In Vitro Reconstitution of CFTR Biogenesis and Degradation

The regulated degradation of cellular proteins occurs primarily through the ATP-dependent ubiquitin/proteasome pathway (1 ). One function of this pathway is to control the quality of nascent proteins (2 ). In this process ubiquitinconjugating enzymes and ubiquitin-protein ligases create isopeptide bonds linking the 8-kDa ubiquitin polypeptide to lysine residues on the target protein. Additional ubiquitin molecules are added via ubiquitin-ubiquitin isopeptide bonds until the resulting chain is recognized by a large multicatalytic protease, the 26S proteasome. This proteolytic machine is formed through the ATP-dependent association of two multimeric components, the 20S proteasome and the 19S regulatory subunit. The 19S subunit recognizes the polyubiquitinated protein, removes the ubiquitin chains, and feeds the protein into the 20S proteasome, a toroidal cylinder composed of four stacked heptameric rings (3 ). The two outer rings are comprised of α subunits that bind the 19S particles. The inner rings consist of � subunits, three of which exhibit distinct protease activities. These Pup1/Z/beta₂ , Pre2/X/beta₅ , and Pre3/Y/beta1 subunits preferentially cleave substrates after basic, hydrophobic, or acidic residues, respectively (4 ,5 ). These cleavage specificities are responsible for the trypsin-like (T-L), chymotrypsin-like (ChT-L), and caspase-like (CP-L) activities characteristic of eukaryotic proteasomes. The hallmark of proteasome-mediated degradation is ATP-dependent conversion of substrate into small peptide fragments 6-20 amino acids in length (6 ).