CONTROL OF CELLULAR PROTEOLYSIS

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Proteins to be degraded via the cytoplasmic ubiquitin-dependent pathway are first covalently linked with ubiquitin. These ubiquitin-protein conjugates are then recognized and degraded by the proteolytic component of the pathway. One of the major thrusts of this proposed research is to identify this proteolytic component and to characterize the proteolytic reaction using ubiquitin-calmodulin as a substrate. In order to determine how an ubiquitin-protein conjugate is recognized and degraded by the proteolytic component of the ubiquitin dependent pathway, it requires a substrate having a known structure, and for which the proteolytic products can be characterized. Dictyostelium calmodulin forms a one-to-one conjugate with ubiquitin and specifically at lysine 115. All other substrate proteins reported to date are conjugated with ubiquitin at multiple sites, and the conjugation sites in these proteins have yet to be defined. Using ubiquitin-calmodulin as a substrate, we have tentatively identified an ATP-dependent protease in reticulocytes which degrades calmodulin only in the conjugated form. The proposed studies include the continued purification of this protease and to characterize the cleavage of ubiquitinated calmodulin. Synthetic peptides containing the isopeptide linkage, as well as small calmodulin fragments conjugated to ubiquitin, will be tested as potential substrates or as inhibitors. These results, together with the mapping of the cleavage sites of conjugated calmodulin, will be used to test the hypothesis that the isopeptide linkage region serves as the recognition site for the protease. Calmodulin isolated from most sources contains a trimethyllysine at position 115 and cannot be conjugated with ubiquitin. Our current hypothesis is that the post-translational methylation of this lysine stabilizes this protein by excluding it from degradation via the ubiquitin-dependent pathway. This hypothesis will be tested by measuring the degradative rates of various forms of microinjected calmodulin in oocytes under normal and under conditions in which the ubiquitin-dependent pathway has been rendered inactive. We will also test whether acetylation of the N-terminus of calmodulin serves the same function by using bacterial expressed chicken calmodulin which contains an unblocked N-terminus.