9506140 Ogrydziak Protein translocation across the endoplasmic reticulum (ER) membrane is a basic biological process in which signal recognition particle (SRP) has a critical role. Mammalian SRP consists of RNA (300 nucleotides) and six protein subunits (Srp9, 14, 19, 54, 68, 72p). Only recently has an in vivo role for SRP in protein translocation been demonstrated. The long term goals of this research are to understand early events in ER protein targeting, with an emphasis on the structures, functions, and interactions of SRP and its components. Specific objectives of this project are 1) to identify key components involved in the process and determine if they are essential for growth, and 2) to develop tools needed for in vivo studies of protein targeting and translocation. The studies will be done with the genetically tractable yeast Yarrowia lipolytica. It secretes high levels of an alkaline extracellular protease (AEP) which will be used as a reporter molecule. Several interrelated lines of research will be conducted. 1) SRP will be purified, sizes of the subunits determined, and N- terminal amino acid sequence of SRP14 homologue gene product determined. The SRP receptor alpha-subunit gene will be cloned, sequenced, and disrupted. In Saccharomyces cerevisiae all SRP components are important but not essential for growth. Unexpectedly, at least one of Y. lipolytica's two functional SRP RNA genes is essential for growth but SRP54 is not. So determining whether or not the SRP receptor alpha subunit gene is essential will be of interest. 2) Because sensititity to loss of SRP components seems to be protein-specific in S cerevisiae, studies on Y. lipolytica Kar2p as an additional reporter molecule will be completed. Several laboratories, including our own, have had difficulties isolating temperature (heat) sensitive mutations in genes coding for SRP polypeptides. Therefore, a system for protein depletion will be developed because it has been found in S. cerevisiae that depletion of SRP components results in more severe translocation defects than does deletion. %%% Of the many fungi upon which people rely for food and drink, yeast is arguably the most important. This project focuses on a yeast species, Yarrowia lipolytica, which is a "high secretor"; its rate of secretion of certain proteins is greater than for other yeast species, such as Saccharomyces cerevisiae. To explore this interesting difference, the initial events involved in secretion are studied. These events include the recognition of the secreted proteins so that they can enter the secretory system efficiently. This is accomplished by a signal recognition particle, SRP, that recognizes a signal sequence on the protein to be secreted. Part of the project is to biochemically and genetically dissect the SRP and to develop more probes to test its functionality. The SRP "sits" on the membrane system, the endoplasmic reticulum, which serves as the entry point for secreted proteins. Where the SRP "sits" is determined by a receptor which recognizes the SRP. This project will test if one of the subunits of the receptor is essential for growth. It will also genetically deplete other components of the SRP to determine if their role in protein delivery is ancillary or direct. The work has broad biotechnological application through providing a system whereby commercial proteins may be more rapidly secreted and through shedding light on the secretion process itself. ***
