In eukaryotic organisms, proteins which are to be exported to the extracellular space, inserted into the plasma membrane, or incorporated into lysosomes, pass through an ordered series of membrane-bounded organelles along the route to their final destinations. Based on in vitro experiments using a reconstituted heterologous system, Walter and Blobel have postulated that targeting to the first of these, the endoplasmic reticulum (ER), is mediated by a ribonucleoprotein, designated Signal Recognition Particle (SRP), which recognizes a sequence of hydrophobic amino acids carried by proteins destined for export from the cytoplasm via this pathway. SRP is composed of six distinct polypeptides and one molecule of 7SL RNA, all of which are required for activity in vitro. I propose to examine the mechanism by which SRP recognizes nascent pre-secretory proteins on ribosomes by first mutationally altering its structure and then determining whether and how this perturbs function in vivo. The yeast Saccharomyces cerevisiae has been chosen for these studies because of the ease with which it can be manipulated genetically. The strategy will be to clone the gene encoding yeast 7SL RNA, mutagenize it in vitro, and test the consequences in cells transformed with altered copies. The first critical question will be whether the function of 7SL RNA is essential for viability in yeast, i.e. is a null allele of the gene lethal to the cell? Subsequently, more subtle lesions will be generated in order to dissect the function of the 7SL molecule. Once point mutations in this RNA have been isolated, it will be possible to identify other components in the cell which interact with this molecule by selecting for second-site suppressors of the secretory defects. Correct protein compartmentalization is of critical importance to cells; defects in secretion produce pathological states in humans, e.g. I-cell disease and Alpha-1 anti-trypsin deficiency. Since the secretory pathway appears to be highly conserved through evolution, the results of my proposed experiments should provide valuable insight into the initial steps of this process in higher eukaryotes, where a genetic approach is not feasible.
| Brennwald, P; Wise, J A (1994) A homologous cell-free system for studying protein translocation across the endoplasmic reticulum membrane in fission yeast. Yeast 10:159-72 |
| Liao, X B; Brennwald, P; Wise, J A (1989) Genetic analysis of Schizosaccharomyces pombe 7SL RNA: a structural motif that includes a conserved tetranucleotide loop is important for function. Proc Natl Acad Sci U S A 86:4137-41 |
| Brennwald, P; Liao, X; Holm, K et al. (1988) Identification of an essential Schizosaccharomyces pombe RNA homologous to the 7SL component of signal recognition particle. Mol Cell Biol 8:1580-90 |
