INVOLVEMENT OF UBIQUITINATED PROTEINS IN AUTOPHAGY

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DESCRIPTION (Adapted from the applicant's abstract): Until recently it was thought that the ubiquitin and lysosomal protein degradation pathways of proteolysis functioned independently. Dr. Dunn and others have recently uncovered a linkage between these degradation pathways in stressed cells. In addition to higher molecular weight ubiquitin conjugates, Dr. Dunn has found two major ubiquitinated proteins (Ub60 and Ub68) within autophagic vacuoles (AVs) of nutrient-stressed cells. However, ts20 cells, defective in protein ubiquitination, are unable to degrade proteins in response to stress resulting in the accumulation of AVs that lack ubiquitinated proteins. The hypothesis to be tested is: The protein ubiquitination pathway is an essential component of autophagy-mediated protein degradation. To test this hypothesis whether or not ubiquitination of substrate proteins and/or proteins that comprise the machinery of autophagy are essential for lysosomal proteolysis will be examined. The acceptor protein of Ub68, ap40, will be cloned and epitope-tagged (c-myc) at its C-terminus, and lysines will be converted to arginines through site-directed mutagenesis. These recombinant proteins will be stably transfected into hepatoma cells and their turnover measured under nonstresses and nutrient-stressed conditions. Biochemical and morphological analyses in vivo and in vitro will evaluate the site of degradation of these proteins and investigate the role of ubiquitination of ap40 in its sequestration and degradation within AVs. The turnover rates of ap40-myc and ap40 lys-arg-myc will be compared in two normal cell lines, two ubiquitination mutant cell lines, and a revertant cell line that have been transfected with the recombinant proteins. Finally, the effects of ubiquitination of AVs isolated from the mutant cells on their ability to sequester and degrade ap40 will be evaluated. These studies will further characterize the relationship between ubiquitin-mediated protein degradation and stress-induced autophagy.