[CANCER RESEARCH 59, 4890 – 4897, October 1, 1999]
Mouse Transporter Protein, a Membrane Protein That Regulates Cellular
Multidrug Resistance, Is Localized to Lysosomes1
Miguel A. Cabrita, Tom C. Hobman, Douglas L. Hogue, Karen M. King, and Carol E. Cass2
MRC Molecular Biology of Membranes Group [M. A. C., K. M. K., C. E. C.] and Departments of Biochemistry [M. A. C., K. M. K., C. E. C.], Cell Biology [T. C. H.], and
Oncology [C. E. C.], University of Alberta, and Cross Cancer Institute [M. A. C., K. M. K., C. E. C.], Edmonton, Alberta T6G 1Z2, Canada, and British Columbia Cancer
Research Centre, Vancouver, British Columbia V5Z 1L3, Canada [D. L. H.]
ABSTRACT
Mouse transporter protein (MTP), a small, highly conserved mammalian intracellular membrane protein with four putative transmembrane
domains, has been implicated in the transport of nucleosides and/or
related molecules across intracellular membranes. The production of
recombinant MTP in Saccharomyces cerevisiae alters sensitivity of yeast
cells to a heterogeneous group of compounds (e.g., antimetabolites, antibiotics, anthracyclines, ionophores, and steroid hormones) by changing
the subcellular compartmentalization of these drugs, suggesting that MTP
functions similarly in higher organisms. The present study was undertaken to define the intracellular location of MTP in mammalian cells.
Native MTP was not detected by indirect immunofluorescence in cell types
that expressed MTP mRNA; therefore, a hemagglutinin (HA) epitopetagged version of MTP was produced in cultured BHK21 cells by transient
transfection, and its distribution within cells was determined by confocal
microscopy using antibodies directed against the HA epitope and various
organellar proteins. Antibodies directed against HA-MTP colocalized with
antibodies against late endosomal and lysosomal proteins but not with
antibodies against either Golgi or early endosomal proteins. Analysis of
subcellular fractions from rat liver by immunoblotting with antibodies
directed against MTP demonstrated the presence of a MTP-like protein in
Golgi- and lysosome-enriched membranes but not in mitochondria. These
results indicate that MTP resides in late endosomes and lysosomes, a
finding that is consistent with the proposed role for MTP in the movement
of a variety of small molecules across endosomal and lysosomal membranes. MTP shares a number of characteristics with other lysosomeassociated proteins. We, therefore, propose that it be redesignated murine
lysosome-associated protein transmembrane 4.
INTRODUCTION
A novel protein with nucleoside transport activity has been identified recently by the ability of a partial cDNA isolated from mouse
leukemia L1210 cells to complement a thymidine transport deficiency
in the yeast Saccharomyces cerevisiae (1). The full-length cDNA
(GenBank accession number U34259) encodes a small hydrophobic
protein named MTP3 with 233 amino acids and four predicted transmembrane domains (1). MTP is highly conserved, with 97% identity
at the amino acid level to a human protein of unknown function,
HUMORF13 (GenBank accession number D14696), suggesting an
Received 4/27/99; accepted 8/9/99.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from the Medical Research Council of Canada
and a Canadian Cancer Society operating grant from the National Cancer Institute of
Canada. M. A. C. was a recipient of a University of Alberta Faculty of Medicine 75th
Anniversary Graduate Studentship. T. C. H. is a Heritage Scholar of the Alberta Heritage
Foundation for Medical Research and a Scholar of the Medical Research Council of
Canada. D. L. H. is a recipient of an Alberta Heritage Foundation for Medical Research
Postdoctoral Fellowship.
2
To whom requests for reprints should be addressed, at Department of Oncology,
Cross Cancer Institute, 11560 University Avenue, Edmonton, Alberta T6G 1Z2, Canada.
Phone: (780) 432-8320; Fax (780) 432-8425; E-mail: [email protected].
3
The abbreviations used are: MTP, mouse transporter protein; BHK, baby hamster
kidney; Cy5, indodicarbocyanine; EST, expressed sequence tag; HA, hemagglutinin; INT,
p-iodonitrotetrazolium violet; LAPTm, lysosome-associated protein transmembrane;
lgp, lysosomal glycoprotein; a-ManII, a-mannosidase II; M6PR, mannose-6-phosphate
receptor.
important role in mammalian cells. Recombinant MTP localized to the
plasma membrane and exhibited low levels of nucleoside transport
activity when produced in truncated form but not when produced in
full-length form in oocytes of Xenopus laevis (1). The functional
results, together with the demonstration of MTP in intracellular membranes of liver subcellular fractions, led to the suggestion by Hogue et
al. (1) that MTP plays a role in the transport of nucleosides and/or
related molecules across intracellular membranes. MTP is structurally
unrelated to the equilibrative nucleoside transporter and concentrative
nucleoside transporter protein families, the molecular identities of
which have been determined by isolation and functional characterization of cDNAs in several heterologous expression systems (reviewed
in Refs. 2– 4).
Recently, full-length MTP was produced in drug-sensitive strains of
S. cerevisiae and found to mediate a multidrug resistance phenotype
(5). Yeast cells with MTP exhibited a collateral: (a) increased resistance toward anthracyclines, carboxylic and neutral ionophores, dihydropyrines, and steroids; and (b) increased sensitivity toward hydrophobic cations (i.e., ethidium and tetraphenylphosphonium),
5-fluorouracil, 5-fluorouridine, and trifluoperazine. MTP was also
shown to alter the subcellular distribution of steroids in yeast. These
observations indicate that the multidrug resistance phenotype resulted
from MTP-mediated alterations in subcellular distribution of drug in
yeast and suggest that a similar role exists for MTP in mammalian
cells (5).
Multidrug resistance can arise in mammalian cells by several different types of biochemical changes that alter sensitivity to cytotoxic
drugs (6). One of the most well-studied and widely accepted mechanisms for the generation of multidrug resistance is the overproduction
of the ATP-dependent drug pump, P-glycoprotein (7). P-glycoprotein
works primarily by mediating the efflux of drugs from cells; however,
many forms of multidrug resistance, including P-glycoprotein-dependent multidrug resistance, have been associated with the sequestration of drugs into subcellular compartments (6, 8 –19). Interestingly, some studies have demonstrated that drug-resistant cell lines
differ from parental cell lines by accumulating drugs in different
subcellular compartments (9 –15, 18). In some cases, lysosomes have
been implicated in the subcellular compartmentalization of the drugs
(9 –14); but in other cases, the vesicular compartment has not been
defined clearly (15, 18). Compartmentalization of cationic amphiphilic drugs into acidic organelles was originally thought to be due only
to the action of proton pumps (20), wherein the transmembrane proton
gradient leads to the intraorganelle trapping of protonated drug. However, recent evidence for the presence of active drug transport processes in lysosomes (21) and the involvement of MTP in the subcellular redistribution of drugs in yeast (5) suggest that protein-mediated
processes are responsible for the accumulation of certain drugs within
acidic vesicles.
In light of the unusual functional characteristics of recombinant
MTP in yeast, the present study was undertaken to define the intracellular location of MTP in mammalian cells. In our previous study
(1), based on the observation that a MTP-like protein was discovered
by immunoblot analysis in subcellular membrane fractions of rat liver
enriched in Golgi membranes, we concluded that MTP normally
4890
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1999 American Association for Cancer
Research.
LOCALIZATION OF MTP (MLAPTM4) TO LYSOSOMES
Fig. 1. Immunoblotting of MTP and HA-MTP in transfected BHK21 cells. Cell lysates
were prepared from BHK21 cells that had been transfected with pcDNA3/HA-MTP,
pcDNA3/MTP3, or pcDNA3 as described in “Materials and Methods.” A, immunoblot
prepared from cell lysates was incubated first with anti-MTP antibodies and then with
horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies. B, the immunoblot
shown in A was stripped, blocked, and incubated with anti-HA antibodies and then with
horseradish peroxidase-conjugated goat anti-rat IgG antibodies. Detection was by ECL as
described in “Materials and Methods.” Left, positions of the protein markers (in thousands).
resides in intracellular membranes. However, MTP is a low-abundance protein and could not be detected by either indirect immunofluorescence of cells or immunoblot analysis of solubilized membranes with MTP-specific antibodies in the cell line (mouse leukemia
L1210) from which its cDNA was initially obtained. To circumvent
this difficulty, we have transiently transfected baby hamster kidney
cells (BHK21) with cDNA encoding an epitope-tagged version of
MTP and then examined for localization by double-label indirect
immunofluorescence using anti-tag antibodies and antibodies to various organellar proteins. In addition, we have examined subcellular
fractions obtained from rat liver for the presence of native MTP by
immunoblotting with anti-MTP antibodies. The highest levels of MTP
were found in late endosomes and lysosomes, indicating that MTP is
a resident protein of lysosomes, thereby substantiating the recent
suggestion (5) that MTP plays a role in the subcellular distribution of
drugs associated with multidrug resistance. Preliminary observations
have been reported previously (22).
MATERIALS AND METHODS
Construction of Expression Vectors. The molecular biology techniques
used are described in Ausubel et al. (23). All DNA primers were synthesized
with a Perkin-Elmer/Applied Biosystems Model 394 DNA synthesizer (Foster
City, CA) using cyanoethyl chemistry. PCR products were sequenced using
PRISM Dye Terminator chemistry with a Perkin-Elmer/Applied Biosystems
Model 373A DNA sequencer (DNA Core Facility, Department of Biochemistry, University of Alberta).
pcDNA3/MTP3. pcDNA3/MTP3 was constructed by ligating the EcoRIMTP3-NotI fragment from pcDMTP3 (1) into the pcDNA3 mammalian expression vector (Invitrogen, Carlsbad, CA).
pcDNA3/HA-MTP. The HA-tagged version of MTP (termed HA-MTP)
was prepared using the PCR. An oligonucleotide primer was used to introduce
a BamHI site (GGATCC), the nonapeptide (Y-P-Y-D-V-P-D-Y-A) HA epitope
tag (24, 25), and a consensus ribosomal binding domain (GCCACCATG; Ref.
26) at the 59 end of the MTP cDNA; this primer (59-GGGATCCGCCACCATGTACCCATACGATGTTCCAGATTACGCTATGGTGTCCATGAGTTTCAAGCGG-39; start site is underlined) was termed MTPHA1. A second
oligonucleotide primer was used to introduce an EcoRI site (GAATTC) after
the stop codon on the 39 end of the MTP cDNA; this primer (59GCAGAGAATTCTCAGGCAGGCAGGTAAGGAGG-39; start site is underlined) was termed MTP-P2. The HA-MTP construct was obtained by amplification from pcDMTP3 (1) using the oligonucleotides MTPHA1 and MTP-P2.
The construct was digested with BamHI and EcoRI and subsequently ligated
into pcDNA3 to produce pcDNA3/HA-MTP.
Cell Culture. BHK21 cells were obtained from the American Tissue Culture Collection (Bethesda, MD) and were cultured in DMEM supplemented
with 10% FCS. Cells were grown as adherent cultures at 37°C in 5% CO2 and
cultured for 20 passages. New cultures were reinitiated from Mycoplasma-free
stock cultures stored in liquid nitrogen. All media and sera were obtained from
Life Technologies (Burlington, Ontario, Canada). Cells were enumerated using
an electronic particle counter (Coulter Electronics, Miami, FL).
Transient Transfections. BHK21 cells were plated at 2 3 105 cells/well in
six-well tissue culture dishes and grown for 18 –24 h until ;60 – 80% confluency was reached. The DNA used in the transfections was purified using Midi
or Mini columns (Qiagen, Mississauga, Ontario, Canada) according to the
manufacturer’s instructions. The cells were transfected with 1 mg of either
pcDNA3/HA-MTP, pcDNA3/MTP3, or pcDNA3 and 5 mg of Lipofectamine
(Life Technologies) according to the manufacturer’s instructions.
Indirect Immunofluorescence. Cultures were grown on 12-mm coverslips
(five coverslips/well) contained in 35-mm wells and transfected exactly as
described above. At 24 – 48 h after transfection, cells were fixed and permeabilized with 100% methanol at 220°C for 6 min. The cells (on coverslips)
were washed twice with PBS and incubated at room temperature for 1 h or 4°C
overnight with 2% goat serum in PBS to block nonspecific binding of antibodies. The cells were then double stained with rat monoclonal anti-HA (from
clone 3F10, 100 ng/ml; Roche Molecular Biochemicals, Laval, Quebec, Canada; Ref. 25) and rabbit polyclonal antibodies against either: (a) a-mannosidase II (anti-a-Man II, diluted 1:500; Ref. 27); (b) mannose-6-phosphate
receptor (anti-M6PR, diluted 1:500; Ref. 28, 29); (c) lysosomal glycoprotein
110 (anti-lgp-110, diluted 1:100; Ref. 30); (d) rab5A (diluted 1:100; Santa
Cruz Biotechnology, Santa Cruz, CA); or (e) a synthetic peptide (TFKRSRSDRFYSTRC) corresponding to residues 5–19 of MTP (anti-MTP, diluted
1:1000; Ref. 1). Cells were washed with 1 ml of PBS three times and incubated
with either: (a) Cy5-conjugated donkey anti-rabbit IgG (diluted 1:250) and
FITC-conjugated goat anti-rat IgG (diluted 1:250); (b) Cy5-conjugated donkey
anti-rabbit IgG (diluted 1:250) and FITC-conjugated goat anti-mouse IgG
(diluted 1:250); or (c) Cy5-conjugated goat-anti-mouse IgG (diluted 1:250)
and FITC-conjugated goat anti-rat IgG (diluted 1:250). The antibody conjugates were from Jackson ImmunoResearch Laboratories (West Grove, PA). All
antibodies were diluted in PBS, and incubations were for 30 min at room
temperature. Coverslips were mounted onto slides in 1 mg/ml paraphenylenediamine/90% glycerol in PBS. Samples were examined with an Axioskop immunofluorescence microscope (Carl Zeiss, Toronto, Ontario, Canada)
or an LSM-510 laser-scanning confocal microscope (Carl Zeiss). Images were
acquired using the LSM-510 software and printed out on an NP-1600 sublimation printer (Codonics, Middleburg Heights, OH).
Subcellular Fractionation. Golgi membranes were isolated according to
the protocol of Morré and Morré (31) with minor modifications. Three freshly
isolated rat livers were placed in ice-cold homogenization buffer [0.5 M
sucrose, 50 mM Tris-HCl (pH 6.9), 5 mM b-mercaptoethanol, and 1% dextran
(Sigma, 500,000 g/mol)], minced with scissors, and homogenized with a
Polytron homogenizer (Brinkmann Instruments, Mississauga, Ontario, Canada). The homogenate was filtered through cheesecloth and centrifuged
(5000 3 g for 15 min at 4°C). Most of the supernatant was discarded, and the
upper one-third of the pellet was resuspended in a small amount of supernatant.
The resulting suspension was layered over a gradient solution (1.2 M sucrose
in distilled water containing 3% dextran) and centrifuged (100,000 3 g for 30
min at 4°C). The Golgi-enriched membranes were removed from the top of the
4891
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1999 American Association for Cancer
Research.
LOCALIZATION OF MTP (MLAPTM4) TO LYSOSOMES
Fig. 2. Presence of MTP and HA-MTP in juxtanuclear vesicles. BHK21 cells were transfected with pcDNA3/HA-MTP, pcDNA3/MTP3, or pcDNA3 as described in “Materials and
Methods.” Cells were fixed, permeabilized, and incubated first with anti-HA antibodies (A, B, and C) or anti-MTP antibodies (D, E, and F), then with FITC-conjugated donkey anti-rat
IgG antibodies (A, B, and C) or Cy5-conjugated goat anti-rabbit IgG antibodies (D, E, and F). Overlapping images are shown in G, H, and I. Bars, 20 mm.
1.2 M sucrose gradient, collected by centrifugation (5000 3 g for 15 min at
4°C), and resuspended in homogenization buffer.
Preparation of lysosomes was performed according to the instructions
accompanying the density gradient media, Nycodenz (Life Technologies) and
according to Graham et al. (32). Freshly isolated rat livers were placed in
ice-cold buffer A [0.25 M sucrose, 5 mM Tris-HCl (pH 7.6), and 1 mM EDTA],
cut into small pieces, and homogenized with a Polytron homogenizer. The
homogenate was filtered through cheesecloth and centrifuged (1,000 3 g for
10 min at 4°C), and the postnuclear supernatant was centrifuged (15,000 3 g
for 10 min at 4°C). The resulting 15,000 3 g pellet was resuspended in 11 ml
of buffer A, which was combined with an equal volume of 50% Nycodenz (w/v
in buffer A). A discontinuous Nycodenz gradient was assembled (0.5 ml of
40% Nycodenz, 1.0 ml of 30% Nycodenz, 3.5 ml of 15,000 3 g pellet in 25%
Nycodenz, 2.0 ml of 23% Nycodenz, 2.0 ml of 20% Nycodenz, 2.0 ml of 15%
Nycodenz, and 1.0 ml of 10% Nycodenz) and centrifuged (52,000 3 g for
1.5 h at 4°C) in a swinging bucket rotor. Lysosome-enriched membranes were
collected from the 15/20% Nycodenz interface.
Mitochondria were isolated from rat livers using the procedure of
Rickwood et al. (33) and Fleischer et al. (34). Freshly isolated livers were
placed in ice-cold homogenization buffer (0.3 M sucrose, 1 mM EGTA, 5
mM 3-(N-morpholino)-propane sulfonic acid, 5 mM potassium phosphate
monobasic, 0.1% BSA (fatty acid free), and 1 mM phenylmethylsulfonyl
fluoride, pH 7.4) and rinsed with homogenization buffer to remove excess
blood. The remainder of the procedure was carried out on ice. The livers
were minced and washed with homogenization buffer. Homogenization
buffer (2 ml/g liver) was added, and the minced liver was homogenized for
1 min using a motorized Polytron homogenizer. The resulting homogenate
was strained through cheesecloth and centrifuged (1,000 3 g for 10 min at
4°C). The supernatant was decanted and then centrifuged (10,000 3 g for
10 min at 4°C), and the supernatant from this centrifugation was discarded.
Next, the crude mitochondrial pellet was separated from the pelleted RBCs
and resuspended in homogenization buffer (35 ml). This centrifugation step
was repeated until the RBC pellet was removed. The crude mitochondrial
pellet was further purified using centrifugation (4,000 3 g for 40 min at
4°C) through a continuous sucrose gradient (0.75–1.75 M sucrose in 1 mM
EGTA, 5 mM 3-(N-morpholino)propanesulfonic acid, 5 mM potassium
phosphate monobasic, and 0.1% BSA, pH 7.4). The resulting fractions were
collected, resuspended in homogenization buffer, centrifuged (10,000 3 g
for 20 min at 4°C) and resuspended at concentrations of 10 –20 mg/ml. The
functionality of the mitochondria was determined by measurement of the
respiratory control ratio using a Clarke oxygen electrode (33).
Golgi and lysosomal preparations were either used immediately or were
frozen in liquid nitrogen and stored at 270°C. Mitochondria were frozen in
liquid nitrogen (in homogenization buffer containing 10 mg/ml BSA) and
stored at 270°C. The samples were tested for b-galactosidase (lysosomespecific enzyme) and succinate INT reductase (mitochondria-specific enzyme)
as described in Graham et al. (32) and for galactosyl transferase (Golgispecific enzyme) as described in Bergeron et al. (35). Protein concentrations
were determined using the bicinchoninic acid assay (36).
Immunoblotting. Untransfected and transfected cells plated in 35-mm
wells as described above were harvested by trypsinization and collected by
centrifugation. Cells were lysed, and membranes were solubilized in 100 ml of
13 sample loading buffer [50 mM Tris-HCl (pH 6.8), 100 mM DTT, 2% SDS,
0.1% bromphenol blue, and 10% glycerol] as described in Maniatis et al. (37).
The DNA in the samples was sheared using a 1-ml syringe and 22-gauge
needle. Cell lysates were collected by centrifugation (15,800 3 g for 3 min at
24°C). Rat liver samples prepared as described above were mixed with an
equal volume of 23 sample loading buffer (37). All samples were heated at
4892
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1999 American Association for Cancer
Research.
LOCALIZATION OF MTP (MLAPTM4) TO LYSOSOMES
failed to detect native MTP in BHK21 and Chinese hamster ovary
cells or in human cervical carcinoma HeLa cells, which were shown
previously to contain MTP-like mRNA (1). Endogenous levels of
MTP are below the level of detection by these methods. Thus, we
asked whether recombinant MTP and an epitope-tagged version,
HA-MTP, could be localized by transient transfection of BHK21 cells
with plasmids that contained the cDNA encoding either full-length
MTP (pcDNA3/MTP3) or the HA-tagged version of MTP (pcDNA3/
HA-MTP). Solubilized membrane preparations of transfected cells
were analyzed by immunoblotting with antibodies directed against a
synthetic peptide derived from the MTP sequence (Fig. 1A) or against
the HA epitope (Fig. 1B). The anti-MTP antibodies recognized a
protein of Mr ;20,000 –22,000 in cells transfected with pcDNA3/
MTP3 and a protein with a slightly higher apparent molecular weight
in cells transfected with pcDNA3/HA-MTP. The anti-HA antibodies,
which did not detect any proteins in cells transfected with pcDNA3/
MTP3, recognized a single band of Mr ;21,000 –23,000 in cells
transfected with pcDNA3/HA-MTP. The anti-MTP and anti-HA antibodies did not detect any proteins in preparations from cells transfected with pcDNA3 alone.
Localization of Recombinant MTP and HA-MTP in Transfected BHK21 Cells. In the experiments of Fig. 2, double-label
indirect immunofluorescence experiments using anti-MTP and anti-HA antibodies were carried out on cells transiently transfected as
described above. These experiments demonstrated that both MTP and
HA-MTP were localized to vesicular structures concentrated in the
perinuclear region (Fig. 2, A, D, E, G, and H). As expected, HA-
Fig. 3. Absence of HA-MTP in the Golgi apparatus of transfected cells. BHK21 cells
were transfected with pcDNA3/HA-MTP or pcDNA3 as described in “Materials and
Methods.” Cells were fixed, permeabilized, and incubated with monoclonal anti-HA
antibodies (A and B) or with polyclonal anti-a-Man II antibodies (C and D), then with
FITC-conjugated goat anti-rat IgG antibodies (A and B) or Cy5-conjugated donkey
anti-rabbit IgG antibodies (C and D). Overlapping images are shown in E and F. Bars, 20
mm.
65°C for 5 min and centrifuged (15,800 3 g for 1 min at 24°C), and the
resulting supernatants were subjected to electrophoresis on 12% SDS-polyacrylamide gels (38) along with prestained broad-range (Mr 6,000 –175,000)
protein markers (New England Biolabs, Mississauga, Ontario, Canada). Proteins were electroblotted onto polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated overnight at
4°C, first with 5% skim milk powder in blocking buffer (Tris-buffered saline,
0.2% Tween 20) and then for 12–18 h with 5% skim milk powder in blocking
buffer that also contained primary antibodies (either anti-MTP diluted 1:10,000
or anti-HA diluted 1:1,000). The membranes were then washed three times
with blocking buffer and incubated for 2 h at room temperature with either goat
anti-rabbit IgG or goat anti-rat IgG conjugated to horseradish peroxidase,
diluted 1:10,000 (Jackson ImmunoResearch Laboratories). Membranes were
washed with blocking buffer three times and visualized using the enhanced
chemiluminescence (ECL) reagents (Amersham Pharmacia Biotech, Baie
d’Urfé, Quebec, Canada) according to the manufacturer’s instructions. Membranes were stripped and reprobed according to the instructions for use with
the ECL reagents.
RESULTS
Production of Recombinant MTP and HA-MTP in BHK21
Cells by Transient Transfection. Native MTP was not detected in
mouse L1210 leukemia cells (the source of the cDNA library from
which the MTP cDNA was obtained) or freshly isolated rat hepatocytes by either immunoblotting of membrane preparations or indirect
immunofluorescence of cells (1). In the present work, immunocytochemical studies (data not shown) with antibodies against MTP also
Fig. 4. Absence of HA-MTP in the early endosomes of transfected cells. BHK21 cells
were transfected with pcDNA3/HA-MTP or pcDNA3 as described in “Materials and
Methods.” Cells were fixed, permeabilized, and incubated with rat anti-HA antibodies (A
and B) or with polyclonal anti-rab5A antibodies (C and D), then with FITC-conjugated
goat anti-rat IgG antibodies (A and B) or Cy5-conjugated donkey anti-rabbit IgG antibodies (C and D). Overlapping images are shown in E and F. Bars, 20 mm.
4893
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1999 American Association for Cancer
Research.
LOCALIZATION OF MTP (MLAPTM4) TO LYSOSOMES
ies (Fig. 3, A, C, D, E, and F). Thus, recombinant HA-MTP was not
detected in the Golgi apparatus by this technique.
The experiments of Figs. 4 and 5 were undertaken to determine
whether MTP could be detected in early or late endosomes, respectively. Antibodies against the early endosomal marker, rab5A (39),
and the late endosomal marker, cation-independent M6PR (28, 29),
were used with the anti-HA antibodies to examine, by double indirect
immunofluorescence, cells that had been transfected with either
pcDNA3/HA-MTP or pcDNA3. The rab5A protein is a member of the
large GTP-binding family that is required for membrane fusion and
regulates transport between the plasma membrane and early endo-
Fig. 5. Presence of HA-MTP in late endosomes of transfected cells. BHK21 cells were
transfected with pcDNA3/HA-MTP or pcDNA3 as described in “Materials and Methods.”
Cells were fixed, permeabilized, and double labeled for HA-MTP (anti-HA; A and B) and
late endosomes (anti-M6PR; C and D). The secondary antibodies were FITC-conjugated
goat anti-rat IgG antibodies (A and B) or Cy5-conjugated donkey anti-rabbit IgG antibodies (C and D). Overlapping images are shown in E and F. Bars, 20 mm.
MTP-containing vesicles were detected by both anti-MTP and anti-HA antibodies (Fig. 2, A, D, and G). No such vesicular staining was
observed in cells transfected with vector alone (Fig. 2, C, F, and I).
These observations suggested that the HA epitope tag did not affect
the targeting of MTP, because the vesicles in both HA-MTP- and
MTP-producing cells were similar in both size and location. Recombinant HA-MTP and MTP were found only in vesicular structures and
were not detected at the plasma membrane. To facilitate the use of
rabbit antiorganelle antibodies for double-label indirect immunofluorescence, experiments were thereafter conducted with cells that produced the epitope-tagged recombinant protein, HA-MTP.
Localization of HA-MTP to Late Endosomes and Lysosomes in
Transfected Cells. The size and location of the MTP- and HA-MTPcontaining vesicular structures suggested localization to endosomes
and/or lysosomes. In addition, treatment of MTP- and HA-MTPtransfected cells with chloroquine by procedures described elsewhere
(29) resulted in the swelling of MTP- and HA-MTP-containing vesicles (data not shown), suggesting that these vesicles were acidic in
nature and thus part of the endosomal/lysosomal pathway.
To verify that the MTP-containing vesicles were not part of other
perinuclear membranes such as Golgi, cells transfected with either
pcDNA3/HA-MTP or pcDNA3 were subjected to double labeling
with anti-HA antibodies and antibodies raised against the Golgi membrane protein, a-ManII (27). The Golgi apparatus in cells transfected
with either plasmid exhibited a normal appearance, being crescentshaped and juxtanuclear (Fig. 3, C and D). The structures labeled with
the anti-HA and the anti-a-ManII antibodies were clearly different,
and consequently there was no colocalization between these antibod-
Fig. 6. Presence of HA-MTP in lysosomes of transfected cells. BHK21 cells were
transfected with pcDNA3/HA-MTP or pcDNA3 as described in “Materials and Methods.”
Cells were fixed, permeabilized, and double labeled for HA-MTP (anti-HA; A and B) and
for lysosomes (anti-lgp110; C and D). The secondary antibodies were FITC-conjugated
goat anti-rat IgG antibodies (A and B) or Cy5-conjugated donkey anti-rabbit IgG antibodies (C and D). Overlapping images are shown in E and F. Bars, 20 mm.
Table 1 Enzyme marker assays of subcellular fractions
Established procedures were used to enrich for lysosomes (32), Golgi (31), and
mitochondria (33, 34). The values (mean 6 SD, n 5 3 except where noted otherwise) are
units/100 mg of protein, where units represent absorbance at 405 mm for b-galactosidase
(lysosomal marker), absorbance at 490 mm for succinate INT reductase (mitochondrial
marker), and cpm for galactosyl transferase (Golgi marker). Enzyme assays were conducted as described in “Materials and Methods,” and background values, which were
obtained using reaction mixtures that did not contain subcellular fractions, were subtracted
from experimental values.
Specific activity of enzyme markers
Subcellular
fractions
b-Galactosidase
(3 103)
Succinate INT
reductase (3 104)
Galactosyl
transferase
(3 1023)
Lysosomes
Golgi
Mitochondria
228 6 15
55.0 6 1.9
46.7 6 2.9
48.6 6 4.9
14.5 6 1.2
117.6 6 17.5
83.1a
221 6 28.3
14.0 6 2.0b
a
b
n 5 2.
n 5 4.
4894
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1999 American Association for Cancer
Research.
LOCALIZATION OF MTP (MLAPTM4) TO LYSOSOMES
transfected with either pcDNA3/MTP3 or pcDNA3 were included as
positive and negative controls, respectively, for the detection of MTP.
A band of Mr ;20,000 –22,000 was observed in the Golgi- and
lysosome-enriched membrane fractions but not in the mitochondriaenriched membrane fraction. These results demonstrated the presence
of the rat equivalent of MTP in the Golgi- and lysosome-enriched
membranes.
DISCUSSION
Fig. 7. Immunoblot of rat liver subcellular fractions. Samples from rat liver and
transfected BHK21 cells were prepared as described in “Materials and Methods.” For
electrophoresis (12% gels), 100 mg of protein from each membrane fraction and 10 ml
from cell lysates for each transfected sample were loaded into the sample wells. The
immunoblot was probed first with anti-MTP antibodies and then with horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies. Detection was by enhanced chemiluminescence as described in “Materials and Methods.” Left and right, positions of the protein
markers (in thousands).
somes (39). The cation-independent M6PR mediates the transport of
soluble lysosomal enzymes from the trans-Golgi network to lysosomes and is concentrated in late endosomes/prelysosomes (29). The
production of HA-MTP did not modify the staining pattern of either
anti-rab5A (Fig. 4, C and D) or anti-M6PR antibodies (Fig. 5, C and
D). In cells transfected with pcDNA3/HA-MTP, the staining pattern
of the anti-HA antibodies partially overlapped with that of the antiM6PR antibodies (Fig. 5, A, C, and E) but not with that of the
anti-rab5A antibodies (Fig. 4, A, C, and E). Thus, although recombinant HA-MTP could not be detected by immunofluorescence in early
endosomes, it was present in late endosomes.
The experiments of Fig. 6, which were undertaken to determine
whether MTP was also present in lysosomes, used anti-lgp-110, an
antibody that recognizes the lysosomal membrane glycoprotein 110
(30). The anti-lgp110 antibodies recognized large vesicular structures
in cells transfected with either plasmid (Fig. 6, C and D). In cells
transfected with pcDNA3/HA-MTP, the anti-HA antibodies labeled
many of the same large juxtanuclear vesicles as the anti-lgp-110
antibodies (Fig. 6, A, C, and E). The anti-HA antibodies did not
recognize any structures in cells transfected with pcDNA3 (Fig. 6B).
These results demonstrated the presence of recombinant HA-MTP
protein in lysosomes.
Identification of a MTP-like Protein in Subcellular Fractions of
Rat Liver. Six rat ESTs exhibiting high homology with MTP and its
human equivalent were isolated in a recent study of the effects of
nerve growth factor on rat cells (40). Two of these ESTs (105428 and
106134) correspond to the NH2 terminus of MTP, the region against
which anti-peptide antibodies were raised, and because they are almost identical to MTP, the anti-MTP polyclonal antibodies should
recognize the rat equivalent of MTP. Thus, we examined subcellular
fractions of rat liver prepared using established protocols (31–34). The
identities of the membrane fractions enriched for lysosomes, mitochondria, and Golgi were verified by measurement of b-galactosidase,
succinate INT reductase, and galactosyl transferase activities, respectively (Table 1). The lysosome-, mitochondria- and Golgi-enriched
membrane fractions each exhibited high activity for the particular
marker enzyme known to be associated with that organelle. The
presence of rat MTP in these fractions was determined by immunoblotting (Fig. 7). Cell lysates from BHK21 cells that were transiently
The objective of this study was to identify the subcellular location
of MTP, a highly conserved membrane protein identified by isolation
of its cDNA from a mouse leukemia L1210/C2 cDNA library by
functional complementation of a thymidine-transport defect in S.
cerevisiae (1). The high degree of conservation between the human
and mouse proteins implies an important function for this protein
family. Interestingly, rat ESTs that exhibit high sequence identity to
MTP were isolated in a study that indicated that the gene encoding the
rat equivalent of MTP can be down-regulated by treatment of pheochromocytoma cells with nerve growth factor (40). MTP and HUMORF13 share features with several proteins with related sequences
that were identified in the GenBank database using the BLAST
algorithm (41). MTP and HUMORF13, like the human and murine
“lysosome-associated protein transmembrane 5” proteins, hLAPTm5
and mLAPTm5 (GenBank accession numbers U51240 and U51239,
respectively; Ref. 42), have COOH termini that contain the tyrosinebased motifs, YXXØ, where Y is tyrosine, X can be any amino acid,
and Ø is a bulky hydrophobic amino acid residue (Fig. 8). This
tyrosine-based motif has been implicated as an internalization and a
lysosomal targeting signal (43– 48). One of the tyrosine-based motifs
in MTP (Fig. 8) is located nine amino acids downstream from the last
putative transmembrane domain that is predicted to be facing the
cytosol, an arrangement that resembles that of the tyrosine-based
lysosomal targeting signal in Lamp1 (47). The importance of the
tyrosine-based motifs in the targeting of MTP to lysosomes has not
been established.
We have demonstrated that MTP resides in late endosomes and
lysosomes by two independent approaches: localization of an epitopetagged version of recombinant MTP in intracellular membranes of
transiently transfected cultured cells by indirect immunofluoresence
and identification of a native MTP-like molecule by immunoblotting
of subcellular fractions from rat liver. In the immunofluoresence
experiments, recombinant HA-MTP and MTP were both found in
large juxtanuclear vesicular structures, indicating that the epitopetagged protein was targeted to the same intracellular location as the
untagged protein. HA-MTP was specifically shown to be present in
late endosomes and lysosomes, but not in the Golgi apparatus nor in
early endosomes. Because both recombinant HA-MTP and MTP were
produced at high levels in BHK21 cells, transient levels of these
proteins are expected in late endosomes en route to lysosomes. In the
Fig. 8. Comparison of the COOH termini of MTP and related proteins. Sequences were
obtained from GenBank and aligned using the PileUp program (Wisconsin Package
Version 9.1, Genetics Computer Group). Identical residues are depicted in bold, the
putative transmembrane regions are underlined, and the YXXØ motifs are enclosed in
boxes.
4895
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1999 American Association for Cancer
Research.
LOCALIZATION OF MTP (MLAPTM4) TO LYSOSOMES
subcellular fractionation experiments, an MTP-like protein was found
in the Golgi- and lysosome-enriched preparations from rat liver. In
both the earlier study (1) and this study, it is likely that the rat
equivalent of MTP was observed in Golgi-enriched fractions because
such preparations are likely to contain late endosomes. Also, some
lysosomal membrane proteins can be localized to late endosomes en
route to their final destination (44). Based upon the observations
reported in this study, we propose that MTP be redesignated
mLAPTm4 and HUMORF13 be designated hLAPTm4. This change
in nomenclature more accurately reflects characteristics of the protein
and allows for simplified cross-species naming.
What is the physiological function of mLAPTm4 (i.e., MTP)?
Lysosomes are responsible for turnover of intracellular macromolecules as well as extracellular material that enters cells through phagocytosis and endocytosis. Human lysosomes have several well-described transport systems that function to release breakdown products
into the cytosol (49), at least one of which is specialized for transport
of nucleosides (50). The lysosomal nucleoside transport process,
which was studied in lysosomes isolated from human fibroblasts, has
a preference for purine nucleosides (e.g., 29-deoxyadenosine and
inosine) over pyrimidine nucleosides (e.g., uridine), exhibits lower
affinities for nucleosides than the plasma membrane nucleoside-transport processes, and is inhibited by the well-known transport inhibitors
dipyridamole and nitrobenzylthioinosine. The evidence that
mLAPTm4 has nucleoside transport activity is based on studies in
heterologous expression systems where thymidine uptake was observed with the truncated proteins, MTP1 and MTPDC (1).
There is no mLAPTm4 homologue in either the S. cerevisiae or the
Caenorhabditis elegans genomic databases, although various ESTs
with extraordinarily high homology have been identified in vertebrate
genomes (e.g., mouse, rat, rabbit, and zebrafish). A recent study (5)
discovered that production of recombinant full-length mLAPTm4 in
yeast confers increased resistance or sensitivity to a wide variety of
drugs. Drug-sensitive yeast producing recombinant mLAPTm4 were
found to have increased resistance to daunorubicin, doxorubicin,
erythromycin, progesterone, and rhodamine-123 and increased sensitivity to 5-fluorouracil, 5-fluorouridine, and trifluoperazine. Several
studies in mammalian cells have demonstrated drug accumulation in
lysosomes (8 –14, 19 –21, 51), probably attributable to a variety of
mechanisms. The demonstration that mLAPTm4 is a resident protein
of mammalian lysosomes, combined with its functional activity when
produced in yeast, suggests that mLAPTm4 plays a role in sequestering structurally unrelated amphiphilic molecules in lysosomes. Drug
compartmentalization and subsequent accumulation in lysosomes is
an important determinant of the multidrug resistance phenotype (6,
8 –14, 16); however, little is known about the resident transporter
proteins of lysosomes that are responsible for this process. Identification of the physiological substrates of mLAPTm4 awaits development of a system in which the transport characteristics of the fulllength, functional protein can be studied in isolation from other
transporters, for example, by functional reconstitution of recombinant
mLAPTm4 in proteoliposomes or by expression in heterologous systems.
ACKNOWLEDGMENTS
REFERENCES
1. Hogue, D. L., Ellison, M. J., Young, J. D., and Cass, C. E. Identification of a novel
membrane transporter associated with intracellular membranes by phenotypic
complementation in the yeast Saccharomyces cerevisiae. J. Biol. Chem., 271: 9801–
9808, 1996.
2. Cass, C. E., Young, J. D., and Baldwin, S. A. Recent advances in the molecular
biology of nucleoside transporters of mammalian cells. Biochem. Cell Biol., 76:
761–770, 1998.
3. Baldwin, S. A., Mackey, J. R., Cass, C. E., and Young, J. D. Nucleoside transporters:
molecular biology and implications for therapeutic development. Mol. Med. Today,
5: 216 –224, 1999.
4. Mackey, J. R., Baldwin, S. A., Young, J. D., and Cass, C. E. Nucleoside transport and
its significance for anticancer drug resistance. Drug Resist. Updates, 1: 310 –324,
1998.
5. Hogue, D. L., Kerby, L., and Ling, V. A mammalian lysosomal membrane protein
confers multidrug resistance upon expression in Saccharomyces cerevisiae. J. Biol.
Chem., 274: 12877–12882, 1999.
6. Simon, S. M., and Schindler, M. Cell biological mechanisms of multidrug resistance
in tumors. Proc. Natl. Acad. Sci. USA, 91: 3497–3504, 1994.
7. Gottesman, M. M., and Pastan, I. Biochemistry of multidrug resistance mediated by
the multidrug transporter. Annu. Rev. Biochem., 62: 385– 427, 1993.
8. Breuninger, L. M., Paul, S., Gaughan, K., Miki, T., Chan, A., Aaronson, S. A., and
Kruh, G. D. Expression of multidrug resistance-associated protein in NIH/3T3 cells
confers multidrug resistance associated with increased drug efflux and altered intracellular drug distribution. Cancer Res., 55: 5342–5347, 1995.
9. Altan, N., Chen, Y., Schindler, M., and Simon, S. M. Defective acidification in human
breast tumor cells and implications for chemotherapy. J. Exp. Med., 187: 1583–1598,
1998.
10. Gervasoni, J. E., Jr., Fields, S. Z., Krishna, S., Baker, M. A., Rosado, M., Thuraisamy,
K., Hindenburg, A. A., and Taub, R. N. Subcellular distribution of daunorubicin in
P-glycoprotein-positive and -negative drug-resistant cell lines using laser-assisted
confocal microscopy. Cancer Res., 51: 4955– 4963, 1991.
11. Hindenburg, A. A., Gervasoni, J. E., Jr., Krishna, S., Stewart, V. J., Rosado, M.,
Lutzky, J., Bhalla, K., Baker, M. A., and Taub, R. N. Intracellular distribution and
pharmacokinetics of daunorubicin in anthracycline-sensitive and -resistant HL-60
cells. Cancer Res., 49: 4607– 4614, 1989.
12. Kramer, D. L., Black, J. D., Mett, H., Bergeron, R. J., and Porter, C. W. Lysosomal
sequestration of polyamine analogues in Chinese hamster ovary cells resistant to the
S-adenosylmethionine decarboxylase inhibitor, CGP-48664. Cancer Res., 58: 3883–
3890, 1998.
13. Slapak, C. A., Lecerf, J. M., Daniel, J. C., and Levy, S. B. Energy-dependent
accumulation of daunorubicin into subcellular compartments of human leukemia cells
and cytoplasts. J. Biol. Chem., 267: 10638 –10644, 1992.
14. Hurwitz, S. J., Terashima, M., Mizunuma, N., and Slapak, C. A. Vesicular anthracycline accumulation in doxorubicin-selected U-937 cells: participation of lysosomes.
Blood, 89: 3745–3754, 1997.
15. Lautier, D., Bailly, J. D., Demur, C., Herbert, J. M., Bousquet, C., and Laurent, G.
Altered intracellular distribution of daunorubicin in immature acute myeloid leukemia
cells. Int. J. Cancer, 71: 292–299, 1997.
16. Beck, W. T. The cell biology of multiple drug resistance. Biochem. Pharmacol., 36:
2879 –2887, 1987.
17. Simon, S., Roy, D., and Schindler, M. Intracellular pH and the control of multidrug
resistance [published erratum appears in Proc. Natl. Acad. Sci. USA, 91: 4101, 1994].
Proc. Natl. Acad. Sci. USA, 91: 1128 –1132, 1994.
18. Cleary, I., Doherty, G., Moran, E., and Clynes, M. The multidrug-resistant human
lung tumour cell line, DLKP-A10, expresses novel drug accumulation and sequestration systems. Biochem. Pharmacol., 53: 1493–1502, 1997.
19. Daniel, W. A., and Wojcikowski, J. Contribution of lysosomal trapping to the total
tissue uptake of psychotropic drugs. Pharmacol. Toxicol., 80: 62– 68, 1997.
20. Moriyama, Y. Membrane energization by proton pumps is important for compartmentalization of drugs and toxins: a new type of active transport. J. Exp. Biol., 199:
1447–1454, 1996.
21. Ishizaki, J., Yokogawa, K., Hirano, M., Nakashima, E., Sai, Y., Ohkuma, S.,
Ohshima, T., and Ichimura, F. Contribution of lysosomes to the subcellular distribution
of basic drugs in the rat liver. Pharm. Res., 13: 902–906, 1996.
22. Cabrita, M. A., Hobman, T. C., Hogue, D. L., and Cass, C. E. Localization of a nucleoside
transporter to late endosomes and/or lysosomes. Mol. Biol. Cell, 7: 135a, 1996.
23. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A.,
and Struhl, K. Current protocols in molecular biology. In: V. B. Chanda (ed.), Current
Protocols. New York: John Wiley & Sons, Inc., 1997.
24. Niman, H. L., Houghten, R. A., Walker, L. E., Reisfeld, R. A., Wilson, I. A., Hogle,
J. M., and Lerner, R. A. Generation of protein-reactive antibodies by short peptides
is an event of high frequency: implications for the structural basis of immune
recognition. Proc. Natl. Acad. Sci. USA, 80: 4949 – 4953, 1983.
25. Wilson, I. A., Niman, H. L., Houghten, R. A., Cherenson, A. R., Connolly, M. L., and Lerner,
R. A. The structure of an antigenic determinant in a protein. Cell, 37: 767–778, 1984.
26. Kozak, M. An analysis of vertebrate mRNA sequences: intimations of translational
control. J. Cell Biol., 115: 887–903, 1991.
27. Velasco, A., Hendricks, L., Moremen, K. W., Tulsiani, D. R., Touster, O., and
Farquhar, M. G. Cell type-dependent variations in the subcellular distribution of
a-mannosidase I and II. J. Cell Biol., 122: 39 –51, 1993.
28. Brown, W. J., and Farquhar, M. G. The mannose-6-phosphate receptor for lysosomal
enzymes is concentrated in cis Golgi cisternae. Cell, 36: 295–307, 1984.
We thank Drs. B. Reaves and P. Luzio for generous gifts of anti-lgp110
antibodies; Dr. M. Farquhar for gifts of anti-a-ManII and anti-M6PR antibodies; Drs. L. Pilarksi and R. Godbout for gifts of Cy5-conjugated secondary
antibodies; Dr. S. Rice for the use of his immunofluorescence microscope; Dr.
W. Colmers for supplying rat livers; Drs. A. Claude and P. Melançon for the
galactosyl transferase assay protocol; Dr. X. J. Sun for assistance with confocal
microscopy; D. Mowles for expert technical assistance; and Dr. I. Coe for
helpful discussions.
4896
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1999 American Association for Cancer
Research.
LOCALIZATION OF MTP (MLAPTM4) TO LYSOSOMES
29. Brown, W. J., Goodhouse, J., and Farquhar, M. G. Mannose-6-phosphate receptors
for lysosomal enzymes cycle between the Golgi complex and endosomes. J. Cell
Biol., 103: 1235–1247, 1986.
30. Reaves, B. J., Bright, N. A., Mullock, B. M., and Luzio, J. P. The effect of
wortmannin on the localisation of lysosomal type I integral membrane glycoproteins
suggests a role for phosphoinositide 3- kinase activity in regulating membrane traffic
late in the endocytic pathway. J. Cell Sci., 109: 749 –762, 1996.
31. Morré, D. J., and Morré, D. M. Isolation of the Golgi apparatus from rat liver. In:
D. L. Spector, R. D. Goldman, and L. A. Leinwand (eds.), Cells: A Laboratory
Manual, Vol. 1, pp. 39.3–39.7. Cold Spring Harbor, NY: Cold Spring Harbor
Laboratory, 1998.
32. Graham, J. M., Ford, T., and Rickwood, D. Isolation of the major subcellular
organelles from mouse liver using Nycodenz gradients without the use of an ultracentrifuge. Anal. Biochem., 187: 318 –323, 1990.
33. Rickwood, D., Wilson, M. T., and Darley-Usmar, V. M. Isolation and characteristics
of intact mitochondria. In: V. M. Darley-Usmar, D. Rickwood, and M. T. Wilson
(eds.), Mitochondria: A Practical Approach. Oxford: IRL Press, Ltd., 1987.
34. Fleischer, S., McIntyre, J. O., and Vidal, J. C. Large-scale preparation of rat liver
mitochondria in high yield. Methods Enzymol., 55: 32–39, 1979.
35. Bergeron, J. J., Rachubinski, R. A., Sikstrom, R. A., Posner, B. I., and Paiement, J.
Galactose transfer to endogenous acceptors within Golgi fractions of rat liver. J. Cell
Biol., 92: 139 –146, 1982.
36. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H.,
Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C.
Measurement of protein using bicinchoninic acid. Anal. Biochem., 150: 76 – 85, 1985.
37. Maniatis, T., Fritsch, E. F., and Sambrook, J. Molecular Cloning: A Laboratory
Manual. Cold Spring Harbor Laboratory, NY: Cold Spring Harbor Laboratory, 1989.
38. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature (Lond.), 227: 680 – 685, 1970.
39. Ferro-Novick, S., and Novick, P. The role of GTP-binding proteins in transport along
the exocytic pathway. Annu. Rev. Cell Biol., 9: 575–599, 1993.
40. Lee, N. H., Weinstock, K. G., Kirkness, E. F., Earle-Hughes, J. A., Fuldner, R. A.,
Marmaros, S., Glodek, A., Gocayne, J. D., Adams, M. D., Kerlavage, A. R., Fraser,
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
C. M., and Ventner, J. C. Comparative expressed-sequence-tag analysis of differential
gene expression profiles in PC-12 cells before and after nerve growth factor treatment. Proc. Natl. Acad. Sci. USA, 92: 8303– 8307, 1995.
Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., and
Lipman, D. J. Gapped BLAST and PSI-BLAST: a new generation of protein database
search programs. Nucleic Acids Res., 25: 3389 –3402, 1997.
Adra, C. N., Zhu, S., Ko, J. L., Guillemot, J. C., Cuervo, A. M., Kobayashi, H.,
Horiuchi, T., Lelias, J. M., Rowley, J. D., and Lim, B. LAPTM5: a novel lysosomalassociated multispanning membrane protein preferentially expressed in hematopoietic
cells. Genomics, 35: 328 –337, 1996.
Honing, S., Griffith, J., Geuze, H. J., and Hunziker, W. The tyrosine-based lysosomal
targeting signal in lamp-1 mediates sorting into Golgi-derived clathrin-coated vesicles. EMBO J., 15: 5230 –5239, 1996.
Hunziker, W., and Geuze, H. J. Intracellular trafficking of lysosomal membrane
proteins. BioEssays, 18: 379 –389, 1996.
Marks, M. S., Woodruff, L., Ohno, H., and Bonifacino, J. S. Protein targeting by
tyrosine- and di-leucine-based signals: evidence for distinct saturable components.
J. Cell Biol., 135: 341–354, 1996.
Marks, M. S., Ohno, H., Kirchausen, T., and Bonifacino, J. S. Protein sorting by
tyrosine-based signals: adapting to the Y’s and wherefores. Trends Cell Biol., 7:
124 –128, 1997.
Rohrer, J., Schweizer, A., Russell, D., and Kornfeld, S. The targeting of Lamp1 to
lysosomes is dependent on the spacing of its cytoplasmic tail tyrosine sorting motif
relative to the membrane. J. Cell Biol., 132: 565–576, 1996.
Sandoval, I. V., and Bakke, O. Targeting of membrane proteins to endosomes and
lysosomes. Trends Cell Biol., 4: 292–297, 1994.
Pisoni, R. L., and Thoene, J. G. The transport systems of mammalian lysosomes.
Biochim. Biophys. Acta, 1071: 351–373, 1991.
Pisoni, R. L., and Thoene, J. G. Detection and characterization of a nucleoside
transport system in human fibroblast lysosomes. J. Biol. Chem., 264: 4850 – 4856,
1989.
Sandoval, R., Leiser, J., and Molitoris, B. A. Aminoglycoside antibiotics traffic to the
Golgi complex in LLC-PK1 cells. J. Am. Soc. Nephrol., 9: 167–174, 1998.
4897
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1999 American Association for Cancer
Research.
Mouse Transporter Protein, a Membrane Protein That
Regulates Cellular Multidrug Resistance, Is Localized to
Lysosomes
Miguel A. Cabrita, Tom C. Hobman, Douglas L. Hogue, et al.
Cancer Res 1999;59:4890-4897.
Updated version
Cited articles
Citing articles
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/59/19/4890
This article cites 48 articles, 24 of which you can access for free at:
http://cancerres.aacrjournals.org/content/59/19/4890.full.html#ref-list-1
This article has been cited by 11 HighWire-hosted articles. Access the articles at:
/content/59/19/4890.full.html#related-urls
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1999 American Association for Cancer
Research.