9604669 Green Technical abstract: Signal peptidase complex (SPC) from the endoplasmic reticulum (ER) is a multisubunit enzyme functioning in the cleavage of N-terminal signal peptides and the degradation of some abnormal membrane proteins. As the bacterial counterpart to the SPC, leader peptidase, is a monomeric enzyme, the specific SPC subunits may have individual roles. To identify these roles, the PI and co-PI, Drs. Green and Fang, will test existing models and a new hypothesis for the presence of a multisubunit signal peptidase. They have cloned the genes encoding three subunits of the yeast SPC and have analyzed these subunits functionally, and they have evidence that two homologous subunits of the mammalian SPC are functionally distinct. The following three objectives are proposed for the next five years of study: 1. Genetically and biochemically examine the function of each subunit of the yeast SPC. The SPC of yeast is comprised of at least four subunits. The gene encoding one of these, Sec11p, has been cloned and sequenced. Recently, the genes encoding the other three subunits have been identified. Specific mutations will be constructed, and cell growth, signal peptidase activity, and protein degradation will be examined in mutant cells. The analysis of mutations in these genes will help to test two existing models: (i) that one or more subunits may be important for protein translocation across the ER membrane, and (ii) that different subunits may be required to cleave signal peptides from non-overlapping sets of precursors. The hypothesis that two subunits of ER signal peptidase perform the role played by leader peptidase will also be tested. 2. Use newly constructed yeast mutants to examine the function of mammalian SPC subunits. The canine SPC contains five subunits, two of which are homologous to Sec11p. Methods will be developed to express genes encodes these proteins in yeast, in order to determine whether the canine proteins function in the heterologous system , and whether one, or both, of the Sec11p homologues functionally replaces Sec11p in yeast. They will also determine whether the two mammalian Sec11p homologues are functionally equivalent, and whether yeast cells containing mammalian subunits of signal peptidase are better hosts for the expression and secretion of mammalian proteins than currently available systems. 3. Functionally characterize new suppressors of conditional defects in the yeast SPC. Five distinct multicopy suppressors of a sec11 mutant have been identified, including SPC1 encoding the smallest subunit of the SPC and SPC3 which encodes the largest subunit of the SPC. Other suppressors do not appear to encode characterized subunits. One of these suppressors encodes a 13 kD membrane protein with a similar membrane topology as Spc1. They will determine whether these suppressors are important for signal peptidase activity, and whether they physically and genetically interact with the SPC. Recently, a spc3-ts mutant was constructed by in vitro random mutagenesis. New suppressors of this conditional mutant will be isolated. The goal of this objective is to identify new proteins interacting with or functionally equivalent to subunits of the SPC. Lay language abstract: The signal peptidase complex (SPC) is a very important piece of the cellular secretion machinery. Proteins destined for secretion carry a "signal peptide" at their amino terminus, which is unrelated to the protein's ultimate function but which contains the targeting information for insertion of that amino terminus of the nascent protein through the membrane of the endoplasmic reticulum. This insertion is the key first step in the secretion pathway. After this insertion, as the protein continues to be synthesized, it continues to be threaded at the insertion site and thereby is translocated from the cytoplasmic side of the membrane (where it is being synthesized) to the interior, or lumenal, side, where further processing steps necessary for its secretion subsequently take place. The SPC is on the lumenal side of the ER membrane, and its role is to remove the signal sequence once it is inside the ER. Drs. Green and Fang have identified the genes encoding the polypeptide subunits that comprise the SPC in yeast, and have begun to define the function of each subunit. The proposed work will greatly expand our understanding of how the SPC functions in yeast and other cells. One of the potential benefits of the results might be the development, by genetic engineering, of improved strains of yeast that are more efficient at overproducing and secreting mammalian proteins of commercial interest. ***
