IDENTIFICATION OF TESTICULAR PROTEINS ASSOCIATED WITH THE GERM CELL-SPECIMC PROTEIN, TEX101 : CELLUBREVIN IS INVOLVED IN TEX101-TRAFFICKING TO THE GERM CELL SURFACE DURING SPERMATOGENESIS IN MICE

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A complete understanding of the germ cell differentiation process requires the elucidation of the molecular functions and spatiotemporal expression of germ cell-specific molecules. Recently, we identified a cell-surface marker protein, designated TEX101, that is unique to male and female germ cells. On/off switching of TEX101 expression in germ cells is closely linked to the kinetics of gametogenesis. In the present study, we isolated testicular proteins by immunoprecipitation with anti-TEX101 antibody and identified the proteins using liquid chromatography/tandem mass spectrometry. Of three proteins identified (annexin 2, ly6k and cellubrevin), a biochemical association between TEX101 and cellubrevin was confirmed by immunoprecipitation-Western blotting experiments. Results of immunohistochemical studies using a cellubrevin-specific antibody indicated that the molecule is abundant on certain germ cells, such as spermatocytes and early-stage spermatids, whereas negligible amounts are found in Sertoli cells, spermatogonia, spermatozoa, and late-stage spermatids. Most of the intracellular cellubrevin appeared to be juxtaposed with intracellular TEX101, and membrane-associated cellubrevin appeared to be docked near TEX101-positive plasma membranes on the cytoplasmic side. This close association was never observed on the outer surface of the plasma membrane. From these results we conclude that cellubrevin-dependent membrane trafficking is involved in transport of de novo TEX101 to the surface of male germ cells. In addition, these findings suggest possible molecular functions for TEX101, and will thus help to increase our understanding of the fundamental mechanisms of spermatogenesis.