940 Research Article ZP2 and ZP3 cytoplasmic tails prevent premature interactions and ensure incorporation into the

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

Summary The zona pellucida contains three (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 the insoluble zona matrix and yet each traffics through without oligomerization. ZP2 and ZP3 were fluorescently tagged and molecular interactions were assayed by fluorescent complementation in CHO cells and growing oocytes. ZP2 and ZP3 traffic independently, but colocalize at the plasma membrane. However, protein–protein interactions were observed only after release and incorporation of ZP2 and ZP3 into the extracellular matrix surrounding mouse oocytes. In the absence of their hydrophilic cytoplasmic tails, ZP2 and ZP3 interacted within the cell and did not participate in the zona pellucida. A heterologous GPI-anchored ‘zona’ domain protein fused with the cytoplasmic tails was integrated into the zona matrix. We conclude that the cytoplasmic tails are sufficient and necessary 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

Introduction This is dependent on the cleavage status of ZP2, rendering the Extracellular matrices present in the interstices of multicellular zona pellucida either permissive (intact ZP2) or non-permissive organisms form three-dimensional networks that provide physical (cleaved ZP2) for sperm binding (Gahlay et al., 2010). Although support and modulate biological function (Aszodi et al., 2006; primarily investigated for its role in fertilization and prevention of

Journal of Cell Science Tsang et al., 2010). Their macromolecular components share a polyspermy, the zona pellucida also is essential for passage of the common challenge in remaining soluble during biosynthesis and early embryo through the oviduct before implantation on the wall intracellular trafficking while retaining the ability to form an of the uterus. Removal of the zona pellucida either biochemically insoluble matrix in the extracellular space. Various strategies have (Modlinski, 1970; Bronson and McLaren, 1970) or genetically evolved, several of which have been characterized in molecular (Rankin et al., 1996; Liu et al., 1996; Rankin et al., 2001) results detail. For example, collagen precursors are synthesized with N- in resorption of eggs and early embryonic loss, with resultant and C-terminal globular domains that are proteolytically removed female sterility. to allow formation of extracellular intermediates that culminate in Each zona is single copy in the mouse genome (Kinloch et formation of mature collagen fibrils (Shoulders and Raines, 2009). al., 1988; Liang et al., 1990; Lunsford et al., 1990; Epifano et al., Likewise, soluble tropoelastin is secreted and deposited on pre- 1995a) and zona transcripts accumulate during oogenesis (Epifano existing microfibrillar matrices where they undergo conformational et al., 1995b). After translation (Greve et al., 1982; Salzmann et al., changes to enable cross-linking and formation of insoluble elastin 1983), the three mouse zona proteins are secreted to form an polymers (Sato et al., 2007). Another set of extracellular proteins extracellular matrix (Bleil and Wassarman, 1980b; Shimizu et al., use a ‘zona domain’ (Bork and Sander, 1992) for polymerization 1983). Commencing with growth, the zona matrix is initially of tissues (Jovine et al., 2005), including the tectorin membrane of observed as discrete patches of amorphous material in the space the inner ear (Richardson et al., 2008) and the zona pellucida between the surface of oocytes and the innermost layer of granulosa formed during folliculogenesis in the mammalian ovary cells. With time, the patches coalesce to become a continuous, (Wassarman, 2008). highly porous matrix that reaches a diameter of ~7 m in fully The newborn mouse ovary is composed primarily of primordial grown, ovulated mouse eggs (Phillips and Shalgi, 1980). follicles in which each oocyte is surrounded by a flattened layer of Genetic ablation of Zp1 indicates that expression of ZP2 and granulosa cells encased in a basement membrane (Brambell, 1928). ZP3 is sufficient to form an extracellular zona matrix robust With cyclic periodicity, cohorts are selected to enter into a 2 week enough for fertilization and early development (Rankin et al., growth phase in which oocytes grow from ~15 to 80 m during 1999). ZP2 (713 aa) and ZP3 (424 aa) share motifs, including a which time they form the extracellular zona pellucida , a ‘zona domain’ (260 aa with eight or ten conserved (Yanagimachi, 1994). The mouse zona is composed of three cysteine residues) and an endoproteinase cleavage site, which is , ZP1, ZP2 and ZP3 (Bleil and Wassarman, 1980a; followed by a transmembrane domain and a short, hydrophilic Boja et al., 2003) and has an important role in gamete recognition. cytoplasmic tail (Ringuette et al., 1988; Liang et al., 1990). The Cytoplasmic tails of zona proteins 941

signal peptide directs individual zona proteins into a secretory pathway and the ectodomain is released by cleavage before its incorporation into the insoluble zona pellucida (Boja et al., 2003). These observations present a mechanistic conundrum. How do zona proteins avoid interacting to form polymers during intracellular trafficking and then oligomerize after secretion to form the insoluble, extracellular zona matrix? Here, we explore the role of the cytoplasmic tails of ZP2 and ZP3 in orchestrating these events.

Results Interactions of ZP2 and ZP3 expressed in heterologous cells To 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 by fluorescence microscopy. Initially, the two zona proteins colocalized in the endoplasmic reticulum, but appeared to traffic independently through the cell before again colocalizing in the plasma membrane (Fig. 1B). The presence of ZP3 at the plasma membrane was confirmed biochemically by a decrease in the abundance of mature isoforms (larger molecular masses) after digestion of intact cells with trypsin to remove extracellular protein domains before lysis. Similar processing of ZP2 was observed, but the larger molecular mass band was much fainter. Each zona protein was subsequently secreted into the culture medium (Fig. 1C). To further characterize the interactions of ZP2Venus and ZP3Cherry after secretion from heterologous cells, medium from stably transfected cells was evaluated by size exclusion chromatography. Individual column fractions were analyzed by immunoblots using antibodies 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 the fluorescent tag on ZP2, it was possible to co-immunoprecipitate

Journal of Cell Science ZP3 from medium of the transfected cells (supplementary material Fig. S2B). This assay was extended for evaluation of individual column fractions and co-immunoprecipitation of ZP2 and ZP3 in the peak fractions (31–28) confirmed interactions between the two secreted zona proteins (supplementary material Fig. S2C).

Cytoplasmic tails direct ZP2 and ZP3 separately to the plasma membrane In contrast to results observed with intact zona proteins, ZP2 and ZP3 truncated before their cytoplasmic tails (Tail, Fig. 1A) Fig. 1. Cytoplasmic tails direct separate trafficking of ZP2 and ZP3 in colocalized during intracellular trafficking and at the plasma CHO cells. (A)ZP2 (713 aa) and ZP3 (424 aa) have a signal peptide, a ‘zona’ membrane before secretion into the medium (Fig. 1B). With the domain, a dibasic cleavage site followed by a transmembrane domain and a short cytoplasmic tail. Using cDNA expression vectors, full-length, truncated additional removal of their transmembrane domains (TM), ZP2 or modified forms of ZP2 and ZP3 were cloned in-frame with Venus or Cherry and ZP3 continued to co-traffic, albeit in smaller vesicles that were fluorescent proteins, respectively. (B)CHO cells were co-transfected with diffusely present throughout the cells (Fig. 1B). Unlike the two ZP2Venus and ZP3Cherry expression vectors encoding full-length (normal), zona proteins that lacked just their cytoplasmic tails, those also truncated proteins lacking cytoplasmic tails (⌬Tail), transmembrane domains lacking their transmembrane domains (ZP2Venus-TM; ZP3Cherry- (⌬TM) or ZP3 with a ZP2 cytoplasmic tail, ZP3–(ZP2 tail). Cells were fixed TM) were not detected at the plasma membrane before secretion and imaged by fluorescence microscopy using ApoTome technology. Higher- (Fig. 1B) and therefore ZP3 was not subject to digestion with magnification inserts provide images of vesicle-like structures when proteins trypsin (Fig. 1C). that lack the cytoplasmic tail or share the same cytoplasmic tail colocalize. These results suggested that the cytoplasmic tails of ZP2 and Merge includes ER-Tracker, blue. Scale bars: 10m. (C)Media and cell ZP3 were sufficient to prevent co-trafficking of the two zona lysates treated without (left panel) or with (right panel) trypsin were assayed by immunoblot using monoclonal antibodies against ZP2 or ZP3. The proteins. To confirm these observations, the ZP3 expression plasmid reduction of signal after treatment of cell lysates with trypsin (red asterisks) was modified to replace the normal tail with that of ZP2 [ZP3– indicates the presence of expressed protein on the plasma membrane. Note that (ZP2 tail)] (Fig. 1A). The two different zona proteins, but with the upper band is not detected in TM proteins and there is no reduction in identical tails, co-trafficked to the plasma membrane, where they signal after treatment with trypsin. Data reflect representative images from colocalized before secretion into the medium (Fig. 1B,C). Taken experiments that were repeated three times. 942 Journal of Cell Science 124 (6)

together, these data indicate that the transmembrane domain is Cytoplasmic tails prevent ZP2–ZP3 polymerization within essential for the presence of zona proteins on the plasma membrane the cell and that distinct cytoplasmic tails ensure that ZP2 and ZP3 traffic To investigate ZP2 and ZP3 interactions before incorporation into independently through the cell. the extracellular zona matrix, we established a fluorescent-based Journal of Cell Science

Fig. 2. See next page for legend. Cytoplasmic tails of zona proteins 943

protein fragment complementation (BiFC) assay (Fig. 2A). BiFC pattern was discretely localized in the cytoplasm, which is is based on the formation of a fluorescent complex from two consistent with complementation within secretory vesicles. separate non-fluorescent fragments, which are brought together by Fluorescence was not observed in the medium (Fig. 2E), indicating the association of two interacting partner proteins fused to the that complementation of ZP2 and ZP3 within the cell precluded fragments (Hu et al., 2002; Magliery et al., 2005; Kerppola, 2006). secretion. However, interactions with ZP2Venus-⌬Tail and ZP3Cherry- The N-terminal fragment (aa 1–156) of Venus was fused to ZP2 ⌬Tail proteins were detected in the medium by immunoprecipitation just after the signal peptide, ZP2NTVenus(N), whereas the C-terminal (Fig. 2F), which indicated that non-complementing zona proteins fragment of Venus (aa 157–239) was fused just after the N-terminal (negative BiFC signal) can be secreted. Thus, we propose that tail- signal peptide of ZP3 [ZP3Venus(C)] (Fig. 2B,C). No fluorescence less (Tail) zona proteins traffic through heterologous cells via was observed in CHO cell lysates or media after expression of two pathways. In the first, premature release of ZP2 and ZP3 from ZP2NTVenus(N) alone, ZP3Venus(C) alone or if the N-terminus of the membrane (perhaps by convertases during passage through the Venus was inserted at the end of the ZP2 zona domain Golgi) results in BiFC complementation, which precludes secretion. [ZP2CTVenus(N), supplementary material Fig. S3B]. However, In the second, ZP2 and ZP3 remain colocalized and tethered to the fluorescence was readily detected in the medium, but not in CHO membrane as they progress to the plasma membrane. Although cells, after co-expression of ZP2NTVenus(N) and ZP3Venus(C). Intact ZP2 and ZP3 are released from their transmembrane domains in zona proteins did not interact within the cell (Fig. 2D–F), but did the absence of their cytoplasmic tails, there are subtle structural fluoresce (positive BiFC) after secretion into the medium (Fig. differences that prevent complementation and formation of a 2E). Only ZP2NTVenus(N) was co-expressed with ZP3Venus(C) in secreted protein complex with a positive BiFC signal. subsequent experiments and was abbreviated to ZP2Venus(N). Similar complementation of BiFC was detected within cells The BiFC assay was extended to zona proteins lacking their expressing ZP2Venus(N)-⌬TM and ZP3Venus(C)-⌬TM, but these cytoplasmic tails. When ZP2Venus(N)-⌬Tail and ZP3Venus(C)-⌬Tail proteins, which also lack their transmembrane domains, remained were co-expressed, fluorescence was observed within cells complemented after secretion into the medium (Fig. 2D,E). There indicating protein–protein interactions of the two zona proteins in was no evidence of either zona protein on the plasma membrane the absence of their cytoplasmic tails (Fig. 2D). The fluorescence (Fig. 1B,C). Thus, ZP2 and ZP3 truncated before their transmembrane domains, appear to follow a constitutive secretory pathway, with no obligatory presence at the plasma membrane. To further investigate Fig. 2. ZP2 and ZP3 cytoplasmic tails prevent premature interactions the role of cytoplasmic tails, ZP2Venus(N)-Normal and ZP3Venus(C)- within cells. (A)Schematic representation of BiFC assay in which the N- and C-termini of Venus fluorescent protein were inserted in-frame just downstream (ZP2tail) (Fig. 2B,C) were co-expressed in CHO cells. The two of the signal peptide of ZP2 and ZP3, respectively. Correct zona protein proteins colocalized in the cell, but did not interact, as determined dimerization resulted in Venus complementation and production of a by the BiFC assay. However, fluorescence complementation was fluorescent signal. (B)Expression vectors encoding ZP2Venus(N) that were full- observed in secreted proteins (Fig. 2E). These observations suggest length (Normal), truncated before the cytoplasmic tail (Tail) or truncated that release from the plasma membrane represents an important step before the transmembrane domain (TM). (C)ZP3Venus(C) with an additional in the physiological interactions of ZP2 and ZP3. Thus, the presence Venus construct encoding ZP3 with a ZP2 cytoplasmic tail, ZP3 (C)–(ZP2 tail). of either cytoplasmic tail is sufficient for physiological interaction of (D)After transfection, fixed cells were imaged by fluorescence microscopy Journal of Cell Science the two proteins after release from the cell surface. These data are using monoclonal antibodies against ZP2 and an antibody that binds to the consistent with a model in which the cytoplasmic tail ensures that C-terminal fragment of Venus in ZP3. The fluorescent signal of ZP2 (blue), ZP3 (red) and Venus complementation (green) were recorded separately and as ZP2 and ZP3 traffic independently in the cell before they colocalize a merged image. Scale bars: 10m. (E)Venus complementation (BiFC) was at the plasma membrane, where their release from the transmembrane quantified by fluorometric analysis of cell suspensions (left) and medium domain provokes structural changes reflected in fluorescent (right). Normal, full-length ZP2 and ZP3 did not complement within cells complementation. (open bars), but did after secretion into the medium (green bars). Zona proteins lacking their transmembrane domains and cytoplasmic tails complemented Cytoplasmic tails are required for incorporation of ZP2 prematurely in the cell and complementation was observed in the medium. and ZP3 into the zona pellucida ZP2 and ZP3, lacking only their cytoplasmic tails, also complemented within To investigate the role of ZP2 and ZP3 cytoplasmic tails in the cell, but complementation was not detected in the medium. Data are the determining cellular localization under more physiological mean ± s.e.m. of three independent experiments. ZP2 and ZP3 were detected conditions, expression plasmids were microinjected into the nucleus by immunoblots of cell suspensions and media. Actin levels documented of isolated mouse oocytes and imaged by confocal microscopy. To protein equivalence among samples. (F)Cell lysates and media were immunoprecipitated (IP) with mouse anti-GFP monoclonal antibody that distinguish localization of peripheral signals between the plasma recognizes ZP2Venus, but not ZP3Cherry. Resultant protein samples were membrane and the closely opposed extracellular zona pellucida separated by SDS-PAGE and analyzed by immunoblot using monoclonal matrix, 50% of the injected oocytes were lysed with non-ionic antibodies against ZP2 and ZP3. Normal, full-length ZP2 and ZP3 did not detergent and high salt to obtain zona ‘ghosts’ which were then interact within the cells but co-immunoprecipitated from the medium. When imaged by confocal microscopy (Shimizu et al., 1983; Zhao et al., truncated to remove their cytoplasmic tails alone (Tail) or in conjunction with 2003). their transmembrane domains (TM), ZP2 and ZP3 interactions were detected The presence of fluorescence in >85% of injected oocytes in both cell lysate and the medium. The upper band (asterisk), identified as the indicated that the plasmid construct encoding ZP2Venus and ZP3Cherry TM isoforms based on their absence in the TM samples, were not well were transcribed and translated into protein. Each protein appeared precipitated in cell lysates expressing proteins lacking their cytoplasmic tails suggesting premature release from the transmembrane domain. After secretion to traffic through the cell independently of the other before detection into the medium, ZP2 and ZP3 interactions were detected in the medium for at the periphery (Fig. 3A) and incorporation into the zona pellucida all constructions. No signal was detected in control cells, transfected with only (Fig. 3B). However, when truncated before their cytoplasmic tails, Venus Cherry ZP3Cherry. Photomicrographs and immunoblots are representative images from ZP2 -Tail and ZP3 -Tail colocalized within the oocyte experiments that were repeated three times. and, although detected in the plasma membrane (Fig. 3A), they 944 Journal of Cell Science 124 (6)

Fig. 3. Distinct cytoplasmic tails are essential for incorporation of ZP2 and ZP3 into the zona pellucida. (A)Oocytes were co-microinjected with ZP2Venus and ZP3Cherry expression vectors encoding full-length (normal), truncated proteins lacking cytoplasmic tails (⌬Tail), transmembrane domains (⌬TM), ZP2 with a ZP3 cytoplasmic 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 confocal microscopy. 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 in presence of 0.5 M NaCl and 1% NP-40 before imaging. Photomicrographs are representative images from experiments that were repeated three times with 10–20 oocytes. Scale bars: 20m.

Journal of Cell Science were not incorporated into the zona pellucida (Fig. 3B). After the fluorescence was observed in the periphery of the cells (Fig. 4A). additional removal of the transmembrane domain of ZP2Venus-TM To determine whether the peripheral signal reflected ZP2–ZP3 and ZP3Cherry-TM, the two proteins continued to colocalize in the interactions in the plasma membrane, the zona pellucida was oocytes, but were not detected in the plasma membrane and were removed biochemically after microinjection. No complementation not incorporated into the zona pellucida (Fig. 3A,B). Following fluorescence (BiFC) of ZP2Venus(N) and ZP3Venus(C) was detected replacement of the ZP3 tail with that of ZP2, ZP3Cherry-(ZP2 tail) (Fig. 4B), although the two proteins trafficked to the plasma colocalized with ZP2Venus in the oocyte and trafficked to the membrane as ZP2Venus and ZP3Cherry (Fig. 4B) and the presence of periphery (Fig. 3A), but was not incorporated into the extracellular the complementation signal in isolated zona ‘ghosts’ indicated zona pellucida (Fig. 3B). Similar results were obtained by replacing incorporation into the extracellular zona pellucida (Fig. 4A). the cytoplasmic tail of ZP2 with that of ZP3 (Fig. 3A,B). These By contrast, ZP2Venus(N) and ZP3Venus(C), lacking their data suggest that not only are cytoplasmic tails necessary for cytoplasmic tails alone or in conjunction with their transmembrane incorporation of ZP2 and ZP3 into the zona matrix, but they need domains, interacted in oocytes as assayed by fluorescent to differ from one another (i.e. they cannot both be ZP2 or ZP3). complementation, but no complementation was observed at the These studies were extended using the BiFC assay. Expression periphery or in isolated zona ‘ghosts’ (Fig. 4A). After replacement of full-length Venus inserted at the end of the ZP2 zona domain of the ZP3 cytoplasmic tail with that of ZP2, the two proteins did was observed in CHO cells and in oocytes (supplementary material not interact to produce a BiFC signal (Fig. 4A) either in the oocyte Fig. S3A,C,E). When the N-terminal fragment of Venus was or in the extracellular zona pellucida, although both were able to similarly positioned [ZP2CTVenus(N)], it was expressed traffic to the plasma membrane (Fig. 3A). Thus, ZP2 and ZP3 (supplementary material Fig. S3B,D), but did not complement traffic independently through the cell and, although they colocalize ZP3Venus(C) in oocytes (supplementary material Fig. S3F). Thus, in the plasma membrane, they only interact to provide a BiFC complementation was only observed when both Venus fragments signal after release from the membrane and incorporation into the were positioned near the N-termini of ZP2 and ZP3 and only these zona pellucida. constructs were used in the experiments described below. After microinjection into oocytes, complementation between -tectorin with a ZP3 cytoplasmic tail is incorporated into ZP2Venus(N) and ZP3Venus(C) was not observed within the the zona pellucida cytoplasm, indicating that the two zona proteins do not interact The tectorin membrane of the inner ear is composed of -tectorin within the endomembrane system. However, complementation and (2155 aa) and -tectorin (329 aa), the latter composed primarily of Cytoplasmic tails of zona proteins 945

a ‘zona’ domain that is analogous to ZP3. Mouse -tectorin has a replacement of its C-terminus with that of ZP3 (Fig. 5A). When signal peptide to direct it into a secretory pathway and a zona expressed in CHO cells, two isoforms were observed by domain by which it interacts with -tectorin. Unlike ZP3, - immunoblot with those of -tectorin–ZP3 tail having greater tectorin lacks a hydrophilic cytoplasmic tail and forms a molecular mass than native -tectorin (Fig. 5B). When - glycosylphosphatidylinositol-linked membrane-bound precursor tectorinVenus was co-injected with ZP3Cherry into oocytes, both that is released into the extracellular space following cleavage at a proteins were observed in the periphery, but only ZP3Cherry was tetrabasic site (Legan et al., 1997). Venus was inserted downstream incorporated into the extracellular zona pellucida (Fig. 5C). of the -tectorin signal peptide and cloned as cDNA into an However, after replacement of the -tectorinVenus C-terminus with expression vector either with its native sequence or after the that of ZP3, -tectorin–ZP3-tail was incorporated into the extracellular zona pellucida (Fig. 5D). Thus, processing by the ZP3 tail and transmembrane domain was sufficient for integration of the zona domain of -tectorin into the mouse zona pellucida.

Discussion Little is known about the intracellular trafficking of the zona pellucida proteins or of the processing that ensures release of N-terminal ectodomains for assembly into the insoluble, extracellular matrix. By tagging zona proteins with different fluorescent markers and assaying molecular interactions using bimolecular fluorescent complementation (BiFC), we have investigated the molecular processing of ZP2 and ZP3 in growing oocytes. Our results suggest a model in which signal peptides direct ZP2 and ZP3 into a secretory pathway where their ectodomains remain tethered to a transmembrane domain. While traversing the endomembrane, cytoplasmic tails prevent interactions between ZP2 and ZP3 and ensure passage through 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 intracellular cytoplasmic tails of ZP2 and ZP3 and releases extracellular domains of each zona protein. This cleavage alters the conformation of ZP2 and ZP3 ectodomains, permitting oligomerization and formation of the insoluble zona pellucida (Fig. 6).

Intracellular trafficking

Journal of Cell Science During protein synthesis and before incorporation into the extracellular zona pellucida, ZP2 (713 amino acids) and ZP3 (424 aa) undergo disulfide bond formation, proteolytic processing and glycosylation. The signal peptide cleavage sites, as well as the C- termini of the released ectodomains, have been defined by microscale mass spectrometry for native ZP2 (Val35-Ser633) and ZP3 (Gln23-Asn351) (Boja et al., 2003). As the two proteins transit the endoplasmic reticulum and Golgi, they are heavily and heterogeneously glycosylated by complex N-glycans and, to a much less extent, O-glycans. Together these post-translational modifications account for roughly one-half of the mass of ZP2 and ZP3 on SDS-PAGE gels (Nagdas et al., 1994; Easton et al., 2000; Boja et al., 2003). The two proteins are observed in peripherally Fig. 4. Cytoplasmic tails prevent ZP2 and ZP3 interaction within growing located multivesicular aggregates (MVAs) consisting of 1–5 m oocytes. (A)Oocytes were co-microinjected with ZP2Venus(N) and ZP3Venus(C) vesicles embedded in an amorphous matrix before transit to the expression vectors encoding full-length (normal), truncated proteins lacking plasma membrane and ultimate incorporation into the extracellular cytoplasmic tails (⌬Tail), transmembrane domains (⌬TM) or ZP3 with a ZP2 zona matrix (Merchant and Chang, 1971; El Mestrah et al., 2002; cytoplasmic tail, ZP3–(ZP2 tail). After 40 hours in culture, oocytes or zona Hoodbhoy et al., 2006). ‘ghosts’ were imaged by confocal microscopy alone (left) or merged with DIC ZP2 and ZP3 with intact transmembrane domains co-expressed images (right). Fluorescence (green) in the BiFC assay indicated protein- in heterologous cells and oocytes are localized in vesicle-like protein of ZP2 and ZP3, rather than mere colocalization. (B)Oocytes were co- structures, but seem more diffuse when transmembrane domains microinjected with full-length ZP2Venus(N) and ZP3Venus(C) expression vectors are absent ( TM). These data are consistent with tethering of ZP2 and the zona pellucida was removed by brief exposure to Tyrode’s acidified  media, before incubation and imaging by confocal for BiFC and DIC and ZP3 to the lipid bilayer of distinct transport vesicles while they microscopy (upper panels). As controls, oocytes were injected with ZP2Venus are trafficking through the cell, and implies the action of a and ZP3Cherry before removal of the zona pellucida, incubation and imaging cytoplasmic regulator(s) that distinguishes between the two (lower panels). Photomicrographs are representative images from experiments proteins. We now implicate the relatively short (9–15 aa) that were repeated three times with 10–20 oocytes. cytoplasmic tails as crucial for the independent trafficking of the 946 Journal of Cell Science 124 (6)

Fig. 6. Model for ZP2 and ZP3 incorporation into the zona pellucida. The signal peptides of intact ZP2 and ZP3 direct each into a secretory pathway. While traversing the endomembrane system, the cytoplasmic tails prevent interactions of the two zona proteins (either directly or indirectly via interactions with binding proteins) and ensure passage through the Golgi without cleavage by resident convertases (e.g. ). 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 the extracellular ectodomain of each. This cleavage alters the conformation of the two proteins permitting their oligomerization and incorporation into the zona pellucida

apical sorting in polarized cells in which Tctex, the dynein light- chain, binds to the cytoplasmic tails of a variety of proteins (Chuang and Sung, 1998; Marzolo et al., 2003; Hodson et al., 2006; Braiterman et al., 2009; Carmosino et al., 2010). Journal of Cell Science Prevention of intracellular polymerization Fig. 5. -Tectorin with the ZP3 cytoplasmic tail is incorporated into the Two short hydrophobic patches have been identified in ZP3, one zona pellucida. (A)-Tectorin (329 aa) has a signal peptide, a ‘zona’ domain, of which is C-terminal to the cleavage site that releases the zona a potential dibasic cleavage site followed by a GPI-anchor site and a ectodomain (Zhao et al., 2003; Jovine et al., 2004). It has been hydrophobic tail that is removed in the mature protein. -TectorinVenus cDNA proposed that the two hydrophobic motifs interact to ensure an was modified by replacing its C-terminus with that of ZP3 and cloned into the ‘inactive’ conformation of individual zona proteins, which prevents expression vector -tectorin–(ZP3 tail). (B) Lysates from CHO cells Venus intracellular polymerization. Once cleaved from the transmembrane transfected with normal (Tectorin) and modified (Tec-ZP3) -tectorin domain, the zona ectodomain retains only one of the hydrophobic were assayed by immunoblot using monoclonal antibody against GFP that patches, which could interact with other zona ectodomains to recognizes Venus. (C)Oocytes were co-injected with -tectorinVenus and ZP3Cherry before imaging oocytes and zona ‘ghosts’ by confocal microscopy. promote polymerization and formation of the extracellular zona Scale bar: 20m. (D) C-terminus of -tectorinVenus was replaced by that of pellucida (Jovine et al., 2004). Consistent with this model, we find ZP3 containing its cytoplasmic tail. Photomicrographs are representative that molecular complementation (BiFC) of ZP2 and ZP3 only images from experiments that were repeated three times with 10–20 oocytes. occurs after release from the plasma membrane and incorporation into the inner aspect of the zona pellucida. However, we also observe that in the absence of their cytoplasmic tails, ZP2 and ZP3 zona proteins. Upon their removal (Tail) or when genetically interact within growing oocytes, suggesting either adventitious engineered to be the same, ZP2 and ZP3 colocalize in the release from their transmembrane domains, which promotes endomembrane system of growing oocytes, including the MVA. protein–protein interaction according to the above model, or an Each cytoplasmic tail contains 3–5 basic residues that might have ability of the two different cytoplasmic tails to physically separate a role in ensuring that the ectodomains of the two proteins do not ZP2 and ZP3 while trafficking within cells. interact precociously and form insoluble zona matrices within the To distinguish between these two possibilities, we genetically oocyte. Similar basic residues in the cytoplasmic tails of other switched the tails and co-expressed proteins sharing the same transmembrane proteins have been implicated (1) in coat assembly, cytoplasmic tails (either both ZP2 or both ZP3). The absence of which is essential for vesicular transport (Dominguez et al., 1998; intracellular molecular interaction assayed by BiFC and the inability Bremser et al., 1999); (2) in providing signals for localization in to co-immunoprecipitate the two proteins from cell lysates indicates the endomembrane system (Schoberer et al., 2009); and (3) in that the cytoplasmic tails do not inhibit polymerization but rather Cytoplasmic tails of zona proteins 947

maintain the zona proteins in an ‘inactive’ conformation. In support anchored ‘zona’ domain containing protein that normally complexes of this, proteins lacking cytoplasmic tails exhibit fluorescent (BiFC) with -tectorin to form the extracellular tectorin membrane in the complementation within the cells, but not at the cell surface vertebrate inner ear (Legan et al., 1997; Petit et al., 2001). When membrane. Together, these data indicate that: (1) the cytoplasmic expressed in oocytes, -tectorin trafficked correctly to the plasma tails prevent cleavage of the ectodomain and premature interactions membrane, but was not incorporated into the zona pellucida. between ZP2 and ZP3 within the endomembrane during However, after genetically replacing its C-terminus with the intracellular trafficking, and (2) the C-terminus of zona proteins transmembrane and cytoplasmic tail of ZP3, -tectorin was (including the external hydrophobic patch, the transmembrane incorporated into the zona pellucida. This suggests that the domain and the cytoplasmic tails) modulate zona protein assembly cytoplasmic tail must be specifically recognized at the plasma required for incorporation into the zona pellucida. membrane for the ectodomain to be released and incorporated into the zona matrix. The importance of cytoplasmic tails has been Processing zona proteins at the plasma membrane documented in other experimental systems including Herpes Upon reaching the plasma membrane, ZP2 and ZP3 colocalize, but simplex Virus 1 glycoproteins H and D which are virion envelope do not interact molecularly until released from the plasma proteins required for fusion with infected cells. Each has a membrane before incorporation into the zona pellucida. A well- transmembrane domain with a short cytoplasmic tail. Insertional R conserved convertase cleavage site (RX[ /K]Rf) has been mutation in the cytoplasmic tail of H completely implicated in the release of the zona ectodomains (Yurewicz et al., abrogate cell fusion and viral infectivity (Jackson et al., 2010) as 1993), but microscale mass spectrometry of native zonae pellucidae does tethering glycoprotein D to the viral envelop with a GPI- defined the C-terminus upstream of a dibasic motif within the anchor sequence (Browne et al., 2003). R same site (RXf[ /K]R) (Boja et al., 2003). Once secreted from heterologous cells, ZP2 and ZP3 interact Conclusion even when lacking their transmembrane domain and/or cytoplasmic We propose a model in which the cytoplasmic tails of ZP2 and tails. The cleavage site is heterogeneous in heterologous cells ZP3 control the release of zona ectodomains to form the (Zhao et al., 2004) and differs from that observed in the native extracellular zona pellucida. Before arrival at the plasma membrane, proteins as documented by changes in gel mobility (current zona tails prevent premature cleavage of the proteins and their manuscript) and definition of the C-terminus by mass spectrometry absence renders ZP2 and ZP3 susceptible to cleavage (perhaps by (Boja et al., 2003). The precision in the cleavage site in the native a convertase in the Golgi) and precocious intracellular ZP2 and ZP3, suggests involvement of oocyte-specific mechanisms polymerization. We speculate that upon arrival at the plasma in processing zona proteins. The proteins lacking their membrane, the ZP2 and ZP3 cytoplasmic tails bind (directly or transmembrane domains and/or cytoplasmic tails are not indirectly) to a putative membrane-associated protease. The incorporated into the zona pellucida of growing oocytes (Jovine et cleavage of each protein would alter the conformation of its al., 2004) (current manuscript). More recently, it has been reported ectodomains, permitting oligomerization and incorporation into that mutations of the EHP and IHP of uromodulin resulted in the the zona matrix. Identification of a molecular basis of these release of monomers unable to assemble into filaments (Schaeffer hypothetical interactions and determining whether specificity is

Journal of Cell Science et al., 2009). Together these data suggest that the absence of tails based on specific protein–protein or protein–phospholipid (Deford- may affect correct cleavage of zona proteins and precise cleavage Watts et al., 2009) interactions will be of considerable interest. may determine the ability of zona proteins to assembly and ZP2 and ZP3 have different cytoplasmic tails, albeit with similar participate in the zona pellucida. basic charges. When their tails are genetically switched to be the Sheddases or secretases release ectodomains from membrane- same, the ZP2 and ZP3 ectodomains were not incorporated into the spanning domains in a variety of proteins (Hooper et al., 1997). extracellular matrix surrounding growing oocytes. This is consistent Particularly well studied are the selectins, three members of the with ZP2 and ZP3 tails being recognized at the plasma membrane CAM (cell adhesion molecules) family identified in endothelial to ensure correct stoichiometry of the two zona proteins for cells (E-selectin), platelets (P-selectin) and leukocytes (L-selectin) incorporation into the zona pellucida. We note that although the (Gonzalez-Amaro and Sanchez-Madrid, 1999). The extracellular zona matrix is generally thought to be formed from heterodimers domains of the three proteins have similar structural features, but of ZP2 and ZP3, a role for homodimers of either protein has not the divergence of their cytoplasmic tails suggests differences in been excluded. To validate these models it will be important to regulation. The 17 residue cytoplasmic tail of L-selectin regulates biochemically identify and genetically confirm the molecular basis proteolytic cleavage, microvillar positioning and the of zona protein processing at the plasma membrane. Understanding tethering/rolling behavior of leucocytes. The tail interacts with at of these molecular details might provide insight into formation of least three proteins, including CaM (calmodulin), -actinin, and other matrices formed by proteins containing ‘zona’ domains the members of the ERM (erzin–radixin–moesin) family of (Jovine et al., 2005), including the tectorin membrane of the inner cytoskeletal proteins (Ivetic and Ridley, 2004; Killock et al., 2009). ear (Cosgrove and Grotton, 2001), as well as other extracellular CaM negatively regulates shedding by interacting with the L- polymers (Aszodi et al., 2006; Larsen et al., 2006). selectin tail and inducing a conformational changes in the extracellular domain that renders the cleavage site resistant to Materials and Methods Construction of expression plasmids proteolysis (Kahn et al., 1998; Diaz-Rodriguez et al., 2000). The C-terminal domains of ZP2 and ZP3 are well conserved among mammals Conversely, when CaM dissociates from the tail, the conformational (supplementary material Fig. S1). To insert Venus at the N-terminus of ZP2, the 5Ј change in the extracellular domain promoted proteolytic cleavage region (nucleotides 21–150) of ZP2, including the signal sequence (aa 1–34), was by a sheddase. amplified by PCR with two primers (supplementary material Table S1), each containing either an NheI or an AgeI site (ZP2IF, ZP2IR). cDNA encoding full- To further assay the requirement for a cytoplasmic tail in length Venus fluorescent protein (aa 1–239) (gift from Eneko Urizar, Center for formation of the zona pellucida, we expressed -tectorin, a GPI- Molecular Recognition, Columbia University College of Physicians and Surgeons, 948 Journal of Cell Science 124 (6)

New York, NY) was amplified by PCR with two primers, each containing an AgeI Junction fragments and PCR products of all plasmids were verified by DNA or an EcoRI recognition site (VenusF, VenusR). The resultant PCR products were sequencing and the presence of recombinant protein was confirmed by immunoblot assembled and subcloned into pcDNA3.1(+) (Invitrogen, Carlsbad, CA), previously after expression in heterologous cells. Expression plasmids were purified with the digested with NheI and EcoRI. To construct ZP2Venus(N), the N-terminal fragment GenEluted Plasmid Kit (Sigma). 1–156 of Venus, Venus(N), was amplified by PCR with a 5Ј primer (VenusF) containing an AgeI recognition site and a 3Ј primer (NVenIR) containing a sequence Expression in heterologous cells coding for a five amino acid linker (GGGGS) and an EcoRI site. The Venus(N) CHO-K1 cells (American Type Culture Collection, Manassas, VA) were grown fragment was subcloned in-frame with the 5Ј region of ZP2 in the pcDNA vector. (37°C, 5% CO2) for 24 hours to 50–70% confluence in F-12 medium supplemented The remainder of ZP2 (bp 151–2201) was amplified by PCR product containing with 10% fetal bovine serum and 100 U/ml penicillin-streptomycin (Gibco- EcoRI and PciI recognition sites (ZP2IIF, ZP2IIR) and the rest of ZP2 (bp 413– Invitrogen). Transient transfections were performed with FuGene HD (Roche Applied 2169) was isolated from ZP2 cDNA after digestion with PciI and EcoRI. These two Science, Indianapolis, IN) in accordance with the manufacturer’s protocol. For each products were assembled and cloned into the EcoRI recognition site of pcDNA. The transfection, 6 l FuGene HD transfection reagent was added to 100 l Opti-MEM resultant EcoRI fragments were isolated and subcloned into the EcoRI site reduced-serum medium (Gibco-Invitrogen) predissolved with 2 g template plasmid downstream and in-frame with full length ZP2 to complete the ZP2Venus and and incubated for 15 minutes at room temperature. The complex was diluted with 2 ZP2Venus(N) plasmids. ml Opti-MEM and overlaid on the growing cells. Transiently transfected cells were To insert Venus and Venus(N) at the C-terminal position of ZP2, the 3Ј region (bp harvested at 24 hours and stable clones were selected after culture for an additional 1921–2201), including the transmembrane domain (aa 684–703) and cytoplasmic 1–2 weeks in the presence of Geneticin (200 g/ml) and Zeocin (350 g/ml). tail (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) Photomicroscopy fragments were amplified by PCR with 5Ј primers (VenusF, NVenIIF) containing Transfected cells expressing fluorescent-tagged proteins were grown on coverslips EcoRI, and 3Ј primers (VenusR, NVenIIR) containing the AgeI site. The resultant for 48 hours after which pre-warmed (37°C) medium (Opti-MEM) containing 20 PCR products were assembled and subcloned into EcoRI and XhoI digested pcDNA M ER-Tracker-Blue-White DPX (Molecular Probes–Invitrogen) was added. The to generate a fusion protein with the C-terminus of ZP2 in-frame and downstream cells were incubated for an additional 30 minutes, fixed with 3% paraformaldehyde of Venus and Venus(N) fragments. The remainder of ZP2 (bp 21–1920) was amplified and imaged at room temperature with an Axioplan 2 fluorescence microscope (Carl by PCR using primers with NheI and EcoRI recognition sites (ZP2IF, ZP2IIIR). The Zeiss, Thornwood, NY) equipped with C-Apochromat 63ϫ/1.2W Corr objective, PCR product was isolated after digestion with NheI-EcoRI and subcloned upstream ApoTome technology, CCD camera and AxioVision 2 software. Venus fluorochrome and in-frame with Venus and Venus(N) fragments. was excited with BP450–490 nm filter and emission detected with BP515–565 nm For ZP3Cherry, pmCherry was cloned by PCR from pmCherry-N1 vector (Clontech, filter; Cherry and Alexa Fluor 568 fluorochromes were excited with BP534–558 nm Mountain View, CA) with two primers (CherryF, CherryR) containing AgeI and filter and was detected through a LP590 nm filter; Alexa Fluor 633 fluorochrome BglII sites, respectively. The PCR product was subcloned into pSEGFP-MoZP3 was excited with HQ590–650 nm filter and emission was detected through a HQ667– (Zhao et al., 2002), replacing EGFP, but remaining in-frame and downstream of the 738 nm filter; and Dapoxyl (DPX) fluorochrome was excited with a G365 and signal sequence of ZP3 and upstream of the remainder of ZP3. For ZP3Venus(C), the emission detected through a BP420–470 nm filter. Venus(C), C-terminus of the Venus fragment (157–239) was amplified by PCR with For BiFC, cells were incubated an additional 2 hours at 30°C to allow maturation a 5Ј primer (CVenF) containing an AgeI recognition site and 3Ј primer (CVenR) of the Venus fluorophore (Hu et al., 2002). Cells were then fixed (3% containing a sequence encoding a five amino acid linker (GGGGS) and a BglII site. paraformaldehyde, 15 minutes), washed in PBS (3ϫ), permeabilized (PBS and The Venus(C) fragment was subcloned into pSEGFP-MoZP3. cDNA encoding ZP3 Triton X-100, 5 minutes), blocked (PBS with BSA, 1 hour) and incubated with rat fused in-frame to pmCherry and the Venus(C) fragments was isolated from the monoclonal antibodies to ZP2 (1:200) and mouse antibodies against (1:250)(Roche original vector after digestion with NheI and XhoI and subcloned into Applied Science), which binds to the C-terminus of Venus (Nyfeler et al., 2005) pcDNA3.1/Zeo(+) vector (Invitrogen). contained in ZP3. Antibody binding was detected with either Alexa-Fluor-633- For ZP3Cherry(ZP2-tail) and ZP3Venus(C) (ZP2-tail), the 5Ј regions of ZP3Cherry and conjugated goat anti-rat antibody (1:100) or Alexa-Fluor-568-conjugated goat anti- ZP3Venus(C) (1938 and 1497 bp, respectively) were amplified by PCR with two mouse (1:100) secondary antibodies (Invitrogen). primers (ZP3ctZP2F, ZP3ctZP2R) each containing either an NheI or an EcoRI Confocal images from growing oocytes were obtained at room temperature with recognition site. The C-terminus of ZP2 (bp 2017–2169) was amplified by PCR an LSM confocal microscope (Carl Zeiss) equipped with differential contrast optics using two primers (ZP2ctF, ZP2ctR), containing EcoRI and XhoI sites. The PCR using a C-Apochromat 63ϫ/1.2W Corr objective and scan zoom of 1.7. The Venus

Journal of Cell Science products were subcloned into pcDNA after digestion with NheI and XhoI sites to fluorochrome was excited with a 488 nm argon laser and emission detected through establish cDNA encoding a protein fusion in which the C-terminus of ZP2 was in- a BP500–550 nm filter and Cherry fluorochrome was excited with 561 nm DPSS frame and downstream of ZP3. laser and detected through a BP575–615 nm filter. For ZP2Venus(ZP3 tail), the 5Ј region of ZP2Venus (2776 bp) was amplified from the above cDNA by PCR using the primers ZP2ctZP3F and ZP2ctZP3R containing NheI Immunoblots and HindIII sites. The C-terminus of ZP3 (bp 1167–1317) was amplified by PCR Supernatants and cells were recovered 48 hours after transfection, after which cells using two primers (ZP3ctF, ZP3ctR) containing HindIII and XhoI sites. The PCR were homogenized with M-PER Mammalian Protein Extraction Reagent (Pierce products were subcloned in pcDNA vector digested with NheI and XhoI generating Biotechnology, Rockford, IL) supplemented with Complete Protease Inhibitor a cDNA encoding a fusion protein with the C-terminus of ZP3 in-frame and Cocktail (Roche Applied Science) according to the manufacturer’s instructions. downstream of the ZP2Venus protein. Following centrifugation (16,000 g, 4°C), cell supernatants and lysates were separated Truncated proteins were generated by inserting a stop codon using specific primers by SDS-PAGE, transferred to PVDF membranes which were probed with monoclonal (supplementary material Table S2) and QuikChange site-directed mutagenesis antibodies to ZP2 (East and Dean, 1984), ZP3 (East et al., 1985) or actin (Santa Cruz (Stratagene, Garden Grove, CA) following the manufacturer’s instructions. Briefly, Biotechnology, Santa Cruz, CA) and visualized by chemiluminescence (Rankin et for ZP2-⌬TM, Asp (673) from ZP2Venus or ZP2Venus(N), was replaced with a stop al., 2003). codon using ZP2⌬TMF and ZP2⌬TMR primers. For the ZP2-⌬Tail, Tyr (704) from ZP2Venus or ZP2Venus(N), was replaced with a stop codon using ZP2⌬CTF and Gel filtration chromatography ZP2⌬CTR primers. For ZP3-⌬TM, Trp (380) from ZP3Cherry and ZP3Venus(C) was Stably transfected cells co-expressing ZP2Venus and ZP3Cherry were grown in 75 cm2 replaced with a stop codon using ZP3⌬TMF and ZP3⌬TMF primers. For ZP3-⌬Tail, tissue culture flasks (Corning Life Sciences, Lowell, MA) to 80% confluence and Val (409) was replaced with a stop codon using ZP3⌬CTF and ZP3⌬CTR primers. serum starved for 48 hours. Cell supernatants were dialyzed and concentrated against For the -tectorinVenus expression plasmid, the 5Ј region (bp 11–181) of -tectorin, 50 mM Tris-HCl, 0.1 M NaCl, 10% glycerol, pH 7.5 before FPLC chromatography including the signal sequence (aa 1–17), was amplified by PCR with two primers on a Superose 6 10/300 GL column (GE Healthcare Life Sciences) in the same (BtecIF, BTecIR), each containing either an NheI or an AgeI recognition site. cDNA buffer. Using a flow rate of 0.5 ml/minute, the protein elution pattern was monitored encoding full-length Venus fluorescent protein (aa 1–239) was cloned as described (A280) and collected fractions (0.5 ml) were precipitated with 20% trichloroacetic above. The resultant PCR products were assembled and subcloned into pcDNA3.1(+) acid. Following cold acetone washes (1.0 ml, 1ϫ), the precipitated samples were digested with NheI and EcoRI. The remainder of -tectorin (bp 182–1120) was analyzed by immunoblot using monoclonal antibodies against ZP2 and ZP3. The amplified by PCR product (151–413) with primers (BTecIIF, BTecIIR) containing data (average of three experiments) were quantified by on a LAS-3000 (Fujifilm EcoRI and XhoI recognition sites. pcDNA was digested with EcoRI and XhoI and Medical Systems, Stamford, CN). Protein standards from Gel Filtration Calibration the products were cloned in-frame and downstream of the Venus protein. For - Kits (GE Healthcare) run in the same buffer were used to calibrate the column. tectorinVenus-(ZP3 tail) the 5Ј region of -tectorinVenus (encoding 1815 aa) was amplified from the above cDNA by PCR using the primers BTecIIF and BTecIIR BiFC assay in CHO cells containing NheI and HindIII sites. The C-terminus of ZP3 (bp 1167–1317) was 48 hours after transfection, medium was recovered and centrifuged to remove amplified by PCR using two primers containing HindIII and XhoI sites (ZP3ctF, detritus. Cells were washed with PBS, harvested by scraping and resuspended in 1 ZP3ctR). The PCR products were subcloned in pcDNA digested with NheI and XhoI ml PBS. 20 l aliquots were counted on a Cellometer Auto T4 (Nexcelom Bioscience, to generate a cDNA encoding fusion protein in which the C-terminus of ZP3 was in- Lawrence, MA) and all samples were normalized to 5ϫ105 cells. After centrifugation frame and downstream of -tectorinVenus. and resuspension in 300 l PBS, the cells and media were transferred to 96-well, Cytoplasmic tails of zona proteins 949

clear-bottom, black microtiter plates (Nunc-Thermo Fischer Scientific). Fluorescence Deford-Watts, L. M., Tassin, T. C., Becker, A. M., Medeiros, J. J., Albanesi, J. P., was determined on a Spectramax GEMINI EM (Molecular Devices, Sunnyvale, CA) Love, P. E., Wulfing, C. and van Oers, N. S. (2009). The cytoplasmic tail of the T using excitation and emission wavelengths of 470 and 535 nm, respectively. The C- cell receptor CD3 epsilon subunit contains a phospholipid-binding motif that regulates terminal fragment of Venus was cloned downstream of the ZP3 zona domain (see T cell functions. J. Immunol. 183, 1055-1064. above). The N-terminal fragment of Venus was cloned downstream of the ZP2 signal Diaz-Rodriguez, E., Esparis-Ogando, A., Montero, J. C., Yuste, L. and Pandiella, A. peptide and of the ZP2 zona domain; fluorescence was observed in the BiFC assay (2000). Stimulation of cleavage of membrane proteins by calmodulin inhibitors. Biochem. with the former, but not the latter. Mock-transfected cells and medium were used as J. 346, 359-367. negative controls. Data from three independent experiments were averaged and Dominguez, M., Dejgaard, K., Fullekrug, J., Dahan, S., Fazel, A., Paccaud, J. P., s.e.m. calculated. Thomas, D. Y., Bergeron, J. J. and Nilsson, T. (1998). gp25L/emp24/p24 protein family members of the cis-Golgi network bind both COP I and II coatomer. J. Cell Biol. Immunoprecipitation 140, 751-765. East, I. J. and Dean, J. (1984). Monoclonal antibodies as probes of the distribution of For immunoprecipitation of ZP2Venus and ZP3Cherry normal and mutated proteins, ZP-2, the major sulfated glycoprotein of the murine zona pellucida. J. Cell Biol. 98, monoclonal antibody specific to GFP (Roche Applied Science) was incubated with ® 795-800. magnetic Dynabeads Protein G (Invitrogen) according to the manufacturer’s East, I. J., Gulyas, B. J. and Dean, J. (1985). Monoclonal antibodies to the murine zona instructions. The complex was incubated with supernatant and lysate from transfected pellucida protein with sperm receptor activity: Effects on fertilization and early cells and analyzed by immunoblot. development. Dev. Biol. 109, 268-273. Easton, R. L., Patankar, M. S., Lattanzio, F. A., Leaven, T. H., Morris, H. R., Clark, Microinjection of isolated oocytes G. F. and Dell, A. (2000). Structural analysis of murine zona pellucida glycans. Oocytes isolated from 11- to 13-day-old mouse ovaries (Millar et al., 1991) were Evidence for the expression of core 2-type O-glycans and the Sd(a) antigen. J. Biol. incubated in M2 medium (Chemicon-Millipore, Billerica, MA) supplemented with Chem. 275, 7731-7742. IBMX (3-isobutyl-1-methylxanthine) at 37°C before microinjection. 10 pl plasmid El Mestrah, M., Castle, P. E., Borossa, G. and Kan, F. W. (2002). Subcellular distribution DNA (50 ng/ml) was injected into the nucleus and surviving oocytes were cultured of ZP1, ZP2, and ZP3 glycoproteins during folliculogenesis and demonstration of their (37°C, 5% CO2) for 48 hours in KSOM medium (Millipore) containing IBMX. The topographical disposition within the zona matrix of mouse ovarian oocytes. Biol. oocytes were then separated into two groups. One group was fixed with 2% Reprod. 66, 866-876. paraformaldehyde for 1 hour at room temperature. The other group was transferred Epifano, O., Liang, L.-F. and Dean, J. (1995a). Mouse Zp1 encodes a zona pellucida into 20 mM Tris-HCl, pH 7.4, containing 1% NP-40 and 0.5 M NaCl and freeze- protein homologous to egg envelope proteins in mammals and fish. J. Biol. Chem. 270, thawed ten times on ethanol in dry ice to isolate zona ‘ghosts’ (Shimizu et al., 1983; 27254-27258. Zhao et al., 2003). To obtain zona-free oocytes after nuclear microinjection, the zona Epifano, O., Liang, L.-F., Familari, M., Moos, M. C., Jr and Dean, J. (1995b). matrix was removed from growing oocytes by brief exposure to acidified Tyrode’s Coordinate expression of the three zona pellucida during mouse oogenesis. medium (Hogan et al., 1994) and fixed as above after 48 hours of incubation. Development 121, 1947-1956. Oocytes co-injected with BiFC fragments were incubated for 48 hours, then Gahlay, G., Gauthier, L., Baibakov, B., Epifano, O. and Dean, J. (2010). Gamete placed at 30°C for 2 hours and fixed. Individual plasmids carrying the Venus recognition in mice depends on the cleavage status of an egg’s zona pellucida protein. Science 329, 216-219. fragments were also microinjected as a negative control of the complementation. Gonzalez-Amaro, R. and Sanchez-Madrid, F. (1999). Cell adhesion molecules: selectins The fixed oocytes and treated zona ‘ghosts’ were washed three times with PBS and and integrins. Crit. Rev. Immunol. 19, 389-429. placed on a slide chambered (20 l cavity) with Gene Frame (Advanced Greve, J. M., Salzmann, G. S., Roller, R. J. and Wassarman, P. M. (1982). Biosynthesis Biotechnologies, Leatherhead, UK). Images were obtained by confocal microscopy. of the major zona pellucida glycoprotein secreted by oocytes during mammalian oogenesis. Cell 31, 749-759. For their help and advice, we thank Lyn Gauthier (microinjection of Hodson, C. A., Ambrogi, I. G., Scott, R. O., Mohler, P. J. and Milgram, S. L. (2006). oocytes) and Boris Baibakov (confocal microscopy). This research Polarized apical sorting of guanylyl cyclase C is specified by a cytosolic signal. Traffic 7, 456-464. was supported by the Intramural Research Program of the National Hogan, B., Beddington, R., Costantini, F. and Lacy, E. (1994). 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