From the Cell Biology and Metabolism Branch and the
Laboratory of Molecular Genetics, NICHD, National Institutes of
Health, Bethesda, Maryland 20892, the
Department of Molecular Genetics, Chiba
University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba
260-8670, Japan, and the §§ Division of
Molecular Membrane Biology, Cancer Research Institute, Kanazawa
University, 13-1 Takaramachi, Kanazawa 920-0934, Japan
Received for publication, November 22, 2000
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ABSTRACT |
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The medium (µ) chains of the adaptor protein
(AP) complexes AP-1, AP-2, and AP-3 recognize distinct subsets of
tyrosine-based (YXXØ) sorting signals found within
the cytoplasmic domains of integral membrane proteins. Here, we
describe the signal-binding specificity and affinity of the medium
subunit µ4 of the recently described adaptor protein complex AP-4. To
elucidate the determinants of specificity, we screened a two-hybrid
combinatorial peptide library using µ4 as a selector protein.
Statistical analyses of the results revealed that µ4 prefers aspartic
acid at position Y+1, proline or arginine at Y+2, and phenylalanine at
Y The heterotetrameric adaptor protein
(AP)1 complexes AP-1, AP-2,
AP-3, and AP-4 are components of protein coats that associate with the
cytosolic face of organelles of the secretory and endocytic pathways
(reviewed in Refs. 1-4). AP-2 is associated with the plasma membrane
and mediates rapid internalization of endocytic receptors, whereas
AP-1, AP-3, and AP-4 are associated with the trans-Golgi
network and/or endosomes and mediate intracellular sorting events. AP
complexes are thought to participate in protein sorting by inducing
the formation of coated vesicles as well as concentration of cargo
molecules within the vesicles. Concentration of integral membrane
proteins is mediated by direct interaction of the AP complexes with
sorting signals present within the cytosolic tails of the proteins.
Several types of cytosolic sorting signals have been described, the
most common of which are referred to as "tyrosine-based" or
"dileucine-based" depending on which residues are critical for
activity (5, 6).
The four AP complexes have a similar structure and are composed of two
large chains ( Our laboratory has been particularly interested in the role of the µ chains in signal recognition. We have previously demonstrated that µ1
and µ2 display a bipartite structure, with the amino-terminal one-third being involved in interactions with the corresponding Although much has been done to characterize the signal-binding
specificity of µ1, µ2, and µ3A, little is known about sequence preferences for the more recently described µ4 (also known as µ-ARP2) (24). Previous studies have shown that µ4 interacts weakly
with YXXØ signals from the lysosomal membrane proteins LAMP-1 (AGYQTI) (18) and CD63 (SGYEVM) (25) and the
trans-Golgi network protein TGN38 (SDYQRL) (18). To
determine whether µ4 might be able to recognize with higher affinity
a defined subset of YXXØ signals, we have undertaken a
yeast two-hybrid screening of a combinatorial YXXØ library.
The results show that µ4 prefers signals with Phe at position Y Recombinant DNA Constructs--
The constructs
Gal4AD-µ1, Gal4AD-µ2, and Gal4AD-µ3A in the
pACTII(LEU2) plasmid (CLONTECH, Palo
Alto, CA) have been described previously (12, 13). The Gal4AD-µ4
construct was prepared by ligating a BamHI-SacI
polymerase chain reaction fragment corresponding to the 5'-part of µ4
and a SacI-PstI cDNA fragment corresponding to the 3'-part of µ4 into the BamHI-XhoI sites
of the pACTII(LEU2) vector using a
PstI-XhoI adaptor. As previously described (14), a DNA fragment encoding the 33-amino acid cytoplasmic tail of TGN38
engineered to contain an EagI site (by introduction of
silent mutations in place of the codons for Arg21 and
Pro22 from the TGN38 cytoplasmic tail) was used to prepare
the pGBT9-TGN Yeast Culture, Transformation, and Two-hybrid
Assays--
The Saccharomyces cerevisiae strain HF7c
(MATa, ura3-52, HIS3-200, lys2-801,
ade2-101, trp1-901, leu2-3,112, GAL4-542, gal80-538,
LYS2::GAL1-HIS3, URA3::(GAL4
17-mers)3-CYC1-lacZ) (CLONTECH) was maintained on yeast extract/peptone/dextrose-agar plates. Transformations were done by the lithium acetate procedure as described
in the instructions for the MATCHMAKER two-hybrid kit (CLONTECH). For colony growth assays, HF7c
transformants were streaked on plates lacking leucine, tryptophan, and
histidine and allowed to grow at 30 °C, usually for 4-5 days, until
colonies were visible. For two-hybrid screening of the combinatorial
library, the yeast cells were first transformed with Gal4AD-µ4 and
plated onto yeast dropout agar plates lacking leucine as described in the protocol for the MATCHMAKER two-hybrid system. Transformants were
re-transformed with the combinatorial DNA library and selected on
plates lacking leucine and tryptophan for selection of co-transformants and lacking histidine for selection of interacting clones;
Leu+Trp+ and His+ colonies were
then tested for Cell Culture and Transfection--
HeLa cells (American Type
Culture Collection, Manassas, VA) were cultured in Dulbecco's modified
Eagle's medium supplemented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin (Biofluids, Inc.,
Rockville, MD) (regular medium). Primary cultures of skin fibroblasts
from AP-3-deficient mocha mice (Jackson Laboratory, Bar
Harbor, ME) were obtained as previously described (27) and maintained
in regular medium. The night before transfection, cells were seeded
onto 6-well plates (Costar Corp., Corning, NY) in 2 ml of
regular medium. The following day, the cells were cotransfected with
the Tac constructs and pCI-NEO (Promega, Madison, WI) using Fugene-6
reagent (Roche Molecular Biochemicals). To obtain stable transfectant
clones, the regular medium from HeLa cells was replaced with fresh
medium containing 1 mg/ml G418 (Calbiochem) 24 hours after
transfection. The clones obtained were analyzed for expression of the
Tac constructs by immunofluorescence microscopy.
Statistical Analyses--
The experimental (observed)
frequency for each residue at each position of the
XXXYXXØ sequence was calculated using the
sequences selected by the µ4 subunit from the combinatorial library.
Preferences were evaluated by calculating the difference between the
observed and expected frequencies ( Site-directed Mutagenesis--
Single amino acid substitutions
were made using the QuickChange mutagenesis kit (Stratagene, La Jolla,
CA). Briefly, 50 ng of plasmid carrying the target cDNA was
incubated with two complementary primers (2 mM each)
containing the desired mutation in the presence of 2 mM
dNTP mixture and 2.5 units of Pfu DNA polymerase for 16 cycles according to the following temperature profile: 0.5 min at
95 °C, 1 min at 55 °C, and 8 or 16 min at 68 °C. After
replication of both vector strands, the methylated parental DNA was
digested for 1 h at 37 °C with 10 units of DpnI
endonuclease, and the nicked vector with the desired mutation was
transformed into Escherichia coli.
In Vitro Binding Assays--
35S-labeled
µ4-(156-453) protein was obtained by in vitro
transcription/translation using the TNT T7 Quick coupled
transcription/translation system (Promega) and EasytagTM
expression protein labeling mixture (PerkinElmer Life Sciences) according to the manufacturers' instructions. In brief, 500 ng of the
pET28a-µ4-(156-453) construct was incubated with 20 µl of TNT
Quick Master Mix and 11 µCi of [35S]methionine in a
total volume of 25 µl at 30 °C for 90 min. The transcription/translation reaction mixture (containing
35S-labeled µ4-(156-453)) was diluted 1:100 in binding
buffer and centrifuged (180,000 × g, 15 min, 4 °C).
500 µl of supernatant was applied to peptide-coupled beads and
incubated for 12 h at 4 °C. The beads were washed three times
at 4 °C with binding buffer without bovine serum albumin, boiled in
Laemmli sample buffer, and separated by SDS-polyacrylamide gel
electrophoresis. The SDS gel was soaked in sodium salicylate and
subjected to autoradiography.
Expression and Purification of µ4-(156-453)--
E.
coli BL21(DE3) cells were transformed with
pET28a-His10-µ4-(156-453); a single colony was picked;
and the presence of the construct was verified. 2 liters of
LB/kanamycin medium was inoculated with 100 ml of preculture and grown
at 37 °C until A600 reached 1.6. Protein
expression then was induced by the addition of
isopropyl- Preparation of Peptide-coupled Beads and Surface Plasmon
Resonance Sensor Chips--
The following peptides were obtained from
Zymed Laboratories Inc. (South San Francisco, CA):
CWKRHHTGYEQF, CWKRHHTGAEQF, CWKRHHTGYEQA, CWRPKETLYRRF, CWRPKETLARRF,
and CWRPKETLYRRA. Peptide-coupled beads for in vitro binding
assays were prepared by coupling the Cys residue of the peptides to
EZ-LinkTM PEO-maleimide-activated biotin (Pierce) in
phosphate-buffered saline (pH 6.9) at peptide and biotin concentrations
of 1 and 1.67 mM, respectively. The reaction was quenched
by the addition of Surface Plasmon Resonance Spectroscopy--
Surface plasmon
resonance permits, in a label-free mode, real-time detection of binding
events on the chip surface and estimation of binding parameters (28).
10 µl of µ4-(156-453) at the indicated concentrations was injected
onto sensor chip surfaces. Dissociation of bound protein was carried
out for 10 min, and then the surface was regenerated by two 30-s
injections of regeneration solution and by two 30-s injections of
running buffer. All experiments were repeated twice on two different
chips. Data transformation and overlay sensorgrams were prepared using
BIAevaluation Version 3.0 software (BIAcore AB). The response from the
reference surface was subtracted from the other three flow cells to
correct for refractive index changes, matrix effects, nonspecific
binding, injection noise, and base-line drift. Using nonlinear
least-squares fitting, the equilibrium dissociation constant
(KD) was evaluated by fitting data to a single site
interaction model (Equation 1),
Antibodies and Immunofluorescence
Microscopy--
Immunofluorescence microscopy of fixed permeabilized
cells and antibody internalization microscopy experiments were done as previously described (27, 29). The following monoclonal antibodies were
used: anti-mouse LAMP-2 monoclonal antibody ABL-93, anti-human LAMP-2
monoclonal antibody H4B4, and anti-human CD63 monoclonal antibody H5C6
(Developmental Studies Hybridoma Bank, University of Iowa, Iowa City,
IA). A polyclonal antiserum to recombinant Tac was raised in rabbits.
Alexa 448 and Cy3-conjugated secondary antibodies were from Jackson
ImmunoResearch Laboratories, Inc. (West Grove, PA).
Signal-binding Specificity of µ4 Determined by Screening of a
Combinatorial XXXYXXØ Yeast Two-hybrid Library--
We have
previously analyzed the signal-binding specificity of µ1, µ2, and
µ3 (A and B isoforms) by screening a
Gal4AD-XXXYXXØ combinatorial library using the
yeast two-hybrid system (14). Here, we have used the same method to
define the signal-binding specificity of the µ4 subunit of AP-4. To
this end, the combinatorial library was coexpressed with a Gal4AD-µ4
construct in yeast cells. Twenty clones that grew in medium lacking
histidine and that tested positive for
Some of the sequences selected by µ4 (DLYYDPM, ETLYRRF, DFYYERL, and
DYCYDRF) were tested for their ability to interact with other µ subunits (Fig. 1C). The results showed that the DLYYDPM sequence was specific for µ4, whereas the ETLYRRF and DYCYDRF sequences interacted with µ2 and µ4, and the DFYYERL sequence interacted with all four µ chains (Fig. 1B). Thus, µ4
shares with the other µ chains the ability to interact with distinct
but overlapping sets of YXXØ-type sequences.
Interaction of µ4 with Naturally Occurring Tyrosine-based Sorting
Signals--
To further characterize the interactions of µ4 with
YXXØ motifs, we used the yeast two-hybrid system to test
for interactions with YXXØ signals found in the cytosolic
tails of some transmembrane proteins. The YXXØ signal of
TGN38 was replaced by the analogous signals from LAMP-1, CD68, CD63,
and LAMP-2, and interactions with µ chains were tested using the
yeast two-hybrid system. A qualitative assay for growth on
histidine-deficient plates revealed that µ4 interacted only with the
YXXØ signal from human LAMP-2 (HTGYEQF) (Fig.
2A). The LAMP-2 signal was not
recognized only by µ4 though, as it bound even more strongly to µ2
and µ3A (Fig. 2, A and B). A salient feature of
this signal is the presence of Phe at the Ø position, which fits the
µ4 preferences deduced from the combinatorial analyses. The
Tyr-to-Ala and Phe-to-Ala variants of the LAMP-2 signal (HTGAEQF and
HTGYEQA, respectively) were unable to interact with µ4 or with any
other µ chain (Fig. 2B).
To verify the yeast two-hybrid results, we performed a binding assay
using in vitro transcribed/translated µ4-(156-453) and chemically synthesized and biotinylated LAMP-2 peptides. The peptides were bound to streptavidin beads and incubated with radioactively labeled µ4-(156-453). Bound µ4 was revealed by SDS-polyacrylamide gel electrophoresis and fluorography. As shown in Fig. 2C,
µ4-(156-453) bound well to the wild-type LAMP-2 sequence (HTGYEQF),
but only barely to the Tyr-to-Ala (HTGAEQF) and Phe-to-Ala (HTGYEQA)
variants of the sequence.
The resolution of the crystal structure of the µ2 signal-binding
domain allowed identification of residues that are directly involved in
interactions with the critical tyrosine residue of the signals (19).
Several of those residues are conserved in the other µ chains,
including µ4 (3). To determine whether interactions of µ4 with
YXXØ signals involved conserved residues in the
tyrosine-binding pocket, we mutated the conserved Asp190 or
Lys438 residue of µ4 to Ala. Two-hybrid assays revealed
that these mutations abrogated interactions of µ4 with the
tyrosine-based signal from LAMP-2 (Fig. 2D). Thus, the
structural bases for the recognition of YXXØ signals by
µ4 appear to be similar to those of µ2.
Characterization of µ4-YXXØ Interactions by Surface Plasmon
Resonance Spectroscopy--
µ4-YXXØ interactions were
further characterized by surface plasmon resonance spectroscopy. In
these studies, we used three biotinylated peptides: CWRPKETLYRRF,
corresponding to one of the sequences selected from the combinatorial
library (Fig. 1B), and its Tyr-to-Ala (CWRPKETLARRF) and
Phe-to-Ala (CWRPKETLYRRA) variants. Preliminary in vitro
binding experiments showed that the CWRPKETLYRRF peptide bound
radiolabeled µ4-(156-453) (Fig.
3A) in a
concentration-dependent manner (Fig. 3B),
whereas CWRPKETLARRF and CWRPKETLYRRA did not (Fig. 3, A and
B). The three biotinylated peptides were loaded onto
separate flow cells of a streptavidin-coated chip. Recombinant µ4-(156-453) was then applied, and binding of the protein was measured by an increase in response units. The signal for the CWRPKETLYRRF peptide at a concentration of 13.4 µM
reached a plateau at ~1000 response units, whereas that of the two
variant peptides only reached 100-150 response units. This was in the
range of the nonspecific binding of µ4-(156-453) to the biotinylated
streptavidin surface without any peptide bound, as shown by the blank
curve. After ending the injection of protein solution at 5 min, the
value for the signal dropped sharply for all samples, indicating that the binding process was mostly reversible. However, ~20% of the binding could not be reversed even after washing for 10 min (data not
shown). We performed an analysis of the interaction of different concentrations of µ4-(156-453) with the CWRPKETLYRRF peptide (Fig. 4A). As expected, the signal
amplitude was dependent on the amount of µ4-(156-453) applied. The
response approached a plateau value (a steady-state level,
RUeq (Equation 1)) after ~4.5 min. A plot of
RUeq against the concentration of µ4 is presented in Fig.
4B. Nonlinear regression analysis of these data yielded an
apparent equilibrium dissociation constant of 7.0 ± 2.5 µM and a maximum binding capacity (RUmax) of
the surface of 1550 ± 165 response units. Although these values
should be considered only estimates, it is nonetheless clear that the
interactions are of low affinity.
Intracellular Localization of a Chimeric Protein Bearing a
µ4-specific Signal--
To gain insights into the possible function
of AP-4, we took advantage of the identification of a YXXØ
signal (DLYYDPM) that was apparently specific for µ4 (Fig.
1B). This signal, as well as its corresponding Tyr-to-Ala
mutant (DLYADPM), was appended to the cytosolic tail of the
transmembrane protein Tac (29). The constructs were stably expressed in
HeLa cells, and their intracellular distribution at steady state was
examined by immunofluorescence microscopy using antibodies to the Tac
luminal domain. We observed that the Tac-DLYYDPM chimera was present in
the Golgi complex and plasma membrane (Fig.
5A). Treatment with the
lysosomal inhibitor leupeptin, however, resulted in accumulation of
Tac-DLYYDPM in intracellular vesicles (Fig. 5B). Some of
these vesicles colocalized with the lysosomal transmembrane proteins
CD63 (Fig. 5, D-F) and LAMP-2 (Fig. 5, G-I),
suggesting that a fraction of the Tac-DLYYDPM chimera was transported
to late endosomes or lysosomes. The vesicular staining and
colocalization of the chimera with LAMP-2 were not affected by the
absence of the AP-3 complex in cells from the mocha mouse
strain (Fig. 5, J-L) (30), consistent with the observation that the DLYYDPM signal does not interact with µ3A (Fig.
1C). The DLYYDPM signal did not mediate internalization of
the chimera from the cell surface (Fig.
6, A and B),
whereas a PLSYTRF signal derived from the transferrin receptor did
(Fig. 6, E and F). As expected, the Tyr-to-Ala
mutant chimera (DLYADPM) and a Tac construct without any tyrosine-based
sorting signal were not significantly internalized (Fig. 6,
C and D, and J and K,
respectively). These observations were in agreement with the inability
of the DLYYDPM signal to interact with µ2 and suggested that the
vesicular localization of the Tac-DLYYDPM chimera was not the result of
internalization from the cell surface.
The results of the experiments reported here show that µ4
shares, with other members of the µ family of AP subunits, the
ability to recognize a subset of YXXØ sorting signals. As
is the case for other µ chains, interactions of µ4 with
YXXØ signals require the Tyr and Ø residues (Figs. 2 and
3) and are saturable (Fig. 4). These properties emphasize the
remarkable structural conservation of the µ chain family of proteins.
Indeed, of 15 residues in µ2 known to be involved in interactions
with Tyr and Ø residues (19), 14 are identical in µ4 (3), with the
remaining one being a conservative Leu173
(µ2)-to-Val187 (µ4) substitution. Mutation of one of
two of the identical amino acids, Asp190 or
Lys438, to Ala abrogates interaction of µ4 with the
signals (Fig. 2D), confirming that µ2 and µ4 recognize
YXXØ signals in a similar fashion.
These structural similarities notwithstanding, the subset of
YXXØ signals recognized by µ4 exhibits some
characteristic features that distinguish it from that of other µ chains. The most salient feature of µ4 specificity is the preference
for aromatic residues (Phe or Tyr) at various positions neighboring the
critical Tyr residue. None of the other µ chains characterized to
date exhibits this preference (14). The preference for Phe residues is
particularly strong at the Y Despite the fact that µ4 prefers certain residues at positions
neighboring the critical Tyr residue, the subset of YXXØ
signals recognized by µ4 overlaps to a significant extent with those
recognized by other µ chains (Fig. 1C). This further
strengthens the previous conclusion that µ chains recognize distinct
but overlapping sets of YXXØ signals (14). Therefore, the
involvement of AP complexes in specific sorting events cannot depend
solely on the specificity of signal recognition by their µ chains.
Rather, the role of signal preferences is likely to "fine-tune" the
efficiency of sorting.
A screening of several naturally occurring YXXØ signals
revealed that the lysosomal targeting signal from LAMP-2 (HTGYEQF) (30)
interacts with µ4 (Fig. 2). This signal has a Phe residue at the Ø position, which could explain why it binds to µ4 (Fig. 1B). Previous studies had demonstrated weak interactions of
µ4 with two other lysosomal membrane proteins, LAMP-1 (18) and CD63
(25). Taken together, these observations suggest a possible role for
the AP-4 complex in sorting to lysosomes. However, the signals from all
of these lysosomal membrane proteins interact better with µ2 and
µ3A than with µ4 (Fig. 2, A and B). To gain insight into the potential function of AP-4, we took advantage of the
identification of a signal (DLYYDPM) that interacts exclusively with
µ4 (Fig. 1C). This signal was placed at the cytosolic
carboxyl terminus of a Tac chimeric construct devoid of other sorting
signals (13). The resulting Tac-DLYYDPM chimera was expressed by stable transfection into HeLa cells, and its localization was determined by
indirect immunofluorescence microscopy. In the absence of protease inhibitors, the protein exhibited a steady-state localization to the
Golgi complex and plasma membrane. However, incubation with the
lysosomal inhibitor leupeptin resulted in accumulation of the protein
in lysosomes, as shown by colocalization with LAMP-2 (Fig. 5). This
indicated that the Tac-DLYYDPM chimera is transported to and degraded
in lysosomes. As expected, this accumulation was dependent on the
critical Tyr residue of the signal. The Tac-DLYYDPM chimera was not
efficiently internalized from the plasma membrane (Fig. 6), in
accordance with its inability to interact with µ2 (Fig.
1C). In addition, the Tac-DLYYDPM chimera was still targeted to lysosomes in AP-3-deficient mocha cells, further
demonstrating that AP-3 does not play a role in the recognition of the
DLYYDPM signal. Even though our two-hybrid results indicated that there is no interaction between the DLYYDPM signal and µ1 (Fig.
1C), we cannot rule out the possibility that AP-1 could
somehow be involved in sorting of the Tac-DLYYDPM chimera. However,
Meyer et al. (31) have suggested that targeting of proteins
to lysosomes is not affected in µ1-deficient cells. These
observations are consistent with the possibility that µ4 and, by
extension, the AP-4 complex are involved in targeting proteins from the
trans-Golgi network to the endosomal-lysosomal system. This
involvement could provide an alternative means of sorting proteins to
lysosomes, the existence of which has been suggested by previous
studies (31-34). The evidence for a role of AP-4 in targeting to the
endosomal-lysosomal system presented here, however, is indirect and
should be considered tentative until it becomes possible to study
protein sorting in AP-4-deficient cells. Attempts to ablate expression
of this complex in mice are underway.
1 and Y+3 (Ø). In addition, we examined the interaction of µ4
with naturally occurring YXXØ signals by both two-hybrid
and in vitro binding analyses. These experiments showed
that µ4 recognized the tyrosine signal from the human lysosomal
protein LAMP-2, HTGYEQF. Using surface plasmon resonance
measurements, we determined the apparent dissociation
constant for the µ4-YXXØ interaction to be in the micromolar range. To gain insight into a possible role of AP-4 in
intracellular trafficking, we constructed a Tac chimera bearing a
µ4-specific YXXØ signal. This chimera was targeted to
the endosomal-lysosomal system without being internalized from the
plasma membrane.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
/
/
and
1-4, 90-130 kDa), a medium chain
(µ1-4, ~50 kDa), and a small chain (
1-4, ~20 kDa), each of
which subserves a different function. Extensive analyses of the
chain of AP-2 have shown that it interacts, either directly or
indirectly, with many regulators of coat assembly and/or vesicle formation (7). By analogy, the
/
/
chains are presumed to interact with other proteins that play similar regulatory roles.
1,
2, and
3 interact with the scaffolding protein, clathrin (8-10).
In addition,
1 and
2 have been found to bind a subset of
dileucine-based sorting signals (11). The µ chains, on the other
hand, function as recognition molecules for signals conforming to the
YXXØ consensus motif (Y is tyrosine, X is any
amino acid, and Ø is leucine, isoleucine, phenylalanine, methionine,
or valine) (12-20). The exact role of the
chains is unknown,
although
1 and
3 are required for the functional integrity of the
AP-1 and AP-3 complexes, respectively (21, 22).
chains and the C-terminal two-thirds being involved in recognition of
YXXØ-type signals (23). X-ray crystallography revealed that the YXXØ-binding domain of µ2 consists of a banana-shaped
all-
structure to which the signals bind in an extended conformation (19). The Tyr and Ø residues fit into hydrophobic pockets on this
domain. Both crystallographic (19) and binding (13-18) studies have
suggested that the identities of the Ø residue and the residues surrounding the critical Tyr residue are important determinants of the
specificity of interaction. Although the subsets of YXXØ signals recognized by µ1, µ2, and µ3A overlap to a significant extent, each chain nonetheless exhibits certain preferences for residues neighboring the critical Tyr residue (14). For example, µ1,
µ2, and µ3A prefer Leu, Leu, and Ile residues at the Ø positions and neutral, basic, and acidic residues at the X positions,
respectively. We have argued that these preferences alone are unlikely
to account for the functional specificity of each AP complex (14).
However, they probably contribute to the selectivity and efficiency of specific signal recognition events.
1,
Asp at Y+1, Pro or Arg at Y+2, and Phe at Y+3 (Ø). A signal that fits
this latter preference is found in the lysosomal membrane protein
LAMP-2, and indeed, we found that the LAMP-2 signal binds to µ4 both
in the yeast two-hybrid system and in vitro. We also found
that a reporter integral membrane protein bearing a µ4-specific
YXXØ signal is delivered to the endosomal-lysosomal system
without being internalized from the plasma membrane.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-EagI construct by ligation into the
EcoRI and XhoI sites of the
pGBT9(TRP1) vector (CLONTECH).
Oligonucleotides encoding either a combinatorial XXXYXXØ peptide library (14) or different
YXXØ-type signals were digested with EagI
and PstI and then ligated into pGBT9-TGN
-EagI cut with EagI and PstI. The amino acid sequence
encoded by the resulting constructs was
Gal4BD-HNKRKIIAFALEGKRSKVTRRPKXXXYXXØ. The
construct Gal4BD-
2 was kindly provided by Dr. M. S. Robinson (University of Cambridge, Cambridge, United Kingdom). All of the other two-hybrid constructs were made by ligation of polymerase chain
reaction products into the pGBT9 or pACTII vector. The construct pET28a-µ4-(156-453) was obtained by cloning nucleotides
466-1362 of the coding sequence of µ4 into pET28a (Invitrogen,
Carlsbad, CA) using NheI and HindIII restriction
sites. pET28a-µ4-(156-453) was digested with NdeI and
BstEII to release a 1066-base pair fragment containing the
amino-terminal His6 tag and ligated with the
NdeI-BstEII fragment of vector pET16b
(Invitrogen) containing the His10 tag. The resulting
construct was named pET28a-His10-µ4-(156-453). Interleukin-2 receptor
subunit (Tac) chimeric constructs were prepared by ligation of complementary oligonucleotides (coding for the
PLSYTRF, DLYYDPM, and DLYADPM sequences) between an XbaI site inserted at the 3'-end of the Tac cDNA and the
BamHI site from the expression vector pCDL-SR
(26).
-galactosidase activity. Colonies expressing
-galactosidase were cultured in dropout medium containing leucine
but lacking tryptophan to obtain cells carrying only the library
plasmid and not the medium subunit plasmid. The resulting cells were
then mated with the yeast strain Y187 (MATa) transformed with Gal4AD-µ4 constructs or with pTD1-1 (SV40 large-T antigen cDNA in pACTII; negative control for histidine auxotrophy and
-galactosidase activity) to test the binding specificity of
library clones.
F) in standard error
units (14). Any
F value above 1 (i.e. favored)
or below
1 (i.e. disfavored) was considered to be
significantly different from 0 (random).
-D-thiogalactopyranoside to a final
concentration of 3 mM, and the cells were incubated at
37 °C for another 4 h. The cells were harvested, resuspended in
buffer A (20 mM Tris-HCl (pH 8.0), 250 mM NaCl,
and 5 mM imidazole), and sonicated. After centrifugation,
the supernatant was loaded onto a Ni2+-nitrilotriacetic
acid Superflow column (QIAGEN Inc., Valencia, CA), and the recombinant
protein was eluted using buffer A with 1 M imidazole.
µ4-(156-453)-containing elution fractions were pooled; dialyzed
against 20 mM Tris-HCl (pH 7.0), 250 mM NaCl, 5 mM EDTA, and 0.5 mM dithiothreitol; and
concentrated by centrifugation in a Centriprep-3 device (Amicon, Inc.,
Beverly, MA).
-mercaptoethanol to a final concentration of 10 mM. 50 µl of ImmunoPure immobilized streptavidin beads
(Pierce) was washed twice with phosphate-buffered saline (pH 6.9),
incubated overnight with 300 µl of biotinylation reaction, and washed
three times with binding buffer (0.05% (w/v) Triton X-100, 50 mM HEPES (pH 7.3), 10% (v/v) glycerol, 100 mM
KCl, 2 mM MgCl2, 0.1 mM
CaCl2, 50 µM dithiothreitol, and 0.1% bovine
serum albumin). Surface plasmon resonance experiments were carried out
on a BIAcore 1000 instrument (BIAcore AB, Uppsala) at 25 °C using
SA sensor chips with streptavidin covalently immobilized on a
carboxymethylated dextran matrix. The chips were conditioned by 10 consecutive 1-min injections of 1 M NaCl, 50 mM
NaOH, and 0.25% (w/v) SDS at a flow rate of 10 µl/min and washed
extensively with Tris-buffered saline (20 mM Tris-HCl (pH
7.0), 250 mM NaCl, 5 mM EDTA, and 0.005% (v/v) polysorbate 20). Biotinylated peptides were injected at a
concentration of 500 nM in Tris-buffered saline running
buffer at a flow rate of 2 µl/min onto the chip surface until the
desired level of immobilization (~150 response unit) was achieved.
Unoccupied streptavidin was blocked by biotin (30 µl of a 10 µM solution at a flow rate of 5 µl/min). The sensor
chip was then washed by five consecutive 1-min injections of
regeneration solution (25 mM NaOH, 500 mM NaCl,
and 0.0005% (w/v) SDS). Flow cell 1 (with biotin-treated streptavidin)
was left blank and used as a reference surface.
where RUeq is the steady-state response level,
RUmax is the maximal capacity of the surface (which was
floated during the fitting procedure), and C is the
concentration of µ4 in micromolar.
(Eq. 1)
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase activity were
isolated, and their amino acid sequences were deduced from DNA
sequencing (Fig. 1A). A
statistical analysis of the residues found at each position is shown in
Fig. 1B; positive or negative
F values
correspond to residues that were favored or disfavored, respectively.
Only residues with
F values equal to or greater than 1 or
equal to or lower than
1 were considered significant. Overall, µ4
seemed to have a distinct preference for aromatic amino acids at
several positions of the XXXYXXØ sequence. The
most commonly found amino acids at each position were Cys, Tyr, Phe,
Tyr, Asp, Pro, and Phe, respectively. Tyr was also favored at Y
1 and
Arg at Y+2. Among the residues at the Ø position, the only preference
was for Phe, whereas Val was strongly disfavored (Fig.
1B).
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Fig. 1.
Two-hybrid screening of a combinatorial
peptide library. A, sequences of
XXXYXXØ clones selected by µ4. A
Gal4BD-XXXYXXØ library was coexpressed with a
Gal4AD-µ4 construct in yeast cells. Co-transformants expressing
interacting Gal4BD and Gal4AD constructs were selected in medium
lacking tryptophan, leucine, and histidine and tested for expression of
-galactosidase activity. A list of the sequences obtained from
library plasmids isolated from those clones is shown (see
"Experimental Procedures" for details). B, statistical
analysis of the library screening results. The preferences of µ4 for
residues within the XXXYXXØ sequence were
inferred from the
F values in S.E. units (y
axis; see "Experimental Procedures" for details) at each
position (panels Y
3 to Y+3). Levels
of significance are indicated by different gray tones, with
the darkest representing the most significant (
2 S.E. also
indicated with **). NS, not significant. C,
cross-reactivity analysis of some sequences selected by µ4. To test
the binding specificity of signals selected by µ4 (indicated at the
top of each panel), the corresponding Gal4BD constructs were
co-transformed with different Gal4AD-µ constructs and tested for
complementation of histidine auxotrophy. Cell growth in liquid medium
lacking histidine was measured as turbidity at 600 nm.
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Fig. 2.
Interaction of µ4
with the YXXØ signal from the lysosomal protein
LAMP-2. A, analysis of the interaction of different µ chains with naturally occurring YXXØ-type signals. The
source of each signal is indicated in parentheses. Yeast
co-transformants expressing the Gal4AD-AP subunit and the Gal4BD signal
constructs indicated were grown on plates lacking leucine and
tryptophan, with or without histidine (+His and
His, respectively). B, yeast
two-hybrid analysis of the interaction of AP chains with the LAMP-2
signal (HTGYEQF) and the HTGAEQF and HTGYEQA variants of this signal.
C, in vitro binding of µ4-(156-453) to the
LAMP-2 signal (HTGYEQF) and the HTGAEQF and HTGYEQA variants of this
signal. The biotinylated CWKRHHTGYEQF, CWKRHHTGAEQF, and CWKRHHTGYEQA
peptides were bound to streptavidin-coated beads and incubated with
in vitro transcribed/translated, radiolabeled
µ4-(156-453). D, analysis of the interaction of µ4
point mutants with the HTGYEQF signal. Mutants of µ4 carrying single
amino acid substitutions of Asp190 or Lys438
with Ala were examined for interaction with HTGYEQF using the yeast
two-hybrid system.
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Fig. 3.
Characterization of the interaction of the
ETLYRRF sequence with recombinant
µ4-(156-453). The binding of
µ4-(156-453) to an ETLYRRF peptide, selected in the combinatorial
screen (see Fig. 1C), was characterized using an in
vitro binding assay (as described for Fig. 2C) and
surface plasmon resonance spectroscopy. A, µ4-(156-453)
binds specifically to the ETLYRRF sequence and not to the Tyr-to-Ala or
Phe-to-Ala variants of the sequence. B, concentration
dependence of the binding of µ4-(156-453) to the ETLYRRF peptide.
Increasing amounts of µ4-(156-453) were added to the biotinylated
CWRPKETLYRRF and CWRPKETLARRF peptides bound to streptavidin beads.
C, results from surface plasmon resonance experiments
performed as described under "Experimental Procedures".
µ4-(156-453) (13.4 µM) was injected onto
streptavidin-coated flow cells previously loaded with the biotinylated
CWRPKETLYRRF, CWRPKETLARRF, or CWRPKETLYRRA peptide. The results of two
determinations for each peptide are shown. Notice that the binding of
µ4-(156-453) to ETLYRRF depends on the presence of the critical Tyr
residue as well as the Phe residue at the Ø position. RU,
response units.
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Fig. 4.
Estimation of the affinity of the ETLYRRF
sequence for µ4-(156-453) by surface plasmon
resonance spectroscopy. A, various concentrations of
µ4-(156-453) as indicated were injected onto streptavidin-coated
flow cells previously loaded with the biotinylated CWRPKETLYRRF
peptide. B, nonlinear least-squares fitting of the data
shown in A yielded an equilibrium dissociation constant
(KD) of 7.0 ± 2.5 µM for the
binding of µ4-(156-453) to the CWRPKETLYRRF peptide. RU,
response units.
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Fig. 5.
Intracellular distribution of a Tac chimera
bearing a µ4-specific signal.
A-I, HeLa cells stably transfected with a Tac-DLYYDPM
chimeric construct were treated with (B-I) or without
(A) leupeptin (Leup; 1 mg/ml) for 4 h.
J-L, primary cultures of fibroblasts from AP-3-deficient
mocha mice were transiently transfected with Tac-DLYYDPM.
Fixed permeabilized cells were incubated with rabbit antiserum to the
luminal domain of Tac and monoclonal antibodies to the lysosomal
membrane proteins CD63 (D--F) and LAMP-2
(G-L), followed by incubation with Alexa 448-conjugated
anti-rabbit and Cy3-conjugated anti-mouse IgG antibodies.
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Fig. 6.
Analysis of the internalization of Tac and
Tac signal chimeras. Live HeLa cells stably expressing Tac
(no signal) or Tac signal chimeras were incubated with
anti-Tac antiserum for 1 h at 4 °C. After washing off the
unbound antibody with phosphate-buffered saline, half of the cells were
incubated at 37 °C for 30 min (B, D,
F, and H) to allow antibody internalization, and
the rest were kept at 4 °C as controls (A, C,
E, and G). All the cells were then fixed, and the
internalized antibody was detected by incubation with Cy3-conjugated
anti-mouse IgG. The presence of the proteins at the plasma membrane was
evidenced by staining of the outline of the cells (A-E and
G-H), whereas internalized proteins were detected as
intracellular vesicles (F).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and Y+3 (Ø) positions. In the case of
the Ø position, this might be explained by the Leu173
(µ2)-to-Val187 (µ4) substitution. The smaller
Val187 residue lining the hydrophobic pocket could allow
accommodation of the large aromatic side chain of Phe while disfavoring
binding of the smaller Val side chain. Another preference specific for µ4 is Asp at position Y+1, whereas other preferences are similar to
those of other µ chains. For instance, the selectivity for Pro at Y+2
appears to be a general characteristic of all the µ chains. This
suggests that a bend in the polypeptide chain imposed by Pro stabilizes
the conformation of the signals for interaction with µ chains. µ4
also favors Arg at Y+2, a preference shared only with µ2 (14). In the
case of µ2, this preference for Arg is due to the establishment of
hydrophobic interactions of the Arg side chain with Trp421
and Ile419 of µ2 and a hydrogen bond between the
guanidinium group of Arg and Lys420 of µ2 (19). Two of
these residues in µ2 (Trp421 and Lys420) are
conserved in µ4 (Trp429 and Lys440,
respectively), but not in the other µ chains (3), which probably explains why only µ2 and µ4 favor Arg at Y+2.
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FOOTNOTES |
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* This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan (to H. O. and T. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
¶ Supported by a grant from the German Academic Exchange Service (DAAD).
** Supported by a National Research Council LMG-NICHD senior research associateship.
¶¶ To whom correspondence should be addressed: Cell Biology and Metabolism Branch, NICHD, Bldg. 18T, Rm. 101, NIH, Bethesda, MD 20892. Tel.: 301-496-6368; Fax: 301-402-0078; E-mail: juan@helix.nih.gov.
Published, JBC Papers in Press, January 3, 2001, DOI 10.1074/jbc.M010591200
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ABBREVIATIONS |
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The abbreviations used are: AP, adaptor protein; Gal4AD, Gal4 transcription activation domain; Gal4BD, Gal4 DNA-binding domain.
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REFERENCES |
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