1999). OSP/claudin-11 appears to be necessary for the for- mation of TJs as OSP/claudin-11–null mice lack intramem- branous junctions in between Sertoli cells in testes and within CNS myelin sheaths, leading to reproductive and neurological deficiencies (Gow et al., 1999). Indeed, OSP/ claudin-11 knockout mice have slowed CNS conduction velocities, suggesting that OSP/claudin-11–containing TJs are necessary for normal axonal function (Gow et al., 1999). It is not yet known if the expression of OSP/claudin- 11 alone is sufficient to establish the TJ molecular barrier or regulate intramembrane diffusion of lipids. Three types of integral membrane proteins that localize to TJs in vivo have been described: occludin (Furuse et al., 1993), claudins (Furuse et al., 1998), and junction adhesion membrane protein (Martin-Padura et al., 1998). It is likely that these proteins are involved in establishing the barrier function of TJs, although OSP/claudin-11 is the only pro- tein to date to be found essential for the formation of some intramembrane strands (Gow et al., 1999). Several other proteins have been identified which localize to the cytoplasmic membrane region of TJs: ZO-1 (Stevenson et al., 1986), ZO-2 (Jesaitis and Goodenough, 1994), ZO-3 (Haskins et al., 1998), cingulin (Citi et al., 1988), 7H6 (Zhong et al., 1993), symplekin (Keon et al., 1996), AF-6/ afadin (Yamamoto et al., 1997), and some Rab proteins (Weber et al., 1994; Zahraoui et al., 1994). These proteins may mediate other proposed functions of TJ proteins such as regulating cell growth, transformation, and differentia- tion (Willott et al., 1993; Li and Mrsny, 2000; Reichert et al., 2000; Ryeom et al., 2000). It has been proposed that the cytoplasmic proteins associate with one or more of the integral TJ membrane proteins, although direct evidence is lacking. ZO-1 can associate with occludin in vitro (Fu- ruse et al., 1994), although it continues to localize to TJs in the occludin-deficient stem cells (Saitou et al., 1998) and in the testes of the OSP/claudin-11 gene knockout mice (Gow et al., 1999). A recent report has demonstrated di- rect binding of ZO-1, ZO-2, and ZO-3 with the COOH termini of claudins 1–8 in vitro (Itoh et al., 1999). This in- teraction appears to be dependent on the PDZ domains of ZO-1, 2, and, 3 and a COOH-terminal YV amino acid se- quence of many of the claudins (Itoh et al., 1999). Unlike other claudins, OSP/claudin-11 terminates with HV, and no proteins have been identified to associate with it. Since known TJ proteins apparently do not associate with OSP/claudin-11, we have used the yeast two-hybrid system to identify associated proteins. A novel member of the tetraspanin super family (TM4SF) was isolated, which interacted with the COOH termini of OSP/claudin-11. The putative interaction was examined by coimmunoprecipita- tion and by immunohistochemistry colocalization. We have named this protein OSP/claudin-11–associated pro- tein 1 (OAP-1). OAP-1 forms a complex with OSP/clau- din-11 and b1 integrin, and evidence is provided to sup- port the role for this complex in regulating proliferation and migration of oligodendrocytes. Materials and Methods Yeast Two-Hybrid Bait and Library Constructions Three OSP/claudin-11 constructs were used as bait for yeast two-hybrid screen: the entire OSP/claudin-11 ORF, the first extracellular domain of OSP/claudin-11 between 35 and 84 amino acids, and the COOH terminus of OSP/claudin-11 between 122 and 207 amino acids. These cDNAs were cloned into the pGBT9 backbone (Matchmaker system; CLONTECH Laboratories, Inc.). GAL4 activation domain cDNA fusion libraries were constructed in modified pGAD GH vector (CLONTECH, Laboratories, Inc.) using mRNA from mouse brain. Plasmids were transfected into JM101-competent bacterial cells and amplified once on agar plates. Yeast Two-Hybrid Screening 100 mg of library DNA and OSP/claudin-11 bait were cotransformed into HF7c-competent yeast cells. Transformation of Saccharomyces cerevisiae was performed using the lithium acetate method (Gietz et al., 1992). Two reporter genes for interacting proteins are contained in the Hfc7 strain: HIS3 and b-galactosidase. Transformants which expressed interacting proteins grew on histidine-dependent medium (His2); all positive clones were transferred to sterile Whatman no. 5 filters, placed on selection Trp2/ Leu2/His2 minimal synthetic dropout (SD) medium agar, incubated for 1–3 d at 308C, and fixed by freezing in liquid nitrogen. Filters were then placed onto filter paper presoaked in buffer Z (100 mM NaH2PO4, pH 7.0, 10 mM KCl, 1 mM MgSO4), supplemented with 50 mM b-mercap- toethanol and 0.07 mg/ml 5-bromo-4-chloro-3-indolyl-b-D-galactopyrano- side (ONPG) (Sigma-Aldrich). Filters were then incubated at 308C and monitored for the appearance of blue colonies. Plasmids from positive yeast colonies were isolated by Yeastmaker (a yeast plasmid isolation kit) (CLONTECH Laboratories, Inc.) method. In brief, yeast cells were spread onto His2/Leu2/Trp2 SD plates and incu- bated at 308C for 3–4 d. 10-mm2 portions of the yeast patch were scraped and resuspended in Lyticase containing Tris-EDTA solution at 378C for 60 min to destroy yeast cell walls, followed by the addition of 20% SDS, vor- texed, and loaded onto Chroma spin-1000 columns. Columns were centri- fuged at 700 g, and elutes containing plasmids were collected and stored at 2208C. Plasmids isolated from positive colonies were individually cotrans- formed with OSP/claudin-11 bait cDNA and plated on His2 SD medium and tested for b-galactosidase activity as described above. Further confir- mation of positive clones was also performed in SFY526 cells using a liquid b-galactosidase assay, using ONPG as substrate. For this assay, SFY526 yeast cells were transformed with candidate and bait plasmids, grown overnight in Leu2/Trp2 SD medium mixed with YPD medium (CLON- TECH Laboratories, Inc.), centrifuged, resuspended in buffer Z, and fro- zen in liquid nitrogen, followed by an incubation with 0.07% ONPG for 2–5 h. Optical densities were measured at wavelengths of 420 and 600 nm. Several controls were used to elucidate false positives. Positive controls included cotransformation of pVA3-murine p53(722390) in pGBT9, with pTD1-SV40 large T-antigen(842708) in pGAD3F and transformation of pCL1 (wild-type full-length GAL4) (Fields and Song, 1989; Chien et al., 1991). Negative controls included cotransformation of candidate clones with pGAD GH library cloning vector, murine MBP in pGAD GH, and pTD1 clone. Cotransformants were assayed for their ability to grow on His2 medium and for b-galactosidase activity. Northern Blot Analysis Total RNA was prepared from different mouse tissues as described by Chomczynski and Sacchi (1987). Total RNA (10 mg/lane) was separated electrophoretically on 1% agarose-formaldehyde gels, transferred to Hy- bond™-N1 nylon membranes, and irreversibly fixed by incubating filters at 808C for 2 h. Prehybridization and hybridization were performed at 658C in solutions containing 7.5% SDS, 0.5 M phosphate buffer, pH 7.0, 1 mM EDTA, and 1% bovine serum albumin. 32P-labeled ORF OAP-1 cDNA probes were generated by isolating cDNA from plasmid DNA and using random primers according to the manufacturer’s protocol (New En- gland Biolabs, Inc.). After hybridization, membranes were washed with 0.23 SSC and 0.1% SDS at 618C before exposure to x-ray film. Northern blots were normalized with radiolabeled 18S probe (Ambion). In Situ Hybridization In situ hybridization was performed on adult mouse brains as described previously (Kornblum et al., 1994). In brief, an adult mouse was perfused fixed before sectioning of the brain. The probes used for localization stud- ies were obtained by in vitro transcription in the presence of [35S]UTP from linearized pGEM 7Zf plasmids containing mouse OAP-1 cDNA. Hybridization was carried out at 608C overnight in a mixture containing 50% formaldehyde, 10% dextran sulfate, 0.7% ficoll, 0.7% polyvinyl pyr- rolidone, 0.7% BSA, 0.15 mg/ml yeast transfer RNA, 0.33 mg/ml dena- tured salmon sperm DNA, 40-mM DTT, and 35S-labeled cRNA at a con- The Journal of Cell Biology, Volume 153, 2001 296