The sorting of membrane proteins to the lysosome
requires tyrosine- or dileucine-based targeting signals. Recycling
receptors have similar signals, yet these proteins seldom enter the
latter stages of the endocytic pathway. To determine how lysosomal and internalization signals differ, we prepared chimeric molecules consisting of the cytoplasmic tails of CD3
-chain, lysosomal acid
phosphatase, and lysosomal-associated membrane glycoprotein-1, each
fused to the transmembrane and extracellular domains of the transferrin
receptor (TR). Each chimera was expressed on the cell surface and
rapidly internalized. Metabolic pulse-chase experiments showed that the
CD3
-chain and lysosomal acid phosphatase chimeras, unlike the
lysosomal-associated membrane glycoprotein chimera, were rapidly
degraded in a post-Golgi compartment following normal glycosylation.
Transplantation of signals from CD3
-chain and lysosomal acid
phosphatase into the TR cytoplasmic tail in place of the native signal,
Y20TRF23, indicated that each signal was
sufficient to promote endocytosis but not lysosomal targeting of the
resulting mutant. Transplantation of two CD3 signals at specific sites
in the TR cytoplasmic tail or a single tyrosine-based signal in a
truncated TR tail, however, was sufficient to promote lysosomal
targeting. Our results therefore suggest that the relative position of
the signal within the cytoplasmic tail is a critical feature that
distinguishes lysosomal targeting signals from internalization
signals.
 |
INTRODUCTION |
The trafficking of integral membrane proteins to lysosomes occurs
independently of the mannose 6-phosphate receptor (1, 2). Newly
synthesized lysosomal membrane proteins are delivered to lysosomes
either by a direct intracellular route or by an indirect route via the
plasma membrane (3-8). Membrane proteins that traffic to lysosomes by
the direct intracellular route are thought to be sorted from the
constitutive biosynthetic pathway in the trans-Golgi via
clathrin-coated vesicles (9). Membrane proteins that are delivered to
the lysosome from the cell surface cluster in clathrin-coated pits and
internalize in a manner indistinguishable from recycling receptors. In
the sorting endosome, however, lysosomally directed membrane proteins
segregate from recycling receptors (10, 11).
Studies on the transferrin receptor recycling pathway demonstrate that
recycling receptors and lipids recycle back to the cell surface with
the same kinetics, suggesting that recycling of membrane proteins
occurs by a bulk flow process (12). Furthermore, deletion of the
TR1 cytoplasmic tail has
dramatic effects on TR internalization, but is without effect on TR
recycling (13, 14). Functional analysis of LAMP-1 mutants clearly
demonstrate that modifications in the cytoplasmic tail convert a
lysosomally targeted protein into a protein trapped in the recycling
pathway (11), suggesting that additional targeting information is
required for entry to the later stages of the endocytic pathway
and recycling receptors lack this information.
Analysis of the trafficking of mutant lysosomal membrane proteins with
altered cytoplasmic tails has identified two classes of sorting signals
involved in lysosomal targeting. One class contains a critical tyrosine
residue that is structurally related to tyrosine-based internalization
signals, while the other contains two adjacent leucine or related large
hydrophobic residues (for reviews, see Refs. 15 and 16). Tyrosine-based
lysosomal-targeting signals have been identified in LAP and LAMP-1 (4,
5, 17, 18). Non-tyrosine-based or dileucine sorting signals have been identified in the cation-independent and -dependent mannose
6-phosphate receptors (19, 20), the T cell receptor CD3
-chain (21), LIMP II (22), FcRII-B2 receptor (23, 24), GLUT-4 (25), the insulin
receptor (26), and major histocompatibility complex class II invariant
chain (27-29). Dileucine-based signals, like tyrosine-based signals,
can function as efficient internalization signals (23-26, 28, 29),
although the two signal types appear to be recognized by distinct
cytosolic factors (30).
Despite the extensive series of studies described above, the structural
requirements for lysosomal targeting have not been clearly defined. For
example, whereas the published literature underscores the close
relationship between tyrosine-based internalization signals and
lysosomal targeting signals, the structural features which distinguish
the two classes of signal have not been revealed by alanine-scanning
mutagenesis. The most striking example of this is the recent analysis
of the structural requirements for lysosomal targeting, endocytosis and
basolateral sorting of rat Lgp120 (LAMP-1) (5). Complete alanine
scanning of the 11-residue cytoplasmic tail of rat Lgp120 indicated
that alteration of only two residues, Tyr-8 and Ile-11, to alanine
decreased the efficiency of lysosomal targeting, endocytosis, and
basolateral sorting, with each sorting step being equally affected.
Mutation of Gly-7 to alanine resulted in a loss of direct targeting,
but not indirect targeting, to the lysosome (5).
In this study we have investigated the structural requirements for
lysosomal targeting by analyzing chimeras consisting of the cytoplasmic
tail of LAP, LAMP-1, or the CD3
-chain each fused to the
transmembrane region and external domain of the TR. We have also
transplanted putative internalization and lysosomal-targeting signals
from each of these proteins into the TR in place of the native TR
internalization signal, YTRF. Tyrosine-based signals from LAMP-1 and
LAP were also transplanted into a truncated TR cytoplasmic domain,
3-18, 29-59. Our studies demonstrate that the position of the
signal within the cytoplasmic tail is a critical feature that
distinguishes lysosomal targeting signals from internalization signals.
 |
EXPERIMENTAL PROCEDURES |
Construction of TR Chimeras and Mutant TRs Containing
Transplanted Signals--
Mutant human TR constructs were prepared as
described previously (13) by the method of Kunkel (31). Mutants were
selected either by differential hybridization or by restriction mapping and cloned into the expression vector, BH-RCAS (32, 33). The mutations
were verified by dideoxynucleotide sequencing (34, 35) of the BH-RCAS
constructs using the Sequenase kit (U.S. Biochemical Corp., Cleveland,
OH) according to the manufacturer's directions.
LAMP-TR and LAP-TR chimeras (Fig. 1A) were prepared by
oligonucleotide-directed mutagenesis using the tailless (
3-59) TR mutant phagemid as a template (13). The CD3
-chain-TR chimera (Fig.
1A) was constructed using the polymerase chain reaction as
described previously (36). A polymerase chain reaction was performed on
the CD3
-chain cDNA (kindly provided by Dr. Bob Hyman,
Department of Cancer Biology, Salk Institute). Our polymerase chain
reaction-generated construct did not contain the carboxyl-terminal three residues (Arg-Lys-Lys) of the CD3
-chain since these residues had previously been shown to be unnecessary for lysosomal targeting, and in the context of a type I membrane protein, acted as an
endoplasmic reticulum retention signal (21). Unique NheI and
AflII sites were introduced in the 5'
(5'-CAT-GCT-AGC-CAG-GAT-GGA-GTT-CGC-3') and 3'
(5'-CAT-CTT-AAG-CAG-TTG-GTT-TCC-TTG-3') primers,
respectively. This polymerase chain reaction-generated fragment was
then subcloned into pBluescript SK+ with a human TR insert
containing these two sites (ATG-ATG-GCT-AGC-CTT-AAG-AGG) encoding a seven residue cytoplasmic tail with the sequence
Met-Met-Ala-Ser-Leu-Lys-Arg ((37); kindly provided by Greg Odorizzi and
Albert Lai (Department of Cancer Biology, The Salk Institute)). The
addition of the two restriction sites adds three residues, Ala, Ser,
Leu to the tailless TR (
3-59; (13)) and the CD3
-chain tail
sequence was inserted between the Ser and Leu residues (Fig.
1A).
The TR mutants containing transplanted signals were prepared by
oligonucleotide-directed mutagenesis (Fig. 1, B and
C). The Y20QTI23, the
Y20RHV23, the
D18KQTLL23, and the
Y20QLPK24 mutants were prepared using the
wild-type TR phagemid as template. The Y4QTI7
and Y4RHV7 mutants (Fig. 1D), were
prepared using the
3-18, 29-59 mutant TR phagemid as template
(38). The
D18KQTLL23,Y31QPLK35
mutant was prepared using the D18KQTLL23 mutant
phagemid as template (Fig. 1C). Preparation of the
Y9TRF12 mutant, the
Y31TRF34 mutant, and the
Y47TRF50 mutant has been previously described
(36).
Expression of Wild-type TR, TR Chimeras, and TR Mutants in
Chicken Embryo Fibroblasts (CEF)--
TR chimeras and mutants were
expressed in CEF as described previously (13). Surface expression
levels of wild-type TR and TR chimeras and mutants were determined by
measuring the binding of 125I-labeled human transferrin
(Tf) at 4 °C (13).
Determination of the Apparent Internalization Efficiencies of TR
Chimeras and Mutants--
Apparent internalization efficiencies of the
wild-type TR, TR chimeras, and TR mutants were estimated from
measurement of the steady-state distribution of receptors at 37 °C
(39) as described previously (28). The internalization efficiencies of
the wild-type TR and TR chimeras and mutants were also determined by
measuring their ability to mediate iron uptake as described previously
(13).
Measurement of Tf Proteolysis after Internalization--
CEF
were plated at 7.5 × 104 cells/cm2 in
24-well Costar tissue culture plates. Twenty-four hours later, the
cells were preincubated in serum-free DMEM at 37 °C for 30 min and
then incubated with 125I-labeled Tf (4 µg/ml) in 0.1%
bovine serum albumin, DMEM at 37 °C for 1 h. The medium was
removed after 1 h, and the cells were washed three times with
ice-cold 0.1% bovine serum albumin, phosphate-buffered saline. Cells
were then incubated at 37 °C with prewarmed DMEM containing 0.1%
bovine serum albumin and 50 µg/ml unlabeled Tf for 0, 15, 30, 60, and
120 min. After incubation, the medium was removed, the protein was
precipitated in 10% trichloroacetic acid, and the acid-soluble and
acid-insoluble radioactivity was counted in a gamma counter. The
surface-bound and internalized Tf in CEF was determined by the acid
wash procedure described in the steady-state distribution assay.
Metabolic Labeling and Immunoprecipitation--
Cells were
plated 24 h before the assay at 2 × 106 cells
into 6-cm plates. The next day, the cells were washed twice with
prewarmed (37 °C) methionine-free DMEM. Cells were then
pulse-labeled for 30 min in 2 ml of methionine- and cysteine-free DMEM
containing 0.12 mCi/ml Tran35S-label (ICN Biomedicals,
Irvine, CA) and 2% dialyzed, defined calf serum. Pulse-labeled cells
were chased for various times up to 24 h in complete medium. In
some experiments, cells were preincubated for 1 h in medium
containing 50 mM NH4Cl, and this inhibitor
concentration was maintained throughout the course of the pulse-chase
experiment. At the end of each time point, cells were solubilized on
ice with 1% Nonidet P-40, phosphate-buffered saline. The lysates were
immunoprecipitated using the B3/25 monoclonal antibody and analyzed on
7.5% SDS-polyacrylamide gels (40). Dried gels were exposed on XAR film
(Eastman Kodak Co.). Quantitation of immunoprecipitates was performed
on a model 425 PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Indirect Immunofluorescence--
CEF and CEF expressing the CD3
-chain-TR, LAP-TR, and LAMP-TR chimeras and the wild-type TR were
plated onto glass coverslips and cultured overnight. Analysis by
indirect immunofluorescence was performed as described previously
(28).
 |
RESULTS |
TR Chimeras Containing LAMP-1, LAP, and the CD3
-Chain
Cytoplasmic Tails Are Expressed on the Cell Surface and Rapidly
Internalized--
To determine if the cytoplasmic tails of lysosomal
membrane proteins were sufficient to promote TR internalization, we
constructed three chimeric proteins consisting of the cytoplasmic
domains of LAMP-1, LAP, or the CD3
-chain, fused to the
transmembrane and extracellular domains of the human TR (Fig.
1A). Each of these chimeras
was expressed in CEF using BH-RCAS, a replication-competent retroviral
vector derived from the Rous sarcoma virus (13, 33). Cell surface
expression of each chimera was confirmed using 125I-labeled
Tf binding at 4 °C (data not shown).

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Fig. 1.
Cytoplasmic tail sequences of LAMP-TR,
LAP-TR, and CD3 -chain-TR chimeric constructs and TR transplantation
constructs. A, schematic representation of the wild-type TR
and of chimeric TR constructs containing the cytoplasmic tails of
LAMP-1, LAP, and the CD3 -chain. Each of the constructs contains the
transmembrane region (TM) and extracellular domain of the
wild-type TR. The endogenous TR internalization signal YTRF is shown
underlined in the wild-type sequence. Amino acid sequences
from each of the cytoplasmic domains of LAMP-1, LAP, and CD3 -chain
(underlined) were inserted into the tailless TR cytoplasmic
tail ( 3-59). Constructs are referred to in the text by the
corresponding names at left. Residues are numbered from the amino
terminus and the position of the transmembrane region is represented by
[TM]. Dashes in the sequence represent
unchanged residues. B, putative lysosomal targeting signals
were inserted in place of the endogenous internalization signal YTRF in
the full-length TR cytoplasmic tail. C, an additional
internalization signal, YTRF, was inserted at various positions in the
TR cytoplasmic tail (36). D, putative signals were inserted
in place of YTRF in a TR cytoplasmic tail deletion mutant ( 2-18,
29-59) that retains wild-type internalization activity (38).
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Internalization efficiencies of the chimeras were determined by
measuring the steady-state distribution of internalized Tf and their
ability to mediate iron uptake (13). As shown in Table I, all of the chimeras were rapidly
internalized with relative activities ranging from 50 to 131%
(compared with the wild-type TR). These data are consistent with
previous studies showing that the cytoplasmic tails of LAMP-1, LAP, and
the CD3
-chain contain internalization signals (4, 7, 18, 21) and
indicate that each of these cytoplasmic tails is sufficient to promote rapid TR endocytosis. They also suggest that the cytoplasmic tails from
type I membrane proteins are sufficient to promote internalization of
type II membrane proteins, as has been previously shown for the
-amyloid precursor cytoplasmic tail (37).
CD3
-Chain-TR and LAP-TR Chimeras Are Degraded in a Post-Golgi
Endocytic Compartment--
To determine whether the cytoplasmic tails
of CD3
-chain, LAP, and LAMP-1 were sufficient to target the
chimeras to the prelysosomal/lysosomal compartment, metabolic
pulse-chase experiments were performed. CEF expressing the TR chimeras
and the wild-type TR were each pulse-labeled with
Tran35S-label for 30 min and chased for various time
periods; wild-type TR and TR chimeras were then isolated by
immunoprecipitation using an antibody to the TR extracellular domain
and analyzed by SDS-polyacrylamide gel electrophoresis. As shown in
Fig. 2 the CD3
-chain-TR and LAP-TR
chimeras were more rapidly degraded (t1/2 < 2 and
10 h, respectively) than the wild-type TR (t
1/2 ~24 h) and the LAMP-TR chimera (t1/2 ~ 22 h), suggesting that only the CD3
-chain and LAP tails
were sufficient to target the TR to the lysosomal compartment. After 2 h (Fig. 2), the Mr of the wild-type TR
and each of the chimeras increased to that of the mature glycoprotein
(40), indicating that all of the chimeras traverse the Golgi
compartment where oligosaccharide processing is completed before
degradation occurs.

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Fig. 2.
Rapid degradation of the CD3 -chain-TR and
LAP-TR chimeras occurs in a post-Golgi endocytic compartment.
Equivalent cell numbers of CEF expressing wild-type (WT) TR,
CD3 -chain-TR chimera, LAP-TR chimera, or LAMP-TR chimera were
pulse-labeled for 30 min with Tran35S-label and chased for
various periods of time (h) as indicated. TR or TR chimeras were then
immunoprecipitated from post-nuclear supernatants and analyzed on
SDS-polyacrylamide gels as described under "Experimental
Procedures." Dried gels were exposed to XAR film overnight (Kodak).
Immunoprecipitates were quantitated on a model 425 PhosphorImager
(Molecular Dynamics). A representative experiment (of five) is
shown.
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To confirm that degradation was occurring in a lysosomal compartment,
we monitored the rate of degradation in the presence of a
lysosomotrophic agent, ammonium chloride. As shown in Fig. 3 the half-lives of the CD3
-chain-TR
and LAP-TR chimeras were extended from 1.5 h to 21 h and
5.5 h to 21 h, respectively, in the presence of 50 mM NH4Cl. Addition of 100 µg/ml protease
inhibitor, leupeptin, also increased the half-lives of the CD3
-chain-TR and LAP-TR chimeras (data not shown).

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Fig. 3.
Ammonium chloride (NH4Cl)
inhibits degradation of CD3 -chain-TR and LAP-TR chimeras.
Equivalent cell numbers of CEF expressing CD3 -chain-TR or LAP-TR
chimeras were preincubated in 50 mM NH4Cl in
medium (+NH4Cl) or medium alone
( NH4Cl) for 1 h at 37 °C and
then pulse-labeled with Tran35S-label and chased in the
presence of NH4Cl in medium (+NH4Cl) or in
media alone ( NH4Cl). The chimeras were then
immunoprecipitated from post-nuclear supernatants and analyzed on
SDS-polyacrylamide gels as described in Fig. 2. A representative
experiment (of three) is shown.
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Next, we compared the intracellular distribution of the chimeras to
LEP100, an endogenous chicken lysosomal integral membrane protein (6,
7, 41). The CD3
-chain-TR and the LAP-TR chimeras both showed
significant co-localization with LEP100 (Fig. 4, A and B,
respectively), whereas the LAMP-TR chimera and wild-type TR did not
(Fig. 4, C and D, respectively), suggesting that
the CD3
-chain-TR and LAP-TR traffic along the prelysosomal segment of the endocytic pathway. The low levels of co-localization of the
wild-type TR and LAMP-TR with LEP100 are consistent with previous results using gold label immunocytochemistry, which demonstrated that
~11% of the wild-type TR is found in the LEP100-containing compartment (28).

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Fig. 4.
Co-localization of CD3 -chain-TR and
LAP-TR chimeras with LEP100. CEF expressing CD3 -chain-TR
chimera (A), LAP-TR chimera (B), LAMP-TR chimera
(C), or wild-type TR (D) were fixed,
permeabilized, and stained with rabbit TR antisera followed by
fluorescein isothiocyanate-labeled goat anti-rabbit Ig or mouse
anti-LEP100 followed by Texas Red-labeled goat anti-mouse IgG.
Co-localization is indicated by yellow fluorescence.
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Transplanted Signals from LAMP-1, LAP, and the CD3
-Chain
Promote TR Internalization but Not Lysosomal Targeting--
To
determine the minimum sorting information required for lysosomal
targeting, we transplanted the tyrosine-based signals from LAMP-1 (the
amino acid residues YQTI) (5, 42), LAP (residues YRHV) (18), and CD3
-chain (residues YQPLK) (21), and the dileucine-based signal
(residues DKQTLL) from the CD3
-chain (21) into the TR cytoplasmic
tail in place of the native TR internalization signal, YTRF (Fig.
1B). As shown in Table II, each of the transplanted signals, DKQTLL (CD3
-chain), YQPLK (CD3
-chain), YQTI (LAMP-1), and YRHV (LAP) were effective at promoting
TR internalization (relative activities of 153, 109, 46, and 80%,
respectively, compared with the wild-type TR), demonstrating that the
dileucine signal was the most effective internalization signal.
To determine if the transplanted signals were sufficient to promote
lysosomal targeting of TR, metabolic pulse-chase experiments were
performed. The results demonstrate that CD3
-chain dileucine signal,
DKQTLL, in contrast to the CD3
-chain-TR chimera, is degraded slowly
(t1/2 = 16 h; Fig.
5). Similarly, the rate of degradation of
the mutant TRs containing the LAP signal, YRHV, or the CD3
-chain
signal, YQPLK, were slow (t1/2 = 15.5 h and
>24 h, respectively) like the wild-type TR (t1/2 > 24 h). This indicated that none of the individual signals were
sufficient to deliver the TR to the prelysosomal/lysosomal
compartment.

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Fig. 5.
TR chimeras containing 4-6-residue signals
from the CD3 -chain and LAP are not rapidly degraded.
Equivalent cell numbers of CEF expressing
D18KQTLL23, Y20QPLK24,
or Y20RHV23 were pulse-chased,
immunoprecipitated from post-nuclear supernatants, and analyzed on
SDS-polyacrylamide gels as described in Fig. 2. A representative
experiment (of six) is shown.
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Two Signals from the CD3
-Chain are Sufficient for Lysosomal
Targeting--
Since the CD3
-chain tail was sufficient for
delivery of the TR to the lysosomal compartment, but the individual
signals were not, we next asked if insertion of two CD3
-chain
signals into the TR tail would reconstitute lysosomal targeting. To
perform this experiment, we took advantage of the fact that
introduction of a YTRF internalization signal at position 31-34 in the
TR cytoplasmic tail restores endocytosis of an
internalization-defective mutant receptor to wild-type levels,
suggesting that a signal in this position is efficiently recognized at
the cell surface (36). Similar results were reported by Pytowski
et al. (43), who demonstrated that introduction of a
tyrosine residue for the first or last residue at position 31-34
created an effective internalization signal. Based on these two
studies, we introduced the CD3
-chain signal, YQPLK, at position
31-35 of the D18KQTLL23 mutant, and analyzed
the resulting mutant,
D18KQTLL23,Y31QPLK35
(Fig. 1C), in a metabolic pulse-chase experiment. As shown
in Fig. 6, the
D18KQTLL23,Y31QPLK35
mutant was similar to the CD3
-chain-TR chimera in that it was rapidly degraded (t1/2 = 2 h) and its
degradation could be inhibited in the presence of NH4Cl
(t1/2 = 17 h). Unexpectedly, the
internalization rate of this mutant was twice that of the CD3
-chain-TR chimera (Table II).

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Fig. 6.
TR chimeras containing two targeting signals
from CD3 -chain are rapidly degraded in a post-Golgi endocytic
compartment. Equivalent cell numbers of CEF expressing the
D18KQTLL23, Y31QPLK35
mutant or wild-type TR were preincubated in 50 mM
NH4Cl in medium (+NH4Cl) or
medium alone ( NH4Cl) for 1 h at
37 °C and then pulse-labeled with Tran35S-label and
chased in the presence of NH4Cl in medium
(+NH4Cl) or in medium alone ( NH4Cl). Analysis
of the immunoprecipitates was the same as described in Fig. 2. A
representative experiment (of three) is shown.
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Transplantation of an Additional YTRF Signal at Position 31-34
Promotes Lysosomal Targeting of the TR--
Since the
D18KQTLL23,Y31QPLK35
mutant promoted lysosomal targeting, we next asked if two
tyrosine-based signals could provide the same sorting information. To
test this, we examined the half-lives of three previously described
mutants that contain an additional copy of the YTRF sequence inserted
at different locations within the TR cytoplasmic domain (36) (Fig.
1C). As shown in Fig. 7, the
Y31TRF34 mutant had a half-life of 6 h,
whereas the Y9TRF12 and
Y47TRF50 mutants had half lives of 17 h
and >24 h, respectively (Fig. 7). Treatment with NH4Cl
increased the half-life of the Y31TRF34 mutant
to 14 h (data not shown). These results suggest that having a
second signal at position 31-34 or 31-35 in the TR cytoplasmic tail
restores lysosomal targeting and that placement of a second signal at
other cytoplasmic tail positions does not.

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Fig. 7.
The Y31TRF34 mutant
is rapidly degraded in a post-Golgi endocytic compartment.
Equivalent cell numbers of CEF expressing
Y9TRF12, Y31TRF34,
Y47TRF50, or wild-type TR were pulse-chased,
immunoprecipitated, and analyzed as described in Fig. 2. A
representative experiment (of three) is shown.
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TR Chimeras with Tyrosine-based Signals Proximal to the
Transmembrane Region Are Rapidly Degraded--
Because a number of
lysosomal membrane proteins have short cytoplasmic tails with
tyrosine-based signals in close proximity to the transmembrane region,
we next asked if transplantation of signals into a truncated TR
cytoplasmic tail,
3-18, 29-59, promoted lysosomal targeting (Fig.
1D).
3-18, 29-59 TR has previously been shown to
promote efficient internalization (13). As shown in Table II, each of
the truncation mutants, Y4QTI7 and
Y4RHV7, promoted TR internalization.
Furthermore, metabolic pulse-chase experiments revealed that each was
rapidly degraded (t1/2 = 3.5, 3, and 3 h, for
the Y4QTI7, Y4RHV7 and
3-18, 29-59 mutants, respectively) (Fig.
8). These results suggest that even the
wild-type TR signal, YTRF, is sufficient for lysosomal targeting if
presented in the proper context.

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Fig. 8.
Mutants containing tyrosine-based signals in
a truncated TR cytoplasmic domain are rapidly degraded in a post-Golgi
endocytic compartment. Equivalent cell numbers of CEF expressing
3-18, 29-59, Y4QTI7,
Y4RHV7, or wild-type TR were pulse-chased,
immunoprecipitated, and analyzed as described in Fig. 2. A
representative experiment (of four) is shown.
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CD3
-Chain-TR and LAP-TR Chimeras, and the
D18KQTLL23, Y31QLPK35,
and Y31TRF34 Mutants That Reach the Cell
Surface Are Targeted to Lysosomes--
Although the metabolic
pulse-chase experiments indicated that several of the TR chimeras were
rapidly degraded in a post-Golgi compartment, we wanted to determine if
the chimeras that reached the cell surface were also delivered to the
lysosomal compartment. To address this, we performed the following
experiment. Cells expressing the chimeras were incubated with
125I-labeled Tf at 37 °C for 1 h to load the
endocytic pathway with receptor-ligand complexes (28). The cells were
then rapidly washed and the reappearance of intact and labeled Tf in
the medium was determined by measuring trichloroacetic acid-insoluble
and soluble radioactive counts. For the CD3
-chain-TR and LAP
chimeras, 31 and 20% of Tf bound, respectively, were degraded before
being released into the medium (trichloroacetic acid-soluble counts; Fig. 9A), whereas only 2.6%
of Tf bound to the wild-type TR was. Thus, a substantial fraction of
CD3
-chain-TR and LAP-TR chimeras are directly targeted to
lysosomes after internalization. Analysis of the
D18KQTLL23 mutant, the
D18KQTLL23,Y31QPLK35
mutant, and
3-18, 29-59 TR indicated that 5, 12, and 16% of Tf
bound was degraded before being released into the medium (Fig. 9B). This result suggested that the
D18KQTLL23,Y31QPLK35
mutant and
3-18, 29-59 TR were delivered to the lysosomal
compartment from the cell surface, albeit less efficiently than the CD3
-chain chimera.

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Fig. 9.
Degradation of Tf bound to TR chimeras and TR
mutants at cell surface. Equivalent cell numbers of CEF, plated in
triplicate for each time point, expressing A, wild-type
(WT) TR, CD3 -chain-TR, or LAP-TR chimeras or
B, D18KQTLL23,
D18KQTLL23, Y31QPLK35,
or 3-18, 29-59 TR mutants were preincubated in serum-free medium
for 30 min at 37 °C, followed by incubation with
125I-labeled Tf for 1 h at 37 °C. The cells were
then washed and reincubated at 37 °C in DMEM containing 50 µg/ml
unlabeled Tf for various times. Trichloroacetic acid
(TCA)-soluble radioactivity ( ) or acid-insoluble
125I-labeled Tf ( ) released into medium, as well as
surface-bound ( ) and internalized 125I-labeled Tf ( )
were determined as described under "Experimental Procedures." Each
experimental point is expressed as a percent of total radioactivity
recovered. Representative experiments (of three (A) and two
(B)) are shown.
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 |
DISCUSSION |
The studies reported here were undertaken to define more precisely
the properties of lysosomal targeting signals. Our data indicate that
the addition of the CD3
-chain and LAP cytoplasmic tails onto a type
II membrane protein, the TR, is sufficient for efficient targeting of
the chimeras to the lysosomal compartment. Interestingly, the LAMP-1
cytoplasmic domain did not confer this same targeting specificity.
Williams and Fukuda (17) first demonstrated that the addition of the
11-residue cytoplasmic tail of LAMP-1 to a reporter molecule, the
soluble human gonadotropin
-chain spliced to the VSV-G protein
transmembrane domain, was sufficient to deliver the chimera to the
lysosomes. They also demonstrated that a tyrosine was a critical part
of the signal and that the position of the tyrosine residue within the
cytoplasmic tail was critical for lysosomal targeting.
Studies by Guarnieri et al. (42) on the murine LAMP-1
extended these findings to show that the lysosomal targeting signal consisted of the last 4 residues in the 11-residue tail that formed the
pattern Y-X-X-hydrophobic. Furthermore, there
appeared to be a strict requirement for the placement of this motif
relative to the transmembrane region since a 7-residue spacer was
necessary for proper recognition (42). Rohrer et al. (11)
demonstrated that the spacer requirement was important for sorting in
the sorting endosome and less so at the cell surface. In our studies,
placement of this 4-residue signal, YQTI, in 61-residue TR tail as well as placement of the entire 11-residue LAMP-1 tail in a 4-residue "tailless" TR tail all produced chimeras that were efficiently internalized but not lysosomally targeted. Interestingly, the only case
in which this motif was recognized as a lysosomal targeting signal was
in a truncated TR cytoplasmic tail, placing the YQTI motif 7 residues
from the transmembrane region. This suggested that the spacer
requirement found in type I membrane proteins (11, 42) is also true for
type II membrane proteins such as the TR. The same spacing requirement,
however, was not important for endocytosis, since chimeras containing
the YQTI motif were internalized with the same efficiency when the
motif was placed at 2, 7, or 38 amino acids from the transmembrane
region.
The evidence that both the CD3
-chain and LAP chimeras are
efficiently delivered to the lysosome comes from both biochemical and
morphological experiments. First, metabolic pulse-chase experiments demonstrated that the chimeras were rapidly degraded in a post-Golgi compartment. Clearly, formation of the maturely glycoslyated chimeras confirmed that they were not simply misfolded proteins that failed to
exit the endoplasmic reticulum. Second, degradation of the chimeras
occurred in a lysosomal compartment since the degradation could be
inhibited with the weak base, ammonium chloride. Third, ~31% of the
CD3
-chain TR chimeras that were displayed on the cell surface were
degraded after internalization. Fourth, the intracellular distribution
of the CD3
-chain-TR and LAP-TR chimeras overlapped significantly
with a marker of the prelysosomal/lysosomal compartment, LEP100,
whereas the wild-type TR and LAMP-TR chimera did not.
Our results on the CD3
-chain-TR chimeras are consistent with those
of Letourneur and Klausner (21) who reported that the CD3
- and
-chains of the T cell antigen receptor cytoplasmic tails contained
two lysosomal-sorting signals. Using chimeras consisting of the
extracellular and transmembrane regions of the Tac antigen (Il-2
receptor
-chain) spliced to the cytoplasmic tails of these subunits,
they identified a tyrosine-based motif, YQLPK, and a dileucine motif,
DKQTLL, that functioned to deliver the chimera to the lysosome without
going to the cell surface. Mutants that contained only one of these
signals were delivered to the cell surface, rapidly internalized, and
subsequently delivered to the lysosomes. The dileucine motif alone,
therefore, served as an effective internalization signal as well as a
lysosomal targeting signal in the context of the CD3
-chain. Our
studies show that the DKQTLL sequence is also sufficient to mediate
rapid internalization of the TR, indicating that tyrosine-based signals and dileucine signals appear to be functionally equivalent. Our results, however, differ in that the dileucine motif alone was not
sufficient to target the TR to the lysosome. The half-life of this
chimera was 18 ± 2.0 h (mean ± S.E. for five
independent experiments), suggesting that additional targeting
information is required.
The other mutant that was delivered to the lysosome, the LAP-TR
chimera, was not as efficiently delivered to the lysosomal compartment
as the CD3
-chain chimera (t1/2 = 8.2 ± 0.6 h (mean ± S.E. of six independent experiments)).
Insertion of the LAP cytoplasmic tail into a "tailless" TR tail
(Fig. 1) placed the YRHV signal 9 residues from the transmembrane.
Interestingly, placement of this signal in the truncated TR tail,
positioned this signal 7 residue from the transmembrane and resulted in
a more rapid degradation (t1/2 = 5.3 ± 1.8 h, mean ± S.E. of three independent experiments). In the
context of the wild-type LAP protein, this signal is 8 residues from
the transmembrane region. That this sequence, YRHV, was not
specifically required for lysosomal targeting in the context of the
truncated TR cytoplasmic tail was demonstrated by the fact that the
LAMP-1 targeting signal, YQTI, as well as the native TR internalization
signal, YTRF, were also sufficient for this sorting process.
Several studies have shown that the direct intracellular route used by
newly synthesized cation-independent mannose 6-phosphate receptors is
from the trans-Golgi to the early endosomal compartment (4,
44). This may represent the pathway taken by lysosomal membrane
proteins, implying that the sorting signals for lysosomal targeting
would need to be recognized at the sorting endosome, as was suggested
in the studies by Rohrer et al. (11) on LAMP-1. Our data
support this idea since all of the mutants successfully reached the
endosome, yet many failed to be delivered further into the endocytic
pathway. For efficient lysosomal delivery, our chimeras contained one
of the following structural features: 1) a dileucine-based signal and a
tyrosine-based signal separated by 5-7 residues, 2) two tyrosine-based
signals separated by 7 residues, or 3) a single tyrosine-based signal
7-9 residues from the transmembrane region. Interestingly, however,
not all two-signal chimeras conferred lysosomal targeting specificity,
suggesting that there was a strict positional requirement of these
signals for proper targeting.
In conclusion, our data support the following model. Constitutively
recycling receptors contain internalization signals that allow them to
enter the endocytic pathway. Once they reach the sorting endosome,
another sorting decision is made. Since the TR lacking a cytoplasmic
domain recycles to the cell surface as effectively as a wild-type TR
(Jing et al. (13)), then recycling would be expected to be
the default pathway. The results presented here demonstrate that the
same signal types that function as efficient internalization signals
can function as lysosomal targeting signals, but the cytoplasmic tail
positional requirements for lysosomal signals appear to be much more
restricted. This suggests that similar motifs have been adapted for use
in endocytosis and lysosomal targeting (and perhaps trans-Golgi network
sorting), and that positional changes of the motif within the
cytoplasmic tail affect recognition at each site differently.