Golgi-localizing, gamma-adaptin ear homology domain, ADP-ribosylation factor-binding (GGA) proteins interact with acidic dileucine sequences within the cytoplasmic domains of sorting receptors through their Vps27p/Hrs/STAM (VHS) domains.

GGA (Golgi-localizing, gamma-adaptin ear homology domain, ARF-binding) proteins are potential effectors of ADP-ribosylation factors, are associated with the trans-Golgi network (TGN), and are involved in protein transport from this compartment. By yeast two-hybrid screening and subsequent two-hybrid and pull-down analyses, we have shown that GGA proteins, through their VHS (Vps27p/Hrs/STAM) domains, interact with acidic dileucine sequences found in the cytoplasmic domains of TGN-localized sorting receptors such as sortilin and mannose 6-phosphate receptor. A mutational analysis has revealed that a leucine pair and a cluster of acidic residues adjacent to the pair are mainly responsible for the interaction. A chimeric receptor with the sortilin cytoplasmic domain localizes to the TGN, whereas the chimeric receptor with a mutation at the leucine pair or the acidic cluster is mislocalized to punctate structures reminiscent of early endosomes. These results indicate that GGA proteins regulate the localization to or exit from the TGN of the sorting receptors.

2491) and cation-dependent (CD)-MPR (aa 212-277) were obtained by PCR amplification of a human liver cDNA library (Life Technologies, Inc.). A cDNA fragment covering the entire cytoplasmic domain of rat LRP3 (aa 521-770) was obtained by PCR amplification of its full-length cDNA (Ref. 17; a kind gift from Dr. Tokuo Yamamoto, Tohoku University, Japan). Mutations of residues within the acidic dileucine sequence of human sortilin were introduced into the cDNA for its cytoplasmic domain (aa 779 -831) by a PCR-based strategy. For two-hybrid assays, the cDNA fragments for the VHS or VHSϩGGAH domains and for the cytoplasmic domains were subcloned into the pGBT9 bait and pACT2 prey vectors (CLONTECH, Palo Alto, CA), respectively. For expression in Escherichia coli as fusion proteins with glutathione S-transferase (GST) or with thioredoxin (TRX)-His 6 , the cDNA fragments for the cytoplasmic domains and for the VHS domains were subcloned into the pGEX-4T (Amersham Pharmacia Biotech, Ltd., Buckinghamshire, UK) and pET-32 (Novagen, Inc., Madison, WI), respectively. Mammalian expression vectors for Tac chimeras were constructed by ligation of the cytoplasmic domain cDNA downstream of the transmembrane domain of the human Tac antigen cDNA (a kind gift from Dr. Hiroshi Ohno, Kanazawa University, Japan), followed by subcloning into the pcDNA3 vector (Invitrogen Corp., Carlsbad, CA). Construction of an expression vector for hemagglutinin (HA)-tagged GGA1 was described previously (8).
Yeast Two-hybrid Screening and Assay-Yeast two-hybrid screening was performed as described previously (18). Briefly, yeast Y190 cells harboring the pGBT9 vector for the VHSϩGGAH domain of GGA1 were transformed with a pACT2-based library of human brain cDNAs (CLONTECH), grown for 5-7 days on synthetic medium containing 25 mM 3-aminotriazole and lacking tryptophan, leucine, and histidine, and subjected to a filter assay for ␤-galactosidase activity. pACT2 plasmids rescued from the positive clones were subjected to sequence analysis. Two-hybrid assays for interactions between the VHS domains and the cytoplasmic domains were performed by essentially the same procedures as described previously (8,18). Briefly, reporter yeast cells harboring the pGBT9-based bait and pACT2-based prey vectors were grown on synthetic medium containing 100 mM 3-aminotriazole and lacking tryptophan, leucine, and histidine or on medium lacking tryptophan and leucine. The cells grown on the latter medium were then subjected to a filter assay for ␤-galactosidase using 5-bromo-4-chloro-3-indolyl ␤-D-galactopyranoside as a substrate.
Pull-down Assay-The VHS domains fused to TRX-His 6 and the wild-type (WT) and mutated cytoplasmic domains fused to GST were expressed in E. coli BL21(DE3) cells and purified using ProBond nickelchelating resin (Invitrogen) and glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech, Ltd.), respectively, as described previously (19). The purified TRX-His 6 -VHS domain fusion protein (ϳ60 g) was incubated with the GST-cytoplasmic domain fusion protein (ϳ20 g) prebound to glutathione-Sepharose beads for 1 h at room temperature. The beads were then washed five times with phosphate-buffered saline. Proteins were eluted from the beads by boiling in SDS-polyacrylamide gel electrophoresis sample buffer, electrophoresed on 12.5% SDS-polyacrylamide gel, and blotted onto an Immobilon-P membrane (Millipore Corp., Bedford, MA). The blot was incubated sequentially with polyclonal rabbit anti-TRX antibody and peroxidase-conjugated anti-rabbit IgG and was detected using a Renaissance chemiluminescence reagent Plus (PerkinElmer Life Sciences).
DNA Transfection and Immunofluorescence Microscopy-DNA transfection and immunofluorescence analysis were performed as described previously (8,18,20). Briefly, HeLa cells grown in wells of 8-well Lab-Tek-II chamber slides (Nunc A/S, Roskilde, Denmark) were transfected with the expression vector for a Tac fusion protein alone or together with that for HA-tagged GGA1 using the FuGene6 transfection reagent (Roche Molecular Biochemicals) and incubated for 10 -16 h. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and incubated sequentially with monoclonal mouse anti-Tac antibody and monoclonal rat anti-HA or polyclonal goat anti-EEA1 antibody and with Alexa488-conjugated and Cy3-conjugated secondary antibodies. The stained cells were observed using a confocal microscope (TCS-SP2, Leica Mikrosysteme Vertrieb GmbH, Bensheim, Germany).

RESULTS
To identify proteins that interact with GGA proteins, we performed a yeast two-hybrid screening with the use of the VHSϩGGAH domain of human GGA1 as bait. By screening a human brain cDNA library, four positive cDNA clones were obtained. Among them, one clone covered a portion of the transmembrane domain and an entire cytoplasmic domain of sortilin (aa 774 -831) and another covered a portion of the cytoplasmic domain of LRP3 (aa 748 -770). As shown in Fig. 1A and summarized in Table I, reporter yeast cells harboring the VHSϩGGAH domain of GGA1 and the cytoplasmic domain of either sortilin or LRP3 were able to grow on a histidine-deficient plate and exhibited ␤-galactosidase activity. When the VHS and GGAH domains were examined separately, only the former showed interactions with the sortilin and LRP3 cytoplasmic domains. We then examined whether the VHS domains of the other two GGAs could also interact with the cytoplasmic domain. The GGA2 VHS domain interacted with the sortilin cytoplasmic domain ( Fig. 1A and Table I) and LRP3 (data not shown). By contrast, the VHS domain of GGA3-short (GGA3S), which lacks a 33-amino acid region within the VHS domain as compared with those of GGA1 and GGA2 (5-8) (see Fig. 1B), did not show a significant interaction. The sequence of GGA3-long (GGA3L), which contains the 33-amino acid region, has been also reported (5, 7) (see Fig. 1B). However, we could not examine whether the GGA3L VHS domain also interact with the cytoplasmic domains because our attempts to isolate its cDNA fragment by PCR of human brain, liver, and kidney libraries were unsuccessful.
The VHS domain is found in various proteins that function in the endocytic pathway and in signal transduction (11) (see Fig.  1B). Therefore, a reasonable speculation was that the VHS domains of other proteins could also interact with the sortilin and LRP3 cytoplasmic domain. However, this was not the case; the VHS domains examined, those of STAM1 and TOM1, failed to interact with the cytoplasmic domains of sortilin ( Fig. 1A and Table I) and LRP3 (data not shown). In the VHS domain, GGA1 shares 64 and 73% of its amino acids with GGA2 and GGA3L, respectively, whereas 29 and 33% are shared with Mutated sortilin sequences are also shown. Acidic residues, leucine pairs, and serine residues that can be phosphorylated by CK-II are shown in red, blue, and purple, respectively. STAM1 and TOM1, respectively (Fig. 1B), suggesting that residues conserved in the GGA family but different from those of other VHS proteins are crucial for the interaction with the cytoplasmic domains.
A primary structural feature common to the cytoplasmic domains of sortilin and LRP3 is that both have a cluster of negatively charged amino acids followed by a leucine pair near their COOH termini (Fig. 1D). In addition, both have a serine residue flanked by acidic residues, which can be phosphorylated by casein kinase-II (CK-II) (21)(22)(23). The "acidic dileucine sequences" are also found in the cytoplasmic domains of CIand CD-MPRs; notably, the acidic dileucine sequence of sortilin is identical to that of CI-MPR (Fig. 1D). We therefore examined whether the cytoplasmic domains of CI-and CD-MPRs were also able to interact with the GGA1 VHS domain using the two-hybrid system. As shown in Fig. 1C and summarized in Table I, the cytoplasmic domain of CI-MPR but not that of CD-MPR showed a significant interaction with the VHS domain. A similar tendency was observed with the VHS domain of GGA2 (data not shown). The acidic dileucine sequence of CD-MPR has an arginine residue within the acidic cluster ( Fig.  1D) such that the positive charge could disturb the interaction requiring the negatively charged cluster. These results led to a speculation that the acidic dileucine sequence is responsible for the interaction with the GGA VHS domains.
To confirm this speculation and to determine sequence requirements for the interaction with the VHS domain, we introduced amino acid substitutions within the acid dileucine sequence of sortilin (Fig. 1D). As shown in Fig. 1C and summarized in Table I, mutations of the leucine residues to alanines (Leu 3 Ala/Leu 3 Ala) and of the three consecutive acidic residues that are proximal to the leucine pair to nonacidic residues (Asp 3 Asn/Glu 3 Gln/Asp 3 Asn) abrogated the interaction with the GGA1 VHS domain. By contrast, a mutation of the two aspartic acids distal to the leucine pair (Asp 3 Asn/Asp 3 Asn) resulted in an extremely reduced but still significant interaction. The results obtained with mutants of the serine residue that can be phosphorylated by CK-II were somewhat complicated. The serine-to-alanine mutation (Ser 3 Ala) extremely weakened, although did not completely abolished, the interaction with the GGA1 VHS domain. The other substitution of the serine with an aspartic acid (Ser 3 Asp), which mimics a phosphorylated serine residue (23), had no obvious effect on the interaction.
The interactions between the GGA VHS domains and the acidic dileucine sequences were also tested biochemically. To this end, GST-fusion proteins of the cytoplasmic domains and TRX-His 6 fusion proteins of the VHS domains expressed in E. coli were purified. Using these fusion proteins, we first examined the specificity of the sortilin cytoplasmic domain to the VHS domains. As shown in Fig. 2A, the VHS domains of GGA1 and GGA2 were pulled down efficiently with the GST-sortilin cytoplasmic domain, whereas the STAM1 VHS domain was not. Essentially the same results were obtained with the CI-MPR cytoplasmic domain fused to GST (Fig. 2A). These data are in good agreement with those obtained by two-hybrid assays. Apparently conflicting with the two-hybrid data was the result that the VHS domain of GGA3S was efficiently pulled down. The discrepancy appeared to be due to a nonspecific interaction of the GGA3S VHS domain with the GST portion of the GST-cytoplasmic domain fusion proteins because the GGA3S VHS domain fused to TRX-His 6 was efficiently pulled down with GST ( Fig. 2A).
We then examined the sequence requirements of the sortilin cytoplasmic domain for interaction with the GGA1 VHS domain by the pull-down assay. As shown in Fig. 2B, the Ser 3 Asp mutant pulled down the GGA1 VHS domain as efficiently as the WT sortilin cytoplasmic domain, and the Ser 3 Ala and Asp 3 Asn/Asp 3 Asn mutants pulled down the VHS domain less efficiently. By contrast, the Asp 3 Asn/Glu 3 Gln/Asp 3 Asn and Leu 3 Ala/Leu 3 Ala mutations almost completely abrogated the interaction with the VHS domain. These data are consistent with the two-hybrid data.
MPRs are known to cycle between the TGN and late endosomes to deliver lysosomal hydrolases through the latter compartment to lysosomes (reviewed in Refs. 24 -26). On the other hand, a previous study has shown that transiently expressed sortilin localizes in the TGN region, and a chimeric protein comprising the luminal and transmembrane domains of the Tac antigen and the cytoplasmic domain of sortilin is colocalized with CI-MPR (27). These data lead to the possibility that the acidic dileucine sequence of sortilin common to MPRs determines the subcellular localization of sortilin. To address this possibility, we examined the localization of the Tac chimera with the WT or mutated sortilin cytoplasmic domain by immunofluorescence microscopy. As shown in Fig. 3, the Tac chimera with the WT sortilin tail localized exclusively in the perinuclear region (panel B) as reported previously (27), Ϫ Ϫ a ϩϩ, ϩ, and Ϫ indicate yeast cells that grew well, grew less but significantly, and did not grow, respectively. b ϩϩ, ϩ, and Ϫ indicate yeast cells that developed blue color in 1-or 15-h incubation with 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside or did not develop blue color, respectively. c ϩϩ, ϩ, and Ϫ indicate interactions comparable with that between GGA1 (VHS) and sortilin (WT), weaker but significant interactions, and no interactions, respectively. The (ϩ) indicates a non-specific interaction of GGA3S (VHS) with GST. ND, not determined.
whereas Tac itself was found largely on the cell surface (panel A). Furthermore, the staining for the Tac chimera was superimposed on that for coexpressed GGA1 (panels E-EЈЈ), indicating that the chimeric protein localized to the TGN. By contrast, the localization of the Tac-sortilin chimera with an Leu 3 Ala/Leu 3 Ala mutation was different; it was found on punctate structures throughout the cytoplasm and in the cell pe-riphery as well as in the perinuclear region (panel C). A similar staining pattern was observed for the Tac-sortilin chimera with an Asp 3 Asn/Glu 3 Gln/Asp 3 Asn mutation (panel D). The Ser 3 Ala, Ser 3 Asp, or Asp 3 Asn/Asp 3 Asn mutation had no obvious effect on the distribution of the Tac chimera (data not shown). Thus, the ability of the sortilin cytoplasmic domain to bind to the GGA VHS domain correlated well with its ability to localize the Tac chimera to the TGN. The punctate staining for the chimera with the Leu 3 Ala/Leu 3 Ala or Asp 3 Asn/Glu 3 Gln/Asp 3 Asn mutation overlapped significantly with that for EEA1 (panels F-FЈЈ and G-GЈЈ, respectively), an early endosome marker, but not with that for Lamp-1 (data not shown), a late endosome/lysosome marker, indicating that these mutations cause mislocalization of the chimera to early endosomes.

DISCUSSION
GGAs are a family of TGN-associated peripheral membrane proteins that have been proposed to function to facilitate protein trafficking between the TGN and lysosomes/vacuole. The data presented here show that GGAs can interact with the cytoplasmic domains of TGN-localized transmembrane proteins including sortilin, CI-MPR, and LRP3. The interactions occur between the VHS domains of GGAs and the conserved acidic dileucine sequences at the COOH termini of the cytoplasmic domains. A mutation of the leucine pair or the cluster of acidic residues proximal to the pair abrogates the interaction and causes mislocalization of a chimeric receptor with the sortilin cytoplasmic tail from the TGN to endosome-like structures. MPRs are known to cycle between the TGN and late endosomes and to function as sorting receptors for lysosomal hydrolases (24 -26). Sortilin has, in its luminal domain, a cysteine-rich region homologous to two corresponding segments of yeast Vps10p (27), which is a sorting receptor for vacuolar proteins (reviewed in Ref. 28), leading to a possibility that sortilin could also function as a sorting receptor for some lysosomal proteins. GGA proteins are recruited onto TGN membranes by virtue of ARFs and bind to clathrin in mammalian cells (5,7,12), facilitating the transport of vacuolar proteins in yeast (6,7,9,10). Taken together with the data presented here, it is likely that GGAs regulate the trafficking of lysosomal proteins by interacting with the sorting receptors.
A Tac chimera containing the sortilin cytoplasmic domain localizes to the TGN. By contrast, a chimera containing the mutated sortilin tail (Leu 3 Ala/Leu 3 Ala or Asp 3 Asn/ Glu 3 Gln/Asp 3 Asn), which cannot interact with GGAs, is mislocalized from the TGN to endosome-like structures. A previous study using systematic alanine-scanning mutagenesis determined that the COOH-terminal acidic dileucine sequence of CI-MPR is the major determinant of sorting of lysosomal enzymes (29). Although that study did not see the localizations of the CI-MPR mutants, their data are in keeping with the idea that the interaction between GGAs and the receptor cytoplasmic domain is crucial for lysosomal protein sorting.
The acidic dileucine sequences are predicted to be able to interact with adaptor molecules other than GGAs. For example, the acidic clusters with serine residues phosphorylated by the CK-II of MPRs were shown to interact with the AP-1 clathrin adaptor complex and proposed to be required for MPR exit from the TGN (reviewed in Ref. 26). The interaction is thought to be mediated by another adaptor protein, PACS-1, which was originally identified as a protein that binds to the phosphorylated acidic cluster of the TGN-localized endoprotease furin (Ref. 30; reviewed in Ref. 31). However, the PACS-1 study suggested that the interaction between PACS-1 and the phosphorylated acidic cluster mediates retrieval of furin from post-TGN compartments rather than its anterograde transport FIG. 2. Pull-down assays. A, the cytoplasmic domain of sortilin or CI-MPR fused to GST or GST alone was prebound to glutathione-Sepharose beads and incubated with the VHS domain of GGA1, GGA2, GGA3S, or STAM1 fused to TRX-His 6 . The materials bound to the beads were subjected to Western blot analysis using anti-TRX antibody as described under "Experimental Procedures." B, the WT or mutated sortilin cytoplasmic domain fused to GST was prebound to glutathione-Sepharose beads and incubated with the GGA1 VHS domain fused to TRX-His 6 . The materials bound to the beads were subjected to Western blot analysis using anti-TRX antibody as described under "Experimental Procedures." from the TGN (23,30,31). Furthermore, the above CI-MPR mutational study suggested that a hydrophilic nature, but not phosphorylation/dephosphorylation cycles, at the serine position is important for lysosomal protein sorting (29), which is compatible with the data presented here. Taking into account the data presented thus far on GGAs, it is tempting to speculate that GGAs regulate the exit of sorting receptors from the TGN by recognizing the acidic dileucine sequences, whereas PACS-1 regulates their retrieval from post-Golgi compartments by recognizing the phosphorylated acidic clusters.
During the review process of this paper, a similar study was reported by Nielsen et al. (32). They identified GGA2 as a protein interacting with the sortilin cytoplasmic tail by twohybrid screening and found that a deletion of the leucine pair abolished the ability of the tail to interact with GGA2 and led a Tac-sortilin chimera to a mild defect in its internalization. These data support our present data. However, apparently in conflict with our data was their finding that a deletion of the acidic cluster had no effect on the internalization of the Tacsortilin chimera. This discrepancy might be because of the difference between our and their experimental systems; we observed the steady-state localization of the Tac-sortilin chimera, whereas they observed its internalization from the cell surface.