Key Players in 40s subunit export

While translation of mRNAs by the ribosome occurs in the cytoplasm, the biosynthesis of ribosomes takes place in the nucleus. The physical separation of the place of synthesis and the place of function of ribosomes requires efficient mechanisms for the export out of the nucleus. Export depends on the presence of a Ran-GTP/GDP gradient across the nuclear membrane. Ribosomal subunits are exported separately through the nuclear pore complexes. 60S subunit export is mediated by the export receptor Crm1, which in the presence of Ran-GTP binds to the NES (nuclear export sequence) of the export adaptor Nmd3, that itself interacts with the pre-60S subunits. Crm1 is responsible for translocation of the subunits, probably through interaction with nuclear pore proteins. In contrast to 60S subunit export, the factors responsible for the export of 40S subunits are not known. Analogously to 60S subunit export, 40S subunit export is also believed to rely on an export receptor, probably also Crm1, and an adaptor bound to the pre-40S subunits. The proposed project aims to identify the key players involved in export of the small ribosomal subunit. Using yeast as a model system, a combination of genetic and biochemical approaches will be employed to identify these export factors and confirm their roles in 40S subunit export. The potential function of Crm1 as an export receptor for small ribosomal subunits will be investigated by examination of the in vitro interaction of pre-40S subunits with Crm1 in the presence of Ran-GTP. Also, the binding of pre-40S subunits to FG-repeat containing nucleoporins will be tested in the presence and absence of Crm1 and Ran-GTP. One factor that is present on pre-40S particles, Ltv1, has already been identified as a NES-element containing protein and could therefore function as an export adaptor. This assumption will be further tested by investigation of an in vitro interaction between Ltv1 and Crm1. Furthermore, as LTV1 is non-essential, I will try to identify other proteins which might function as export adaptors, possibly in a redundant manner with LTV1. If there are several export adaptors with redundant functions, the combination of mutations in these factors should lead to a synthetically lethal phenotype. To identify such factors, I will perform synthetic lethality screens with LTV1. To find novel factors which might not have been identified on pre-40S subunits due to their substoichometric binding, conditional mutants will be used to block 40S export and the protein composition of the accumulating late nuclear particles will be characterized. The 40S export factors identified in these approaches will be characterized further with respect to their exact function in export.