A comprehensive analysis of yeast Elongator phosphorylation and its functional consequences

The addition and removal of phosphate groups at specific points within protein molecules ('protein phosphorylation') is a universal means by which protein function can be regulated. Phosphate groups are added by enzymes termed protein kinases and removed by opposing activities termed protein phosphatases. We are interested in a complex of proteins termed 'Elongator' that is found in all higher cells including those of plants and animals such as ourselves. We have been using yeast cells as a model system for these studies because they provide an experimental system in which both genetic and biochemical approaches to biological questions can be combined in a particularly powerful way. Elongator has been proposed to play a number of cellular roles but most recently it has been discovered that it is important for the correct functioning of transfer RNAs, special molecules that are required for cells to make more protein molecules using their genetic templates, and this is probablyElongator's primary function. We have found that one of the components of the Elongator complex appears to be regulated by phosphorylation, even though this might mean that Elongator is not fully active under all circumstances. Thus the cell's ability to synthesis specific proteins may be controlled through this mechanism. In our proposed work we will combine the expertise of our two laboratories to study both how and why Elongator may be regulated by phosphorylation. We will examine whether changes in Elongator activity lead to effects on the synthesis of specific proteins in the cell and identify both the protein kinases involved in phosphorylating Elongator and the specific sites in the component proteins of Elongator that are modified by these protein kinases. By defining individual sites of phosphorylation we can generate mutants that either cannot be phosphorylated at all or that mimic the presence of a phosphate group. We will use such mutants to build up a detailed picture of how specific phosphhorylation events affect Elongator function.

Elongator is a six-subunit protein complex that is highly conserved in eukaryotic cells and that has been ascribed a number of different cellular roles, although recent work has demonstrated that its principal if not only role is in the modification of uridine residues in tRNA molecules at the anticodon wobble position. Such modified uridines are found in a small subset of tRNAs and the modification is needed for optimal functioning of these tRNAs in translation. Through our work on the K. lactis zymocin we have found that Elongator is required for zymocin inhibition of S. cerevisiae yeast cells, consistent with zymocin having a tRNA anticodon nuclease activity that cleaves certain tRNA molecules at their wobble uridine residues but which requires that they carry their Elongator-dependent modification. We have additionally discovered that mutations in other cellular components that affect zymocin sensitivity cause major changes in the phosphorylation state of Elp1p, the largest Elongator subunit. These data therefore argue both that Elongator is phosphorylated and that such phosphorylation affects its activity. In the proposed work, we seek to carry out a comprehensive analysis of how and why Elongator is subject to regulation by phosphorylation, focussing initially on Elp1p (which we know is phosphorylated) but extending our analysis to examine all six Elongator polypeptides. Key questions to be addressed are the nature of the kinase(s) that phosphorylate Elongator, identification of the functionally important phosphorylation sites, how phosphorylation at specific sites affects Elongator function, the roles of the regulatory components that we have already identified as important (Kti12p, Sit4p phosphatase, Hrr25p kinase) and the potential of Elongator regulation to exert effects by influencing the translation of specific gene products. In this way we anticipate being able to generate a detailed picture of how and why Elongator is regulated in this way.