Surface localization of the sea urchin egg receptor for sperm.
The sea urchin egg receptor for sperm is thought to be involved in species-specific sperm-egg interactions at the egg surface. Recent revisions in the deduced amino acid sequence of the cloned cDNAs indicate that the protein encoded does not possess the common structural hallmarks of a membrane protein. Thus, investigation of the localization and association of the protein with the egg surface is crucial. We describe and characterize a new monoclonal antibody raised against recombinant sperm receptor protein. This antibody, in conjunction with several polyclonal antibodies, was used to study the receptor protein in eggs. Immunoprecipitation studies indicated that the antibodies recognize the high Mr (ca. 350 K) sperm receptor protein which copurified with egg plasma membrane-vitelline layer complexes. The sperm receptor protein was solubilized only by detergents and not by treatments designed to solubilize peripherally associated or lipid-anchored membrane proteins, suggesting a tight association with the membrane fraction. Confocal immunofluorescence microscopy of live eggs indicated surface staining. Finally, lysylendoproteinase C treatment of live eggs resulted in a loss of the high Mr receptor protein epitopes, and the concomitant release of a 70-kDa proteolytic fragment, which correlated with a reduced ability of the eggs to be fertilized. Taken together, these data indicate that at least some fraction of the sperm receptor protein is present on the egg surface, a requisite locale for a sperm binding protein.
Giusti
A F
AF
Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara 93106-9610, USA.
Hoang
K M
KM
Foltz
K R
KR
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002462:Cell Membrane / Q000737:chemistry; D018076:DNA, Complementary; D010450:Endopeptidases; D000939:Epitopes / Q000032:analysis; D005260:Female; D008297:Male; D008970:Molecular Weight; D010063:Ovum / Q000737:chemistry*; D012333:RNA, Messenger / Q000032:analysis; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins; D012617:Sea Urchins; D013084:Sperm-Ovum Interactions; D014817:Vitelline Membrane / Q000737:chemistry
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D000939:Epitopes; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C035027:egg surface sperm receptor; D010450:Endopeptidases