Extracellular matrix proteins are packaged into four groups of vesicles in the sea urchin oocyte for secretion at fertilization and early development. This proposal will study the origins of these proteins during oogenesis, and the mechanisms by which the proteins are trafficked to specific vesicles. Diadema antillarum will be used because oogenesis in this species is synchronous and on a lunar cycle. The localization and timing of synthesis of the proteins will be determined by immunofluorescence and radioimmunoprecipitation using existing monoclonal and polyclonal antibodies. The antibodies will also be used to screen expression libraries to obtain cDNA probes for the proteins' mRNAs (two such probes are already available). These probes will be used in situ hybridization experiments to determine whether the oocytes or somatic cells synthesize the mRNAs. The mechanism of protein trafficking will be studied by using the frog oocyte system, which efficiently translates exogenous mRNAs and secretes the new proteins if they bear an appropriate amino acid sequence. To test if sorting sequences are used to direct hyaline layer proteins to different vesicles, mRNAs will be injected into frog oocytes. Proteins of the same vesicle class should be packaged into the same vesicle if sorting sequences are important to localization. The goal is to learn how the egg packages these secretory proteins in distinct compartments.
