Newly emerging evidence demonstrates that the degradation of aberrant and unassembled proteins retained in the endoplasmic reticulum (ER) involves a novel protein exporting system from ER lumen to the cytosol for the proteasome-mediated proteolysis. The goal of the proposed study is to identify this novel pathway of ER dislocation involves retrograde translocation of polypeptides from ER lumen back to the cytosol via the Sec61p-formed translocation channel; several transacting proteins play crucial roles during this Sec61p-mediated dislocation process. In these proposed studies, yeast cells will be used as the research model. Both genetic and biochemical approaches will be taken to isolated sec61 mutants specifically defective in retrograde translocation, and to identify the distinct domains within Sec61p regulating the retrograde translocation event. The transacting proteins will be identified by transformation of the interesting genes and by screening for complementation suppressors. In vivo pulse-chase experiments and in vitro translocation-translocation- degradation studies will be carried out to illustrate this chaperone- aided. Sec61p-conducted ER dislocation process.
