Networks of protein interactions in cells must be regulated and coordinated based on both internal signals and extracellular stimuli. An example of a complex molecular system that involves a series of coordinated protein interactions and macromolecular assembly events is the synthesis of the ribosome. Ribosome assembly requires the activity of as many as 150 trans-acting factors that interact with the pre-ribosome in different cellular compartments. Because ribosome assembly is coupled to other major cellular events such as DNA replication, proteasome biogenesis, and cell division, we need to understand ribosome synthesis in order to grasp a variety of regulatory mechanisms in cell. We propose experiments to identify and characterize protein-protein interactions in yeast using the recently developed Bimolecular Fluorescence Complementation (BiFC). This approach is based on reconstitution of fluorescent proteins split into two fragments that are attached to two potentially interacting partners. The fluorescent signal develops only if the two non-fluorescent fragments are brought together by the interaction of their associated proteins. This approach complements other protein-protein interaction techniques, and offers the advantages that microscopic analyses can be performed on living cells. In addition, this approach simultaneously allows visualization of sites in the cell where the interactions occur. An increasingly detailed picture of the ribosome assembly has emerged as a result of recent proteomics efforts and many of the intermediate complexes have been characterized. It has been difficult, however, to resolve the in vivo spatial and temporal protein interactions during ribosome assembly. The long-term goals of this study are to characterize these interactions. We start by identifying reliable binary protein interactions, and work our way up by using statistical and computational approaches to predict the order of assembly and the composition of larger macromolecular complexes. ? ? ?
