From the Department of Cell Biology, University of Alabama at
Birmingham, Birmingham, Alabama 35294-0005
The invariant chain (Ii) targets newly
synthesized major histocompatibility complex class II complexes to a
lysosome-like compartment. Previously, we demonstrated that both the
cytoplasmic tail (CT) and transmembrane (TM) domains of Ii were
sufficient for this targeting and that the CT contains two di-leucine
signals, 3DQRDLI8 and
12EQLPML17 (Odorizzi, C. G., Trowbridge,
I. S., Xue, L., Hopkins, C. R., Davis, C. D., and
Collawn, J. F. (1994) J. Cell Biol. 126, 317-330). In the present study, we examined the relationship between
signals required for endocytosis and those required for lysosomal
targeting by analyzing Ii-transferrin receptor chimeras in quantitative transport assays. Analysis of the Ii CT signals indicates that although
3DQRDLI8 is necessary and sufficient for
endocytosis, either di-leucine signal is sufficient for lysosomal
targeting. Deletions between the two signals reduced endocytosis
without affecting lysosomal targeting. Transplantation of the DQRDLI
sequence in place of the EQLPML signal produced a chimera that
trafficked normally, suggesting that this di-leucine sequence coded for
an independent structural motif. Structure-function analysis of the Ii
TM region showed that when Ii TM residues 11-19 and 20-29 were
individually substituted for the corresponding regions in the wild-type
transferrin receptor, lysosomal targeting was dramatically enhanced,
whereas endocytosis remained unchanged. Our results therefore
demonstrate that the structural requirements for Ii endocytosis and
lysosomal targeting are different.
 |
INTRODUCTION |
Major histocompatibility complex class II molecules are cell
surface molecules that function to bind peptide antigens and present
them to CD4+ helper T cells. Newly synthesized major
histocompatibility complex class II 
complexes are targeted to
lysosome-like compartments (1-3) by the invariant chain
(Ii)1 (reviewed in Ref. 4).
Upon arrival, Ii is degraded, and 
chains acquire peptide
antigens (5-11). 
Ii complexes are delivered to the lysosomal
compartment from the TGN directly (11) or indirectly from the cell
surface, where they are rapidly endocytosed (6, 12, 13). Both direct
and indirect pathways are utilized for delivery of class II to a
processing compartment (6, 11, 13, 14).
Support for the idea that Ii is sorted to the latter stages of the
endocytic pathway comes from studies in which the Ii cytoplasmic tail
was replaced with the TR cytoplasmic tail (15). Newly synthesized class
II molecules containing this TR-Ii chimera are delivered to the cell
surface and efficiently internalized, but Ii proteolysis is blocked,
and class II antigen presentation is inhibited (15). This suggests that
the TR cytoplasmic tail, which contains a tyrosine-based internalization signal (16, 17), is not sufficient for delivery of the
class II complexes to a lysosome-like processing compartment and that
additional sorting information is required.
Two Ii cytoplasmic tail targeting signals have been identified using
Ii-neuraminidase chimeras (18) and Ii-TR chimeras (14), Leu7-Ile8 and
Met16-Leu17. These di-leucine signals were
first identified in the CD3
-chain and the
cation-dependent mannose 6-phosphate receptor and shown to
be important for lysosomal and late endosomal targeting, respectively (19, 20). They consist of two hydrophobic residues, usually leucine-leucine or leucine-isoleucine, that are often preceded by four
hydrophilic residues (19). For Ii, residues amino-terminal to the
di-leucine-like motifs are important for sorting (14, 21, 22),
suggesting that Ii di-leucine signals like those found in the CD3
-
and
-chains consist of six residues,
3DQRDLI8 and
12EQLPML17.
NMR structural data on the Ii cytoplasmic tail suggests that the
membrane-distal signal lies within a nascent helix, whereas the
membrane-proximal motif is a part of a turn (22). Tyrosine-based signals have been proposed to be turn structures as well (16, 23-25),
and have been shown to form independent structural motifs because one
tyrosine-based signal can often substitute for another (26-28).
Whether di-leucine-based signals form independent structural motifs is
unknown. Furthermore, how tyrosine- and di-leucine-based signals are
related is unclear, especially because they appear to be recognized by
distinct cytosolic factors (29).
Athough native Ii is a trimer, studies of mixed trimers containing
wild-type and tailless Ii demonstrate that trimers containing at least
two wild-type Ii molecules are targeted properly in fibroblasts (30),
illustrating that molecules containing two or three Ii cytoplasmic
tails traffic the same. Furthermore, the quaternary structural
requirements for Ii sorting appear to be flexible because tetramers
containing the Ii cytoplasmic tail spliced to the transmembrane and
extracellular domains of neuraminidase traffic the same as wild-type Ii
trimers (18).
The purpose of this study was to define how internalization signals
differ from those required for delivery to the latter stages of the
endocytic pathway. We examined two regions of the Ii that had
previously been shown to be important for targeting to the lysosomal
compartment, the cytoplasmic tail (CT) and the transmembrane region
(TM) (14). We took advantage of the fact that we could quantitatively
monitor 1) internalization using radiolabeled transferrin (Tf), and 2)
lysosomal targeting by monitoring the half-lives of the Ii-TR chimeras
(14). Mutational and functional analysis of Ii CT suggests that some
mutations affected endocytosis, some affected lysosomal targeting, and
some affected both. Analysis of the Ii TM region indicated that two
distinct regions within the TM were sufficient to confer lysosomal
targeting on a reporter molecule without affecting internalization
rates. The results demonstrate that the structural requirements for Ii
endocytosis and lysosomal targeting are not the same.
 |
EXPERIMENTAL PROCEDURES |
Human Ii-TR Constructs--
Mutants were prepared as described
previously (14) by the method of Kunkel (31). Polymerase chain reaction
fragments encoding the various cytoplasmic tail deletion mutants were
subcloned into a tailless TR construct that contained BglII
and NheI sites 5' to the start site and at the beginning of
the transmembrane region (amino acid position 64-65 in the TR
sequence), respectively (CCL-1). Introduction of the NheI
site created a substitution mutation (Ala for Gly at position 64, GGA-AGT
GCT-AGC). This substitution had no effect on
internalization or expression levels. Mutants were screened and
selected by restriction mapping or sequencing and cloned into the
expression vector BH-RCAS (32). All mutations were verified by
dideoxynucleotide sequencing of the entire cytoplasmic or transmembrane
domain in BH-RCAS constructs (33, 34).
Expression of Ii Chimeras and Wild-type TR--
Ii chimeras and
wild-type TR were expressed in chicken embryo fibroblasts (CEFs) as
described previously (35). Surface expression of Ii chimeras and
mutants was determined by measuring the binding of
125I-labeled Tf at 4 °C (35).
Internalization Assay--
The rate of Tf internalization was
determined using the IN/SUR method (36). Diferric human Tf (Miles
Scientific, Naperville, IL) was labeled with 125I to a
specific activity of 2-4 µCi/µg using Enzymobeads (Bio-Rad) according to the manufacturer's directions. CEFs were plated in triplicate at a density of 7.5 × 104
cells/cm2 in 24-well tissue culture plates 24 h before
the assay (Costar Corp., Cambridge, MA). Cells were incubated in
serum-free Dulbecco's modified Eagle's medium for 1 h at
37 °C. Prewarmed (37 °C) 125I-labeled Tf (4 µg/ml)
in 0.15 ml of 0.15 M NaCl, 0.01 M sodium phosphate buffer (pH 7.4) containing 0.1% bovine serum albumin (BSA-PBS) was added to each well. After incubation for 0, 2, 4, 6 or 8 min, the incubation medium was removed, and the cells were placed on
ice and rinsed with ice cold BSA-PBS three times. The cells were then
incubated twice for 3 min with 0.5 ml of 0.2 M acetic
acid-0.5 M sodium chloride (pH 2.4) to remove surface bound 125I-labeled Tf (37). Cells were then lysed with 1 M NaOH. Radioactivity in the acid wash (SUR) and in the
cell lysate (IN) was determined. The ratio of internalized (IN) to
surface (SUR) ligand (IN/SUR) yields a biphasic curve when plotted as a
function of time (36). The slope was calculated by linear regression to
determine the endocytic rate constant ke.
Measurement of Proteolysis after
Internalization--
Measurement of 125I-labeled Tf was
determined as described previously (14).
Metabolic Labeling and Immunoprecipitation--
Metabolic
labeling of CEFs expressing the various Ii chimeras and
immunoprecipitation of the Ii chimeras were performed as described
previously (14).
Calculation of the Surface-accessible Counts--
To estimate
the relative amount of chimera that is delivered to the cell surface,
we used the following calculation: Surface-accessible pool (%) = (Surface-labeled countschimera/Surface-labeled
countsTR) × (Relative biosynthetic
rateTR/Relative biosynthetic ratechimera) × (Half-lifeTR/Half-lifechimera) × 100.
To determine the surface-labeled counts, we incubated cells with
125I-labeled Tf for 1 h to label the endocytic
pathway. The cells were then washed with BSA-PBS at 4 °C to remove
the unbound Tf, lysed in 1% Nonidet P-40, and counted in a gamma
counter. Comparison between the individual cell lines gave an estimate
of the total amount of chimeras on the surface and in endosomal
compartments (surface-labeled counts). To determine the relative
biosynthetic rate, we pulsed cells with
35S-trans label for 30 min. The relative
biosynthetic rate refers to the relative protein expression of each of
the constructs compared with the wild-type TR. TR or Ii-TR chimeras
were then immunoprecipitated from postnuclear supernatants and
quantitated on SDS-polyacrylamide gels by PhosphorImager analysis. To
determine the relative degradation rate, we measured the relative
half-lives of each of the constructs using a metabolic pulse-chase
procedure. The relative biosynthetic and degradation rates were then
compared with the wild-type TR control.
For example, if the surface-labeled counts of the chimera are 25% that
of the TR, the relative biosynthetic rate is 4 times that of the TR,
and the relative degradation rate is 8 times (t1/2 chimera = 3 h; t1/2 TR = 24 h),
then the percentage of the chimeras reaching the cell surface
(surface-accessible pool) is 50% (1/4 × 1/4 ×
× 100%). In this case, 50% of the chimeras traffic
directly to the lysosome, and 50% are delivered to the cell surface
before delivery to the lysosome. This calculation assumes that all of
the TR that is synthesized reaches the cell surface and that little
degradation of the chimeras occurs during the 1 h of labeling
(125I-labeled Tf) from the surface (surface-labeled
counts).
 |
RESULTS |
Ii-TR Chimeras Are Expressed on the Cell Surface and Rapidly
Internalized--
To determine whether the structural features of Ii
that are important for endocytosis are the same as those required for
targeting to the latter stages of the endocytic pathway, we constructed Ii-TR chimeras consisting of either the wild-type or mutant Ii cytoplasmic tails spliced to the transmembrane and extracellular domains of TR (Fig. 1). Wild-type human
TR and Ii-TR chimeras were stably expressed in CEFs using BH-RCAS, a
replication-competent retroviral vector derived from the Rous sarcoma
virus (38). Binding studies at 4 °C using 125I-labeled
human Tf indicated that all of the Ii-TR chimeras were expressed on the
cell surface, although at lower levels than the wild-type TR (data not
shown).

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 1.
Cytoplasmic tail and transmembrane sequences
of the TR, Ii, and Ii-TR chimeras. A, schematic diagram
of wild-type (WT) TR, TR 2-59, and WT Ii molecules. The
amino acid sequences of the cytoplasmic tails and transmembrane regions
of each are shown. B, schematic diagram of Ii-TR chimera and
cytoplasmic tail sequences of mutant Ii-TR chimeras.
Unshaded areas represent regions of Ii-TR derived from TR;
shaded areas represent regions derived from Ii.
EC, extracellular domain. Constructs are referred to in text
by the corresponding names shown at the left.
|
|
Internalization rates of the Ii-TR chimeras were monitored using the
IN/SUR method of Wiley and Cunningham (36). Analysis of the chimera
with the wild-type Ii chain cytoplasmic tail (IiCT) indicated that it was internalized as rapidly (ke = 0.117 min
1) as the wild-type TR (ke = 0.107 min
1; Table I). This
internalization rate compares favorably with human TR internalization
rates determined in other cell types (25). A tailless TR (
2-59),
for comparison, was internalized poorly (ke = 0.006 min
1; Table I).
Residues 20-29 of the Ii Cytoplasmic Tail Are Not Required for
Endocytosis or Lysosomal Targeting--
Two regions of the Ii chain
cytoplasmic tail, residues 3-8 and 12-17, have been reported to be
important for sorting in the endocytic pathway (14, 18, 21, 22, 39).
Additional signals may be contained in the membrane-proximal region of
the Ii cytoplasmic tail (15, 40). To determine whether the first 19 residues of the Ii chain cytoplasmic tail were sufficient for both
endocytosis and lysosomal targeting, we prepared three deletion
mutants, IiCT
20-29, IiCT
20-24, and
IiCT
25-29 (Fig. 1B). Each deletion mutant
was tested in internalization assays and found to have nearly wild-type
internalization activity (74-120%; Table I), indicating that residues
20-29 were not required.
Because Ii is transported to an acidic endocytic compartment, where it
is rapidly degraded (5-8), we next determined the effect of these
deletions on the half-lives of the chimeras in metabolic pulse-chase
experiments. CEFs expressing either TR, IiCT, Ii
CT
20-29, Ii CT
20-24, or the Ii
CT
25-29 mutant were pulse-labeled with
35S-trans label for 30 min and chased in
complete medium for the indicated periods of time; TR and Ii-TR
chimeras were then isolated by immunoprecipitation and analyzed by
SDS-PAGE (Fig. 2). The IiCT
chimera was rapidly degraded with an average half-life of 3.1 ± 0.4 h (Table II, average ± S.E.), similar to the half-life of native Ii in antigen presenting
cells (41), whereas the Ii CT
20-29 chimera was
degraded with a half-life of 6.7 ± 0.2 h (Table II,
average ± S.E.). Interestingly, the Ii-TR chimeras with smaller deletions were degraded more rapidly (IiCT
20-24,
t1/2 ~3.0 ± 0.1 h;
IiCT
25-29, t1/2 ~3.7 ± 0.9 h), suggesting that none of the residues from 20 to 29 per se were important for targeting to the processing
compartment. In contrast, the half-life of the wild-type TR was greater
than 20 h (t1/2 = 21.2 ± 1.6 h,
Table II). After 2 h (Fig. 2), the Mr of TR
and Ii-TR chimeras increased to that of the mature glycoprotein (42), indicating that the Ii-TR chimeras traverse the Golgi where
glycosylation is complete and are degraded in a post-Golgi compartment.
Degradation of the IiCT chimera occurred within the
endocytic pathway because treatment of the cells with 50 mM
NH4Cl during the labeling and chase period increased the
half-life dramatically (data not shown).

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 2.
Residues 20-29 of the invariant chain
cytoplasmic tail are not required for efficient lysosomal
targeting. Equivalent cell numbers of CEFs expressing wild-type
TR, IiCT, IiCT 20-29,
IiCT20-24, or IiCT25-25 chimeras were
pulse-labeled for 30 min with 35S-trans label
and chased with complete medium for various periods of time as
indicated (in hours). Wild-type TR or IiCT chimeras were
then immunoprecipitated from postnuclear 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 three) is
shown.
|
|
The Membrane-distal Signal, DQRDLI, Is Necessary for Endocytosis
and Sufficient for Lysosomal Targeting--
Two di-leucine-like
signals, Leu7-Ile8 and
Met16-Leu17, are reported to promote Ii
internalization (14, 18). These studies, however, relied on
radiolabeled bivalent antibody uptake (18) or steady-state
distributions (14) to monitor endocytosis. To monitor internalization
rates more precisely, we measured 125I-labeled Tf uptake
for the IiCT Ala7-Ala8 mutant and
the IiCT Ala16-Ala17 mutant (Fig.
1B). Comparison of the internalization rates indicated that
IiCT Ala7-Ala8 mutant was
internalized poorly (ke = 0.013), whereas the
IiCT Ala16-Ala17 mutant had
significant activity (ke = 0.066) (Fig.
3, A versus B). The
IiCT Ala16-Ala17 mutant had 65%
activity (IiCT ke = 0.101, Fig.
3B), whereas the IiCT
Ala7-Ala8 mutant had ~13% activity, similar
to the background activity of a tailless TR (~8% activity,
ke = 0.008, Fig. 3C). The
IiCT mutant was internalized at essentially the same rate as the wild-type TR (Fig. 3B, ke = 0.101 versus 0.114, respectively). The results indicate that the
membrane-distal signal, Leu7-Ile8, is required
for efficient endocytosis, whereas the membrane-proximal signal,
Met16-Leu17, is not.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 3.
Comparisons of the internalization rates of
Ii-TR chimeras containing various cytoplasmic tail mutations. CEFs
expressing the various Ii-TR chimeras were incubated with prewarmed
(37 °C) 125I-labeled Tf for the indicated times. The
amounts of internalized (Internal Tf) and surface-associated
(Surface Tf) radiolabel were determined as described under
"Experimental Procedures." Data are plotted using the IN/SUR
method, in which the slope of the line equals the endocytic rate
constant ke (36). Three representative experiments
are shown.
|
|
We next determined the effect of the Ala7-Ala8
and Ala16-Ala17 mutations on lysosomal
targeting by analyzing these chimeras in metabolic pulse-chase
experiments. The results indicate that both chimeras were rapidly
degraded (t1/2 ~ 5.2 h (Fig.
4A) and
t1/2 ~ 7.0 h (Fig. 4B),
respectively), although neither was degraded as rapidly as the
wild-type Ii chimera (average = 3.1 h, Table II). This
demonstrated that either region was sufficient for lysosomal
targeting.

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 4.
IiCT
Ala7-Ala8 and IiCT
Ala16-Ala17 chimeras are rapidly degraded in a
post-Golgi compartment. Equivalent cell numbers of CEFs expressing
wild-type TR or IiCT Ala7-Ala8
chimeras (A) or wild-type TR, IiCT, or
IiCT Ala16-Ala17 chimeras
(B) were pulse-chased, immunoprecipitated from postnuclear
supernatants, and analyzed as described in Fig. 2. Representative
experiments (of three) are shown.
|
|
To determine whether the cell surface pool of IiCT
Ala7-Ala8 chimeras was delivered to the
lysosomal compartment despite its poor internalization activity, we
incubated cells expressing IiCT Ala7-Ala8 IiCT, or wild-type TR
with 125I-labeled Tf for 1 h at 37 °C. This
procedure loaded the endocytic pathway with receptor-ligand complexes
(14). The cells were then rapidly washed, and the reappearance of
intact and degraded Tf in the medium was monitored by measuring TCA
insoluble and soluble radioactivity, respectively. As expected, apo-Tf
released into the medium from cells expressing the wild-type TR was
undegraded (Fig. 5), because TR-apo-Tf
complexes are efficiently recycled back to the cell surface through
sorting and recycling compartments (35, 37, 43, 44). Only about 2% of
the counts were in the TCA soluble fraction after 2 h (TCA soluble
counts). In contrast, ~16% of the 125I-labeled Tf
released from cells expressing the IiCT
Ala7-Ala8 chimera was degraded, implying that
this percentage of chimeras traffics directly from the cell surface to
the lysosomal compartment where they are degraded. Although this is
less efficient degradation than the IiCT chimera (~32%),
these results demonstrate that the surface-expressed IiCT
Ala7-Ala8 chimera is also efficiently delivered
to the lysosomal compartment, albeit with reduced kinetics. This
suggests three things. First, endocytosis does not appear to be the
rate-limiting step in lysosomal transport of Ii. Second, only one of
the cytoplasmic tail signals, either LI or ML, is necessary for
efficient lysosomal delivery. Third, the two signals appear to be
additive with regard to lysosomal targeting efficiency.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 5.
Degradation of Tf bound to Ii-TR
chimeras. Equivalent cell numbers of CEFs expressing either
wild-type TR, IiCT, or IiCT
Ala7-Ala8 chimeras were preincubated in
serum-free Dulbecco's modified Eagle's medium for 30 min at 37 °C
and then incubated with 125I-labeled Tf for 1 h at
37 °C. The cells were then washed and reincubated at 37 °C in
Dulbecco's modified Eagle's medium containing 50 µg/ml unlabeled Tf
for various times. Acid-soluble radioactivity ( ) or acid-insoluble
125I-labeled Tf ( ) released into the medium, as well as
surface-bound 125I-labeled Tf ( ) and internalized
125I-labeled Tf ( ), were determined as described under
"Experimental Procedures" and are expressed as a percentage of
total radioactivity recovered. Each data point is the
average of triplicate determinations from a representative experiment
(of two).
|
|
The Membrane-distal Signal, DQRDLI, Is Necessary for Sorting at the
TGN--
Because the majority of major histocompatibility complex
class II complexes are delivered from the TGN to the lysosomal
compartment without appearing on the cell surface, we tested to see
whether the mutations that affected endocytosis also affected sorting at the TGN. Cells expressing wild-type TR, IiCT,
IiCT Ala7-Ala8, IiCT
Ala16-Ala17, IiCT
20-29,
IiCT
20-24, and IiCT
25-29 were
incubated with 125I-labeled Tf for 1 h to label the
endocytic pathway. The cells were then washed with BSA-PBS at 4 °C
to remove the unbound Tf, lysed in 1% Nonidet P-40, and counted in a
gamma counter. Comparison between the individual cell lines gave an
estimate of the total amount of chimeras on the surface and in
endosomal compartments (Table III,
surface-labeled counts). In companion dishes, cells were pulsed-labeled
with 35S-trans label for 30 min. TR or Ii-TR
chimeras were then immunoprecipitated from postnuclear supernatants and
quantitated on SDS-polyacrylamide gels by PhosphorImager analysis to
determine the relative biosynthetic rates of each of the constructs. In
a third set of dishes, we measured the relative half-lives of each of
the constructs using a metabolic-pulse-chase procedure. The relative
biosynthetic rates and degradation rates were then compared with the
wild-type TR control. Based on the assumption that all the wild-type TR
reaches the cell surface and is labeled with Tf, we estimate that only ~34% of the IiCT is delivered to the cell surface
(surface-accessible pool; see under "Experimental Procedures" for
details of the calculation) (Table III). The only mutation which
increases the surface-accessible pool, is the
Ala7-Ala8 mutation, the same one that disrupted
endocytosis, suggesting that the signals for TGN sorting and
endocytosis are closely related.
The Membrane-distal Sequence, DQRDLI, Can Substitute for the
Membrane-proximal Signal without Affecting Endocytosis or Lysosomal
Targeting--
The di-leucine signals in Ii appear to be related to
the identified six-residue signal in the CD3
-chain, DKQTLL (19). To
test whether the two Ii di-leucine related sequences, DQRDLI and
EQLPML, coded for independent structural motifs, such as those that
have been identified for tyrosine-based signals (25-28, 45), we
replaced one sequence, EQLPML, with the other, DQRDLI (IiCT 12DQRDLI17). We also prepared a mutant in which
the two sequences were substituted for each other (IiCT
3EQLPML8, 12DQRDLI17,
Fig. 1B).
Analysis of these two mutants in internalization assays indicated that
IiCT 12DQRDLI17 and
IiCT 3EQLPML8,
12DQRDLI17 were both endocytosed (Table I,
ke = 0.077 and 0.050, respectively), although
neither was equivalent to the wild-type Ii (Table I,
ke = 0.117). Analysis of the IiCT
12DQRDLI17 mutant in a metabolic pulse-chase
experiment indicated that it was rapidly degraded
(t1/2 ~ 3 h, Fig.
6A). Whereas the
IiCT 3EQLPML8,
12DQRDLI17 mutant had an extended half-life
(t1/2 > 12 h, Fig. 6A). These results demonstrated that the DQRDLI sequence can substitute for the
EQLPML signal without a significant loss of either sorting event,
whereas swapping the two sequences results in a partial loss of
internalization activity and an almost total loss of lysosomal targeting. This suggests that the sequence EQLPML could not substitute for the DQRDLI signal.

View larger version (52K):
[in this window]
[in a new window]
|
Fig. 6.
Structural requirements of the invariant
chain cytoplasmic tail necessary for lysosomal targeting.
Wild-type TR, IiCT 12DQRDLI17, or
IiCT 3EQLPML8,
12DQRDLI17 (A) or wild-type TR,
IiCT 9, IiCT 9-10, or IiCT
9-11 (B) chimeras were pulse-chased, immunoprecipitated
from postnuclear supernatants, and analyzed on SDS-polyacrylamide gels
as described in Fig. 2. A representative experiment (of three) is
shown.
|
|
Deletion of Ser9 (
9),
Ser9-Asn10 (
9-10), or
Ser9-Asn10-Asn11 (
9-11)
Inhibits Endocytosis without affecting Lysosomal Targeting--
To
determine whether residues between the two di-leucine signals were
required for either targeting event, we prepared mutants in which one,
two, or three residues were deleted (IiCT
9,
IiCT
9-10, and IiCT
9-11, respectively;
Fig. 1B). Analysis of these mutants indicates that these
deletions result in a 32, 52, and 36% loss of internalization
activity, respectively (Table I, ke = 0.079, 0.061, and 0.075, respectively). Analysis in metabolic pulse-chase experiments
indicated that the half-lives remain unchanged (Fig. 6B;
IiCT
9, t1/2 ~ 3.0 h,
IiCT
9-10, t1/2 = 2.8 h, and
IiCT
9-11, t1/2 ~ 3.5 h,
suggesting that these modifications affected endocytosis without
corresponding effects on lysosomal targeting).
Two 10-Amino Acid Regions from the Ii Transmembrane Are Sufficient
to Promote Lysosomal Targeting of the TR--
To localize the
lysosomal targeting signal in the Ii TM domain (14), we prepared Ii-TR
chimeras that contained ~10 amino acid segments from the Ii TM region
transplanted into the corresponding regions of the wild-type TR (Fig.
7). Each of these chimeras was expressed
in CEFs using BH-RCAS. Binding studies at 4 °C using 125I-labeled human Tf indicated that all of the chimeras
were expressed on the cell surface of transfected CEFs, but at lower
levels than the wild-type TR (data not shown).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 7.
Schematic diagram of the invariant
chain-transferrin receptor transmembrane chimeras. Schematic
representation of the transferrin receptor with various regions
replaced by the corresponding sequence from the invariant chain.
Unshaded areas represent regions of Ii-TR derived from TR;
shaded areas represent regions derived from Ii. The sequence
shown is the invariant chain transmembrane sequence. Residues 1-10,
11-19, and 20-29 of the transferrin receptor were replaced with the
corresponding sequences from the invariant chain. Constructs are
referred to in text by the corresponding names shown at the
left.
|
|
Analysis of the chimeras in metabolic pulse-chase experiments indicated
that two regions within the Ii TM region were sufficient to target the
TR to a processing compartment, residues 11 though 19 and residues 20 though 29 (Fig. 8; IiTM
11-19, t1/2 ~ 3.0 h; IiTM
20-29, t1/2 ~ 3.5 h). For comparison, the
half-lives of the wild-type TR and IiTM 1-10 chimera were
24 and 17 h, respectively. The chimera that contained Ii TM
residues 1-10 and 20-29 had an average half-life of 2.5 h (Table
II), suggesting that the TM 20-29 mutation was sufficient in either
context for lysosomal targeting. As before, the Ii-TR chimeras were
maturely glycosylated, indicating that they had traversed the Golgi
complex and were not simply misfolded proteins. To confirm that
proteolysis of the transmembrane chimeras was occurring in a lysosomal
compartment, we determined their half-lives in the presence of ammonium
chloride. The results indicate that the half-life of the
IiTM 20-29 chimera was extended from 1.5 h to
approximately ~8 h (Fig. 9), indicating that the degradation was occurring within the endocytic pathway. Ammonium chloride treatment extended the half-life of the
IiTM 11-19 chimera as well (data not shown). A summary of
the half-lives of the Ii-TR chimeras is shown in Table II. Analysis of
the TM chimeras in internalization assays demonstrated that none of the mutations dramatically affected internalization (Table
IV, 86-129% activities), indicating
that the increased degradation of the chimeras was not caused by a
corresponding increase in internalization.

View larger version (59K):
[in this window]
[in a new window]
|
Fig. 8.
Residues 11-19 and 20-29 of the invariant
chain transmembrane region are sufficient to mediate targeting of the
TR to a post-Golgi processing compartment. Equivalent cell numbers
of CEFs expressing the WT TR, IiTM 1-10, IiTM
11-19, or IiTM 20-29 chimeras were pulse-chased,
immunoprecipitated from postnuclear supernatants, and analyzed on
SDS-polyacrylamide gels as described in Fig. 2. A representative
experiment (of three) is shown.
|
|

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 9.
Ammonium chloride inhibits degradation of the
IiTM 20-29 chimera. CEFs expressing the
IiTM 20-29 chimera were preincubated for 1 h at
37 °C in medium (control) or medium plus 50 mM NH4Cl (+50 mM
NH4Cl) and then pulse-labeled with
35S-trans label and chased in the presence
(+50 mM NH4Cl) or absence
(control) of NH4Cl. The chimeras were then
immunoprecipitated and analyzed on SDS-polyacrylamide gels as described
in Fig. 2. A representative experiment (of two) is shown.
|
|
View this table:
[in this window]
[in a new window]
|
Table IV
Comparisons of the internalization rates of the transferrin
receptor-invariant chain transmembrane chimeras
|
|
 |
DISCUSSION |
By analyzing Ii-TR chimeras in quantitative assays, we report that
the two cytoplasmic tail di-leucine signals appear to be recognized at
distinct cellular sites. We provide evidence that the membrane-distal
signal, which includes Leu7-Ile8, is important
for endocytosis and lysosomal targeting, whereas the membrane-proximal
signal, which includes Met16-Leu17, appears to
be important for lysosomal targeting (Fig.
10). The distinction between the two
signals is best illustrated by the fact that modification of the
membrane-distal signal, Leu7-Ile8 to
Ala7-Ala8, resulted in a 9-fold reduction in
internalization activity, whereas modification of the membrane-proximal
signal, Met16-Leu17 to
Ala16-Ala17, had less than a 2-fold effect.
Interestingly, Leu7-Ile8 signal also appeared
to be required for sorting at the TGN because its disruption resulted
in a 3-fold increase in the amount of chimera that reached the cell
surface. In contrast to sorting at the TGN and plasma membrane, both
di-leucine signals appear to promote lysosomal targeting, although
neither alone was as efficient as both in combination.

View larger version (7K):
[in this window]
[in a new window]
|
Fig. 10.
Summary of the structural features of the
internalization and lysosomal targeting signals within the Ii
cytoplasmic tail and transmembrane region. The cytoplasmic tail of
Ii contains one region that is necessary for efficient endocytosis
(overlined) and two regions that are required for efficient
lysosomal targeting (underlined). Mutation of either region
decreases lysosomal targeting efficiency by approximately 50%.
Deletions between the two regions inhibit endocytosis (up to 50%)
without affecting lysosomal targeting. The sequence DQRDLI can replace
EQLPML without a significant loss of internalization or lysosomal
targeting activity. The position of the two regions relative to the TM
region influences both internalization and lysosomal targeting. Two
regions within the Ii transmembrane domain are important for lysosomal
targeting (underlined). Each TM region is individually
sufficient to promote lysosomal of the TR. Neither TM region has any
effect on endocytosis.
|
|
Met16-Leu17 has been reported to be important
for internalization (14, 18, 21, 40, 46), but these studies were based on bivalent antibody binding or measurement of steady-state
distributions. Bivalent antibodies have been shown to promote lysosomal
targeting of receptors that normally recycle (47-51), including the TR
(37, 43). In the present study, we directly measured transferrin uptake, a process that does not affect the endocytosis or trafficking of the TR (52). This allowed us to differentiate internalization signals from lysosomal signals because we followed the fate of the
chimeras with a monovalent ligand (53), transferrin. Furthermore, because steady-state distribution measurements for estimation of
internalization rates are based on the assumption that all of the cell
surface protein efficiently recycles (14) (a requirement that is not
met with lysosomally directed proteins, as is the case here), we
measured the internalization rates directly.
One surprising finding regarding lysosomal targeting was the fact that
although the IiCT Ala7-Ala8 chimera
was poorly internalized, it was still rapidly degraded. The half-life
of this chimera was the same as the IiCT
Ala16-Ala17 chimera, which was internalized
rapidly. This suggests that the rate-limiting step for lysosomal
targeting is not endocytosis. Further support for this idea comes from
studies on mutant hemagglutinins by Zwart et al. (54), who
demonstrated that targeting to the latter stages of endocytic pathway
does not simply correlate with the concentration of mutant
hemagglutinins in the early endosome (54). With their hemagglutinin
mutants, they demonstrated that there was no correlation between
internalization rate and degradation rate and, furthermore, that the
signal for these two processes were distinct. Although the
IiCT Ala7-Ala8 chimera most
dramatically illustrated the demarcation between these two processes, a
number of the other Ii mutants (
9,
9-10,
9-11) had
compromised internalization activity without any lysosomal targeting
loss. Studies on P-selectin and the epidermal growth factor receptor
also support the idea that distinct regions of these two molecules are
required for endocytosis and lysosomal targeting (55, 56). These
studies, as well as our own on Ii, suggest, therefore, that monitoring
endocytosis of cell surface molecules is not a reliable method for
monitoring down-regulation or lysosomal targeting.
By monitoring the relative amounts of Ii-TR chimeras that reached the
cell surface, we show that the same signal important for
internalization, Leu7-Ile8, is also important
for recognition at the TGN. A number of studies have suggested that
di-leucine signals can be recognized at the cell surface (19, 57) and
the TGN (20, 58). How recognition at the two cellular sites differs
remains unclear. One attractive model is that the position of the
signal within the tail specifies at which site it is recognized (59).
Dietrich et al. (59) propose that receptors containing
membrane-distal di-leucine signals are sorted directly from the TGN to
the endosome/lysosome, whereas receptors containing membrane-proximal
di-leucine signals require phosphorylation for internalization and
lysosomal targeting. Consistent with this model, the Ii cytoplasmic
tail is phosphorylated on a serine residue, although the site is not
known (60). Although our study does not support this model because the
membrane-distal signal appears to be required for sorting at both
sites, it does support the idea that the position of the signal
influences how and where it is recognized.
One of the goals of this study was to determine whether the two
di-leucine signals in Ii were equivalent. Although position clearly
influenced how each was being recognized, we also wondered whether the
particular sequences themselves were specific. To address this point,
we substituted the sequence from the membrane-distal signal, DQRDLI,
for the membrane-proximal signal, EQLPML. Our results demonstrated that
this mutation did not disrupt either targeting event, suggesting that
there was nothing unique about the second signal. These results also
suggested that di-leucine signals, like tyrosine-based signals, can
often substitute for each other. Interestingly, however, swapping the
signals resulted in a loss of both targeting events. The most likely
explanation for this comes from the NMR data on a 27-residue peptide
corresponding to the Ii cytoplasmic tail, which suggests that the first
14 residues lie within a nascent helix, whereas the membrane-proximal
signal which includes
Pro15-Met16-Leu17 is a part of a
turn (22). Thus, placing a proline residue at position 6 of the Ii
cytoplasmic tail probably disrupted the secondary structure, thereby
inhibiting recognition for all sorting events.
Deletion of residues 20-29 of the Ii cytoplasmic tail indicated that
none of these residues were specifically required for endocytosis or
lysosomal targeting. The mutant lacking residues 20-29, however, was
degraded more slowly than either of the two deletions encompassing this
region, suggesting two things. First, none of the residues per
se were required for lysosomal targeting; second, the position of
the two di-leucine regions relative to the transmembrane region
influenced lysosomal targeting. This is consistent with studies on
LAMP-1 that suggest a strict positional requirement of a signal for
lysosomal targeting signals (61). Interestingly, the membrane-proximal
region of Ii has also been reported to be recognized by the basolateral
sorting machinery in the biosynthetic pathway in MDCK cells (40). Our
studies suggest that this region is not necessary for TGN recognition in fibroblasts because the relative amounts of this chimera that reached the cell surface were the same as the wild-type Ii (Table III).
Our analysis of the Ii transmembrane domain indicates that two 10-amino
acid regions within this domain, residues 11-19 and residues 20-29,
are sufficient to target the TR to lysosomal compartment without
affecting the relative internalization rate. The structural features
within these regions that mediate this effect are not known. We
demonstrated that the chimeras were properly glycosylated, which
suggests that none of them were misfolded proteins. We also showed that
the degradation of the chimeras occurred within the endocytic pathway
because it could be inhibited with weak bases. The only other known
signals to be identified in transmembrane regions are Golgi retention
signals (reviewed in Ref. 62). Interestingly, the important feature of
Golgi localization signals appears to be the presence of uncharged
polar residues (Asn, Thr, and Gln), which when mutated result in the
loss of Golgi retention (63, 64). The Ii has an usual number of polar
residues, although they are not lined along one face of the predicted
helix as they are in the case of the cis-Golgi protein M
(64). The implication from Golgi localization studies is that polar
residues mediate protein-protein interactions resulting in
oligomerization of protein complexes within the lipid bilayer (62).
Oligomerization of the TM chimeras could result in a similar retention
mechanism within the endocytic pathway, resulting in a loss of
recycling efficiency.
Because the three-dimensional structure of the nonameric complex of the
major histocompatibility complex class II molecule is not known, the
significance of a signal within the transmembrane domain is not clear.
However, because the interaction site between the
-
chains and
the Ii is in the extracellular domain and proteolysis of the Ii
extracellular domain occurs first, one possibility is that the
transmembrane signal operates once the extracellular portions of Ii are
gone and the
-
chains have been released. This would ensure that
the residual portions of Ii would be degraded in the lysosome. Clearly,
degradation of the Ii-TR chimera proceeds more rapidly when the Ii
transmembrane region is included (14).
In conclusion, the results reported here suggest that the relative
location of the di-leucine signal within the Ii cytoplasmic tail
influences how it is recognized by the sorting machinery of the cell.
Furthermore, our study suggests that TR chimeras containing Ii
cytoplasmic tail or transmembrane signals are efficiently delivered to
the lysosomal compartment. Because the cellular machinery required for
sorting in the endosomal compartment has yet to be identified, it is
unclear how signals from the two different domain structures would be
recognized.
We thank C. Wright for preparation of the
CCL-1 construct and Christie Brouillette, Fred Maxfield, Doug Cyr, and
Elizabeth Sztul for helpful discussions and for critical reading of the manuscript.