DE3P HRD1P IS REQUIRED FOR ENDOPLASMIC RETICULUM-ASSOCIATED DEGRADATION OF MISFOLDED LUMENAL AND INTEGRAL MEMBRANE-PROTEINS/

We have studied components of the endoplasmic reticulum (ER) proofread ing and degradation system in the yeast Saccharomyces cerevisiae. Usin g a der3-1 mutant defective in the degradation of a mutated lumenal pr otein, carboxypeptidase yscY (CPY), a gene was cloned which encodes a 64-kDa protein of the ER membrane. Der3p was found to be identical wi th Hrd1p, a protein identified to be necessary for degradation of HMG- CoA reductase. Der3p contains five putative transmembrane domains and a long hydrophilic C-terminal tail containing a RING-H2 finger domain which is oriented to the ER lumen. Deletion of DER3 leads to an accumu lation of CPY inside the ER due to a complete block of its degradatio n. In addition, a DER3 null mutant allele suppresses the temperature-d ependent growth phenotype of a mutant carrying the sec61-2 allele. Thi s is accompanied by the stabilization of the Sec61-2 mutant protein. I n contrast, overproduction of Der3p is lethal in a sec61-2 strain at t he permissive temperature of 25 degrees C. A mutant Der3p lacking 114 amino acids of the lumenal tail including the RING-H2 finger domain is unable to mediate degradation of CPY and Sec61-2p. We propose that D er3p acts prior to retrograde transport of ER membrane and lumenal pro teins to the cytoplasm where they are subject to degradation via the u biquitin-proteasome system. Interestingly, in ubc6-ubc7 double mutants , CPY accumulates in the ER, indicating the necessity of an intact cy toplasmic proteolysis machinery for retrograde transport of CPY. Der3 p might serve as a component programming the translocon for retrograde transport of ER proteins, or it might be involved in recognition thro ugh its lumenal RING-H2 motif of proteins of the ER that are destined for degradation.