Targeted protein degradation through the ubiquitin/26S proteasome pathway represents a fundamental strategy for eukaryotic cellular regulation in which proteins are marked for degradation by assembly of specific polyubiquitin degradation signals on lysine and a-amino groups that are recognized by the 26S proteasome. A hierarchy of parallel ubiquitin targeting pathways organized within a common enzymatic mechanism has evolved to meet the conflicting demands of global specificity within the context of precise spatial and temporal regulation. Aberrant targeting by these pathways explicates a growing list of developmental and medical abnormalities related to cell transformation, tumorigenesis, viral infection, hypertension, and birth defects. Progress during the current funding period has identified sequence motifs and features contributing to the protein interactions that define selected substrate specificity, recognition of cognate components of individual targeting pathways, and the mechanism for assembling the polyubiquitin degradation signals. Proposed studies to extend and exploit these findings during the next funding period comprise four Specific Aims. Specific Aim 1 will use point mutagenesis and kinetic analysis to test specific predictions regarding putative active site residues of ubiquitin activating enzyme (El), based on structure-sequence modeling of the related MoeB-MoaD heterodimer of molybdopterin synthase. Specific Aim 2 will use kinetic and equilibrium studies rigorously to test predictions of the principal investigator's recent V(max) model for N-end rule specificity by using a defined dihydrofolate reductase model substrate whose amino terminus can be genetically manipulated. Specific Aim 3 will use kinetic methods to examine the mechanism of Mdm2- and MdmX-catalyzed autoubiquitination and p53 conjugation, the linkage specificity of the non-canonical polyubiquitin degradation signals formed by these ligases, and the validity of extrapolating the principal investigator's recent two-site model for polyubiquitin chain formation to a general functional context for Ring H2 ligases. Specific Aim 4 will examine the role of the mammalian-specific ubiquitin conjugating enzyme E2(epf) in the targeted degradation of aCPI/hnRNP-EI/PCBP1, the 3'-UTR polyC(U) mRNA binding protein responsible for developmentally-programmed translational silencing. Overall, the proposed studies seek to apply conventional biochemical and genetic methods to the detailed examination of key processes within ubiquitin-dependent 26S proteasome targeting at the molecular level.