PROJECT SUMMARY This application features a close collaboration between bioanalytical (Dr. Reilly, Indiana University) and bio- chemistry (Dr. Matouschek, University of Texas, Austin) research groups aimed at improving our understanding of the ubiquitin-proteasome protein degradation system. The investigators contribute complementary expertise and experience in a variety of disciplines ranging from proteomics, protein bioinformatics, analytical chemistry and instrumentation, to biochemistry, molecular biology and cell biology. At Indiana University, cross-linking mass spectrometry will be combined with biochemical and genetic assays to characterize the interaction between the proteasome and its substrates to provide candidates for novel substrate receptors on the proteasome. At The University of Texas at Austin, the biological relevance of candidates will then be tested in biochemical pro- teasome assays and genetic yeast experiments. These assays will define the contribution of newly identified components to substrate recognition and degradation by the proteasome and elucidate the molecular and struc- tural origin of substrate selection and specificity. Overall, we believe that this proposal will result in significant advances in cross-linking methodology and in our understanding of how the proteasome recognizes and digests substrates.