Cycloheximide, a new tool to dissect specific steps in ER-associated degradation of different substrates

To study the degradation requirements of unassembled immunoglobulin (Ig) ch ains, we heterologously expressed a cDNA encoding the secretory form of mur ine mu in the yeast S. cerevisiae, We found that mu chains were translocate d into and retained in the endoplasmic reticulum (ER) as they were N-glycos ylated and bound to the yeast homolog of BIP, Kar2p. Similar to mutant yeas t carboxypeptidase Y (CPY*), known to undergo cytosolic degradation, mu pro tein is stabilized in yeast mutants lacking the ubiquitinating enzymes Ubc6 p and Ubc7p or in cells overexpressing mutant ubiquitin, Unexpectedly, the translation inhibitor cycloheximide (CHX), but not puromycin, led to the ac cumulation of polyubiquitinated mu chains that were still glycosylated. By contrast, degradation of CPY* was not impaired by CHX, indicating that the drug affects a substrate-specific degradation step. In contrast to the situ ation for CPY*, the ER-transmembrane protein Der1p is not essential far mu degradation. Strikingly, however, the CHX-induced accumulation of polyubiqu itinated Ig mu, chains was stronger in Delta der1-mutants as compared to wi ld-type cells, indicating an additive effect of two inhibitory conditions. The results support a previously unknown activity of CHX, i.e. impairing th e degradation of transport-incompetent secretory mu chains. Moreover, this activity wilt allow to dissect substrate-specific steps in ER associated pr otein degradation.