THE MECHANISM UNDERLYING CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR TRANSPORT FROM THE ENDOPLASMIC-RETICULUM TO THE PROTEASOME INCLUDES SEC61-BETA AND A CYTOSOLIC, DEGLYCOSYLATED INTERMEDIARY

Endoplasmic reticulum (ER) degradation pathways can selectively route proteins away from folding and maturation. Both soluble and integral m embrane proteins can be targeted from the ER to proteasomal degradatio n in this fashion. The cystic fibrosis transmembrane conductance regul ator (CFTR) is an integral, multidomain membrane protein localized to the apical surface of epithelial cells that functions to facilitate Cl (-)transport. CFTR was among the first membrane proteins for which a r ole of the proteasome in ER-related degradation was described. However , the signals that route CFTR to ubiquitination and subsequent degrada tion are not known. Moreover, limited information is available concern ing the subcellular localization of polyubiquitinated CFTR or mechanis ms underlying ret regrade dislocation of CFTR from the ER membrane to the proteasome either before or after ubiquitination. In the present s tudy, we show that proteasome inhibition with clasto-lactacystin beta- lactone (4 mu M, 1 h) stabilizes the presence of a deglycosylated CFTR intermediate for up to 5 h without increasing the core glycosylated ( band B) form of CFTR, Deglycosylated CFTR is present under the same co nditions that result in accumulation of polyubiquitinated CFTR, Moreov er, the deglycosylated form of both wild type and Delta F508 CFTR can be found in the cytosolic fraction. Both the level and stability of cy tosolic, deglycosylated CFTR are increased by proteasome blockade. Dur ing retrograde translocation from the ER to the cytosol, CFTR associat es with the Sec61 trimeric complex. Sec61 is the key component of the mammalian co-translational protein translocation system and has been p roposed to function as a two way channel that transports proteins both into the ER and back to the cytosol for degradation. We show that the level of the Sec61.CFTR complexes are highest when CFTR degradation p roceeds at the greatest rate (approximately 90 min after pulse labelin g). Quantities of Sec61.CFTR complexes are also increased by inhibitio n of the proteasome, Based on these results, we propose a model in whi ch complex membrane proteins such as CFTR are transported through the Sec61 trimeric complex back to the cytosol, escorted by the beta subun it of Sec61, and degraded by the proteasome or by other proteolytic sy stems.