Signal-binding Specificity of the µ4 Subunit of the Adaptor Protein Complex AP-4*

Ruben C. AguilarDagger §, Markus BoehmDagger §, Inna Gorshkova||**, Robert J. Crouch||, Kazuhiro TomitaDagger Dagger , Takashi SaitoDagger Dagger , Hiroshi Ohno§§, and Juan S. BonifacinoDagger ¶¶

From the Dagger  Cell Biology and Metabolism Branch and the || Laboratory of Molecular Genetics, NICHD, National Institutes of Health, Bethesda, Maryland 20892, the Dagger Dagger  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



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (alpha /gamma /delta /epsilon and beta 1-4, 90-130 kDa), a medium chain (µ1-4, ~50 kDa), and a small chain (sigma 1-4, ~20 kDa), each of which subserves a different function. Extensive analyses of the alpha  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 gamma /delta /epsilon chains are presumed to interact with other proteins that play similar regulatory roles. beta 1, beta 2, and beta 3 interact with the scaffolding protein, clathrin (8-10). In addition, beta 1 and beta 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 sigma  chains is unknown, although sigma 1 and sigma 3 are required for the functional integrity of the AP-1 and AP-3 complexes, respectively (21, 22).

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 beta  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-beta 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.

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-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
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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REFERENCES

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-TGNDelta -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-TGNDelta -EagI cut with EagI and PstI. The amino acid sequence encoded by the resulting constructs was Gal4BD-HNKRKIIAFALEGKRSKVTRRPKXXXYXXØ. The construct Gal4BD-beta 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 alpha  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-SRalpha (26).

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 beta -galactosidase activity. Colonies expressing beta -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 beta -galactosidase activity) to test the binding specificity of library clones.

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 (Delta F) in standard error units (14). Any Delta F value above 1 (i.e. favored) or below -1 (i.e. disfavored) was considered to be significantly different from 0 (random).

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-beta -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 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).

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 beta -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.

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),


[<UP>RU<SUB>eq</SUB></UP>]=<UP>RU</UP><SUB><UP>max</UP></SUB>/1+(K<SUB>D</SUB>/C) (Eq. 1)
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.

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).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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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 beta -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 Delta F values correspond to residues that were favored or disfavored, respectively. Only residues with Delta 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 beta -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 Delta 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.

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).


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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.

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.


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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.

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.


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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

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-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.

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.

    FOOTNOTES

* 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

    ABBREVIATIONS

The abbreviations used are: AP, adaptor protein; Gal4AD, Gal4 transcription activation domain; Gal4BD, Gal4 DNA-binding domain.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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