that MTP does not provide a cellular resistance to steroid hormone toxicity by tightly binding and sequestering these molecules, but rather by mediating a change in the subcellular localization of steroid hormones that prevents their interference in pathways of sterol transport or biosynthesis.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

MTP is a member of the mammalian 4-transmembrane spanning protein superfamily, which includes the "tetraspanin" family (37), the PMP22 family (38), the "synaptic vesicle" family (39, 40), the "gap/tight junction" family (41, 42), TWIK-1 (43), and micosomal epoxide hydrolase (10). Members of the synaptic vesicle and gap/tight junction families participate in the formation of pores, TWIK-1 functions as a K+ channel, and microsomal epoxide hydrolase can transport bile acids. However, the physiological functions of the majority of these four-transmembrane-spanning proteins have not yet been defined, and most are postulated to be involved in roles ranging from pore formation to cell proliferation and differentiation. The present study, which employed in vivo yeast reporter systems, has demonstrated that MTP expressed in intracellular organelles can function to regulate the cytotoxicity and subcellular localization of molecules that include cationic amphiphilic drugs, hydrophobic anions and cations, hydrophobic steroids, and the more hydrophilic charged nucleobases/nucleoside analogs.

Transport processes that mediate the flux of small hydrophilic molecules across the membranes of lysosomes and endosomes in mammalian cells are relatively well documented (44, 45). In contrast, the mechanisms of lysosomal and endosomal compartmentalization and/or retention of small hydrophobic molecules are not as well understood. Cationic amphiphilic drugs, such as anthracyclines, nicardipine, trifluoperazine, and erythromycin, are concentrated within lysosomes and endosomes due to the passive diffusion of drug across the organelle membrane and their subsequent protonation into a membrane impermeable form (17, 46-48). Hydrophobic anions (i.e. nigericin and monensin) and cations (i.e. TPP, CCCP, and ethidium) also become concentrated inside endosomes and lysosomes (22, 49-53). The mechanism responsible for localization of these molecules is apparently driven by the activity of V-type H+-ATPases (54), but there also appears to be a second, as yet unknown, mechanism for uptake and/or binding of these types of molecules in lysosomes (46). Progesterone and other steroid hormones can block the transport of cholesterol from lysosomes by an unknown mechanism (55-57), implying the presence of steroid hormones within membranes of these organelles.

These limited studies on the interaction of hydrophobic molecules with lysosomes and endosomes suggest that their compartmentalization arises, at least in part, through mediated transport processes. The ability of MTP, a membrane protein localized to lysosomes and endosomes, to regulate the distribution of these hydrophobic molecules in yeast reporter systems suggests that MTP may be involved in regulating the localization and/or retention of these hydrophobic molecules within these organelles in mammalian cells.

In light of the apparent interaction of MTP with hydrophobic small molecules, the ability of MTP to regulate the cytotoxicity of 6-aza-adenine, 5-fluorouracil, and 5-fluorouridine is intriguing. Nucleobases and nucleosides, including 5-fluorouracil and 5-fluorouridine, are rapidly metabolized into their respective nucleotide forms upon uptake into mammalian cells (58, 59); therefore, these predominant nucleotide forms should be the substrates for any subcellular compartmentalization event. The extent of distribution of these nucleotides into lysosomes and endosomes is unclear, although evidence for an energy-dependent vesicular nucleotide transporter exists (60). MTP has previously been shown to mediate an increased membrane permeability toward nucleosides (15); however, these studies were preformed under in vivo conditions that may have allowed for conversion of nucleoside to its nucleotide form. In addition, the nucleobases and nucleoside employed in the present study differ from their physiological counterparts by the presence of a positive or negative charge on their purine or pyrimidine moieties, which in itself may be an important factor for regulation by MTP. Clearly, further studies are required to define the interaction of MTP with nucleotides, nucleosides, and nucleobases.

The MDR phenotype in mammalian cells that arises due to the overexpression of P-glycoprotein can be blocked by MDR modulators, the action of which has been attributed to their ability to bind to P-glycoprotein and inhibit its function (32, 35). It also appears these modulators can induce subtle changes in the inherent drug sensitivity of non-drug-resistant or "normal" cells (61, 62), but their mechanisms of action under these conditions have not been defined. It is relevant that MDR modulators can block MTP-mediated resistance to several different drugs in our yeast reporter system, because this observation suggests that MDR modulators may be capable of altering the inherent drug sensitivity in mammalian cells by interfering with the function of MTP in lysosomes or endosomes.

Most MDR modulators can induce an in vivo inhibition of lysosomal function (47, 63, 64), but there is clear evidence for a specific interaction of MDR modulators in the transport of cholesterol from lysosomes. Cholesterol is transported bidirectionally between the lysosomes and the plasma membrane (65), and it has been suggested that this transport occurs via mediated processes based upon the ability of (i) steroid hormones and cationic amphiphilic MDR modulators to block these transport processes and induce a lysosomal accumulation of cholesterol (55-57, 65), and (ii) oxysterols to reverse this cholesterol accumulation in a manner independent of lysosomal pH (65). Proteins involved in this homostasis of intracellular cholesterol transport would be expected highly conserved and expressed in all tissues and cell types. One such protein is NPC1, a membrane protein implicated in Niemann-Pick C disease (66). An absence of NPC1 leads to the disruption of intracellular cholesterol transport; however, its actual function and subcellular location are unknown (67). It is interesting that there exists an interaction between MTP, a highly conserved and widely expressed lysosomal/endosomal protein, and both steroid hormones and MDR modulators. Our study suggests that MTP may be involved in the homostasis of intracellular transport of steroid hormones or cholesterol.

MTP may regulate the intracellular compartmentalization of small molecules in the yeast reporter systems and mammalian cells by one of several mechanisms. The protein may be a transporter that interacts directly with small molecules to facilitate their flux across the membranes of lysosomes and endosomes. Conversely, the presence of MTP may regulate the function of other transporters by direct protein-protein interaction or by indirectly altering the character of the membrane lipid, the transmembrane H+ gradient, or the lumenal contents of the lysosome/endosome. Future studies will be necessary to define the mechanistic nature of MTP, but whichever mechanism is responsible, this study clearly demonstrates that MTP is capable of regulating the subcellular compartmentalization of small hydrophobic molecules.

    FOOTNOTES

* This study was supported by the National Cancer Institute of Canada and the Alberta Heritage Foundation for Medical Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of an Alberta Heritage Foundation for Medical Research Fellowship. To whom correspondence should be addressed: BC Cancer Research Centre, 601 W. 10th Ave., Vancouver, British Columbia V5Z 1L3, Canada. Tel.: 604-877-6000; Fax: 604-877-6155; E-mail: dhouge{at}bccancer.bc.ca.

2 C. Cass, personal communication.

3 D. Hogue, unpublished results.

    ABBREVIATIONS

The abbreviations used are: MTP, mouse transporter protein; beta -gal, beta -galactosidase; CCCP, carbonyl cyanide m-chlorophenyl-hydrazone; MDR, multidrug-resistant; TPP, tetraphenylphosphonium; MOPS, 4-morpholinepropanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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