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¹ Graduate School of Science and Technology,
² Research Center for Environmental Genomics, Kobe University, Nada-ku, Kobe 657-8501, Japan

A single-transmembrane protein uroplakin III (UPIII) and its tetraspanin binding-partner uroplakin Ib (UPIb) are members of the UP proteins that were originally identified in mammalian urothelium. In Xenopus laevis eggs, these proteins: xUPIII and xUPIb, are components of the cholesterol-enriched membrane microdomains or "rafts" and involved in the sperm–egg membrane interaction and subsequent egg activation signaling via Src tyrosine kinase at fertilization. Here, we investigate whether the xUPIII-xUPIb complex is in close proximity to CD9, a tetraspanin that has been implicated in the sperm–egg fusion in the mouse and GM1, a ganglioside typically enriched in egg rafts. Preparation of the egg membrane microdomains using different non-ionic detergents (Brij 98 and Triton X-100), chemical cross-linking, co-immunoprecipitation, in vitro kinase assay and in vitro fertilization experiments demonstrated that GM1, but not CD9, is in association with the xUPIII-xUPIb complex and contributes to the sperm-dependent egg activation. Transfection experiments using HEK293 cells demonstrated that xUPIII and xUPIb localized efficiently to the cholesterol-dependent membrane microdomains when they were co-expressed, whereas co-expression of xUPIII and CD9, instead of xUPIb, did not show this effect. Furthermore, xUPIII and xUPIb were shown to suppress kinase activity of the wild type, but not a constitutively active form of, Xenopus Src protein co-expressed in HEK293 cells. These results provide novel insight into the molecular architecture of the egg membrane microdomains containing xUPIII, xUPIb and Src, which may contribute to the understanding of sperm–egg interaction and signaling during Xenopus fertilization.

^aContributed equally to this work.

^bPresent address: Department of Applied Chemistry, School of Science, Northwestern Polytechnical University, Xi’an 710072, People's Republic of China

^cPresent address: Department of Reproductive Biology and Pathology, National Research Institute for Child Health and Development, Setagaya-ku, Tokyo 157-8535, Japan

^dPresent address: Division of Diabetes, Digestive, and Kidney Disease, Kobe University School of Medicine, Chuo-ku, Kobe 650-0017, Japan

^* Correspondence: E-mail: kksato{at}kobe-u.ac.jp