.1. Human Sperm Membrane Protein Ywk-ii Function Using Yeast Two-hybrid System To Find Tsh3p13 Hsd-3 Encoded Protein Conjugates

The functional study of sperm-related proteins plays a key role in discovering the molecular mechanisms of spermatogenesis and fertilization. The functions of three proteins were studied by different ways in this dissertation.In a previous study, a novel membrane protein of human sperm, designated as YWK-II was isolated and characterized. Its open reading frame consists of 763 codons encoding a polypeptide characteristic of a single transmembrane protein established by hydropathicity analysis. Although the YWK-II gene has 70% homology to Alzheimer amyloid precursor protein (APP) within the transmembrane and cytoplasmic domains, the extracellular domain of YWK-II protein is distinctly different from that of APP and is shown to possess an anti-fertility activity. Our recent study revealed that YWK- II protein may act as a Go-coupled receptor on the sperm membrane since it cytoplasmic domain binds to Go, a GTP-binding protein. So identification of the ligand of the extracellular domain of YWK- II protein is essential to prove the hypothesis. The preliminary results showed that the extracellular domain of YWK- II protein was able to interact with the 303-376 amino acids segment of the rat Mullerian inhibiting substance (rMIS) in a yeast two-hybrid system.To confirm the interaction between YWK- II protein and MIS, and to reveal the physiological significance of the interaction, the segments of both YWK- II protein and rMIS that interacts in the yeast two-hybrid system were expressed in E coli. The 226 amino acids segment and the 63 amino acids active segment of YWK- II protein that bind MIS in the yeast two-hybrid system were expressed as fusion proteins, namely, His₆-tagged HSD226 and GST-HSD63. And the 303-376 amino acids segment of rMIS was expressed as His₆-tagged rMIS(303-376) and GST-rMIS(303-376), separately. The recombinant proteins were purified with affinity columns by the 6 histidines tag (His₆) or Glutathione-S-Transferase (GST) they were fused to.Several experiments were carried out to confirm the interaction between the YWK- II protein and MIS with the purified recombinant proteins. The in vitro binding experiment showed that the His₆-tagged HSD226 was able to interact with the GST-rMIS(303-376) bound to glutathione Sepharose 4B, which was consistent with the result in yeast two-hybrid assay. In pull-down assay, GST-HSD63 was found to bind specifically with...