Poster Sessions

Mol-6

Maria Jimenez-Movilla

M. Jimenez-Movilla, J. Dean

Intracellular Trafficking of ZP2 and ZP3 to Form the Mouse Zona Pellucida

The zona pellucida surrounding the egg and pre-implantation embryo is composed of three glycoproteins (ZP1, ZP2, ZP3) and is essential for normal fertilization and early development. The glycosylated zona pellucida proteins share a conserved ‘zona’ domain of ~280 amino acids with 8 conserved cysteine residues which plays a role in protein polymerization. Only ZP2 (713 AAs) and ZP3 (424 AAs) are required to form a zona matrix sufficiently robust to serve as binding site for sperm and provide protection of the embryo during preimplantation development. However, little is known about the interaction of these two glycoproteins during intracellular trafficking or where they assembly to form the zona pellucida. Here we adapt a fluorescent-based protein fragment complementation assay (BiFC) to detect ZP2-ZP3 interactions in the secretory pathway. BiFC is based on the formation of a fluorescent complex by two non-fluorescent fragments of Venus (an enhanced yellow fluorescent protein). One non-fluorescent fragment is fused to each of two zona proteins. The interaction of the zona proteins result in complementation and the intrinsic fluorescence of the bimolecular complex readily allows live visualization of the complex in mammalian cells. In the current study, the N-terminal fragment (AAs 1-156) of Venus was fused to ZP2 in three different positions: 1) after the signal peptide; 2) before the ‘zona’ domain; and 3) after the ‘zona’ domain near the carboxyl terminus of the mature protein. The C-terminal fragment of Venus (AAs 157-239) was then fused to the ZP3. In addition, intact Venus and mCherry (modified from dsRED) were inserted just after the signal peptide of ZP2 and ZP3, respectively. Expression plasmids encoding the ZP2-Venus and ZP3-Cherry fusion proteins were co-transfected into CHO cells and imaged by confocal microscopy. Initially, the two zona proteins co-localized in the endoplasmic reticulum, but appeared to traffick independently through the cell prior to again co-localizing on the plasma membrane. Each protein was subsequently secreted and could be detected in culture media by immunoblots using monoclonal antibodies to ZP2 and ZP3. Continuing BiFC analysis of co-expressed ZP2 (N-terminus of Venus) and ZP3 (C-terminus of Venus) in heterologous cells and growing oocytes should define the molecular orientation and cellular location of zona protein interactions required for the formation of the insoluble extracellular zona pellucida matrix.