Role of ubiquitin in proteasomal degradation of mutant alpha(1)-antitrypsin Z in the endoplasmic reticulum

A delay in intracellular degradation of the mutant al-antitrypsin (alpha(1) AT)Z molecule is associated with greater retention within the endoplasmic r eticulum (ER) and susceptibility to liver disease in a subgroup of patients with alpha(1)AT deficiency. Recent studies have shown that alpha(1)ATZ is ordinarily degraded in the ER by a mechanism that involves the proteasome, as demonstrated in intact cells using human fibroblast cell lines engineere d for expression of alpha(1)ATZ and in a cell-free microsomal translocation assay system programmed with purified alpha(1)ATZ mRNA. To determine wheth er the ubiquitin system is required for proteasomal degradation of alpha(1) ATZ and whether specific components of the ubiquitin system can be implicat ed, we have now used two approaches. First, we overexpressed a dominant-neg ative ubiquitin mutant (UbK48R-G76A) by transient transfection in the human fibroblast cell lines expressing alpha(1)ATZ. The results showed that ther e was marked, specific, and selective inhibition of alpha(1)ATZ degradation mediated by UbK48R-G76A, indicating that the ubiquitin system is at least in part involved in ER degradation of alpha(1)ATZ. Second, we subjected ret iculocyte lysate to DE52 chromatography and tested the resulting well-chara cterized fractions in the cell-free system. The results showed that there w ere both ubiquitin-dependent and -independent proteasomal mechanisms for de gradation of alpha(1)ATZ and that the ubiquitin-conjugating enzyme E2-F1 ma y play a role in the ubiquitin-dependent proteasomal mechanism.