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Molecular and Cellular Biology, December 2003, p. 8982-8991, Vol. 23, No. 24
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.24.8982-8991.2003

Mutation of a Conserved Hydrophobic Patch Prevents Incorporation of ZP3 into the Zona Pellucida Surrounding Mouse Eggs

Ming Zhao,¹ Lyn Gold,¹ Heidi Dorward,¹ Li-fang Liang,¹ Tanya Hoodbhoy,¹ Emily Boja,² Henry M. Fales,³ and Jurrien Dean^1*

Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases,¹ Laboratory of Biophysical Chemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892²

Received 21 March 2003/ Returned for modification 17 June 2003/ Accepted 22 September 2003

Three glycoproteins (ZP1, ZP2, and ZP3) are synthesized in growing mouse oocytes and secreted to form an extracellular zona pellucida that mediates sperm binding and fertilization. Each has a signal peptide to direct it into a secretory pathway, a "zona" domain implicated in matrix polymerization and a transmembrane domain from which the ectodomain must be released. Using confocal microscopy and enhanced green fluorescent protein (EGFP), the intracellular trafficking of ZP3 was observed in growing mouse oocytes. Replacement of the zona domain with EGFP did not prevent secretion of ZP3, suggesting the presence of trafficking signals and a cleavage site in the carboxyl terminus. Analysis of linker-scanning mutations of a ZP3-EGFP fusion protein in transient assays and in transgenic mice identified an eight-amino-acid hydrophobic region required for secretion and incorporation into the zona pellucida. The hydrophobic patch is conserved among mouse zona proteins and lies between a potential proprotein convertase (furin) cleavage site and the transmembrane domain. The cleavage site that releases the ectodomain from the transmembrane domain was defined by mass spectrometry of native zonae pellucidae and lies N-terminal to a proprotein convertase site that is distinct from the hydrophobic patch.

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* Corresponding author. Mailing address: Laboratory of Cellular and Developmental Biology, NIDDK, Bldg. 50, Rm. 3134, National Institutes of Health, Bethesda, MD 20892-8028. Phone: (301) 496-2738. Fax: (301) 496-5239. E-mail: jurrien{at}helix.nih.gov.

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Molecular and Cellular Biology, December 2003, p. 8982-8991, Vol. 23, No. 24
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.24.8982-8991.2003

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