ZP2 and ZP3 cytoplasmic tails prevent premature interactions and

940 Research Article

IntroductionExtracellular matrices present in the interstices of multicellularorganisms form three-dimensional networks that provide physicalsupport and modulate biological function (Aszodi et al., 2006;Tsang et al., 2010). Their macromolecular components share acommon challenge in remaining soluble during biosynthesis andintracellular trafficking while retaining the ability to form aninsoluble matrix in the extracellular space. Various strategies haveevolved, several of which have been characterized in moleculardetail. For example, collagen precursors are synthesized with N-and C-terminal globular domains that are proteolytically removedto allow formation of extracellular intermediates that culminate information of mature collagen fibrils (Shoulders and Raines, 2009).Likewise, soluble tropoelastin is secreted and deposited on pre-existing microfibrillar matrices where they undergo conformationalchanges to enable cross-linking and formation of insoluble elastinpolymers (Sato et al., 2007). Another set of extracellular proteinsuse a ‘zona domain’ (Bork and Sander, 1992) for polymerizationof tissues (Jovine et al., 2005), including the tectorin membrane ofthe inner ear (Richardson et al., 2008) and the zona pellucidaformed during folliculogenesis in the mammalian ovary(Wassarman, 2008).

The newborn mouse ovary is composed primarily of primordialfollicles in which each oocyte is surrounded by a flattened layer ofgranulosa cells encased in a basement membrane (Brambell, 1928).With cyclic periodicity, cohorts are selected to enter into a 2 weekgrowth phase in which oocytes grow from ~15 to 80 m duringwhich time they form the extracellular zona pellucida(Yanagimachi, 1994). The mouse zona is composed of threeglycoproteins, ZP1, ZP2 and ZP3 (Bleil and Wassarman, 1980a;Boja et al., 2003) and has an important role in gamete recognition.

This is dependent on the cleavage status of ZP2, rendering thezona pellucida either permissive (intact ZP2) or non-permissive(cleaved ZP2) for sperm binding (Gahlay et al., 2010). Althoughprimarily investigated for its role in fertilization and prevention ofpolyspermy, the zona pellucida also is essential for passage of theearly embryo through the oviduct before implantation on the wallof the uterus. Removal of the zona pellucida either biochemically(Modlinski, 1970; Bronson and McLaren, 1970) or genetically(Rankin et al., 1996; Liu et al., 1996; Rankin et al., 2001) resultsin resorption of eggs and early embryonic loss, with resultantfemale sterility.

Each zona gene is single copy in the mouse genome (Kinloch etal., 1988; Liang et al., 1990; Lunsford et al., 1990; Epifano et al.,1995a) and zona transcripts accumulate during oogenesis (Epifanoet al., 1995b). After translation (Greve et al., 1982; Salzmann et al.,1983), the three mouse zona proteins are secreted to form anextracellular matrix (Bleil and Wassarman, 1980b; Shimizu et al.,1983). Commencing with oocyte growth, the zona matrix is initiallyobserved as discrete patches of amorphous material in the spacebetween the surface of oocytes and the innermost layer of granulosacells. With time, the patches coalesce to become a continuous,highly porous matrix that reaches a diameter of ~7 m in fullygrown, ovulated mouse eggs (Phillips and Shalgi, 1980).

Genetic ablation of Zp1 indicates that expression of ZP2 andZP3 is sufficient to form an extracellular zona matrix robustenough for fertilization and early development (Rankin et al.,1999). ZP2 (713 aa) and ZP3 (424 aa) share motifs, including asignal peptide, a ‘zona domain’ (260 aa with eight or ten conservedcysteine residues) and an endoproteinase cleavage site, which isfollowed by a transmembrane domain and a short, hydrophiliccytoplasmic tail (Ringuette et al., 1988; Liang et al., 1990). The

SummaryThe zona pellucida contains three proteins (ZP1, ZP2, ZP3), the precursors of which possess signal peptides, ‘zona’ domains and short(9–15 residue) cytoplasmic tails downstream of a transmembrane domain. The ectodomains of ZP2 and ZP3 are sufficient to form theinsoluble zona matrix and yet each protein traffics through oocytes without oligomerization. ZP2 and ZP3 were fluorescently taggedand molecular interactions were assayed by fluorescent complementation in CHO cells and growing oocytes. ZP2 and ZP3 trafficindependently, but colocalize at the plasma membrane. However, protein–protein interactions were observed only after release andincorporation of ZP2 and ZP3 into the extracellular matrix surrounding mouse oocytes. In the absence of their hydrophilic cytoplasmictails, ZP2 and ZP3 interacted within the cell and did not participate in the zona pellucida. A heterologous GPI-anchored ‘zona’ domainprotein fused with the cytoplasmic tails was integrated into the zona matrix. We conclude that the cytoplasmic tails are sufficient andnecessary to prevent intracellular oligomerization while ensuring incorporation of processed ZP2 and ZP3 into the zona pellucida.

Key words: Cytoplasmic tails, Zona pellucida, Oocytes, Intracellular trafficking, Extracellular matrix, ZP2, ZP3, -Tectorin

Accepted 10 November 2010Journal of Cell Science 124, 940-950 © 2011. Published by The Company of Biologists Ltddoi:10.1242/jcs.079988

ZP2 and ZP3 cytoplasmic tails prevent prematureinteractions and ensure incorporation into the zonapellucidaMaria Jimenez-Movilla and Jurrien Dean*Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA*Author for correspondence ([email protected])

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signal peptide directs individual zona proteins into a secretorypathway and the ectodomain is released by cleavage before itsincorporation into the insoluble zona pellucida (Boja et al., 2003).These observations present a mechanistic conundrum. How dozona proteins avoid interacting to form polymers duringintracellular trafficking and then oligomerize after secretion toform the insoluble, extracellular zona matrix? Here, we explorethe role of the cytoplasmic tails of ZP2 and ZP3 in orchestratingthese events.

ResultsInteractions of ZP2 and ZP3 expressed in heterologouscellsTo investigate intracellular trafficking of the zona proteins,expression plasmids encoding ZP2Venus and ZP3Cherry fusion proteins(Fig. 1A) were co-transfected into CHO cells and imaged byfluorescence microscopy. Initially, the two zona proteins colocalizedin the endoplasmic reticulum, but appeared to traffic independentlythrough the cell before again colocalizing in the plasma membrane(Fig. 1B). The presence of ZP3 at the plasma membrane wasconfirmed biochemically by a decrease in the abundance of matureisoforms (larger molecular masses) after digestion of intact cellswith trypsin to remove extracellular protein domains before lysis.Similar processing of ZP2 was observed, but the larger molecularmass band was much fainter. Each zona protein was subsequentlysecreted into the culture medium (Fig. 1C).

To further characterize the interactions of ZP2Venus and ZP3Cherry

after secretion from heterologous cells, medium from stablytransfected cells was evaluated by size exclusion chromatography.Individual column fractions were analyzed by immunoblots usingantibodies against ZP2 and ZP3. A peak with a molecular mass of~240 kDa was observed and contained each zona protein(supplementary material Fig. S2A). Using an antibody against thefluorescent tag on ZP2, it was possible to co-immunoprecipitateZP3 from medium of the transfected cells (supplementary materialFig. S2B). This assay was extended for evaluation of individualcolumn fractions and co-immunoprecipitation of ZP2 and ZP3 inthe peak fractions (31–28) confirmed interactions between the twosecreted zona proteins (supplementary material Fig. S2C).

Cytoplasmic tails direct ZP2 and ZP3 separately to theplasma membraneIn contrast to results observed with intact zona proteins, ZP2 andZP3 truncated before their cytoplasmic tails (Tail, Fig. 1A)colocalized during intracellular trafficking and at the plasmamembrane before secretion into the medium (Fig. 1B). With theadditional removal of their transmembrane domains (TM), ZP2and ZP3 continued to co-traffic, albeit in smaller vesicles that werediffusely present throughout the cells (Fig. 1B). Unlike the twozona proteins that lacked just their cytoplasmic tails, those alsolacking their transmembrane domains (ZP2Venus-TM; ZP3Cherry-TM) were not detected at the plasma membrane before secretion(Fig. 1B) and therefore ZP3 was not subject to digestion withtrypsin (Fig. 1C).

These results suggested that the cytoplasmic tails of ZP2 andZP3 were sufficient to prevent co-trafficking of the two zonaproteins. To confirm these observations, the ZP3 expression plasmidwas modified to replace the normal tail with that of ZP2 [ZP3–(ZP2 tail)] (Fig. 1A). The two different zona proteins, but withidentical tails, co-trafficked to the plasma membrane, where theycolocalized before secretion into the medium (Fig. 1B,C). Taken

941Cytoplasmic tails of zona proteins

Fig. 1. Cytoplasmic tails direct separate trafficking of ZP2 and ZP3 inCHO cells. (A)ZP2 (713 aa) and ZP3 (424 aa) have a signal peptide, a ‘zona’domain, a dibasic cleavage site followed by a transmembrane domain and ashort cytoplasmic tail. Using cDNA expression vectors, full-length, truncatedor modified forms of ZP2 and ZP3 were cloned in-frame with Venus or Cherryfluorescent proteins, respectively. (B)CHO cells were co-transfected withZP2Venus and ZP3Cherry expression vectors encoding full-length (normal),truncated proteins lacking cytoplasmic tails (�Tail), transmembrane domains(�TM) or ZP3 with a ZP2 cytoplasmic tail, ZP3–(ZP2 tail). Cells were fixedand imaged by fluorescence microscopy using ApoTome technology. Higher-magnification inserts provide images of vesicle-like structures when proteinsthat lack the cytoplasmic tail or share the same cytoplasmic tail colocalize.Merge includes ER-Tracker, blue. Scale bars: 10m. (C)Media and celllysates treated without (left panel) or with (right panel) trypsin were assayedby immunoblot using monoclonal antibodies against ZP2 or ZP3. Thereduction of signal after treatment of cell lysates with trypsin (red asterisks)indicates the presence of expressed protein on the plasma membrane. Note thatthe upper band is not detected in TM proteins and there is no reduction insignal after treatment with trypsin. Data reflect representative images fromexperiments that were repeated three times.

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together, these data indicate that the transmembrane domain isessential for the presence of zona proteins on the plasma membraneand that distinct cytoplasmic tails ensure that ZP2 and ZP3 trafficindependently through the cell.

942 Journal of Cell Science 124 (6)

Cytoplasmic tails prevent ZP2–ZP3 polymerization withinthe cellTo investigate ZP2 and ZP3 interactions before incorporation intothe extracellular zona matrix, we established a fluorescent-based

Fig. 2. See next page for legend.

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protein fragment complementation (BiFC) assay (Fig. 2A). BiFCis based on the formation of a fluorescent complex from twoseparate non-fluorescent fragments, which are brought together bythe association of two interacting partner proteins fused to thefragments (Hu et al., 2002; Magliery et al., 2005; Kerppola, 2006).The N-terminal fragment (aa 1–156) of Venus was fused to ZP2just after the signal peptide, ZP2NTVenus(N), whereas the C-terminalfragment of Venus (aa 157–239) was fused just after the N-terminalsignal peptide of ZP3 [ZP3Venus(C)] (Fig. 2B,C). No fluorescencewas observed in CHO cell lysates or media after expression ofZP2NTVenus(N) alone, ZP3Venus(C) alone or if the N-terminus ofVenus was inserted at the end of the ZP2 zona domain[ZP2CTVenus(N), supplementary material Fig. S3B]. However,fluorescence was readily detected in the medium, but not in CHOcells, after co-expression of ZP2NTVenus(N) and ZP3Venus(C). Intactzona proteins did not interact within the cell (Fig. 2D–F), but didfluoresce (positive BiFC) after secretion into the medium (Fig.2E). Only ZP2NTVenus(N) was co-expressed with ZP3Venus(C) insubsequent experiments and was abbreviated to ZP2Venus(N).

The BiFC assay was extended to zona proteins lacking theircytoplasmic tails. When ZP2Venus(N)-�Tail and ZP3Venus(C)-�Tailwere co-expressed, fluorescence was observed within cellsindicating protein–protein interactions of the two zona proteins inthe absence of their cytoplasmic tails (Fig. 2D). The fluorescence


pattern was discretely localized in the cytoplasm, which isconsistent with complementation within secretory vesicles.Fluorescence was not observed in the medium (Fig. 2E), indicatingthat complementation of ZP2 and ZP3 within the cell precludedsecretion. However, interactions with ZP2Venus-�Tail and ZP3Cherry-�Tail proteins were detected in the medium by immunoprecipitation(Fig. 2F), which indicated that non-complementing zona proteins(negative BiFC signal) can be secreted. Thus, we propose that tail-less (Tail) zona proteins traffic through heterologous cells viatwo pathways. In the first, premature release of ZP2 and ZP3 fromthe membrane (perhaps by convertases during passage through theGolgi) results in BiFC complementation, which precludes secretion.In the second, ZP2 and ZP3 remain colocalized and tethered to themembrane as they progress to the plasma membrane. AlthoughZP2 and ZP3 are released from their transmembrane domains inthe absence of their cytoplasmic tails, there are subtle structuraldifferences that prevent complementation and formation of asecreted protein complex with a positive BiFC signal.

Similar complementation of BiFC was detected within cellsexpressing ZP2Venus(N)-�TM and ZP3Venus(C)-�TM, but theseproteins, which also lack their transmembrane domains, remainedcomplemented after secretion into the medium (Fig. 2D,E). Therewas no evidence of either zona protein on the plasma membrane(Fig. 1B,C). Thus, ZP2 and ZP3 truncated before their transmembranedomains, appear to follow a constitutive secretory pathway, with noobligatory presence at the plasma membrane. To further investigatethe role of cytoplasmic tails, ZP2Venus(N)-Normal and ZP3Venus(C)-(ZP2tail) (Fig. 2B,C) were co-expressed in CHO cells. The twoproteins colocalized in the cell, but did not interact, as determinedby the BiFC assay. However, fluorescence complementation wasobserved in secreted proteins (Fig. 2E). These observations suggestthat release from the plasma membrane represents an important stepin the physiological interactions of ZP2 and ZP3. Thus, the presenceof either cytoplasmic tail is sufficient for physiological interaction ofthe two proteins after release from the cell surface. These data areconsistent with a model in which the cytoplasmic tail ensures thatZP2 and ZP3 traffic independently in the cell before they colocalizeat the plasma membrane, where their release from the transmembranedomain provokes structural changes reflected in fluorescentcomplementation.

Cytoplasmic tails are required for incorporation of ZP2and ZP3 into the zona pellucidaTo investigate the role of ZP2 and ZP3 cytoplasmic tails indetermining cellular localization under more physiologicalconditions, expression plasmids were microinjected into the nucleusof isolated mouse oocytes and imaged by confocal microscopy. Todistinguish localization of peripheral signals between the plasmamembrane and the closely opposed extracellular zona pellucidamatrix, 50% of the injected oocytes were lysed with non-ionicdetergent and high salt to obtain zona ‘ghosts’ which were thenimaged by confocal microscopy (Shimizu et al., 1983; Zhao et al.,2003).

The presence of fluorescence in >85% of injected oocytesindicated that the plasmid construct encoding ZP2Venus and ZP3Cherry

were transcribed and translated into protein. Each protein appearedto traffic through the cell independently of the other before detectionat the periphery (Fig. 3A) and incorporation into the zona pellucida(Fig. 3B). However, when truncated before their cytoplasmic tails,ZP2Venus-Tail and ZP3Cherry-Tail colocalized within the oocyteand, although detected in the plasma membrane (Fig. 3A), they

Fig. 2. ZP2 and ZP3 cytoplasmic tails prevent premature interactionswithin cells. (A)Schematic representation of BiFC assay in which the N- andC-termini of Venus fluorescent protein were inserted in-frame just downstreamof the signal peptide of ZP2 and ZP3, respectively. Correct zona proteindimerization resulted in Venus complementation and production of afluorescent signal. (B)Expression vectors encoding ZP2Venus(N) that were full-length (Normal), truncated before the cytoplasmic tail (Tail) or truncatedbefore the transmembrane domain (TM). (C)ZP3Venus(C) with an additionalconstruct encoding ZP3 with a ZP2 cytoplasmic tail, ZP3Venus(C)–(ZP2 tail).(D)After transfection, fixed cells were imaged by fluorescence microscopyusing monoclonal antibodies against ZP2 and an antibody that binds to theC-terminal fragment of Venus in ZP3. The fluorescent signal of ZP2 (blue),ZP3 (red) and Venus complementation (green) were recorded separately and asa merged image. Scale bars: 10m. (E)Venus complementation (BiFC) wasquantified by fluorometric analysis of cell suspensions (left) and medium(right). Normal, full-length ZP2 and ZP3 did not complement within cells(open bars), but did after secretion into the medium (green bars). Zona proteinslacking their transmembrane domains and cytoplasmic tails complementedprematurely in the cell and complementation was observed in the medium.ZP2 and ZP3, lacking only their cytoplasmic tails, also complemented withinthe cell, but complementation was not detected in the medium. Data are themean ± s.e.m. of three independent experiments. ZP2 and ZP3 were detectedby immunoblots of cell suspensions and media. Actin levels documentedprotein equivalence among samples. (F)Cell lysates and media wereimmunoprecipitated (IP) with mouse anti-GFP monoclonal antibody thatrecognizes ZP2Venus, but not ZP3Cherry. Resultant protein samples wereseparated by SDS-PAGE and analyzed by immunoblot using monoclonalantibodies against ZP2 and ZP3. Normal, full-length ZP2 and ZP3 did notinteract within the cells but co-immunoprecipitated from the medium. Whentruncated to remove their cytoplasmic tails alone (Tail) or in conjunction withtheir transmembrane domains (TM), ZP2 and ZP3 interactions were detectedin both cell lysate and the medium. The upper band (asterisk), identified as theTM isoforms based on their absence in the TM samples, were not wellprecipitated in cell lysates expressing proteins lacking their cytoplasmic tailssuggesting premature release from the transmembrane domain. After secretioninto the medium, ZP2 and ZP3 interactions were detected in the medium forall constructions. No signal was detected in control cells, transfected with onlyZP3Cherry. Photomicrographs and immunoblots are representative images fromexperiments that were repeated three times.

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were not incorporated into the zona pellucida (Fig. 3B). After theadditional removal of the transmembrane domain of ZP2Venus-TMand ZP3Cherry-TM, the two proteins continued to colocalize in theoocytes, but were not detected in the plasma membrane and werenot incorporated into the zona pellucida (Fig. 3A,B). Followingreplacement of the ZP3 tail with that of ZP2, ZP3Cherry-(ZP2 tail)colocalized with ZP2Venus in the oocyte and trafficked to theperiphery (Fig. 3A), but was not incorporated into the extracellularzona pellucida (Fig. 3B). Similar results were obtained by replacingthe cytoplasmic tail of ZP2 with that of ZP3 (Fig. 3A,B). Thesedata suggest that not only are cytoplasmic tails necessary forincorporation of ZP2 and ZP3 into the zona matrix, but they needto differ from one another (i.e. they cannot both be ZP2 or ZP3).

These studies were extended using the BiFC assay. Expressionof full-length Venus inserted at the end of the ZP2 zona domainwas observed in CHO cells and in oocytes (supplementary materialFig. S3A,C,E). When the N-terminal fragment of Venus wassimilarly positioned [ZP2CTVenus(N)], it was expressed(supplementary material Fig. S3B,D), but did not complementZP3Venus(C) in oocytes (supplementary material Fig. S3F). Thus,complementation was only observed when both Venus fragmentswere positioned near the N-termini of ZP2 and ZP3 and only theseconstructs were used in the experiments described below.

After microinjection into oocytes, complementation betweenZP2Venus(N) and ZP3Venus(C) was not observed within thecytoplasm, indicating that the two zona proteins do not interactwithin the endomembrane system. However, complementation and


fluorescence was observed in the periphery of the cells (Fig. 4A).To determine whether the peripheral signal reflected ZP2–ZP3interactions in the plasma membrane, the zona pellucida wasremoved biochemically after microinjection. No complementationfluorescence (BiFC) of ZP2Venus(N) and ZP3Venus(C) was detected(Fig. 4B), although the two proteins trafficked to the plasmamembrane as ZP2Venus and ZP3Cherry (Fig. 4B) and the presence ofthe complementation signal in isolated zona ‘ghosts’ indicatedincorporation into the extracellular zona pellucida (Fig. 4A).

By contrast, ZP2Venus(N) and ZP3Venus(C), lacking theircytoplasmic tails alone or in conjunction with their transmembranedomains, interacted in oocytes as assayed by fluorescentcomplementation, but no complementation was observed at theperiphery or in isolated zona ‘ghosts’ (Fig. 4A). After replacementof the ZP3 cytoplasmic tail with that of ZP2, the two proteins didnot interact to produce a BiFC signal (Fig. 4A) either in the oocyteor in the extracellular zona pellucida, although both were able totraffic to the plasma membrane (Fig. 3A). Thus, ZP2 and ZP3traffic independently through the cell and, although they colocalizein the plasma membrane, they only interact to provide a BiFCsignal after release from the membrane and incorporation into thezona pellucida.

-tectorin with a ZP3 cytoplasmic tail is incorporated intothe zona pellucidaThe tectorin membrane of the inner ear is composed of -tectorin(2155 aa) and -tectorin (329 aa), the latter composed primarily of

Fig. 3. Distinct cytoplasmic tails are essential for incorporation of ZP2 and ZP3 into the zona pellucida. (A)Oocytes were co-microinjected with ZP2Venus andZP3Cherry expression vectors encoding full-length (normal), truncated proteins lacking cytoplasmic tails (�Tail), transmembrane domains (�TM), ZP2 with a ZP3cytoplasmic tail, ZP2–(ZP3 tail) or ZP3 with a ZP2 cytoplasmic tail, ZP3–(ZP2 tail). After 40 hours in culture, the oocytes were fixed and imaged by confocalmicroscopy. Fluorescent signal of ZP2Venus (green) and ZP3Cherry (red) were imaged individually and merged with and without differential interference contrast(DIC) microscopy. Images are representative of three independent experiments, each with 20–30 oocytes. (B)Zona ‘ghosts’ were obtained by freeze-thawing inpresence of 0.5 M NaCl and 1% NP-40 before imaging. Photomicrographs are representative images from experiments that were repeated three times with 10–20oocytes. Scale bars: 20m.

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a ‘zona’ domain that is analogous to ZP3. Mouse -tectorin has asignal peptide to direct it into a secretory pathway and a zonadomain by which it interacts with -tectorin. Unlike ZP3, -tectorin lacks a hydrophilic cytoplasmic tail and forms aglycosylphosphatidylinositol-linked membrane-bound precursorthat is released into the extracellular space following cleavage at atetrabasic site (Legan et al., 1997). Venus was inserted downstreamof the -tectorin signal peptide and cloned as cDNA into anexpression vector either with its native sequence or after the


replacement of its C-terminus with that of ZP3 (Fig. 5A). Whenexpressed in CHO cells, two isoforms were observed byimmunoblot with those of -tectorin–ZP3 tail having greatermolecular mass than native -tectorin (Fig. 5B). When -tectorinVenus was co-injected with ZP3Cherry into oocytes, bothproteins were observed in the periphery, but only ZP3Cherry wasincorporated into the extracellular zona pellucida (Fig. 5C).However, after replacement of the -tectorinVenus C-terminus withthat of ZP3, -tectorin–ZP3-tail was incorporated into theextracellular zona pellucida (Fig. 5D). Thus, processing by theZP3 tail and transmembrane domain was sufficient for integrationof the zona domain of -tectorin into the mouse zona pellucida.

DiscussionLittle is known about the intracellular trafficking of the zona pellucidaproteins or of the processing that ensures release of N-terminalectodomains for assembly into the insoluble, extracellular matrix.By tagging zona proteins with different fluorescent markers andassaying molecular interactions using bimolecular fluorescentcomplementation (BiFC), we have investigated the molecularprocessing of ZP2 and ZP3 in growing oocytes. Our results suggesta model in which signal peptides direct ZP2 and ZP3 into a secretorypathway where their ectodomains remain tethered to a transmembranedomain. While traversing the endomembrane, cytoplasmic tailsprevent interactions between ZP2 and ZP3 and ensure passagethrough the Golgi complex without cleavage by resident convertases.At the plasma membrane, a hypothetical transmembrane protease(single protein or part of a complex) recognizes the intracellularcytoplasmic tails of ZP2 and ZP3 and releases extracellular domainsof each zona protein. This cleavage alters the conformation of ZP2and ZP3 ectodomains, permitting oligomerization and formation ofthe insoluble zona pellucida (Fig. 6).

Intracellular traffickingDuring protein synthesis and before incorporation into theextracellular zona pellucida, ZP2 (713 amino acids) and ZP3 (424aa) undergo disulfide bond formation, proteolytic processing andglycosylation. The signal peptide cleavage sites, as well as the C-termini of the released ectodomains, have been defined bymicroscale mass spectrometry for native ZP2 (Val35-Ser633) andZP3 (Gln23-Asn351) (Boja et al., 2003). As the two proteins transitthe endoplasmic reticulum and Golgi, they are heavily andheterogeneously glycosylated by complex N-glycans and, to amuch less extent, O-glycans. Together these post-translationalmodifications account for roughly one-half of the mass of ZP2 andZP3 on SDS-PAGE gels (Nagdas et al., 1994; Easton et al., 2000;Boja et al., 2003). The two proteins are observed in peripherallylocated multivesicular aggregates (MVAs) consisting of 1–5 mvesicles embedded in an amorphous matrix before transit to theplasma membrane and ultimate incorporation into the extracellularzona matrix (Merchant and Chang, 1971; El Mestrah et al., 2002;Hoodbhoy et al., 2006).

ZP2 and ZP3 with intact transmembrane domains co-expressedin heterologous cells and oocytes are localized in vesicle-likestructures, but seem more diffuse when transmembrane domainsare absent (TM). These data are consistent with tethering of ZP2and ZP3 to the lipid bilayer of distinct transport vesicles while theyare trafficking through the cell, and implies the action of acytoplasmic regulator(s) that distinguishes between the twoproteins. We now implicate the relatively short (9–15 aa)cytoplasmic tails as crucial for the independent trafficking of the

Fig. 4. Cytoplasmic tails prevent ZP2 and ZP3 interaction within growingoocytes. (A)Oocytes were co-microinjected with ZP2Venus(N) and ZP3Venus(C)expression vectors encoding full-length (normal), truncated proteins lackingcytoplasmic tails (�Tail), transmembrane domains (�TM) or ZP3 with a ZP2cytoplasmic tail, ZP3–(ZP2 tail). After 40 hours in culture, oocytes or zona‘ghosts’ were imaged by confocal microscopy alone (left) or merged with DICimages (right). Fluorescence (green) in the BiFC assay indicated protein-protein of ZP2 and ZP3, rather than mere colocalization. (B)Oocytes were co-microinjected with full-length ZP2Venus(N) and ZP3Venus(C) expression vectorsand the zona pellucida was removed by brief exposure to Tyrode’s acidifiedmedia, before incubation and imaging by confocal for BiFC and DICmicroscopy (upper panels). As controls, oocytes were injected with ZP2Venus

and ZP3Cherry before removal of the zona pellucida, incubation and imaging(lower panels). Photomicrographs are representative images from experimentsthat were repeated three times with 10–20 oocytes.

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zona proteins. Upon their removal (Tail) or when geneticallyengineered to be the same, ZP2 and ZP3 colocalize in theendomembrane system of growing oocytes, including the MVA.Each cytoplasmic tail contains 3–5 basic residues that might havea role in ensuring that the ectodomains of the two proteins do notinteract precociously and form insoluble zona matrices within theoocyte. Similar basic residues in the cytoplasmic tails of othertransmembrane proteins have been implicated (1) in coat assembly,which is essential for vesicular transport (Dominguez et al., 1998;Bremser et al., 1999); (2) in providing signals for localization inthe endomembrane system (Schoberer et al., 2009); and (3) in


apical sorting in polarized cells in which Tctex, the dynein light-chain, binds to the cytoplasmic tails of a variety of proteins (Chuangand Sung, 1998; Marzolo et al., 2003; Hodson et al., 2006;Braiterman et al., 2009; Carmosino et al., 2010).

Prevention of intracellular polymerizationTwo short hydrophobic patches have been identified in ZP3, oneof which is C-terminal to the cleavage site that releases the zonaectodomain (Zhao et al., 2003; Jovine et al., 2004). It has beenproposed that the two hydrophobic motifs interact to ensure an‘inactive’ conformation of individual zona proteins, which preventsintracellular polymerization. Once cleaved from the transmembranedomain, the zona ectodomain retains only one of the hydrophobicpatches, which could interact with other zona ectodomains topromote polymerization and formation of the extracellular zonapellucida (Jovine et al., 2004). Consistent with this model, we findthat molecular complementation (BiFC) of ZP2 and ZP3 onlyoccurs after release from the plasma membrane and incorporationinto the inner aspect of the zona pellucida. However, we alsoobserve that in the absence of their cytoplasmic tails, ZP2 and ZP3interact within growing oocytes, suggesting either adventitiousrelease from their transmembrane domains, which promotesprotein–protein interaction according to the above model, or anability of the two different cytoplasmic tails to physically separateZP2 and ZP3 while trafficking within cells.

To distinguish between these two possibilities, we geneticallyswitched the tails and co-expressed proteins sharing the samecytoplasmic tails (either both ZP2 or both ZP3). The absence ofintracellular molecular interaction assayed by BiFC and the inabilityto co-immunoprecipitate the two proteins from cell lysates indicatesthat the cytoplasmic tails do not inhibit polymerization but rather

Fig. 5. -Tectorin with the ZP3 cytoplasmic tail is incorporated into thezona pellucida. (A)-Tectorin (329 aa) has a signal peptide, a ‘zona’ domain,a potential dibasic cleavage site followed by a GPI-anchor site and ahydrophobic tail that is removed in the mature protein. -TectorinVenus cDNAwas modified by replacing its C-terminus with that of ZP3 and cloned into theexpression vector -tectorin–(ZP3 tail). (B) Lysates from CHO cellstransfected with normal (Tectorin) and modified (Tec-ZP3) -tectorinVenus

were assayed by immunoblot using monoclonal antibody against GFP thatrecognizes Venus. (C)Oocytes were co-injected with -tectorinVenus andZP3Cherry before imaging oocytes and zona ‘ghosts’ by confocal microscopy.Scale bar: 20m. (D) C-terminus of -tectorinVenus was replaced by that ofZP3 containing its cytoplasmic tail. Photomicrographs are representativeimages from experiments that were repeated three times with 10–20 oocytes.

Fig. 6. Model for ZP2 and ZP3 incorporation into the zona pellucida. Thesignal peptides of intact ZP2 and ZP3 direct each into a secretory pathway.While traversing the endomembrane system, the cytoplasmic tails preventinteractions of the two zona proteins (either directly or indirectly viainteractions with binding proteins) and ensure passage through the Golgiwithout cleavage by resident convertases (e.g. furin). At the plasma membrane,a hypothetical transmembrane protease (single protein or part of a complex)recognizes the intracellular cytoplasmic tails of ZP2 and ZP3 and releases theextracellular ectodomain of each. This cleavage alters the conformation of thetwo proteins permitting their oligomerization and incorporation into the zonapellucida

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maintain the zona proteins in an ‘inactive’ conformation. In supportof this, proteins lacking cytoplasmic tails exhibit fluorescent (BiFC)complementation within the cells, but not at the cell surfacemembrane. Together, these data indicate that: (1) the cytoplasmictails prevent cleavage of the ectodomain and premature interactionsbetween ZP2 and ZP3 within the endomembrane duringintracellular trafficking, and (2) the C-terminus of zona proteins(including the external hydrophobic patch, the transmembranedomain and the cytoplasmic tails) modulate zona protein assemblyrequired for incorporation into the zona pellucida.

Processing zona proteins at the plasma membraneUpon reaching the plasma membrane, ZP2 and ZP3 colocalize, butdo not interact molecularly until released from the plasmamembrane before incorporation into the zona pellucida. A well-conserved convertase cleavage site (RX[R/K]Rf) has beenimplicated in the release of the zona ectodomains (Yurewicz et al.,1993), but microscale mass spectrometry of native zonae pellucidaedefined the C-terminus upstream of a dibasic motif within thesame site (RXf[R/K]R) (Boja et al., 2003).

Once secreted from heterologous cells, ZP2 and ZP3 interacteven when lacking their transmembrane domain and/or cytoplasmictails. The cleavage site is heterogeneous in heterologous cells(Zhao et al., 2004) and differs from that observed in the nativeproteins as documented by changes in gel mobility (currentmanuscript) and definition of the C-terminus by mass spectrometry(Boja et al., 2003). The precision in the cleavage site in the nativeZP2 and ZP3, suggests involvement of oocyte-specific mechanismsin processing zona proteins. The proteins lacking theirtransmembrane domains and/or cytoplasmic tails are notincorporated into the zona pellucida of growing oocytes (Jovine etal., 2004) (current manuscript). More recently, it has been reportedthat mutations of the EHP and IHP of uromodulin resulted in therelease of monomers unable to assemble into filaments (Schaefferet al., 2009). Together these data suggest that the absence of tailsmay affect correct cleavage of zona proteins and precise cleavagemay determine the ability of zona proteins to assembly andparticipate in the zona pellucida.

Sheddases or secretases release ectodomains from membrane-spanning domains in a variety of proteins (Hooper et al., 1997).Particularly well studied are the selectins, three members of theCAM (cell adhesion molecules) family identified in endothelialcells (E-selectin), platelets (P-selectin) and leukocytes (L-selectin)(Gonzalez-Amaro and Sanchez-Madrid, 1999). The extracellulardomains of the three proteins have similar structural features, butthe divergence of their cytoplasmic tails suggests differences inregulation. The 17 residue cytoplasmic tail of L-selectin regulatesproteolytic cleavage, microvillar positioning and thetethering/rolling behavior of leucocytes. The tail interacts with atleast three proteins, including CaM (calmodulin), -actinin, andthe members of the ERM (erzin–radixin–moesin) family ofcytoskeletal proteins (Ivetic and Ridley, 2004; Killock et al., 2009).CaM negatively regulates shedding by interacting with the L-selectin tail and inducing a conformational changes in theextracellular domain that renders the cleavage site resistant toproteolysis (Kahn et al., 1998; Diaz-Rodriguez et al., 2000).Conversely, when CaM dissociates from the tail, the conformationalchange in the extracellular domain promoted proteolytic cleavageby a sheddase.

To further assay the requirement for a cytoplasmic tail information of the zona pellucida, we expressed -tectorin, a GPI-


anchored ‘zona’ domain containing protein that normally complexeswith -tectorin to form the extracellular tectorin membrane in thevertebrate inner ear (Legan et al., 1997; Petit et al., 2001). Whenexpressed in oocytes, -tectorin trafficked correctly to the plasmamembrane, but was not incorporated into the zona pellucida.However, after genetically replacing its C-terminus with thetransmembrane and cytoplasmic tail of ZP3, -tectorin wasincorporated into the zona pellucida. This suggests that thecytoplasmic tail must be specifically recognized at the plasmamembrane for the ectodomain to be released and incorporated intothe zona matrix. The importance of cytoplasmic tails has beendocumented in other experimental systems including Herpessimplex Virus 1 glycoproteins H and D which are virion envelopeproteins required for fusion with infected cells. Each has atransmembrane domain with a short cytoplasmic tail. Insertionalmutation in the cytoplasmic tail of glycoprotein H completelyabrogate cell fusion and viral infectivity (Jackson et al., 2010) asdoes tethering glycoprotein D to the viral envelop with a GPI-anchor sequence (Browne et al., 2003).

ConclusionWe propose a model in which the cytoplasmic tails of ZP2 andZP3 control the release of zona ectodomains to form theextracellular zona pellucida. Before arrival at the plasma membrane,zona tails prevent premature cleavage of the proteins and theirabsence renders ZP2 and ZP3 susceptible to cleavage (perhaps bya convertase in the Golgi) and precocious intracellularpolymerization. We speculate that upon arrival at the plasmamembrane, the ZP2 and ZP3 cytoplasmic tails bind (directly orindirectly) to a putative membrane-associated protease. Thecleavage of each protein would alter the conformation of itsectodomains, permitting oligomerization and incorporation intothe zona matrix. Identification of a molecular basis of thesehypothetical interactions and determining whether specificity isbased on specific protein–protein or protein–phospholipid (Deford-Watts et al., 2009) interactions will be of considerable interest.

ZP2 and ZP3 have different cytoplasmic tails, albeit with similarbasic charges. When their tails are genetically switched to be thesame, the ZP2 and ZP3 ectodomains were not incorporated into theextracellular matrix surrounding growing oocytes. This is consistentwith ZP2 and ZP3 tails being recognized at the plasma membraneto ensure correct stoichiometry of the two zona proteins forincorporation into the zona pellucida. We note that although thezona matrix is generally thought to be formed from heterodimersof ZP2 and ZP3, a role for homodimers of either protein has notbeen excluded. To validate these models it will be important tobiochemically identify and genetically confirm the molecular basisof zona protein processing at the plasma membrane. Understandingof these molecular details might provide insight into formation ofother matrices formed by proteins containing ‘zona’ domains(Jovine et al., 2005), including the tectorin membrane of the innerear (Cosgrove and Grotton, 2001), as well as other extracellularpolymers (Aszodi et al., 2006; Larsen et al., 2006).

Materials and MethodsConstruction of expression plasmidsThe C-terminal domains of ZP2 and ZP3 are well conserved among mammals(supplementary material Fig. S1). To insert Venus at the N-terminus of ZP2, the 5�region (nucleotides 21–150) of ZP2, including the signal sequence (aa 1–34), wasamplified by PCR with two primers (supplementary material Table S1), eachcontaining either an NheI or an AgeI site (ZP2IF, ZP2IR). cDNA encoding full-length Venus fluorescent protein (aa 1–239) (gift from Eneko Urizar, Center forMolecular Recognition, Columbia University College of Physicians and Surgeons,

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New York, NY) was amplified by PCR with two primers, each containing an AgeIor an EcoRI recognition site (VenusF, VenusR). The resultant PCR products wereassembled and subcloned into pcDNA3.1(+) (Invitrogen, Carlsbad, CA), previouslydigested with NheI and EcoRI. To construct ZP2Venus(N), the N-terminal fragment1–156 of Venus, Venus(N), was amplified by PCR with a 5� primer (VenusF)containing an AgeI recognition site and a 3� primer (NVenIR) containing a sequencecoding for a five amino acid linker (GGGGS) and an EcoRI site. The Venus(N)fragment was subcloned in-frame with the 5� region of ZP2 in the pcDNA vector.The remainder of ZP2 (bp 151–2201) was amplified by PCR product containingEcoRI and PciI recognition sites (ZP2IIF, ZP2IIR) and the rest of ZP2 (bp 413–2169) was isolated from ZP2 cDNA after digestion with PciI and EcoRI. These twoproducts were assembled and cloned into the EcoRI recognition site of pcDNA. Theresultant EcoRI fragments were isolated and subcloned into the EcoRI sitedownstream and in-frame with full length ZP2 to complete the ZP2Venus andZP2Venus(N) plasmids.

To insert Venus and Venus(N) at the C-terminal position of ZP2, the 3� region (bp1921–2201), including the transmembrane domain (aa 684–703) and cytoplasmictail (aa 704–713), was amplified by PCR with two primer (ZP2IVF, ZP2IVR)containing AgeI and XhoI sites. cDNA encoding full-length Venus and Venus(N)fragments were amplified by PCR with 5� primers (VenusF, NVenIIF) containingEcoRI, and 3� primers (VenusR, NVenIIR) containing the AgeI site. The resultantPCR products were assembled and subcloned into EcoRI and XhoI digested pcDNAto generate a fusion protein with the C-terminus of ZP2 in-frame and downstreamof Venus and Venus(N) fragments. The remainder of ZP2 (bp 21–1920) was amplifiedby PCR using primers with NheI and EcoRI recognition sites (ZP2IF, ZP2IIIR). ThePCR product was isolated after digestion with NheI-EcoRI and subcloned upstreamand in-frame with Venus and Venus(N) fragments.

For ZP3Cherry, pmCherry was cloned by PCR from pmCherry-N1 vector (Clontech,Mountain View, CA) with two primers (CherryF, CherryR) containing AgeI andBglII sites, respectively. The PCR product was subcloned into pSEGFP-MoZP3(Zhao et al., 2002), replacing EGFP, but remaining in-frame and downstream of thesignal sequence of ZP3 and upstream of the remainder of ZP3. For ZP3Venus(C), theVenus(C), C-terminus of the Venus fragment (157–239) was amplified by PCR witha 5� primer (CVenF) containing an AgeI recognition site and 3� primer (CVenR)containing a sequence encoding a five amino acid linker (GGGGS) and a BglII site.The Venus(C) fragment was subcloned into pSEGFP-MoZP3. cDNA encoding ZP3fused in-frame to pmCherry and the Venus(C) fragments was isolated from theoriginal vector after digestion with NheI and XhoI and subcloned intopcDNA3.1/Zeo(+) vector (Invitrogen).

For ZP3Cherry(ZP2-tail) and ZP3Venus(C) (ZP2-tail), the 5� regions of ZP3Cherry andZP3Venus(C) (1938 and 1497 bp, respectively) were amplified by PCR with twoprimers (ZP3ctZP2F, ZP3ctZP2R) each containing either an NheI or an EcoRIrecognition site. The C-terminus of ZP2 (bp 2017–2169) was amplified by PCRusing two primers (ZP2ctF, ZP2ctR), containing EcoRI and XhoI sites. The PCRproducts were subcloned into pcDNA after digestion with NheI and XhoI sites toestablish cDNA encoding a protein fusion in which the C-terminus of ZP2 was in-frame and downstream of ZP3.

For ZP2Venus(ZP3 tail), the 5� region of ZP2Venus (2776 bp) was amplified from theabove cDNA by PCR using the primers ZP2ctZP3F and ZP2ctZP3R containing NheIand HindIII sites. The C-terminus of ZP3 (bp 1167–1317) was amplified by PCRusing two primers (ZP3ctF, ZP3ctR) containing HindIII and XhoI sites. The PCRproducts were subcloned in pcDNA vector digested with NheI and XhoI generatinga cDNA encoding a fusion protein with the C-terminus of ZP3 in-frame anddownstream of the ZP2Venus protein.

Truncated proteins were generated by inserting a stop codon using specific primers(supplementary material Table S2) and QuikChange site-directed mutagenesis(Stratagene, Garden Grove, CA) following the manufacturer’s instructions. Briefly,for ZP2-�TM, Asp (673) from ZP2Venus or ZP2Venus(N), was replaced with a stopcodon using ZP2�TMF and ZP2�TMR primers. For the ZP2-�Tail, Tyr (704) fromZP2Venus or ZP2Venus(N), was replaced with a stop codon using ZP2�CTF andZP2�CTR primers. For ZP3-�TM, Trp (380) from ZP3Cherry and ZP3Venus(C) wasreplaced with a stop codon using ZP3�TMF and ZP3�TMF primers. For ZP3-�Tail,Val (409) was replaced with a stop codon using ZP3�CTF and ZP3�CTR primers.

For the -tectorinVenus expression plasmid, the 5� region (bp 11–181) of -tectorin,including the signal sequence (aa 1–17), was amplified by PCR with two primers(BtecIF, BTecIR), each containing either an NheI or an AgeI recognition site. cDNAencoding full-length Venus fluorescent protein (aa 1–239) was cloned as describedabove. The resultant PCR products were assembled and subcloned into pcDNA3.1(+)digested with NheI and EcoRI. The remainder of -tectorin (bp 182–1120) wasamplified by PCR product (151–413) with primers (BTecIIF, BTecIIR) containingEcoRI and XhoI recognition sites. pcDNA was digested with EcoRI and XhoI andthe products were cloned in-frame and downstream of the Venus protein. For -tectorinVenus-(ZP3 tail) the 5� region of -tectorinVenus (encoding 1815 aa) wasamplified from the above cDNA by PCR using the primers BTecIIF and BTecIIRcontaining NheI and HindIII sites. The C-terminus of ZP3 (bp 1167–1317) wasamplified by PCR using two primers containing HindIII and XhoI sites (ZP3ctF,ZP3ctR). The PCR products were subcloned in pcDNA digested with NheI and XhoIto generate a cDNA encoding fusion protein in which the C-terminus of ZP3 was in-frame and downstream of -tectorinVenus.

Junction fragments and PCR products of all plasmids were verified by DNAsequencing and the presence of recombinant protein was confirmed by immunoblotafter expression in heterologous cells. Expression plasmids were purified with theGenEluted Plasmid Kit (Sigma).

Expression in heterologous cellsCHO-K1 cells (American Type Culture Collection, Manassas, VA) were grown(37°C, 5% CO2) for 24 hours to 50–70% confluence in F-12 medium supplementedwith 10% fetal bovine serum and 100 U/ml penicillin-streptomycin (Gibco-Invitrogen). Transient transfections were performed with FuGene HD (Roche AppliedScience, Indianapolis, IN) in accordance with the manufacturer’s protocol. For eachtransfection, 6 l FuGene HD transfection reagent was added to 100 l Opti-MEMreduced-serum medium (Gibco-Invitrogen) predissolved with 2 g template plasmidand incubated for 15 minutes at room temperature. The complex was diluted with 2ml Opti-MEM and overlaid on the growing cells. Transiently transfected cells wereharvested at 24 hours and stable clones were selected after culture for an additional1–2 weeks in the presence of Geneticin (200 g/ml) and Zeocin (350 g/ml).

PhotomicroscopyTransfected cells expressing fluorescent-tagged proteins were grown on coverslipsfor 48 hours after which pre-warmed (37°C) medium (Opti-MEM) containing 20M ER-Tracker-Blue-White DPX (Molecular Probes–Invitrogen) was added. Thecells were incubated for an additional 30 minutes, fixed with 3% paraformaldehydeand imaged at room temperature with an Axioplan 2 fluorescence microscope (CarlZeiss, Thornwood, NY) equipped with C-Apochromat 63�/1.2W Corr objective,ApoTome technology, CCD camera and AxioVision 2 software. Venus fluorochromewas excited with BP450–490 nm filter and emission detected with BP515–565 nmfilter; Cherry and Alexa Fluor 568 fluorochromes were excited with BP534–558 nmfilter and was detected through a LP590 nm filter; Alexa Fluor 633 fluorochromewas excited with HQ590–650 nm filter and emission was detected through a HQ667–738 nm filter; and Dapoxyl (DPX) fluorochrome was excited with a G365 andemission detected through a BP420–470 nm filter.

For BiFC, cells were incubated an additional 2 hours at 30°C to allow maturationof the Venus fluorophore (Hu et al., 2002). Cells were then fixed (3%paraformaldehyde, 15 minutes), washed in PBS (3�), permeabilized (PBS andTriton X-100, 5 minutes), blocked (PBS with BSA, 1 hour) and incubated with ratmonoclonal antibodies to ZP2 (1:200) and mouse antibodies against (1:250)(RocheApplied Science), which binds to the C-terminus of Venus (Nyfeler et al., 2005)contained in ZP3. Antibody binding was detected with either Alexa-Fluor-633-conjugated goat anti-rat antibody (1:100) or Alexa-Fluor-568-conjugated goat anti-mouse (1:100) secondary antibodies (Invitrogen).

Confocal images from growing oocytes were obtained at room temperature withan LSM confocal microscope (Carl Zeiss) equipped with differential contrast opticsusing a C-Apochromat 63�/1.2W Corr objective and scan zoom of 1.7. The Venusfluorochrome was excited with a 488 nm argon laser and emission detected througha BP500–550 nm filter and Cherry fluorochrome was excited with 561 nm DPSSlaser and detected through a BP575–615 nm filter.

ImmunoblotsSupernatants and cells were recovered 48 hours after transfection, after which cellswere homogenized with M-PER Mammalian Protein Extraction Reagent (PierceBiotechnology, Rockford, IL) supplemented with Complete Protease InhibitorCocktail (Roche Applied Science) according to the manufacturer’s instructions.Following centrifugation (16,000 g, 4°C), cell supernatants and lysates were separatedby SDS-PAGE, transferred to PVDF membranes which were probed with monoclonalantibodies to ZP2 (East and Dean, 1984), ZP3 (East et al., 1985) or actin (Santa CruzBiotechnology, Santa Cruz, CA) and visualized by chemiluminescence (Rankin etal., 2003).

Gel filtration chromatographyStably transfected cells co-expressing ZP2Venus and ZP3Cherry were grown in 75 cm2

tissue culture flasks (Corning Life Sciences, Lowell, MA) to 80% confluence andserum starved for 48 hours. Cell supernatants were dialyzed and concentrated against50 mM Tris-HCl, 0.1 M NaCl, 10% glycerol, pH 7.5 before FPLC chromatographyon a Superose 6 10/300 GL column (GE Healthcare Life Sciences) in the samebuffer. Using a flow rate of 0.5 ml/minute, the protein elution pattern was monitored(A280) and collected fractions (0.5 ml) were precipitated with 20% trichloroaceticacid. Following cold acetone washes (1.0 ml, 1�), the precipitated samples wereanalyzed by immunoblot using monoclonal antibodies against ZP2 and ZP3. Thedata (average of three experiments) were quantified by on a LAS-3000 (FujifilmMedical Systems, Stamford, CN). Protein standards from Gel Filtration CalibrationKits (GE Healthcare) run in the same buffer were used to calibrate the column.

BiFC assay in CHO cells48 hours after transfection, medium was recovered and centrifuged to removedetritus. Cells were washed with PBS, harvested by scraping and resuspended in 1ml PBS. 20 l aliquots were counted on a Cellometer Auto T4 (Nexcelom Bioscience,Lawrence, MA) and all samples were normalized to 5�105 cells. After centrifugationand resuspension in 300 l PBS, the cells and media were transferred to 96-well,

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clear-bottom, black microtiter plates (Nunc-Thermo Fischer Scientific). Fluorescencewas determined on a Spectramax GEMINI EM (Molecular Devices, Sunnyvale, CA)using excitation and emission wavelengths of 470 and 535 nm, respectively. The C-terminal fragment of Venus was cloned downstream of the ZP3 zona domain (seeabove). The N-terminal fragment of Venus was cloned downstream of the ZP2 signalpeptide and of the ZP2 zona domain; fluorescence was observed in the BiFC assaywith the former, but not the latter. Mock-transfected cells and medium were used asnegative controls. Data from three independent experiments were averaged ands.e.m. calculated.

ImmunoprecipitationFor immunoprecipitation of ZP2Venus and ZP3Cherry normal and mutated proteins,monoclonal antibody specific to GFP (Roche Applied Science) was incubated withmagnetic Dynabeads® Protein G (Invitrogen) according to the manufacturer’sinstructions. The complex was incubated with supernatant and lysate from transfectedcells and analyzed by immunoblot.

Microinjection of isolated oocytesOocytes isolated from 11- to 13-day-old mouse ovaries (Millar et al., 1991) wereincubated in M2 medium (Chemicon-Millipore, Billerica, MA) supplemented withIBMX (3-isobutyl-1-methylxanthine) at 37°C before microinjection. 10 pl plasmidDNA (50 ng/ml) was injected into the nucleus and surviving oocytes were cultured(37°C, 5% CO2) for 48 hours in KSOM medium (Millipore) containing IBMX. Theoocytes were then separated into two groups. One group was fixed with 2%paraformaldehyde for 1 hour at room temperature. The other group was transferredinto 20 mM Tris-HCl, pH 7.4, containing 1% NP-40 and 0.5 M NaCl and freeze-thawed ten times on ethanol in dry ice to isolate zona ‘ghosts’ (Shimizu et al., 1983;Zhao et al., 2003). To obtain zona-free oocytes after nuclear microinjection, the zonamatrix was removed from growing oocytes by brief exposure to acidified Tyrode’smedium (Hogan et al., 1994) and fixed as above after 48 hours of incubation.

Oocytes co-injected with BiFC fragments were incubated for 48 hours, thenplaced at 30°C for 2 hours and fixed. Individual plasmids carrying the Venusfragments were also microinjected as a negative control of the complementation.The fixed oocytes and treated zona ‘ghosts’ were washed three times with PBS andplaced on a slide chambered (20 l cavity) with Gene Frame (AdvancedBiotechnologies, Leatherhead, UK). Images were obtained by confocal microscopy.

For their help and advice, we thank Lyn Gauthier (microinjection ofoocytes) and Boris Baibakov (confocal microscopy). This researchwas supported by the Intramural Research Program of the NationalInstitutes of Health, NIDDK. Deposited in PMC for release after 12months.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/124/6/940/DC1

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ZP2 and ZP3 cytoplasmic tails prevent premature interactions and

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