2365Journal of Cell Science 111, 2365-2376 (1998)Printed in Great Britain © The Company of Biologists Limited 1998JCS4561

Subcellular targeting and cytoskeletal attachment of SAP97 to the epithelial

lateral membrane

Hongju Wu, Susanne M. Reuver, Sven Kuhlendahl, Wook Joon Chung and Craig C. Garner*

Department of Neurobiology, University of Alabama at Birmingham, 1719 Sixth Ave. S, Birmingham, AL 35294-0021, USA*Author for correspondence (E-mail: garner@nrc.uab.edu)

Accepted 15 June; published on WWW 30 July 1998

The synapse-associated protein SAP97 is a member of anovel family of cortical cytoskeletal proteins involved in thelocalization of ion channels at such membranespecializations as synaptic junctions. These multidomainproteins have binding sites for protein 4.1,GKAPs/SAPAPs, voltage- and ligand-gated ion channelsand cell-adhesion molecules containing C-terminal T/SXVmotifs. In this study, we evaluated the contribution ofindividual domains in SAP97 to its selective recruitmentand attachment to the cortical cytoskeleton in epithelialcells. We find that the PDZ, SH3 and GK domains, as wellas the I3 insert in SAP97, are not essential for subcellular

targeting, though both PDZ1-2 domains and the I3 insertaffect the efficiency of localization. Instead, we show thatthe first 65 amino acid residues in SAP97, which are absentfrom SAP90/PSD-95 and SAP102, direct the selectivesubcellular localization and can mediate at least one pointof attachment of SAP97 to the cytoskeleton assembled atsites of cell-cell contact. Our data demonstrate that it is thesequences unique to SAP97 that direct its subcellulartargeting to the epithelial lateral membrane.

Key words: Actin, Cell-cell contact, Cytoskeleton, MAGUK, PDZdomain

SUMMARY

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INTRODUCTION

Members of the SAP97 family of synapse-associated prote(SAPs), which includes SAP90/PSD-95, SAP102 aChapsyn-110/PSD-93, have recently emerged as cenplayers in the molecular organization of synapses (reviewedBudnik, 1996; Garner and Kindler, 1996; Sheng, 1996). SAare scaffold proteins involved in linking ligand- and voltaggated ion channels to the cytoskeleton assembled at varmembrane specializations (reviewed by Budnik, 1996; Garand Kindler, 1996; Sheng, 1996). In neuronal cells, SAPs differentially localized either to the pre- and/or postsynapsites of excitatory or inhibitory synapses, along unmyelinataxons or dendritic profiles (Brenman et al., 1996; Hunt et 1996; Kim et al., 1996; Kistner et al., 1993; Müller et al., 1991996). In addition to the synaptic localization of SAP97 in rbrain, it, as well as its human homolog hDlg, are expressedepithelial cells, where they are localized along the latemembrane at cell-cell adhesion sites (Lue et al., 1996; Müet al., 1995; Reuver and Garner, 1998). At present, the cellmechanisms regulating the attachment of SAPs to the cortcytoskeleton and their distinct spatial distribution are unknow

An alignment of the deduced amino acid sequencesSAP97/hDlg, SAP90/PSD-95, SAP102 and Chapsy110/PSD-93 reveals the presence of five regions of hsequence homology flanked by unrelated regions. Thomologous regions represent five domains that include thamino-terminal PDZ (PSD-95/DLG/ZO1) domains, an src

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homology 3 (SH3) region, and a carboxyl-terminal guanylakinase (GK)-like domain (Fig. 2). These domains are sites protein-protein interactions (reviewed by Garner and Kindle1996). For example, the PDZ domains bind with high affinitto the C-terminal peptide motif T/S-X-V/I (T/SXV-motif) in anumber of proteins, including the voltage-gated Shaker K+

channels and the NR2 subunits of the NMDA recepto(reviewed by Sheng, 1996). In vivo, these interactions arequired to cluster ion channels and cell adhesion moleculessynaptic junctions (Tejedor et al., 1997; Thomas et a1997a,b). SH3 domains are classical sites of protein-protinteraction first identified in src protein tyrosine kinase(Musacchio et al., 1992), though in SAPs, SH3 bindinpartners are not known. The GK domain in SAPs exhibitsstriking sequence similarity to low molecular mass guanylakinases, but it does not encode an active guanylate kin(Kistner et al., 1995; Kuhlendahl et al., 1998). Instead, the Gdomain has evolved to interact with a family of synaptiproteins called GKAPs, for guanylate kinase-associatproteins (Kim et al., 1997), or SAPAPs, for SAP90/PSD-95associated proteins (Takeuchi et al., 1997). Although boGKAPs/SAPAPs and channel proteins can promote ttranslocation of SAP90/PSD-95 to the plasma membranetransfected fibroblasts (Takeuchi et al., 1997), the functionsignificance of these interactions for the localization of SAPto sites of cell-cell contact is unclear.

Studies on SAP97/hDlg in non-neuronal cells have providthe first clues to how SAPs may be recruited to speci

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membrane specializations. In epithelial cells, E-cadhermediated cell-cell adhesion induces the recruitment SAP97/hDlg to the plasma membrane and its association wthe cortical cytoskeleton at cell-cell contact sites (Reuver aGarner, 1998). In vitro, specific isoforms of SAP97/hDlcontaining an I3 insert situated between the SH3 and domains, bind the erythrocyte cytoskeletal protein 4.1 viaand sequences situated between PDZ1 and PDZ2 (Lue e1994, 1996). This has led to the hypothesis that thinteractions are important for the subcellular targeting SAP97/hDlg in epithelial cells (Lue et al., 1996).

Clearly, the identification of regions in SAPs that direct thesite-specific localization and attachment to the corticcytoskeleton is fundamental to defining how their differentdistribution at cell-cell contact sites is achieved. Here, we hanalyzed the requirement of individual domains in SAP97 forsubcellular targeting and association with the corticcytoskeleton at the lateral membrane in epithelial cells. The 65 amino acid residues of SAP97 (S97N1-65) were found to directthe selective recruitment of SAP97 to sites of cell-cell contaand the PDZ1-2 domains and I3 insert to affect the efficiencylocalization. Furthermore, we show that S97N1-65 mediates atleast one point of attachment of SAP97 to the corticcytoskeleton at lateral membranes. Our data provide critical cto the domains in SAPs that direct their site-specific localizati

MATERIALS AND METHODS

DNA constructionGreen fluorescent protein (GFP)-tagged SAP90/PSD-95, SAP97SAP102 were constructed by subcloning the coding sequenceSAP90/PSD-95, SAP97 and SAP102 (Kistner et al., 1993; Mülleral., 1995; 1996) into the pEGFP-C1 vector (Clontech) after beamplified by polymerase chain reaction (PCR) as described bymanufacturers (GIBCO-BRL). Sense oligonucleotide primecontained the start codon and an EcoRI site incorporated at the 5′ end(S-EcoRI). Antisense primers included the native stop codon anKpnI site incorporated at the 5′ end (AS-KpnI). The SAP97 deletionmutants (∆S97N, ∆GK) and segments of SAP97 (S97N-PDZ1-3PDZ1-3, PDZ1-2, SH3-I3-GK and GK) were constructed by PCwith pairs of primers flanking the region to be amplified. EcoR1 orKpnI sites were incorporated at the 5′ end of sense and antisensprimers, respectively. p31f2, which contains an I3 insert, was usethe DNA template. The PCR products were all ligated into tpCRTM2.1 vector (Invitrogen, TA cloning kit) and then subcloned inthe EcoRI and KpnI sites of the pEGFP-C1 vector or the pEGFP-Cvector (Clontech).

SAP97 deletion mutants (∆PDZ1, ∆PDZ1-2, ∆PDZ1-3, ∆I3 and∆SH3) were constructed by PCR using primers that flanked regions of SAP97 to be deleted. These primers contained a MluI siteincorporated at the 5′ end and were used with either S-EcoRI or ASKpnI. The PCR products were cloned into pCRTM2.1 vector and thensubcloned into pEGFP-C1 in a three-piece ligation.

Chimeric molecules between SAP97 and SAP90/PSD-95 SAP102 were also constructed by PCR. The S97N was amplified a pair of primers in which an EcoRI site or MluI site were incorporatedat the 5′ end of sense and antisense primers, respectively. Theterminal segments of SAP90/PSD-95 or SAP102 were amplifiedPCR with a pair of primers in which an MluI site or KpnI site wereincorporated at the 5′ end of sense and antisense primers, respectivAll of the PCR products were cloned into pCRTM2.1 vector andsubcloned into the EcoRI and KpnI sites of pEGFP-C1 (Clontech) asthree-part ligations. The expression of all constructs was assesse

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western blot analysis of transiently transfected CACO-2 cells (sbelow).

Cell culture, transfection, Latrunculin B treatment andTriton extraction of live cellsThe human colon carcinoma cell line CACO-2 obtained from thAmerican Type Culture Collection (ATCC# HTB 37) was maintainein MEM supplemented with 20% fetal bovine serum (FBS), at 37°Cunder a 5% CO2 atmosphere.

GFP-tagged DNA constructs were transiently transfected inCACO-2 cells (grown to 60-70% confluency) using lipofectamine adescribed by the manufacturer (GIBCO-BRL). In brief, 2.1 µg DNAand 18 µl lipofectamine were applied to cells grown on 22×22 mmcoverslips (in 3.5 cm dishes). Both DNA and lipofectamine werdiluted into 150 µl Opti-MEM (GIBCO BRL), then mixed togetherand incubated at room temperature for 30-45 minutes to allow DNlipid complexes to form. 600 µl Opti-MEM was added, mixed gentlyand then overlaid onto the Opti-MEM pre-washed cells. Cells weincubated for 5 hours at 37°C and then the medium replaced withculture medium. Cells were fixed with 3.7% formaldehyde 24 houlater. Coverslips were mounted with Vectashield mounting mediu(Vector) and fluorescent images taken with a Nikon Diaphot 30microscope equipped with a Photometrics CH250 CCD-cameDigitally stored images were combined and displayed with IP laSpectrum or Adobe Photoshop.

To disrupt filamentous-actin (F-actin), 24 hours after transfectio5 µM Latrunculin B (Calbiochem) in DMSO was added directly tothe culture medium of transfected CACO-2 cultures and incubated 5 minutes at 37°C. Control cells were treated with DMSO alonCultures were immediately fixed with 3.7% formaldehyde anprocessed for epifluorescence microscopy.

To assess the association of GFP-tagged constructs with the cortcytoskeleton, transfected cells were extracted with Triton X-100. brief, cells were cultured and transfected in glass-bottom Microwe(MatTek Corporation). 24 hours after transfection, the cells weextracted by perfusion with 0.5% Triton X-100 in phosphate-bufferesaline (PBS) under the microscope at room temperature. This allowreal-time imaging of small groups of transfected cells. Cells weinitially perfused for 3 minutes with PBS, for 1.5 minutes with 0.5%Triton X-100 in PBS, and then with PBS again. Images of selectcells were taken prior to perfusion with 0.5% Triton X-100 in PBSand then at 1-minute intervals. After 10 minutes, the loss or retentiof fluorescence at sites of cell-cell contact was examined neighboring cells. When retained, fluorescence was still visible minutes after extraction.

Antibodies, immunoprecipitation and immunoblotting The mouse monoclonal antibodies mAb-197.4 and mAb-11(produced by the hybridoma core facility at UAB) were generateagainst the N-terminal 163 and 119 amino acid residues of SAP97 aSAP102, respectively. The rabbit polyclonal antibodies rAb-63 anrAb-PDZ12 were generated against the N-terminal 63 amino acresidues or PDZ1-2 of SAP90/PSD-95 fused to glutathione-transferase (GST), respectively. The mouse polyclonal antibody AGK was generated against the GK domain of SAP97 fused to GSThe GFP monoclonal antibody mAb-GFP was purchased froClontech, the mouse monoclonal antibody anti-human PSD-95 froUpstate Biotechnology and rabbit Kv1.4 polyclonal from AlomonLabs.

Immunoprecipitation was performed essentially as described Kim and Sheng (1996). Transfected and nontransfected cells whomogenized in RIPA buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 1mM EDTA, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS) anincubated on ice for 1 hour to solubilize proteins. After centrifugatioat 16,000 g for 30 minutes, the supernatant was precleared with BSAtreated Protein-A sepharose for 1 hour at 4°C, followed by incubatiwith antibody bound to Protein-A sepharose for 2 hours at 4°

2367Subcellular targeting of SAP97

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Fig. 1.Expression and distribution of endogenous and GFP-taggedSAP90/PSD-95, SAP97 and SAP102 in the epithelial cell lineCACO-2. (A) Western blots of rat brain homogenates (lanes 1, 4 and7), total cellular extracts from untransfected CACO-2 cells (lanes 2,5 and 8), or CACO-2 cells transfected with GFP-SAP90/PSD-95(lane 3), GFP-SAP97 (lane 6) or GFP-SAP102 (lane 9). Antibodiesagainst SAP90/PSD-95 (lanes 1-3), SAP97 (lanes 4-6) or SAP102(lanes 7-9) were used to probe the blots. The asterisk marks a SAP97breakdown product. (B) Confluent monolayers of CACO-2 cellswere incubated with antibodies against SAP90/PSD-95, SAP97 orSAP102, followed by FITC-conjugated secondary antibodies, toreveal the distribution of endogenous SAP90/PSD-95 (endo SAP90),SAP97 (endo-SAP97) or SAP102 (not shown). The distributions ofGFP-tagged SAP90/PSD-95, (GFP-SAP90), SAP97 (GFP-SAP97),SAP102 (GFP-SAP102) or GFP alone were assessed by GFPepifluorescence with FITC filters, 24 hours after transfection of semi-confluent CACO-2 cells. Note the diffuse pattern for bothSAP90/PSD-95 and SAP102 compared to the lateral membranelocalized pattern for SAP97.

Immunoprecipitates were washed, separated by SDS-PAGE analyzed by immunoblotting.

For immunoblotting, transfected and nontransfected cells cultuin 60-mm dishes were scraped off with a rubber policeman in presence of 1× SDS sample buffer. Samples were boiled for 5 minutand separated on 10% SDS polyacrylamide gels, then transferrenitrocellulose membrane (Micron Separations Inc.). Western blwere blocked with 5% skimmed milk, 0.5% Nonidet P-40 in Tribuffered saline (TBS) (buffer A) for 30 minutes before incubating f2 hours at room temperature with primary antibodies diluted 1:500TBS/3% FBS. After three washes in TBS and reblocking in buffer blots were incubated for 2 hours at room temperature with alkalphosphatase-conjugated goat anti-mouse antibodies (Sigma) (dilu1:2000). Membranes were washed twice with TBS, 0.5% Tweenand once with TBS. Immunoreactive bands were detected as descpreviously (Müller et al., 1995).

RESULTS

Differential distribution of SAPs in epithelial cellsIn neuronal cells, SAPs are differentially distributed (See Garand Kindler, 1996). In epithelial cells, SAP97 is localized alothe lateral membrane at sites of cell-cell contact (Müller et 1995; Reuver and Garner, 1998). We therefore investigawhether SAP90/PSD-95 and/or SAP102 were also expresseepithelial cells and whether this cell system could be useddefine protein sequences directing the subcellular localizationSAPs. Western blots of CACO-2 cell extracts were stained wSAP-specific antibodies (Fig. 1A). Rabbit antibody againSAP90/PSD-95 detected a single band at 95 kDa with the srelative mobility as SAP90/PSD-95 immunoreactive banpresent in rat brain extracts (Fig. 1A). Antibody against SAP1reacted with a 102 kDa band in brain extracts but failed to dea similar molecular mass protein in CACO-2 cell extracts (F1A). Antibodies against SAP97 recognized a doublet at 120 kin both CACO-2 and brain extracts (Fig. 1A). The expressionSAPs in CACO-2 cells was also assessed immunofluorescence microscopy. Confluent monolayers CACO-2 cells stained with antibody against SAP97 showedintense fluorescence at sites of cell-cell contact (Fig. 1B).contrast, cells stained with antibody against SAP90/PSDexhibited a diffuse pattern (Fig. 1B). No immunofluorescenlabeling was observed with the SAP102 antibody (not showThese data indicate that while SAP90/PSD-95 is expressed awith SAP97 in CACO-2 cells, it is not co-recruited with SAP9to sites of cell-cell contact in these cells.

The ability of SAP97 to become localized to the latermembrane in CACO-2 cells, in contrast to SAP90, wanalyzed further by expressing recombinant SAP97 aSAP90/PSD-95 as well as SAP102 in CACO-2 celRecombinant proteins were distinguished from endogenouexpressed SAPs by epitope tagging either with a 10 amino apeptide from c-myc (Munro and Pelham, 1987) or with thgreen fluorescent protein (GFP). Similar to endogenoSAP90, GFP-tagged SAP90/PSD-95 and SAP102 (Fig. 1exhibited a diffuse cytoplasmic distribution in cells expressiboth low and high levels of recombinant proteins. At sites cell-cell contact between two high-expressing cells somincrease in fluorescence was observed. However, sinceincrease was observed at sites of cell-cell contact with ntransfected cells, a non-localization phenotype was scored

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contrast, GFP-tagged SAP97 became localized to the latmembrane at sites of cell-cell contact between adjacent c(Fig. 1B). Myc-tagged recombinant SAPs showed a simidistribution (data not shown). The presence of recombinprotein of the correct molecular mass was assessed analyzing cell extracts of transfected cells on western bl(Fig. 1A). As expected, GFP-tagged SAP90/PSD-95, SAPand SAP102 were found to be approx. 27 kDa larger than endogenous epithelial and/or brain proteins (Fig. 1A). Thedata show that SAP97, as compared to SAP90/PSD-95 SAP102, is selectively localized at specific membrane regioin epithelial cells and that GFP-tagged recombinant protecan be used to identify regions in SAPs that direct thsubcellular distribution.

The N-terminal domain directs the selective lateralmembrane localization of SAP97A high degree of sequence similarity between SAPs (Kistnet al., 1993; Müller et al., 1995; 1996) and the latermembrane localization of SAP97 in CACO-2 cells, in contrato the cytoplasmic distribution of SAP90/PSD-95 and SAP10allowed a chimeric approach to be used to identify regions

Fig. 2.Spatial distribution of chimericSAP97/SAP90 and SAP97/SAP102 moleculesin CACO-2 cells. (A) Schematic diagrams ofthe GFP-tagged SAP97, SAP90/PSD-95,SAP102 and chimeric molecules. The basicorganization of the PDZ, SH3 and GKdomains in each SAP as well as the I3 insertin SAP97 are indicated as gray boxes. Exceptfor I3, the regions flanking boxes exhibit alow degree of sequence similarity (5-30%).The amino acid boundaries of SAP97 andSAP90 or SAP102 are indicated for eachchimeric molecule. The localization efficiencywas measured as the percentage (±s.d.) oftransfected cells showing clear lateralmembrane localization. Note that onlytransfected cells exhibiting a low to moderatelevel of protein were included in this analysis.Data shown are an average of threeindependent experiments with approximately200 cells per experiment counted. (B) Spatialdistribution of GFP-tagged SAP90/PSD-95,SAP102, S97N/S90PDZ1-GK andS97N/S102PDZ1-GK of transfected CACO-2cells, assessed by GFP epifluorescence.

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SAP97 that direct its subcellular localization. Initial chimeriproteins were made between SAP97 and SAP90/PSD-95replacing C-terminal segments of SAP97 with the analogoregions from SAP90/PSD-95. The cross-over points weselected at boundaries between conserved and unisequences (Fig. 2A). All three GFP-tagged chimeras wefound to localize to the epithelial lateral membrane in a patteidentical to endogenous and GFP-tagged SAP97 (Fig. 2Common to all three were the N-terminal 186 amino-acresidues of SAP97 that precede the PDZ1 domain (Fig. 2suggesting that this region (henceforth termed S97N) important for the selective targeting of SAP97 in epithelicells. This conclusion was supported by the ability of S97Nalso direct the lateral membrane localization of SAP102 (F2B).

PDZ1-2 and the I3 domain contribute to the efficientlocalization of SAP97A comparison of the efficiency of lateral membranlocalization of different S97-S90 and S97-S102 chimeras (F2A) revealed that S97N/S90PDZ1-GK (33%) and S97NPDZ2/S90PDZ3-GK (37%) were less effective at localizatio

2369Subcellular targeting of SAP97

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than S97N-I3/S90GK (65%), SAP97 (72%) oS97N/S102PDZ1-GK (60%). Localization efficiency waassessed by comparing the percentage of transfected showing clear lateral membrane localization. Only transfeccells expressing low to moderate levels were counted, si

Fig. 3.Localization efficiency of GFP-tagged SAP97 deletion mutastudy. The amino acid boundaries of each deletion are indicated.transfected cells showing clear lateral membrane localization. NDAlso summarized are the abilities of individual constructs to remaafter being treated with 0.5% Triton X-100 for 1.5 minutes. Similafluorescence in small groups of cells was monitored in real time d(B) Western blots of protein extracts from CACO-2 cells transfecteindividual GFP-fusion proteins were assayed by immunoblotting w197.4), the GK domain of SAP97 (Ab-GK) or against GFP (mAb-G

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localization could not be assessed unequivocally in higexpressing cells. These data suggest that one or more dommay play a role in mediating the efficient lateral membranlocalization of SAP97. A series of GFP-tagged deletioconstructs were therefore created (Fig. 3A). Deletion of PD

nts. (A) Schematic diagram of deletion constructs of SAP97 used in this The localization efficiency was measured as the percentage (±s.d.) of, not determined, due to the susceptibility of expressed protein to degradation.in localized at sites of cell-cell contact (+) or to be extracted (−) 10 minutesr results were obtain in three independent experiments in which GFPuring the first 5 minutes and on the remainder of the dish after 10 minutes.d with the GFP-tagged SAP97 deletion constructs. The molecular masses ofith either a monoclonal antibody against the S97N domain of SAP97 (mAb-FP). The position of endogenous SAP97 is indicated with an arrow.

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(∆PDZ3) from SAP97 had no effect on localization efficienc(70%). In contrast, deleting PDZ1-2 (∆PDZ1-2) or PDZ1-3(∆PDZ1-3) caused a reduction of approx. 50% in tlocalization efficiency as compared to full-length SAP97 (Fi3A). This suggests that PDZ1-2 contributes to the efficielocalization of SAP97. A specific role for PDZ1 could not banalyzed since transfection of ∆PDZ1 mutant constructs didnot lead to the synthesis of full-length proteins (Fig. 3A,B).

Removing the SH3 (∆SH3) or GK (∆GK) domains had noeffect on the localization efficiency of GFP-tagged SAP9(Figs 3A, 4), indicating that these two domains are dispensafor localization of SAP97. However, when the SH3 and Gdomains were deleted together with intervening sequen(S97N-PDZ1-3), a significant effect on the localizatioefficiency (43%) was observed. These observations suggthat sequences situated between the SH3 and GK domainsa role in localization. This region of SAP97 has previousbeen shown to be a site of sequence variation that arisesalternative splicing (Lue et al., 1994; Müller et al., 1995). TGFP-SAP97 used in this study contains the I3 insert. Whenwas deleted (∆I3), a significant decrease in efficiency olocalization (40%) was observed as compared to GFP-SAP(70%) (Fig. 3A). These data indicate that the I3 insert as was PDZ1-2 play a role in the efficient localization of SAP9However, the deletion of neither domain abolished latemembrane localization of SAP97. We therefore deleted bPDZ1-3 and I3 (S97N-GK) to examine whether these domaact synergistically to localize SAP97 efficiently. This mutalocalized with the same efficiency as ∆PDZ1-2, ∆PDZ1-3, ∆I3and S97N-PDZ1-3 (Fig. 3A), indicating that there is nsynergism between these regions. Furthermore, we examwhether either region had an innate ability to localize to tlateral membrane of CACO-2 cells. GFP-tagged PDZ1

Fig. 4.Distribution of GFP-tagged SAP97 deletionconstructs. Semi-confluentmonolayers of CACO-2 cellswere transfected with GFP-tagged SAP97 constructs asindicated and 24 hours latervisualized by direct GFPfluorescence with FITC filterset. Note that selected imagesdo not necessarily reflectlocalization efficiency, due tothe nature of the transcienttransfection assay. Forlocalization efficiency ofindividual constructs, see Fig.3. Interestingly, SH3-I3-GKexhibits a nuclear pattern.

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PDZ1-3, GK and SH3-I3-GK all exhibited a diffuse pattern(Figs 3A, 4). Taken together, these data show that the PDZ1and I3 domains appear to contribute to the localizatioefficiency of SAP97 at the epithelial lateral membrane, yet thedo not contain the localization signals.

The N-terminal segment of SAP97 is required forlocalizationSignificantly, all deletion constructs capable of localizing to thlateral membrane contained the amino-terminal 186 aminacid residues of SAP97. Furthermore, the addition of S97N those constructs which by themselves could not localize leda precise localization to the lateral membrane (Figs 3A, 4). Ttest whether this region is essential for the localization oSAP97, we deleted S97N (∆S97N) from GFP-SAP97. This∆S97N mutation abolished the lateral membrane localizatioof SAP97 (8%) (Fig. 3A), demonstrating that this segment oSAP97 was critical for its subcellular targeting. We alsexamined whether S97N was sufficient to direct lateramembrane localization of SAP97. When S97N was fuseeither up- (S97N-GFP) or down- (GFP-S97N) stream of GFa diffuse pattern was observed, though proteins of thappropriate molecular mass were expressed. However, addition of one half of PDZ1 (PDZ1/2) or glutathione Stransferase (GST) to GFP-S97N (S97N-PDZ1/2; S97N-GSrespectively) resulted in lateral membrane localization (Figs 4). These data demonstrate that S97N is both necessary sufficient to direct the subcellular localization of SAP97 inepithelial cells and that the presence of additional C-terminsequences appears to help maintain it in an acticonformation.

The S97N domain can be divided into two regions (Fig. 5AThe first region (residues 1-104) (S97N1-104) is unique to

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Fig. 5.The first 65 amino acid residues inSAP97 direct its localization to the lateralmembrane. (A) Alignment of the deducedN-terminal amino acid sequences precedingPDZ1 of SAP97, SAP90/PSD-95, SAP102and Chapsyn-110/PSD-93. Identical aminoacid residues are indicated by gray boxes.Cysteine residues involved in themultimerization of SAP90/PSD-95 andChapsyn-110/PSD-93 as well as the twofound in SAP97 are marked with asterisks.Arrows also label the start of the first PDZdomain in each protein as well as theboundaries of the S97N domain fused to theSAP97 GK domain. (B) Schematic diagramof S97N-GK deletion constructs. The aminoacid boundaries of each deletion areindicated. Tabulated are the localizationefficiency and stability of each construct toextraction by Triton X-100. (C) Distributionof S97N1-104-GK, S97N1-65-GK, S97N105-186-GK and S97N66-186-GK in transfectedCACO-2 cells analyzed by GFPfluorescence.

SAP97. The second (residues 105-186) (S97N105-186) sharesabout 60% similarity with the N-terminal regions oSAP90/PSD-95, SAP102 and Chapsyn-110/PSD-93 (Fig. 5This latter region in SAP90/PSD-95 and Chapsyn-110/PSDhas been shown to promote ion channel clustering throughead to head multimerization (Hsueh et al., 1997). In boSAPs, ion channel clustering in HEK293 cells (Hsueh et a1997; Topinka and Bredt, 1998) and visualization of multimeon non-reducing SDS gels (Hsueh et al., 1997) are mediaby a pair of cysteine residues situated in the N terminus. Githe importance of S97N in the selective localization of SAPto the epithelial lateral membrane, we examined whether tpotential multimerization domain and/or the two cysteinresidues (C66 and C73) in the S97N domain of SAP97 (F5A) play a role in the localization of SAP97 to the latermembrane. These possibilities were initially investigated examining whether removing amino acid residues 105-1containing the putative multimerization domain, could blocS97N-mediated localizaiton of SAP97. This was accomplishby attaching the GK domain as a stabilizer to S97N1-104(S97N1-104-GK) or S97N105-186 (S97N105-186 -GK). Only

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S97N1-104-GK became localized to the lateral membrane (Fi5B,C). These data demonstrate that sequences in S9homologous to the multimerization domain in SAP90/PSD-9are not essential for the lateral membrane localization SAP97 and that the targeting signal is present in S97N1-104.Next, we tested whether SAP97 can lead to the formationstable multimers in non-reducing conditions, as reported SAP90/PSD-95 (Hsueh et al., 1997). Whereas high molecumass immunoreactive bands (180 kDa), indicative multimers of the 95 kDa SAP90/PSD-95, could be observwith recombinant SAP90 under non-reducing conditions, tmobility of SAP97 immuno-reactive bands remained at 12kDa (data not shown), indicating that SAP97 does not formultimers that can be stabilized by disulfide bridges. Tdirectly test whether the two cysteine residues (C66 and Care required for the localization of SAP97, two additiondeletion molecules were constructed. The first, S97N1-65-GK,lacks these two residues while the second, S97N66-186-GK,contains them. Here, only S97N1-65-GK was localized to theepithelial lateral membrane (Fig. 5B,C). These dademonstrate that the localization of SAP97 is not mediated

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Fig. 6.Localization of the GFP-taggedS97N is not mediated by association withthe endogenous SAP97. (A) Western blots ofimmunoprecipitates from transfected COS-7cells. Extracts from transfected cells wereimmunoprecipitated either with mAb-GFP(lanes 2-4) or anti-Kv1.4 (lane 5) antibodiesand western blot-probed with anti-SAP97(mAb-1974) (lanes 1-4) or anti-SAP90(rAb-63) (lane 5). Lane 1, total cell extractsfrom nontransfected COS 7 cells; lane 2, cell extracts from nontransfected cells; lane 3, cells transfected with GFP-tagged SAP97; lane 4, cellstransfected with GFP-S97N-PDZ1/2; lane 5, cells doubly transfected with SAP90/PSD-95 and Kv1.4. The arrow marks the position ofendogenous SAP97. (B) Western blots of GFP-tagged S97N1-65-GK transfected CACO-2 cells immunoprecipitated with rAb-PDZ1-2, whichrecognizes the PDZ1-2 domain of SAP97. Blots from supernatant (S) or immunoprecipitates (P) were incubated either with mAb-197.4(SAP97) (upper panel) or mAb-GFP (S97N1-65-GK) (lower panel).

a mechanism that involves its amino-terminal cysteines putative multimerization domain. As a final test of whethS97N-mediated localization could be due to the interactionthis domain with endogenous SAP97 in CACO-2 celimmunoprecipitation experiments were conducted. In the fiGFP-tagged SAP97 and S97N-PDZ1/2 were transfected COS-7 cells and cell extracts subjected to immunoprecipitatwith GFP antibody followed by immuno-blotting with mAb197.4. Here only GFP-fusion proteins and not endogenSAP97 could be detected in the immunoprecipitates (Fig. 6In the second experiment, CACO-2 cells were transfected wGFP-tagged S97N1-65-GK and cell extracts were immunoprecipitated with antibody rAb-PDZ12, which recognizes thendogenous SAP97 but not S97N1-65-GK. Only theendogenous SAP97 was detected in the immunoprecipit(Fig. 6B). GFP-S97N1-65-GK stayed only in the supernatanAs a positive control, extracts of COS-7 cells doubtransfected with SAP90 and Kv1.4 were immunoprecipitatwith Kv1.4 antibody. In western blots incubated with SAP9antibody, a 95 kDa band is visible (Fig. 6A), indicating thconditions used for immunoprecipitation permit proteinprotein interactions to be maintained. Thus these data showthe localization of GFP S97N fusion proteins is not mediatby stable association with the endogenous SAP97 and other proteins mediate its localization.

S97N1-65 directs attachment of SAP97 to the lateralmembrane A striking feature of SAP97 in epithelial cells is its stabassociation with the cortical cytoskeleton along the latemembrane (Reuver and Garner, 1998). To identify whidomains in SAP97 play a role in its attachment to the corticytoskeleton, two assays were employed. In the first, live cwere briefly extracted with 0.5% Triton X-100, a condition thextracts soluble and most integral membrane proteins leaves the cortial cytoskeleton intact (Kaiser et al., 198Nelson et al., 1990; Nelson and Veshnock, 1986; 1987; Reuand Garner, 1998). Under these conditions, GFP-tagSAP97 (Fig. 7) as well as S97N-containing molecules, such∆PDZ1-3, ∆SH3, ∆I3, ∆GK, S97N-PDZ1-3, S97N-GK andS97N-PDZ1/2, were retained at sites of cell-cell containdicating that they were also associated with the Triton X-10resistant cortical cytoskeleton (Figs 3A, 7). Unexpectedconstructs lacking S97N (∆S97N, GK, PDZ1-3 and SH3-I3-GK) exhibited a cytoplasmic distribution and were readi

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extracted by Triton X-100 perfusion (Fig. 3A), even thougconstructs like PDZ1-3 and SH3-I3-GK contain a protein 4binding site. These data indicate that without S97N, protein 4binding does not occur in vivo. In addition, we found thaS97N1-65-GK was also retained at sites of cell-cell contact aftTriton-X-100 extraction, yet S97N66-186-GK was easilyremoved (Figs 5B, 7), indicating that the first 65 amino aciprovide an attachment point for SAP97 to the corticcytoskeleton as well as a localization signal.

As a second measure of the ability of S97N to mediate tassociation of SAP97 with the actin-cortical cytoskeleton, cewere treated with Latrunculin B, a drug that binds globulaactin with high affinity and disrupts F-actin (Spector et al1989). Treatment of CACO-2 cells for 5 minutes with 5 µMLatrunculin B caused a profound disruption of cortical F-actand a redistribution of endogenous SAP97 from sites of cecell contact to an even cytoplasmic pattern (Reuver and Garn1998). GFP-tagged SAP97 as well as S97N1-65-GK (Fig. 8),S97N-PDZ1/2 and S97N-GST (data not shown) becamdiffuse in the cytoplasm after treatment with Latrunculin Bdemonstrating that the interaction of S97N with the epithelilateral membrane requires an intact cortical cytoskeletoTaken together, these data show that S97N1-65 is not onlyrequired for the localization but also is an attachment point fSAP97 to the cortical cytoskeleton.

DISCUSSION

Selective subcellular targeting of SAP97In this study, we have examined the contribution of individudomains to the localization of SAP97 to the epithelial latermembrane. We show that amino acid residues preceding PDare required to direct the localization of SAP97 to sites of cecell contact as well as to provide at least one point of attachmto the cortical cytoskeleton. This region alone can direct tlateral membrane localization of individual SAP97 domains well as heterologous protein GST. Furthermore, it can provia lateral membrane localization signal to two non-localizemembers of the SAP97 family: SAP90/PSD-95 and SAP10Taken together, these findings indicate that the N terminusSAP97 plays a critical role in the selective association of SAPwith its specific membrane specializations.

Our analysis of SAP97 revealed that the PDZ, SH3 and Gdomains, as well as the I3 insert, do not contain functiona

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independent epithelial lateral membrane localization signNonetheless, we observed that removal of either PDZ1-2 ocaused about a 50% decrease in localization efficiency. Thregions do not appear to act synergistically, since no furtdecrease in efficiency was detected when both were del(S97N-GK). Presumably, their effect on localization is duethe ability of these regions to interact with components of lateral membrane. This conclusion is consistent with recenvitro studies on the human homologue of SAP97, hDshowing that PDZ1-2 and I3, but not PDZ3, SH3 or GK, habinding sites available to them at the lateral membranepermeabilized epithelial cells (Lue et al., 1996). Potentlateral membrane binding partners for PDZ1-2 and I3 regioof SAP97/hDlg are non-erythroid isoforms of protein 4.which are present at the epithelial lateral membrane (Lue et1994, 1996; Schischmanoff et al., 1997). In vitro, erythroprotein 4.1 binds the I3 insert of SAP97/hDlg as well as hinge region between PDZ1 and PDZ2 (Lue et al., 1994, 19Marfatia et al., 1996). Nonetheless, data presented hdemonstrate that these protein 4.1 binding sites are not cri

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for directing the lateral membrane localization in epitheliacells but may facilitate the stable association of SAP97/hDwith the lateral membrane.

In erythrocytes, where SAP97/hDlg is also expresse(Marfatia et al., 1996) the situation is less clear. Here, prote4.1 plays a critical role in maintaining membrane structurintegrity via its stabilization of spectrin-actin linkage at theplasma membrane (Discher et al., 1993) and its association wglycophorin C, p55 and SAP97/hDlg (Ruff et al., 1991; Marfatiet al., 1995; 1996). In erythrocytes from patients with hereditaelliptocytosis lacking protein 4.1, neither glycophorin C, p55 noSAP97/hDlg are present at the plasma membrane (Chishti et1996; Marfatia et al., 1996). This suggests that protein 4.1 plaa vital role in maintaining the association of SAP97/hDlg witthe erythrocyte plasma membrane. However, it remains unclewhether the loss of SAP97/hDlg from the plasma membranedue to a general effect on cytoskeletal structure or to the lossprotein 4.1 as a binding site.

At the epithelial septate junction in Drosophila, a differentmechanism appears to be used to direct the localization of D

Fig. 7. Triton X-100 extraction of CACO-2 cellstransfected with SAP97 deletion constructs. Small clustersof CACO-2 cells transfected with either GFP-taggedSAP97 (a, b), S97N-PDZ1/2 (c, d), S97N1-65-GK (e, f) orS97N66-186-GK (g, h) were imaged with FITC filter seteither prior to (−TX-100) or after (+TX-100) perfusingcells for 1.5 minutes with 0.5% Triton X-100.

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Fig. 8.Effect of disassembling F-actin on the retention of GFP-tagged SAP97 or S97N1-65-GK at the epithelial lateral membrane.CACO-2 cells grown on coverslips were transfected with either Gtagged SAP97 or S97N1-65-GK. 24 hours later, cultures were treatewith either 5 µM Latrunculin B (LaB) in DMSO or DMSO alone for5 minutes at 37°C before being fixed with 3.7% formaldehyde. Cewere stained with rhodamine-conjugated phalloidin to detect F-acLatrunculin B caused F-actin, in stress fibers and at the lateralmembrane, to breakdown into small clumps. Under these conditioGFP-tagged SAP97 and S97N1-65-GK relocalized from the lateralmembrane to the cytoplasm.

a Drosophila homolog of SAP97/hDlg (Hough et al., 1997Here, two domains, PDZ2 and HOOK, were found to actconcert to localize Dlg to septate junctions. PDZ2 was shoto possess the septate junction targeting signal. The HOregion, which is homologous to I3 and situated between SH3 and GK domains, is required for association with tplasma membrane. The N terminus of Dlg, which is quite sh(20 amino acid residues), is apparently non-essential subcellular targeting (Hough et al., 1997). Given that PDZ1and I3 are not necessary for subcellular targeting of SAPmoving the targeting signal to the N terminus allows seque

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variations to occur at least at the I3 site (Lue et al., 199Müller et al., 1995; C. C. Garner, unpublished data), which turn may provide an increased repertoire of binding partnersthis position. Moreover, utilizing the unique sequences SAPs, i.e. in the N terminus, for subcellular targeting raththan a conserved domain, such as PDZ2, provides a flexisystem to differentially localize individual SAPs.

Significantly, replacing the N-terminal domains of SAP10and SAP90 with S97N, directed these SAPs to the epithellateral membrane. However, whereas S97N/S102PDZ1-Glocalized with a similar efficiency to SAP97 (approx. 60%)only about 33% of the cells transfected with S97N-S90PDZGK exhibited a clear lateral membrane localization. Thsuggests that like SAP97/hDlg, SAP102 contains an elemewhich acts to increase its localization efficiency that is missinin SAP90. Although we did not map this element in SAP10there are several indications that it is situated between its Sand GK domains. The most compelling is that SAP10contains inserts between its SH3 and GK domains whiresemble those in some isoforms of SAP97/hDlg (Lue et a1994; Muller et al., 1995, 1996). In contrast, SAP90 lackinserts at this site and localizes with a similar low efficiency tthe ∆I3 deletion construct of SAP97 (42%).

Attachment of SAP97 to the epithelial lateralmembraneIn a recent study, we have found that cell-cell adhesion betweepithelial cells mediated by E-cadherin induces thtranslocation of SAP97/hDlg from cytoplasmic pools to thplasma membrane at sites of cell-cell contact (Reuver aGarner, 1998). The attachment of SAP97/hDlg to the latermembrane was found to be mediated by its tight associatwith the cortical actin cytoskeleton, requiring an F-actilinkage to tether it to the E-cadherin adhesion complex (Reuvand Garner, 1998). In this study, we have sought to identify tregions that not only target SAP97/hDlg to the lateral plasmmembrane but also mediate its association with the Triton 100-resistant cortical cytoskeleton, a feature characteristiccortical cytoskeletal proteins (Luna and Hitt, 1992; Nelson al., 1990; Reuver and Garner, 1998). Surprisingly, the putatprotein 4.1 binding sites, I3 and hinge region between PDZand PDZ2, were not required to maintain SAP97 at the latemembrane of CACO-2 cells extracted with Triton X-100Furthermore, the three PDZ domains as well as the SH3 aGK domains were found to be non-essential for the associatof SAP97 with the Triton X-100-resistant cytoskeletonInstead, this characteristic was found to be mediated by S97This conclusion is based on the ability of SAP97 deletioconstructs containing S97N to (1) be retained at sites of cecell contact after extraction with Triton X-100 and (2) bereleased into the cytoplasm after disrupting cortical actin wiLatrunculin B. Interestingly, deletion analysis of S97N reveathat both the lateral membrane targeting signal and the regthat mediates an association with the cytoskeletal are situawithin the first 65 N-terminal amino acid residues of SAP97This indicates that the receptor for S97N at the latermembrane is likely to be a component of the corticacytoskeleton or an integral membrane protein tightly tetherto this structure.

The concept that the N termini of SAPs confer fundamentproperties to this protein family is not restricted to SAP97

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Recent studies on SAP90/PSD-95 and Chapsyn-110/PSDhave revealed that sequences preceeding their first PDZ do(residues 1-60 (S90N) and 1-93 (C110N), respectivemediate clustering of ion channels by these SAPs in transfecells (Kim et al., 1996; Hsueh et al., 1997; Topinka and Bre1998). Mechanistically, clustering is mediated by twproperties of these N termini. The first is membrane attachmthrough palmitoylation of a pair of cysteine residues at C3 aC5 in S90N or C5 and C7 in C110N (Topinka and Bredt, 199(Fig. 5A). The second is multimerization (Hsueh et al., 199Topinka and Bredt, 1998), which can either be homo- heteromer between SAP90/PSD-95 and Chapsyn-110/PSDand is thus thought to be mediated by the region of shahomology between these two N termini (S90N1-60; C1101-9(Fig. 5A). Interestingly, multimerization can be observed cell extracts run on non-reducing SDS-gels (Hsueh et 1997), due to the labile nature of palmitoylation and the raformation of intramolecular disulfide bridges (Hsueh et a1997; Topinka and Bredt, 1998). Although SAP97 shares ab60% sequence homology to the multimerization domainsSAP90/PSD-95 and Chapsyn-110/PSD-93 (residues 100-1and has two cysteine residues (C66 and C73) (Fig. 5A), it dnot cluster ion channels in heterologous cells (Kim and She1996) nor form heteromeric (Hsueh et al., 1997) or homomemultimers, as evaluated on non-reducing SDS-geFurthermore, recombinant GFP-tagged SAP97 construcould not be co-immunoprecipitated with endogenousexpressed SAP97 in COS or CACO-2 cells. Thus, in contrto SAP90/PSD-95 and Chapsyn-110/PSD-93, the N termiof SAP97 does not appear to be involved in the formationmultimers. Instead, data presented here demonstrate that Sin particular residues 1-65, confers a novel property to SAPas a lateral membrane localization signal. Significantly, tsignal lies outside of its putative multimerization domain, donot require its N-terminal cysteines and appears to interact wa component of the Triton X-100-resistant cortical accytoskeleton.

In epithelial cells, four proteins have been characterized tshare a similar domain structure to SAP97/hDlg. Three, ZOZO-2 and ZO-3, contain three PDZ domains, an SH3 doma guanylate kinase-like domain and a long C-terminextension. They are exquisitely restricted in polarizepithelial to tight junctions, where they interact with each othwith actin filaments and the cytoplasmic domain of occlud(Haskins et al., 1998). At present, tight junction targetidomains in this subfamily of membrane-associated guanykinase homologs (MAGUKs) have not been described, given their distinct spatial distribution from SAP97 (Reuvand Garner, 1998), it appears unlikely that they interact wSAP97 or affect its localization along the lateral membraThe fourth protein, CASK, contains a single PDZ domain, SH3 domain, a GK domain and an N-terminal calmoduldependent protein kinase-like domain (CaMK), and is reporto exhibit a similar lateral membrane localization to SAP(Brecher Cohen et al., 1997). The mechanism underling localization of CASK is not known. Interestingly, a shosegment of CASK situated between residues 350-400 sharmoderate degree of sequence similarity to residues 30-5SAP97 (data not shown). Whether this is just a coincidencerepresents a lateral membrane targeting signal in CAremains to be tested.

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Together, these data indicate that the N domains in SAconfer unique fundamental properties to each SAP. In the caof SAP90/PSD-95 and Chapsyn/PSD-93, the N domaiappear to play a central role in the clustering of channelsfeature that is essential to achieve a high density of ligand avoltage-gated channels within the postsynaptic density (PSDAlternatively, S97N appears to perform a primary role in thattachment of SAP97 to the cortical cytoskeleton. Based on localization along the epithelial lateral membrane and alonunmyelinated axons (Müller et al., 1995), this feature wouallow the even distribution of channels and other proteins alothe length of these membrane specializations. An important bunresolved issue is whether the N-domains in SAP9SAP90/PSD-95, SAP102 and Chapsyn-110/PSD-93 also pa role in their differential localization in neurons or whetheother regions are required.

We would like to thank Stefan Kindler for critical reading of themanuscript. This work was supported by Keck Foundation (93136and the National Institutes of Health (P50 HD32901; AG 12978-0AG 06569-09).

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