Regulated protein degradation by the ubiquitin system controls the concentrations of many regulatory proteins. The targets of the N-end rule pathway, one pathway of the Ub system, include proteins bearing destabilizing N-terminal residues. The functions of the N-end role pathway include fidelity of chromosome segregation. Perturbations of this pathway are therefore expected to influence the frequency of emergence of malignant phenotypes in the mammalian cell lineages. Among destabilizing residues in the N-end role, N-terminal Asp, Glu, and Cys are unique in that they are conjugated to Arg before the recognition of a substrate protein by the N-end rule pathway. The arginylation of N-terminal Asp, Glu, and Cys is mediated by ATE1-encoded Arg-tRNA protein transferases. The Varshavsky lab has demonstrated that mouse ATE1 -/- strains, lacking N-terminal arginylation, die as embryos, owing to defects in heart development and angiogenic remodeling. In addition, arginylation of N-terminal Cys was discovered to require its prior oxidation, suggesting that the arginylation branch of the N-end role may function as an oxygen sensor. I will carry out molecular genetic studies in the mouse, using techniques that allow conditional ablation of N-terminal arginylation in specific cell lineages, to dissect, in detail, the cardiovascular and other functions of this major branch of the N-end rule pathway. I will also identify physiological substrates of R-transferases and analyze chromosome stability in ATE1 -/- ceils.
