(Received for publication, March 1, 1995; and in revised form, June 8, 1995)
From the
Recent reports have suggested that major histocompatibility complex class II molecules load peptide through a specialized compartment of the endocytic pathway and are targeted to this pathway by association with invariant chain (Iip31). Therefore we used a site-directed mutagenesis approach to determine whether Iip31 possesses novel protein targeting signals. Our results indicate that two di-leucine-like pairs mediate Iip31 targeting and that an acidic amino acid residue four or five residues N-terminal to each Iip31 di-leucine-like pair is required for endocytic targeting. Results from additional testing with hybrid Iip31 molecules indicate that the acidic residues N-terminal to di-leucine pairs are critical for accumulation of these molecules in large endocytic vesicles and in some cases provide a structure favorable for internalization. The acidic residues N-terminal to di-leucine pairs are important in some sequence contexts in providing a structure favorable for internalization, whereas in other contexts an acidic residue is critical for targeting to, and formation of, large endocytic vesicles. Although our results do not support the idea that Iip31 possesses unique protein targeting motifs, they do suggest that di-leucine motifs may be recognized as part of a larger secondary structure. In addition, our data imply that the targeting motif requirements for internalization may differ from the requirements for further transport in the endocytic pathway.
The major histocompatibility complex (MHC) ()class II
molecules are expressed on antigen-presenting cells such as B cells,
macrophages, and dendritic cells and present primarily antigenic
peptides derived from exogenous proteins to helper T cells (for review
see Neefjes and Ploegh, 1992; Germain and Margulies, 1993; Germain,
1994; and Cresswell, 1994). The Iip31 molecule is a type II
transmembrane protein that associates with MHC class II molecules in
the endoplasmic reticulum and limits binding of these molecules to
antigenic peptides (Roche and Cresswell, 1990; Teyton et al.,
1990) while escorting them (Bakke and Dobberstein, 1990; Lotteau et
al., 1990) to a specialized endosomal compartment (Peters et
al., 1991; Amigorena et al., 1994; Tulp et al.,
1994; West et al., 1994; Qui et al., 1994). In this
compartment Ii is released, and class II molecules bind peptides before
appearing at the cell surface. We wished to determine the precise
targeting information in the Iip31 cytoplasmic tail and in particular
whether Iip31 contains a novel targeting motif that may explain the
transport of MHC class II molecules to a specialized endocytic
compartment dedicated to peptide loading.
Previous studies have demonstrated that information contained in the Iip31 cytoplasmic tail is both necessary and sufficient for Iip31 targeting to the endosomal/lysosomal compartment. Deletion analyses showed that the first 16 amino acid residues were required for Iip31 endosomal/lysosomal targeting (Bakke and Dobberstein, 1990; Lotteau et al., 1990). Site-directed mutagenesis localized two signals in the Iip31 cytoplasmic tail which could independently target invariant chain to the endosomal system; one signal contains leucine and isoleucine at positions 7 and 8 of the tail (Pieters et al., 1993). The second signal contains methionine and leucine at positions 16 and 17 (Odorizzi et al., 1994; Bremnes et al., 1994).
These Ii targeting motifs are part of a larger
family of di-leucine-related targeting motifs that mediate lysosomal
targeting and internalization from the cell surface (for review see
Sandoval and Bakke, 1994). Di-leucine-related signals have been
described for T lymphocyte antigen receptor CD3 and
chains
(Letourneur and Klausner, 1992), cation-dependent and -independent
mannose 6-phosphate receptors (CD-M6PR and CI-M6PR, respectively;
Johnson and Kornfeld, 1992a, 1992b; Chen et al., 1993),
interferon-
receptor (Farrar and Schreiber, 1993), lysosomal
integral membrane protein (LIMP) II (Ogata and Fukuda, 1994; Sandoval et al., 1994), glucose transporter 4 (Verhey and Birnbaum,
1994), T lymphocyte cell surface CD4 molecule (Aiken et al.,
1994), and Iip31 (Odorizzi et al., 1994; Bremnes et
al., 1994). Di-leucine-related variants that permit different
degrees of LIMP II lysosomal targeting include Leu-Ile, Leu-Val, and
Ile-Ile, whereas Val-Ile does not target LIMP II to lysosomes (Ogata
and Fukuda, 1994; Sandoval et al., 1994). Substitution of
alanine for one or both hydrophobic residues abrogates lysosomal
localization and internalization.
In this report we use alanine scan mutagenesis to ascertain the sequence requirements for Iip31 targeting of class II molecules to a specialized endosomal compartment. Our analysis confirmed earlier studies demonstrating the importance of two di-leucine-like motifs for internalization of Ii from the cell surface and intracellular targeting to large vesicular endosomal structures. In addition, we uncovered a second critical component: an acidic amino acid residue four or five residues N-terminal to each of these di-leucine-like signals which is required for efficient targeting. By constructing hybrid molecules of Iip31 containing heterologous di-leucine-based signals and using alanine substitution analyses we have shown that acidic residues are required for the formation of the large endocytic vesicles induced by Iip31 expression. Thus, the di-leucine-based pair is part of a larger internalization and endocytic targeting motif that includes an N-terminal acidic residue. In accordance with the tyrosine-based internalization motifs, we suggest that the secondary structure of the di-leucine-containing cytoplasmic tails is an important part of the recognition motif. Targeting of Ii to the compartment for peptide loading is discussed in light of these findings.
Two
days after transfection, HeLa cells in 100-mm dishes were treated with
1 ml of trypsin-EDTA (Life Technologies, Inc.) and resuspended with 9
ml of cDMEM. One-ml aliquots were distributed into six-well plates
(Corning, Corning, NY), and cells were cultured for 12-18 h.
Cells were washed on ice three times with 3 ml of ice-cold PBS
containing Ca and Mg
(Irvine
Scientific, Santa Ana, CA), then incubated with 5
10
cpm of
I-labeled BU45 in cold PBS with 1% bovine
serum albumin, 1 ml/well, for 1 h. Cells were washed four times with 3
ml of cold PBS, then incubated with Dulbecco's modified
Eagle's medium containing 1% bovine serum albumin, 2 mM
glutamine, 10 mM HEPES at 37 °C, 5% CO
, or on
ice. After incubation at 37 °C, plates were placed on ice, and the
cells were washed once with cold PBS. Incubation with cold acid (0.5 M acetic acid in 0.15 M NaCl, pH 2.5; 0.5 ml/well)
for 7 min removed the remaining cell surface-bound
Ilabeled BU45. Addition of 2 ml of PBS neutralized the
acid. The acid wash was removed, and cells were washed with 1 ml PBS,
which was added to the original acid wash. Cells were lysed with 1 N NaOH. For each transfectant at each time point, the
percentage of internalized
I-BU45 was calculated as
((A - B)/(A - B + C))
100, where A = cpm in the cell
lysate of acid-washed cells incubated at 37 °C, B =
cpm in the cell lysate of the corresponding acid-washed cells incubated
at 4 °C, and C = cpm in the acid wash of cells
incubated at 37 °C. In all cases, the acid wash removed >95% of
the initial cell surface-bound
I-BU45. Background binding
of
I-labeled BU45 to nontransfected HeLa cells in 35-mm
dishes at 4 °C was always less than 1,000 cpm.
The Iip31 cytoplasmic tail contains two independent
di-leucine-based pairs, Leu-Ile and Met-Leu, which direct
internalization and endocytic targeting of Iip31 or heterologous
extracytoplasmic domains ()(Odorizzi et al., 1994;
Bremnes et al., 1994). To define further the sequence
requirements for di-leucine-based internalization and endocytic
targeting, we selectively altered the Iip31 cytoplasmic tail sequence
and used two assays to measure the targeting of Iip31 cytoplasmic tail
mutants to the endocytic compartment. First, we measured Iip31
internalization from the cell surface of transiently transfected HeLa
cells by quantitating the uptake of cell surface-bound
I-labeled BU45 antibody to Ii as described under
``Experimental Procedures.'' We used intact BU45 antibody, as
it has been shown that internalization of
I-labeled BU45
and of
I-labeled BU45 Fab fragments is equivalent (Roche et al., 1993). Second, we analyzed the subcellular
localization of wild-type and mutant Iip31 molecules by indirect
immunofluorescence microscopy. The precise location in the
endosomal/lysosomal pathway in which Iip31 accumulates at steady state
remains controversial. The observation that Iip31 colocalizes with
endocytic markers (Romagnoli et al., 1993) and with
lysosomal-associated membrane protein (Pieters et al., 1993) (
)suggests that it is located in late endosomes or
lysosomes.
Figure 1:
Internalization of Iip31 molecules
containing cytoplasmic tail deletions or mutations of the
di-leucine-based signals. Internalization of I-BU45 was
measured as described under ``Experimental Procedures.'' One
representative experiment of three or more is shown for each construct. Del, delta.
Indirect immunofluorescence microscopy showed that the wild-type
Iip31 molecules localized to large vesicles (Fig. 2A)
and the cell surface (data not shown). The 2-7Ii molecules
were present predominantly in small vesicles and the cell surface,
although some cells contained
2-7Ii molecules in large
vesicles (data not shown). The
2-11Ii molecules were present
in small vesicles (data not shown) in accordance with previous reports
for
2-11Ii molecules (Bakke and Dobberstein, 1990). The
2-13Ii,
2-16Ii, and
2-26Ii molecules
were not detected in vesicles but were present at the cell surface
(data not shown). The presence of
2-11Ii molecules in small
but not large vesicles suggests that Iip31 cytoplasmic tail sequences,
in addition to lumenal domain sequences
(Bremnes et
al., 1994), are necessary for large vesicle formation.
Figure 2:
Indirect immunofluorescence microscopy of
HeLa cells expressing Iip31 molecules containing wild-type (panel
A) or mutant cytoplasmic tails (panels B-H). Cells
were stained with C351 rabbit antibodies to the Ii lumenal domain
followed by fluorescein-conjugated antibodies to rabbit IgG. Panel
B, hybrid LIMP II-Ii molecules. The first 17 residues of the
cytoplasmic tail are MDERAPLISNNEQLPSS. Panel C, Ii molecules
with the first 17 cytoplasmic tail residues mutated to
MAARAPLISNNEQLPSS. Panel D, hybrid CI-M6PR-Iip31 molecules.
The first 17 cytoplasmic tail residues are MDDRVGLVSNNEQLPSS. Panel
E, hybrid CD3 -Ii molecules. The first 17 cytoplasmic tail
residues are MSDQKTLLSNNEQLPSS. Panel F, hybrid CD3
-Ii
molecules with Asp
mutated to alanine. The first 17
cytoplasmic tail residues are MSAQKTLLSNNEQLPSS. Panel G,
hybrid CD4-Ii molecules. The first 17 cytoplasmic tail residues are
MSQIKRLLSNNEQLPSS. Panel H, hybrid CD4-Ii molecules with
Ser
and Gln
mutated to aspartic acid
residues.
Substitution of alanine for Iip31 residues Asp,
Leu
, or Ile
abolished internalization, whereas
mutation of Asp
to alanine dramatically decreased the
internalization rate mediated by the Leu-Ile pair (Fig. 3A). In contrast, the internalization rates of
Iip31 molecules containing an alanine substitution at residue
Gln
, Arg
, Asp
, or Ser
was comparable to the internalization rate of the parent
M16S,L17S Iip31 molecule (Fig. 3A). Similarly, for the
Met-Leu pair, replacement of residue Glu
with alanine
eliminated internalization, but replacement of residue Ser
,
Asn
, Gln
, or Leu
with alanine
resulted in mutant Iip31 molecules that were internalized (Fig. 3B). Replacing residue Asn
or Pro15
with alanine decreased the L7S,I8S Ii internalization rate by
40-50% relative to wild-type Iip31 (Fig. 3B).
Indirect immunofluorescence showed that all internalized mutant Iip31
molecules in the alanine scan series were present in large vesicles
(data not shown), whereas noninternalized mutant Iip31 molecules
concentrated at the cell surface and were not detected in vesicles
(data not shown). Thus, to function as internalization and endosomal
targeting signals for Iip31, residues Asp
and Asp
are essential for the Leu-Ile pair, and residue Glu
is essential for the Met-Leu pair.
Figure 3:
Internalization of Iip31 molecules
containing alanine substitutions at residues 2-9 with the Met-Leu
pair mutated to Ser-Ser (panel A) or at residues 9-15
with the Leu-Ile pair mutated to Ser-Ser (panel B).
Internalization of I-BU45 was measured as described under
``Experimental Procedures.'' One representative experiment of
two or more is shown for each construct.
To assess further the
requirements for Iip31 internalization, we tested the effects of
conservative substitutions, and substitution of polar but uncharged
residues, for the acidic amino acid residues. The internalization rates
of mutants D2E and D3E containing only the Leu-Ile pair, and of mutant
E12D containing only the Met-Leu pair (Table 1), did not differ
from the parent M16S,L17S and L7S,I8S mutants, respectively (Fig. 4). Thus, aspartic acid and glutamic acid are functionally
interchangeable at positions 2, 3, and 12 in the Iip31 cytoplasmic
tail. The D2N,M16S,L17S and D3N,M16S,L17S mutants were not internalized (Fig. 4), indicating that an isoteric polar, uncharged residue
is not permissible at position 2 or 3. Likewise, substitution of
glutamine for Glu abolished internalization mediated by
the Met-Leu pair (Fig. 4). Substitution of polar, uncharged
residues for the acidic residues yielded molecules expressed
predominantly at the cell surface with no detectable vesicular
localization (data not shown). Thus, isoteric polar residues cannot
substitute for the critical acidic residues necessary for recognition
of the two Iip31 internalization and endosomal targeting signals.
Figure 4:
Internalization of Iip31 molecules
containing substitutions of the acidic residues at position 2 or 3 with
mutation of the Met-Leu pair to Ser-Ser, and at position 12 with
mutation of the Leu-Ile pair to Ser-Ser. Internalization of I-BU45 was measured as described under
``Experimental Procedures.'' One representative experiment of
two or more is shown for each construct.
Figure 5:
Panel A, internalization of Iip31
molecules containing LIMP II-Ii hybrid cytoplasmic tails. Panel
B, internalization of Iip31 molecules containing multiple alanine
substitutions in the LIMP II-Ii hybrid cytoplasmic tail. In each case,
amino acid residues 2-8 of the Iip31 sequence were replaced by
the indicated sequence, and the Met-Leu pair was mutated to Ser-Ser.
Internalization of I-BU45 was measured as described under
``Experimental Procedures.'' One representative experiment of
two or more is shown for each construct.
We made further mutations in the LIMP II signal to determine which features of this signal are critical for internalization. Having found that the sequence MAARAPLISNNEQLPSS gave the wild-type internalization rate, we made the mutants MAARAALISNNEQLPSS and MAAAAPLISNNEQLPSS and found that Ii hybrid molecules containing these mutant cytoplasmic tails were internalized at approximately half the rate of hybrid molecules containing the wild-type LIMP II signal (Fig. 5B). Mutation of residues 2-6 to alanines, resulting in the mutant MAAAAALISNNEQLPSS, abrogated internalization (Fig. 5B). Thus, MAARAP constitutes a permissive, minimal sequence N-terminal to the Leu-Ile pair required to achieve the wild-type Iip31 internalization rate.
Figure 6: Internalization of Iip31 molecules containing the CI-M6PR-Ii hybrid cytoplasmic tail or alanine scan mutations. See the Fig. 5legend for details. One representative experiment of two or more is shown for each construct.
Figure 7:
Internalization of Iip31 molecules
containing CD3 chain-Ii hybrid cytoplasmic tail or alanine scan
mutations. See the Fig. 5legend for details. One representative
experiment of three or more is shown for each
construct.
Figure 8: Panel A, internalization of Iip31 molecules containing CD4-Ii hybrid cytoplasmic tail or alanine scan mutations. Panel B, internalization of CD4-Ii chimeric molecules containing aspartic acid substitutions in the CD4 di-leucine-based motif. See the Fig. 5legend for details. One representative experiment of two or more is shown for each construct.
In this report, we performed extensive alanine scan mutagenesis of sequences N-terminal to di-leucine-like pairs in the MHC class II-associated Iip31 molecule, and on four other cell surface receptors. Our results suggest that internalization and targeting within the endocytic pathway require different targeting motifs. Mutation to alanine of an acidic residue, but not the other residues immediately N-terminal to the di-leucine pair, abolished large endocytic vesicle formation and, in some cases, internalization. These results show that residues N-terminal to the di-leucine-based pairs are part of a larger motif recognized by the intracellular targeting machinery and that the sequences surrounding the di-leucine pair, in particular N-terminal acidic residues, are involved in targeting within the endocytic compartment.
Our data show that residues 2-8 of
the Iip31 cytoplasmic tail constitute one internalization motif, and
residues 12-17 constitute a second motif. Indeed, Iip31
cytoplasmic tail residues 1-11 and 12-30 can separately
confer internalization to hybrid molecules with the neuraminidase
transmembrane and lumenal domains (Bakke O. et al., 1993).
Furthermore, we note similarities in the two Iip31 signals (Table 2). We have also shown that when the Met-Leu pair is
mutated to Ser-Ser, mutation of amino acid residues Asp or
Asp
to alanine abrogates internalization with respect to
the Leu-Ile pair. Although residue Glu
is present, it
apparently cannot substitute for the Asp
or Asp
residues. Similarly, when amino acid residue Glu
is
mutated to alanine and the Leu-Ile pair is mutated to Ser-Ser, residues
Asp
and Asp
cannot provide the acidic residue
needed for internalization mediated by the Met-Leu pair. Thus, the two
Iip31 internalization motifs are functionally separate.
MHC class II and Iip31 molecules are targeted to a specialized endosomal compartment where class II molecules bind antigenic peptides (see the Introduction). We were interested in determining if the Iip31 cytoplasmic tail has a novel signal for targeting to this compartment. Our results show that the LIMP II and CI-M6PR di-leucine-based signals can mediate targeting to the same or similar vesicular compartments as wild-type Iip31. Therefore, we suggest that the Iip31 cytoplasmic tail does not contain novel sequence information for targeting to the compartment for peptide loading and therefore that other molecules with di-leucine-based signals have access to this compartment. Whether class II molecules influence targeting of class II molecule-Iip31 complexes to this compartment remains to be tested.
One hypothesis to explain these contrasting results is that different cell types have differing abilities to recognize two di-leucine motifs in one cytoplasmic tail. This theoretical difference regarding internalization motifs across cell types and species has been demonstrated experimentally. For example, the tyrosine motif analog NPVF mediates low density lipoprotein receptor internalization in Chinese hamster ovary cells (Davis et al., 1987) but not in Madin-Darby canine kidney cells (Matter et al., 1994). A second hypothesis is that the transferrin receptor and Ii lumenal or transmembrane domains themselves influence the internalization rate. Precedence for this hypothesis is suggested by data showing that the M6PR lumenal domain influences receptor recycling and residence time within endosomes (Dintzis et al., 1994).
Cytoplasmic tail acidic residues in other sequence contexts may be involved in other intracellular targeting processes. For example, basolateral targeting, but not internalization, mediated by cytoplasmic tail sequences of the type I transmembrane protein low density lipoprotein receptor is influenced by acidic residues C-terminal to its tyrosine-based targeting motif (Matter et al., 1994). This observation suggests that although basolateral targeting and internalization sequences may overlap, the motifs for the two processes are distinct. These results are analogous to our observations that N-terminal acidic residues are not always required for di-leucine-based internalization but are required for large vesicle formation. Interestingly, basolateral targeting mediated by the Fc receptor II di-leucine-based signal does not appear to need acidic residues, as determined by analyses of truncation and alanine scan mutants (Hunziker and Fumey, 1994).
An alternative hypothesis is that the acidic residues are not required for Iip31 targeting to a specialized endocytic compartment per se but that they are required for large vesicle formation after Ii entry into an endocytic subcompartment. Immunofluorescence studies have shown previously that large vesicles staining for Iip31 are also enriched with late endosomal or lysosomal markers such as CI-M6PR or lysosomal-associated membrane protein molecules (Lotteau et al., 1990; Romagnoli et al., 1993; Pieters et al., 1993). Large vesicle formation induced by Iip31 expression is time-dependent after Iip31 transfection (Pieters et al., 1993), indicating that a critical level of Iip31 accumulation must be reached before large vesicles form. The mechanism by which these large endocytic vesicles form is not known. The Iip31 molecule may alter endosome/lysosome morphology, perhaps by multimerizing and interfering with protein trafficking as suggested previously (Romagnoli et al., 1993). We note that overexpression of rab5 (Bucci et al., 1992) or rab22 (Olkkonen et al., 1993) proteins, which are localized to early and late endosomes and the plasma membrane, also causes large vesicle formation, perhaps by inducing endosomal clustering and fusion. Therefore, another possible mechanism for large vesicle formation in Iip31-expressing cells is that acidic residues in the Iip31 tail mimic a signal recognized for fusion of vesicles in the endosomal compartment. Knowledge of the mechanism by which these large vesicles form may provide insight into the regulation of protein trafficking within the endocytic compartment.
The Iip31 molecules might enter the
endosomal/lysosomal compartment either directly from the TGN or by
internalization from the cell surface. The relative contributions of
these two routes for Iip31 is presently not known. Odorizzi et
al.(1994) estimate that more than 80% of hybrid molecules
containing the Iip31 cytoplasmic and transmembrane domains and the
transferrin receptor lumenal domain are delivered intracellularly from
the TGN to the endocytic pathway. For the CI-M6PR and CD-M6PR, which
contain tyrosine-based and di-leucine-based targeting motifs, mutation
or deletion of the di-leucine pair did not alter their internalization
rates, but drastically reduced correct sorting of lysosomal enzymes
(Lobel et al., 1989; Johnson and Kornfeld, 1992a). These
results suggest that di-leucine motifs mediate direct sorting of
CD-M6PR from the TGN to late endosomes. In this report and others
(Letourneur and Klausner, 1992; Ogata and Fukuda, 1994; Pieters et
al., 1993; Aiken et al., 1994), di-leucine-based motifs
were shown to mediate internalization from the cell surface. Moreover,
internalization of the CD4 molecule and of the CD3 -Tac hybrid is
likely to proceed via clathrin-coated pits (Pelchen-Matthews et
al., 1991; Letourneur and Klausner, 1992). Thus, it is likely that
the di-leucine motif is recognized at the TGN and at the cell surface.
This suggestion raises further questions. Are the proteins recognizing
the same di-leucine motifs at the TGN as at the cell surface? Are these
proteins the same as the adaptin complexes that recognize aromatic
residue-based internalization motifs (Glickman et al., 1989).
The results described here provide insight into the minimal
requirements for Iip31 internalization and endocytic targeting mediated
by its two di-leucine-based signals. Our results show that acidic
residues are required for targeting of Ii within the endosomal
compartment, that internalization is not determined solely by the
di-leucine-like pairs, and that nearby residues also contribute to
efficient internalization. If all di-leucine-based motifs are
recognized by the same targeting molecules, it is probable that a
common secondary structure exists. Future studies will determine the
validity of this hypothesis. The sequence requirements for the
di-leucine-based signals are strikingly different from the aromatic
residue-based signals, although parallels may yet exist between the
di-leucine-based motif structure and the aromatic residue-based motif
-turn or nascent
-helical structure. The discovery that
di-leucine-based signals responsible for internalization and endosomal
targeting involve a larger motif helps explain the specificity of the
molecular recognition events required for protein sorting.