GENETIC AND BIOCHEMICAL-ANALYSIS OF THE FISSION YEAST RIBONUCLEOPROTEIN PARTICLE CONTAINING A HOMOLOG OF SRP54P

Mammalian signal recognition particle (SRP), a complex of six polypept ides and one 7SL RNA molecule, is required for targeting nascent prese cretory proteins to the endoplasmic reticulum (ER). Earlier work ident ified a Schizosaccharomyces pombe homolog of human SRP RNA and showed that it is a component of a particle similar in size and biochemical p roperties to mammalian SRP. The recent cloning of the gene encoding a fission yeast protein homologous to Srp54p has made possible further c haracterization of the subunit structure, subcellular distribution, an d assembly of fission yeast SRP. S.pombe SRP RNA and Srp54p co-sedimen t on a sucrose velocity gradient and coimmunoprecipitate, indicating t hat they reside in the same complex. In vitro assays demonstrate that fission yeast Srp54p binds under stringent conditions to E.coli SRP RN A, which consists essentially of domain IV, but not to the full-length cognate RNA nor to an RNA in which domain III has been deleted in an effort to mirror the structure of bacterial homologs. Moreover, the as sociation of S.pombe Srp54p with SRP RNA in vivo is disrupted by condi tional mutations not only in domain IV, which contains its binding sit e, but in domains I and III, suggesting that the particle may assemble cooperatively. The growth defects conferred by mutations throughout S RP RNA can be suppressed by overexpression of Srp54p, and the degree t o which growth is restored correlates inversely with the severity of t he reduction in protein binding. Conditional mutations in SRP RNA also reduce its sedimentation with the ribosome/membrane pellet during cel l fractionation. Finally, immunoprecipitation under native conditions of an SRP-enriched fraction from [S-35]-labeled fission yeast cells su ggests that five additional polypeptides are complexed with Srp54p; ea ch of these proteins is similar in size to a constituent of mammalian SRP, implying that the subunit structure of this ribonucleoprotein is conserved over vast evolutionary distances.