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Vol. 17, Issue 8, 3397-3408, August 2006
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*Institute of Cell Biology and Neuroscience, Johann-Wolfgang Goethe University of Frankfurt, D-60323 Frankfurt am Main, Germany; and Institute of Biochemistry II, University Clinic of Frankfurt, D-60590 Frankfurt am Main, Germany
Submitted November 10, 2005;
Revised May 3, 2006;
Accepted May 5, 2006
Monitoring Editor: Jennifer Lippincott-Schwartz
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In addition to distinct proteins, the lipid composition within these two membrane domains also differs significantly (Yeaman et al., 1999). To achieve the characteristic spatial expression patterns of proteins, epithelial cells display a variety of sorting mechanisms to ensure correct routing of proteins to the appropriate plasma membrane region. As shown for polarized epithelial Madin-Darby canine kidney (MDCK) cells, apical and basolateral transmembrane proteins are sorted into distinct vesicles that derive from the trans-Golgi network (TGN). These vesicles carry the proteins to the correct target plasma membrane domain (Ikonen and Simons, 1998; Matter, 2000; Mostov et al., 2000). Despite these observations and many additional studies, our understanding of how cells perform polarized sorting of proteins to different plasma membrane domains is still fragmentary. In particular, routing to the apical membrane is more poorly understood than basolateral targeting.
For basolateral sorting of transmembrane proteins, it has been established with several examples that targeting signals located in the cytoplasmic domains of proteins are required to mediate efficient sorting (Mellman, 1993; Ikonen and Simons, 1998). As opposed to other sorting information (e.g., GAT-2 GABA transporter; Brown et al., 2004), these so-called classical basolateral consensus sorting signals within the cytoplasmic domain of membrane proteins are tyrosine based (YXX
, where Y is tyrosine, X is any amino acid, and is a bulky hydrophobic residue) and dileucine motifs (Bonifacino and Traub, 2003). In some cases, however, these sorting motifs are not necessarily colinear but apparently arise through folding (Orsel et al., 2000).
These short peptide motifs can be recognized by a variety of adaptor proteins (APs) peripherally associated with the cytosolic membrane. One family of APs includes AP-1 to AP-4 and Golgi-localized, 
ear-containing, ADP ribosylation factor-binding proteins (GGAs) (Kirchhausen, 1999; Bonifacino, 2004). Each of the AP family members exists as a heterotetramer consisting of two large (
, , 
, or 
, and 
1-4), one medium (µ1-4), and one small (
1-4) subunit, whereas GGAs are monomeric proteins (Hirst and Robinson, 1998; Bonifacino and Dell’Angelica, 1999; Bonifacino, 2004). In addition to the adaptor proteins, a set of accessory proteins is involved in the assembly of clathrin coats and vesicle budding (Pearse and Robinson, 1990; Kirchhausen, 2000; Slepnev and De Camilli, 2000). Several reports have indicated that the interaction between APs and their cargo may be regulated by phosphorylation and dephosphorylation events.
Our previous studies indicated that shrew-1, a type I transmembrane protein, is localized in the basolateral membrane of polarized epithelial MDCK cells and integrates specifically into E-cadherin-mediated adherens junctions (Bharti et al., 2004). These results raised questions concerning the signals mediating this targeting and the protein(s) possibly involved.
Here, we show that the cytoplasmic domain of shrew-1 indeed contains basolateral sorting signals, which are functional in polarized epithelial cells. Systematic mutation of single amino acids in these putative sorting signals revealed three critical tyrosines and a dileucine motif within the cytoplasmic domain. These mutants localize apically instead of basolaterally and thus the mutated amino acids play an important role in shrew-1 targeting. By using tannic acid, we present evidence that the apically localized mutants were first targeted to the basolateral membrane and were then redistributed to the apical membrane. Furthermore, we demonstrate that µ1B, a subunit of adaptor protein AP-1B is important for retaining shrew-1 at the basolateral membrane, implying the involvement of postendocytic sorting machinery.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-catenin and E-cadherin were stained with rabbit anti--catenin or anti-E-cadherin polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). To visualize the proteins, fluorescent-conjugated antibodies derived from goat Alexa-Fluor 488 anti-mouse and Alexa-Fluor 594 anti-rabbit (Invitrogen, Carlsbad, CA) were used. The nuclei were counterstained with ToPro-3. The sections were analyzed using confocal microscope LSM 510 (Carl Zeiss, Jena, Germany). x,z and three-dimensional (3D) reconstructions were generated using Imaris 4.0.4 software (Bitplane, Zurich, Switzerland).
Construction of cDNAs Encoding Shrew-1-GFP Mutants
pEGFP-N3-Shrew-1 (Bharti et al., 2004) (Clontech, Mountain View, CA) was used as a source of cDNA encoding human shrew-1 (GenBank accession no. AY282806). The deletion mutants were derived by PCR using sets of primers obtained from Sigma-Genosys (Cambridge, United Kingdom). The newly introduced restriction sites HindIII and Acc65I were used to reclone the mutant shrew-1 cDNA into pEGFP-N3 vector. A GeneTailor site-directed mutagenesis kit (Invitrogen, Karlsruhe, Germany) was used to introduce substitutions within the shrew-1 cDNA. The different shrew-1 constructs generated are listed in Table 1. All constructs were subsequently sequenced to verify the intended mutation. DNA sequencing was performed by GENterprice (Mainz, Germany).
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Cell Transfection
To obtain cell lines stably expressing wild-type or mutant shrew-1-GFP, MDCK and LLC-PK1 cells were grown on 10-cm plates until 40% confluent and transfected with wild-type or mutant pEGFP-shrew-1 applying magnet-assisted transfection (MaTra) (IBA, Goettingen, Germany). Stable cell lines were selected by adding the eukaryotic selection marker G418 (Invitrogen) at a final concentration of 5 mg/ml 48 h after transfection. This concentration of G418 was maintained until single colonies formed. Twenty to 25 colonies were isolated, expanded, and grown in the presence of 0.5 mg of G418 per milliliter of medium. Double-transfected LLC-PK1 cells were obtained as described for LLC-PK1 cells. Additionally, pCB6-µ1B-HA or pCB6-µ1A-HA were cotransfected. The expression of µ1B-HA was then determined in vivo and in vitro using a monoclonal anti-hemagglutinin (HA) antibody (Covance, Berkeley, CA) at an appropriate dilution. For colocalization studies of shrew-1 with endocytic markers, MCF-7 cells were transfected with pEGFP-shrew-1 plasmid using MaTra. Cells were fixed after 24 h, and for endosome staining polyclonal anti-rab5 and anti-rab11 antibodies (Santa Cruz Biotechnology) and secondary anti-rabbit Alexa-Fluor 594 were used. The cells were then processed for immunofluorescence.
Confocal Microscopic Identification of Site of Expression of Shrew-1-GFP Mutants
MDCK and LLC-PK1 cells expressing wild-type or mutant shrew-1-GFP were grown for at least 5 d after becoming confluent on Transwell filter inserts (Corning Glassworks, Corning, NY). Staining of tight junctions was achieved using polyclonal rabbit anti-occludin antibody (Zymed, Invitrogen, Karlsruhe, Germany) and subsequent incubation with anti-rabbit Alexa-Fluor 594 antibody. Confocal images were acquired using Leica TCS NT and Leica TCS SP5 scanner (Leica). x,z and 3D reconstructions were generated using Imaris 4.5.1 software (Bitplane, Zurich).
Analysis of Surface Shrew-1-GFP Content by Surface-specific Biotinylation
MDCK cells stably expressing wild-type or mutant shrew-1-GFP were grown on Transwell inserts for at least 7 d in six-well plates. Cell monolayers were biotinylated with EZ-Link Sulfo-NHS-SS-Biotin (Pierce, Perbio Science, Bonn, Germany) that was added from either the apical or basal side. After washing, cells were lysed, and the membrane was solubilized using 10 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, and 5 mM EDTA. Cell lysates were clarified by centrifugation (15,000 x g; 10 min). Samples containing 10% of supernatant were analyzed by SDS-PAGE to estimate the total amount of shrew-1-GFP in the supernatant. To isolate biotinylated proteins, the rest of each supernatant was incubated with 50 µl of Neutravidin beads (Pierce, Perbio Science) in a total volume of 500 µl of lysis buffer for 1 h at 4°C with continuous rotation. The beads were washed three times with lysis buffer, and the proteins were eluted from the beads by incubation in 25 µl of Roti-Load1 sample buffer (Carl Roth, Karlsruhe, Germany) 5 min at 95°C, separated by SDS-PAGE, and analyzed by Western blot using monoclonal anti-green fluorescent protein (GFP) antibody (Roche Diagnostics, Mannheim, Germany) and as secondary antibody an anti-mouse IgG conjugated to alkaline phosphatase (ALP) (Jackson ImmunoResearch, Dianova, Hamburg, Germany). Immunoblots were quantified by densitometry and the amount of surface shrew-1-GFP was normalized to the total labeled shrew-1-GFP using Scion image software (Scion, Frederick, MD). The representative immunoblots are shown in Figures 2 and 4.
Coimmunoprecipitation
For coimmunoprecipitations, a 3-cm dish of cells was incubated in 200 µl of lysis buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and 60 mM octyl--D-glucopyranoside) supplemented with protease inhibitor mixture (Complete, protease inhibitor cocktail; Roche Diagnostics) at 4°C for 30 min. Insoluble material was removed by centrifugation at 10,000 x g at 4°C for 10 min. Lysate was incubated with 0.5 µg of anti-HA monoclonal antibody (mAb) (Covance) and immobilized protein G-agarose beads at 4°C for 4 h. Beads were then washed four times with cold lysis buffer. SDS-PAGE sample buffer was added to the immunoprecipitates, and samples were heated to 95°C for 5 min, separated by SDS-PAGE, and analyzed by Western blot using monoclonal anti-GFP antibody (Roche Diagnostics). Staining was achieved using anti-mouse IgG conjugated to ALP.
Tannic Acid Treatment
Tannic acid treatment was performed as described previously (Polishchuk et al., 2004). Briefly, stably expressing wild-type or mutant shrew-1-GFP MDCK cells were grown on Transwell inserts for at least 7 d to obtain proper tight junction development and electrically tight monolayers (>100 
cm–2). Cells were then incubated at 20°C for 4 h to accumulate shrew-1-GFP within the Golgi apparatus. A temperature shift to 37°C was performed to release protein from the Golgi. During the shift from 20 to 37°C, 0.5% (wt/vol) tannic acid (Sigma-Aldrich, Munich, Germany) was added either to apical or basal growth medium. Tannic acid-treated cells were then fixed after 45 min and examined by confocal microscopy.
Antibody Uptake
MDCK and LLC-PK1 cells stably expressing wild-type or mutant shrew-1-GFP were grown on Transwell inserts for 7 d. The basal chamber was washed with PBS and refilled with DMEM medium supplemented with shrew-1 monoclonal antibodies (nanoTools, Tenningen, Germany) in a concentration of 10 µg/ml. The cells were incubated at 4°C for 30 min and either fixed or the medium was changed and the cells were incubated at 37°C for 60 min and then fixed. The cells were incubated in blocking buffer (10% FCS in PBS) for 1 h and stained with secondary anti-mouse Alexa-Fluor 594 antibodies (Molecular Probes) for an additional hour. The cells were then washed and processed for immunofluorescence.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). To show that the ectopic expression of shrew-1 reflects the expression in vivo, endogenous shrew-1 localization was analyzed in frozen sections of human endometrium using polyclonal rat shrew-1 antibodies (Figure 1, A and D, green fluorescence). The results suggested that shrew-1 also localizes at the plasma membrane in tissue. To substantiate this interpretation more in detail, the sections were costained with antibody directed against either E-cadherin (Figure 1E, red fluorescence), the transmembrane protein characterizing adherens junctions of epithelial cells, or with antibody against -catenin (Figure 1B, red fluorescence), which is a cadherin-binding protein and thus also shows plasma membrane localization. The merged pictures of shrew-1 with either E-cadherin or -catenin as revealed by confocal microscopy (merged pictures in Figure 1, C and F) clearly indicate plasma membrane localization of shrew-1 in vivo. Therefore, these data imply that the previously reported targeting of shrew-1 to adherens junctions in polarized epithelial cells is indeed reflected by the in vivo situation in this cell type.
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).
Based on these findings, MDCK cells stably expressing shrew-1-GFP, here wild-type shrew-1, were plated on Transwell filters and grown for 7 d to ensure proper polarization. The spatial distribution of shrew-1-GFP between apical and basolateral membranes was visualized by confocal microscopy and by performing optical sectioning. This analysis once more confirmed that shrew-1-GFP wild type is sorted to the basolateral side of the cell and hardly any apical localization could be detected (Figure 2B). To support this observation with a biochemical method, surface selective biotinylation was performed. This allows quantitative comparisons of the amounts of shrew-1-GFP in apical and basolateral membrane domains. Cell monolayers were surface biotinylated from either the apical or basolateral side. Biotinylated proteins were purified with Neutravidin beads and analyzed by Western blots stained with GFP antibodies to visualize shrew-1-GFP. Quantification of the Western blots (Figure 2E) revealed that 
90% of the total surface shrew-1-GFP was present in the basolateral domain and 10% in the apical domain of the polarized MDCK cells.
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Microscopy analysis was confirmed by using a surface selective biotinylation assay of either the apical or basolateral side of the polarized MDCK cells (Figure 2E). Quantification of the biotinylated protein isolated from the basolateral and apical side of shrew-1-TCD30 expressing MDCK cells revealed a distribution of 80% of shrew-1 protein at the basolateral and 20% at the apical side. Biochemical analysis of shrew-1-NTD5 in polarized MDCK cells showed almost the reverse distribution, namely, 95% of the protein at the apical side and only 5% in the basolateral region.
Altogether, these findings imply that the cytoplasmic tail of shrew-1 is of fundamental importance for sorting and that the amino acid region between 303 and 411 of the shrew-1 protein sequence should contain essential targeting signals. In line with this, the ectodomain should not contain any basolateral targeting signal because deletion of this domain does not affect basolateral sorting. At this point, we can, however, not fully exclude that the ectodomain of shrew-1 contains some hidden apical sorting signals.
Basolateral Sorting Motifs in Shrew-1
Because we know that the cytoplasmic domain must harbor the signal for basolateral targeting, we focused on this domain to identify specific signals. First, we screened the cytoplasmic domain sequence for potential basolateral sorting signals that might resemble known signals. This analysis revealed five putative signals (Figure 3). Shrew-1 contains a single leucine at position 303, correlating with a basolateral sorting signal described in transmembrane protein CD147 (Deora et al., 2004). In addition, three putative tyrosine-based signals at positions 350, 368, and 380 resemble, but do not perfectly match, consensus basolateral sorting motifs YXX and YXXX deduced from the analysis of several membrane proteins (Ikonen and Simons, 1998; Nelson and Yeaman, 2001; Mostov et al., 2003; Nelson, 2003; Deora et al., 2004; see Introduction). Finally, we detected an acidic dileucine motif at position 396/397, which was also described as a basolateral signal for several proteins (Ikonen and Simons, 1998; Nelson and Yeaman, 2001; Mostov et al., 2003; Nelson, 2003; Deora et al., 2004).
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). Finally, glutamate at position 359 was changed to alanine, again as a control to exclude the possibility that the effect on sorting might be caused by mutation of any amino acid in the cytoplasmic domain. As with the deletion mutants, the steady-state distribution of shrew-1 point mutants stably expressed in MDCK was examined by confocal microscopy and confirmed by surface selective biotinylation. For better distinction between apical and basolateral membranes in optical sections and to confirm polarization, we stained tight junctions using anti-occludin antibody (Figure 4, A–F, red staining).
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10–35% in the basolateral membrane of the polarized MDCK cells (Figure 4, A–F, and Table 1). The strongest missorting of shrew-1 to the apical membrane was caused by mutation of the tyrosine-based targeting signals. Interestingly, of these Y368A showed the strongest apical targeting (Figure 4C). The L396A and LI396/397HV mutants also displayed apical localization. It seems, however, that the misrouting of the double mutant LI396/397HV (Figure 4F and Table 1; 65% apical, 35% basolateral) was less than that of the single mutant L396A (Figure 4E and Table 1; 79% apical, 19% basolateral) but comparable with that of L303A (Figure 4A and Table 1; 65% apical, 35% basolateral).
As expected, the exchange of lysine at position 304 to alanine did not alter the membrane localization of shrew-1 with respect to wild type (Figure 4G), nor did substitution E359A have any visible effect (Figure 4H). They were mainly found, both microscopically and biochemically, in the basolateral part of the cell membrane (Figure 4, G and H, and Table 1).
These data demonstrate that the tyrosines and specific leucines within the cytoplasmic domain of shrew-1 are involved in the control of trafficking and positioning of the protein: mutation of these amino acids leads to an alternative distribution of shrew-1 in the polarized cell membrane of epithelial cells.
Expression of Wild-Type and Mutant Shrew-1 in LLC-PK1 Cells
An alternative epithelial cell type, LLC-PK1, was used to examine possible discrepancies in sorting of wild-type and mutant shrew-1. It is known that LLC-PK1 cells in contrast to MDCK cells exhibit different protein sorting of, for example, H+,K+-ATPase -subunit (Duffield et al., 2004), low-density lipoprotein receptor (LDLR) and transferrin receptor (Matter et al., 1992), (Odorizzi and Trowbridge, 1997; Folsch et al., 1999). One reason is that these cells lack the subunit µ1B of adaptor protein AP-1 (Folsch et al., 2001). To check shrew-1 sorting in these cells, LLC-PK1 cells were plated on Transwell filters, transfected 24 h later with wild-type shrew-1-GFP, and allowed to achieve polarization for 5 d. After fixation, the cells were analyzed by confocal microscopy. Analysis of these cells revealed that wild-type shrew-1-GFP was localized predominantly at the apical plasma membrane (Figure 5A). However, we also observed that newly synthesized shrew-1-GFP also localized to the basolateral surface and was then distributed to the apical membrane (Figure 9, E and F). The shrew-1 mutant shrew-1-NTD5-GFP, also transiently transfected into LLC-PK1 cells, showed an apical steady-state distribution similar to that found for wild-type shrew-1 (Figure 5C).
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Furthermore, we tested whether shrew-1 and µ1 subunits interact in LLC-PK1 cells by performing coimmunoprecipitation with anti-HA antibody. Western blots were then probed with GFP antibody. We could demonstrate that µ1B subunit formed a complex with shrew-1, whereas subunit µ1A did not. In addition, mutant shrew-1-NTD5 did not show any complex formation with µ1B (Figure 5F). In conclusion, µ1B subunit, which is incorporated into the AP-1B complex, is active in LLC-PK1 cells and recognizes signals for basolateral targeting within the cytoplasmic domain of shrew-1.
Double immunofluorescence microscopy experiments in MCF-7 cells transfected with shrew-1-GFP demonstrated colocalization of shrew-1-GFP with endogenous recycling endosome marker rab11 (Figure 6C). However, shrew-1-GFP displayed no colocalization with endogenous early endosome marker rab5 (Figure 6F).
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Use of Tannic Acid to Analyze Trafficking of Shrew-1 in Polarized MDCK Cells
Tannic acid is a fixative that disturbs the fusion of membrane vesicles derived from the TGN, or subsequent compartments, with the plasma membrane. Because tannic acid cannot pass through tight junctions it can be added selectively to either the apical or the basolateral side of polarized cells. This approach (Figure 7A) is rather useful for testing the order in which membrane proteins reach particular membrane domains (Polishchuk et al., 2004).
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MDCK cells stably expressing wild-type shrew-1 or mutant shrew-1-NTD5 were plated on Transwell filters, propagated for 7 d, and then treated for 45 min with tannic acid added to either apical or basal growth medium. After fixation and staining of the tight junctions, the cells were analyzed by confocal microscopy, and the localization of shrew-1-GFP and shrew-1-NTD5-GFP was determined. We ensured the impermeability of confluent MDCK monolayers by measuring the electrical resistance before and after administration of tannic acid. In both cases, the electrical resistance was higher than 100 cm–2. Furthermore, to exclude membrane depolarization after treatment with tannic acid, we performed E-cadherin staining. The analysis of E-cadherin localization revealed that E-cadherin remained restricted to basolateral surface. Tannic acid administered to the apical medium resulted in normal basolateral distribution of wild-type shrew-1 (Figure 8C). The shrew-1 mutant previously localizing in the apical membrane could not longer be observed there, but in the lateral membrane and we could observe carrier accumulation close to the tight junction zone (Figure 7G). This suggested that the mutant shrew-1 was initially transported to the basolateral membrane and that subsequent protein resorting to the apical membrane was disrupted by tannic acid. Tannic acid added to the basal growth medium diminished the appearance of wild-type shrew-1 in the basolateral membrane. We observed protein accumulation near tight junctions, indicating that the Golgi-derived vesicles containing shrew-1 were unable to fuse with the plasma membrane (Figure 7D). Neither basolateral nor apical localization was observed when examining the shrew-1 mutant for polarized distribution after basal treatment with tannic acid (Figure 7H), but protein carriers accumulated in the proximity of tight junctions and the apical membrane domain.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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So far, the best characterized tyrosine-based protein sorting motifs are those with the consensus sequence YXX, described as endosomal sorting signals for a number of transmembrane proteins (Bonifacino and Traub, 2003). Furthermore, there is evidence that a reversed motif YXX, namely, XXY, may also be an efficient functional sorting motif (Roush et al., 1998). The latter occurs twice in shrew-1 as FTAY350 and VPVY368. In line with this, a tyrosine-based motif, YXX, inverted in the transferring receptor and in the LDL receptor reduced, but did not abolish internalization (Gironès et al., 1991).
A sorting motif similar to YXX is also present in shrew-1, but with a different number of residues between Y and (YKSTF). Although structural studies have indicated that the spacing between Y and is crucial for recognition by adaptor proteins (Owen and Evans, 1998), it seems that YXXX can also be an efficient sorting signal, as supported by internalization studies of homomeric receptor P2X4 (Royle et al., 2005). Thus, the number of amino acids between Y and in tyrosine-based sorting motifs might not be as critical as previously postulated, and some variation in number and composition between residues Y and is permitted.
Further analysis of shrew-1 cytoplasmic domain revealed that a single leucine (residue 303), but not the adjacent lysine (residue 304), is necessary for basolateral sorting. This is reminiscent of a basolateral sorting motif in CD147 where mutation of the juxtamembrane leucine but not the adjacent lysine also leads to apical instead of basolateral localization in polarized MDCK cells (Deora et al., 2004). This suggests a strikingly similar basolateral targeting signal in both shrew-1 and CD147. Nevertheless, we cannot exclude that mutation L303A and the equivalent in CD147 might, for example, modulate the protein hydrophobicity in both cases, possibly affecting their secondary and tertiary structure, and thus influencing their trafficking in a nonspecific manner (discussed in Deora et al., 2004). In addition, accessibility of leucine residue 303 by an adaptor protein is difficult to imagine because it localizes close to the membrane.
Mutations of the leucines in the "acidic cluster dileucine" DXXLL of shrew-1 resulted in apical distribution in polarized MDCK cells. Such "acidic cluster dileucines," including a crucial aspartate 2 positions upstream have been described as recognition motifs for GGAs. These bind to, and hence are involved in, the sorting of cargo proteins from the TGN to endosomal compartments (Johnson and Kornfeld, 1992; Chen et al., 1997; Bonifacino, 2004). Furthermore, it is known that a dileucine motif in the C-terminal cytoplasmic domain of proteins is required for endocytosis (Bonifacino and Traub, 2003) and sometimes also for basolateral sorting (Bello et al., 2001; El Nemer et al., 1999). In contrast, another report postulates that dileucine motifs target proteins from the TGN to endosomal/lysosomal compartments (Sandoval and Bakke, 1994).
Evidently, the DXXLL motif is part of a potential casein kinase 2 phosphorylation site due to the presence of three serines before the aspartate. It has been shown that phosphorylation of serine residues upstream of the acidic dileucine signal results in tighter binding to the VHS domain of GGA adaptors (Kato et al., 2002). Thus, the presence of the three serines emphasizes the potential of this region for being a signal (Meggio et al., 1984; Boldyreff et al., 1994). Preliminary data from our laboratory suggesting serine phosphorylation of shrew-1 (our unpublished data) also support such an idea.
Interestingly, comparison of cytoplasmic amino acids between human, mouse, and zebrafish shrew-1 revealed a high degree of conservation (Figure 3B). In particular, the three tyrosines described above are conserved, including their positions. Zebrafish shrew-1 has an additional tyrosine at a position comparable with amino acid 360 in human and mouse. It remains to be seen whether this tyrosine is of functional importance for targeting in zebrafish. Also, the targeting motif DRHLI in human and mouse shrew-1 is almost identical in zebrafish, except this motif contains another negatively charged amino acid, glutamate, instead of aspartate (Figure 3B).
Together, the identification of several functionally and evolutionary conserved basolateral targeting signals in shrew-1 is rather unexpected. It is possible that the sum of all putative basolateral signals in the cytoplasmic tail of shrew-1 is necessary for proper routing to the target membrane, and, importantly, for retaining the protein there. Alternatively, it is conceivable that the different targeting signals provide distinct but overlapping functions.
MDCK cells use two different routes to target newly synthesized apically localized proteins to the apical surface: a direct route from the Golgi complex and an indirect or transcytotic route that involves an intermediate stop at the basolateral surface (Nelson and Yeaman, 2001). The mechanisms and proteins enabling transcytosis are generally unknown. Epithelial cell-specific sorting adaptor AP-1B, which is localized to recycling endosomes, plays a role in transcytotic or postendocyic sorting (Folsch et al., 1999; Gan et al., 2002) in MDCK cells (Traub and Apodaca, 2003). However, it has been shown for apically localized neuronglia cell adhesion molecule and vesicular stomatitis virus glycoprotein that these AP-1B–dependent cargo proteins are targeted from the TGN to the basolateral surface before endocytosis and then distributed to the apical surface (Ang et al., 2004; Polishchuk et al., 2004; Anderson et al., 2005). These findings suggest that AP-1B–dependent sorting, where AP-1B seems to function as a filter occurs in recycling endosomes (Ang et al., 2004) and that the recycling endosome plays an important role in polarized sorting in the endocytic pathway.
Two lines of evidence are in favor of the idea that targeting of shrew-1 is achieved via postendocytic sorting. First, routing of shrew-1-NTD5 (lacking the cytoplasmic domain) in the presence of tannic acid led to its accumulation at the basolateral surface, implying that it is initially attached to the basolateral membrane, and then redistributed to the apical membrane. This supports the view that wild-type shrew-1 is first delivered to the basolateral membrane where it is then retained by recognition of specific amino acid sequence motifs not present in mutant shrew-1-NTD5.
Additional evidence for postendocytic sorting was obtained by expressing wild-type shrew-1 and mutant shrew-1-NTD5 (cytoplasmic domain deletion) in LLC-PK1 cells lacking subunit µ1B of adaptor protein AP-1. In LLC-PK1 cells both wild-type and mutant shrew-1-NTD5 were predominantly found at the apical surface. This apical targeting could be reversed to basolateral targeting for shrew-1 wild type, but not for the mutant lacking basolateral sorting signals, by ectopic expression of µ1B. These results correlate with previous observations showing that AP-1B (containing subunit µ1B) prevents proteins (e.g., LDLR, which is delivered to the basolateral membrane) from switching to the apical membrane domain via postendocytic sorting mechanisms (Girotti and Banting, 1996; Gan et al., 2002).
In conclusion, our data provide evidence for a scenario (Figure 10) in which shrew-1 is primarily delivered to the basolateral membrane by a so far unknown mechanism. Once there, adaptor protein complex AP-1B is involved in retaining shrew-1 at the basolateral membrane. Sorting signals involved in this process may nevertheless differ from the signals involved in sorting at the TGN (Odorizzi and Trowbridge, 1997).
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Anna Starzinski-Powitz ( starzinski-powitz{at}em.uni-frankfurt.de)
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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