Posttranslational elimination of incorrectly folded polypeptides from the endoplasmic reticulum ensures that only functional proteins are deployed to dista1 compartments of the secretory pathway. It is now recognized that by """"""""silencing"""""""" the posttranslational expression of mutant gene products, conformation-based quality control functions as a common basis for a diverse set of heritable pathologic conditions, each of which is caused by the hindered intracellular transport of a distinct mutant protein. Chronic destruction of lung connective tissue, resulting from heritable plasma alpha1-antitrypsin (AAT) deficiency, is caused by the impaired secretion of incompletely folded genetic variants of the protein from liver hepatocytes. The long-term goal of this research program is to gain molecular insight into the conformation-based quality control of AAT secretion. To this end, it is now understood that the processing of asparagine-linked oligosaccharides can facilitate the folding of a polypeptide to which they are attached by promoting a physical interaction with lectin-like molecular chaperones of the endoplasmic reticulum. Recent evidence indicates that the removal of mannose from asparagine- linked oligosaccharides functions as an obligatory step in the intracellular disposal of misfolded AAT, indicating that an overlap may exist between protein folding and degradation machinery. The hypothesis to be tested is that modification by intracellular mannosidases sorts genetic variant PIZ for non-proteasomal elimination by promoting specific sequential interactions with the glycoprotein folding sensor UDP- glucose:glycoprotein glucosyltransferase and a novel 95 kDa phosphorylated protein. Metabolic radiolabeling, coimmunoprecipitation of protein complexes, glycosidase inhibition, protein purification from transgenic mouse liver, and molecular cloning will be employed in three specific aims which are to: (1) elucidate how modification by intracellular mannosidases generates a nonproteasomal """"""""degradation signal"""""""", (2) determine whether reversible phosphorylation of a novel 95 kDa protein (pp95) regulates its physical interaction with variant PI Z, and (3) characterize pp95 and elucidate its obligatory role in PI Z disposal through the molecular cloning and expression of the recombinant molecule. Results from this study will identify the combinatorial roles of protein folding and quality control machinery in heritable plasma AAT deficiency plus enhance our understanding of how these fundamental processes participate in normal cell physiology.
