Cytoplasmic tails direct separate trafficking of ZP2 and ZP3 in CHO 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 a short cytoplasmic tail. Using cDNA expression vectors, full-length, truncated or modified forms of ZP2 and ZP3 were cloned in-frame with Venus or Cherry fluorescent proteins, respectively. (B) CHO cells were co-transfected with ZP2^Venus and ZP3^Cherry expression vectors encoding full-length (normal), truncated proteins lacking cytoplasmic tails (ΔTail), transmembrane domains (ΔTM) or ZP3 with a ZP2 cytoplasmic tail, ZP3–(ZP2 tail). Cells were fixed and imaged by fluorescence microscopy using ApoTome technology. Higher-magnification inserts provide images of vesicle-like structures when proteins that lack the cytoplasmic tail or share the same cytoplasmic tail colocalize. Merge includes ER-Tracker, blue. Scale bars: 10 μm. (C) Media and cell lysates treated without (left panel) or with (right panel) trypsin were assayed by immunoblot using monoclonal antibodies against ZP2 or ZP3. The reduction of signal after treatment of cell lysates with trypsin (red asterisks) indicates the presence of expressed protein on the plasma membrane. Note that the upper band is not detected in ΔTM proteins and there is no reduction in signal after treatment with trypsin. Data reflect representative images from experiments that were repeated three times.

ZP2 and ZP3 cytoplasmic tails prevent premature interactions 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 of the signal peptide of ZP2 and ZP3, respectively. Correct zona protein dimerization resulted in Venus complementation and production of a fluorescent signal. (B) Expression vectors encoding ZP2^Venus(N) that were full-length (Normal), truncated before the cytoplasmic tail (ΔTail) or truncated before the transmembrane domain (ΔTM). (C) ZP3^Venus(C) with an additional construct encoding ZP3 with a ZP2 cytoplasmic tail, ZP3^Venus(C)–(ZP2 tail). (D) After transfection, fixed cells were imaged by fluorescence microscopy using monoclonal antibodies against ZP2 and an antibody that binds to the C-terminal fragment of Venus in ZP3. The fluorescent signal of ZP2 (blue), ZP3 (red) and Venus complementation (green) were recorded separately and as a merged image. Scale bars: 10 μm. (E) Venus complementation (BiFC) was quantified 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 proteins lacking their transmembrane domains and cytoplasmic tails complemented prematurely in the cell and complementation was observed in the medium. ZP2 and ZP3, lacking only their cytoplasmic tails, also complemented within the cell, but complementation was not detected in the medium. Data are the mean ± s.e.m. of three independent experiments. ZP2 and ZP3 were detected by immunoblots of cell suspensions and media. Actin levels documented protein equivalence among samples. (F) Cell lysates and media were immunoprecipitated (IP) with mouse anti-GFP monoclonal antibody that recognizes ZP2^Venus, but not ZP3^Cherry. Resultant protein samples were separated by SDS-PAGE and analyzed by immunoblot using monoclonal antibodies against ZP2 and ZP3. Normal, full-length ZP2 and ZP3 did not interact within the cells but co-immunoprecipitated from the medium. When truncated to remove their cytoplasmic tails alone (ΔTail) or in conjunction with their transmembrane domains (ΔTM), ZP2 and ZP3 interactions were detected in both cell lysate and the medium. The upper band (asterisk), identified as the TM isoforms based on their absence in the ΔTM samples, were not well precipitated in cell lysates expressing proteins lacking their cytoplasmic tails suggesting premature release from the transmembrane domain. After secretion into the medium, ZP2 and ZP3 interactions were detected in the medium for all constructions. No signal was detected in control cells, transfected with only ZP3^Cherry. Photomicrographs and immunoblots are representative images from experiments that were repeated three times.

Distinct cytoplasmic tails are essential for incorporation of ZP2 and ZP3 into the zona pellucida. (A) Oocytes were co-microinjected with ZP2^Venus and ZP3^Cherry expression vectors encoding full-length (normal), truncated proteins lacking cytoplasmic tails (ΔTail), transmembrane domains (ΔTM), 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 ZP2^Venus (green) and ZP3^Cherry (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 μm.

Cytoplasmic tails prevent ZP2 and ZP3 interaction within growing oocytes. (A) Oocytes were co-microinjected with ZP2^Venus(N) and ZP3^Venus(C) expression vectors encoding full-length (normal), truncated proteins lacking cytoplasmic tails (ΔTail), transmembrane domains (ΔTM) or ZP3 with a ZP2 cytoplasmic tail, ZP3–(ZP2 tail). After 40 hours in culture, oocytes or zona ‘ghosts’ were imaged by confocal microscopy alone (left) or merged with DIC images (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 ZP2^Venus(N) and ZP3^Venus(C) expression vectors and the zona pellucida was removed by brief exposure to Tyrode's acidified media, before incubation and imaging by confocal for BiFC and DIC microscopy (upper panels). As controls, oocytes were injected with ZP2^Venus and ZP3^Cherry before removal of the zona pellucida, incubation and imaging (lower panels). Photomicrographs are representative images from experiments that were repeated three times with 10–20 oocytes.

β-Tectorin with the ZP3 cytoplasmic tail is incorporated into the zona pellucida. (A) β-Tectorin (329 aa) has a signal peptide, a ‘zona’ domain, a potential dibasic cleavage site followed by a GPI-anchor site and a hydrophobic tail that is removed in the mature protein. β-Tectorin^Venus cDNA was modified by replacing its C-terminus with that of ZP3 and cloned into the expression vector β-tectorin–(ZP3 tail). (B) Lysates from CHO cells transfected with normal (Tectorin) and modified (Tec-ZP3) β-tectorin^Venus were assayed by immunoblot using monoclonal antibody against GFP that recognizes Venus. (C) Oocytes were co-injected with β-tectorin^Venus and ZP3^Cherry before imaging oocytes and zona ‘ghosts’ by confocal microscopy. Scale bar: 20 μm. (D) C-terminus of β-tectorin^Venus was replaced by that of ZP3 containing its cytoplasmic tail. Photomicrographs are representative images from experiments that were repeated three times with 10–20 oocytes.

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. 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 the extracellular ectodomain of each. This cleavage alters the conformation of the two proteins permitting their oligomerization and incorporation into the zona pellucida